University Of Pennsylvania

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Pre-eclampsia is an obstetric condition that affects 5–8% of all pregnancies and is a leading cause of maternal mortality worldwide. Pre-eclampsia is characterized by the onset of maternal hypertension after 20 weeks of gestation, stemming from insufficient remodeling of the uterine spiral arteries that supply blood flow to the placenta and fetus. Clinical treatments for pre-eclampsia, such as antihypertensive drugs to manage blood pressure and anticonvulsants to prevent seizures, address the associated symptoms, yet no drug or therapeutic has been developed to slow the progression of pre-eclampsia. The only curative treatment option for pre- eclampsia is the early delivery of the placenta and fetus, which often resolves maternal hypertension within a few days, but can cause fetal morbidity and mortality, especially in cases where fetal growth restriction occurs concurrently with pre-eclampsia. Therefore, a significant unmet need exists for novel therapeutic strategies that can facilitate vasodilation in the placenta during pre-eclampsia to resolve maternal hypertension and improve fetal health. With no drug available to slow disease progression, engineering ionizable lipid nanoparticles (LNPs) for extrahepatic mRNA delivery to the placenta is an attractive therapeutic platform to treat pre-eclampsia. The goal of the proposed work is to develop a targeted, placenta-tropic LNP platform for the delivery of mRNA cargo to the placenta to treat pre-eclampsia during pregnancy. First, high-throughput in vivo screening will be utilized to evaluate a large library of LNPs with novel ionizable lipid structures and excipient compositions for extrahepatic tropism to the placenta. Lead candidates from this high-throughput screen will be functionalized with active targeting motifs, namely placenta-specific antibodies, for selective delivery to trophoblasts and endothelial cells in vivo. Following enrichment analysis using next generation sequencing, LNPs demonstrating placental tropism will be functionalized with placental-specific antibodies to promote active targeting to key cell types in the placenta — trophoblasts and endothelial cells. In vitro, ex vivo, and in vivo expression of therapeutic mRNA cargos (VEGF, PlGF, and eNOS) encapsulated in LNPs will then be evaluated in primary human and mouse placentas. The lead mRNA LNP candidates will then be utilized to rescue maternal hypertension, fetal health, and immunophenotype in an induced model of severe, early onset pre-eclampsia in mice. The work proposed here is the first to evaluate the therapeutic efficacy of a pro-angiogenic mRNA LNPs for treating pre-eclampsia, a placental disorder during pregnancy for which currently no curative treatment options exist. Due to the modular nature of LNPs and the ability to readily swap mRNA cargoes, the platform developed through the completion of these studies can be used for treating not only pre-eclampsia, but also a wide range of placental disorders occurring during pregnancy.

NIH Research Projects · FY 2025 · 2024-08

Abstract Mechanical forces exerted by fetal movement during development influence skeletal morphogenesis. Thus, fetal akinesia (insufficient movement), caused by low amniotic fluid volume, breech position or impaired muscle development, can cause skeletal disorders such as hip dysplasia, arthrogryposis, and impaired bone development. We found that maternal exercise (via wheel-running) rescued both bone and joint development in “muscle-less limb” (Spd) mice and that bioreactor-based mechanical loading of explanted Spd limbs rescued key aspects of limb development, directly implicating mechanical cues. To enable development of maternal exercise-based interventions for fetal akinesia, we need to know how maternal exercise rescues akinesia- impaired bone and joint development. Therefore, the goals of this application are to determine the cells that respond to maternal exercise to rescue akinesia-impaired development and to define the underlying signaling mechanisms. This will provide new insights into fetal akinesia and potentially identify maternal exercise as a therapeutic intervention.

NIH Research Projects · FY 2024 · 2024-08

Abstract Immunosuppression management follows standard protocol that is aimed at predetermined drug levels, with minimal adjustments over the lifetime of the recipient. While successful in preventing rejection, the high burden of suppression is associated with an increased incidence of drug-related side effects impacting the well-being of the recipient. The absence of objective assays to evaluate immune activation poses a challenge in determining the optimal dosage of immunosuppressive drugs and the ability to personalize therapy. Our NIH Immune Tolerance Network trial (ITN030ST) demonstrated that many of the recipients can maintain good allograft function with less immunosuppression, starting at a relatively early stage after transplantation. However, tapering immunosuppression based on clinical observation of allograft function risks the development of acute rejection, and the need for additional treatment to reverse rejection and rescue allograft function. Mechanistic studies using ITN030ST serum samples retrieved prior to, during, and after tapering of immunosuppression demonstrated that the trajectories of serum miRNAs are sensitive, specific, prognostic, and diagnostic of acute rejection. Taken together we hypothesize that highly sensitive and specific miRNA profiles can detect early molecular activation of immune response and allograft injury and will greatly improve safe management and allow personalizing immunosuppression while reducing the risk of rejection. The R34 application outlines a design for a prospective randomized study to assess whether serum miRNA profiling can assist in safe tapering of immunosuppression when compared to clinically guided management. The 3-phase approach to the design involve establishing trial procedures, followed by protocol design towards implementation, and concludes with an outcome assessment plan. A subsequent clinical study will assess whether miRNA profiles can be utilized as a predictor of rejection and aid in personalizing immunosuppression. We also propose the design of mechanistic studies that will leverage clinical samples and data to improve prediction of rejection. A novel technology developed by our investigators for methylation-based determination the cell/tissue origins of cfDNA. Using this method, it will be possible to interrogate both liver pathology and the activation of specific immune and inflammatory cell types, to identify early allograft injury and expose the underlying causes of dysfunction and immune activation when tapering immunosuppression. In the long run, this technology can get incorporated into protocols for personalized management of immunosuppression. The proposed application takes us one step closer towards developing a clinical study aimed at implementing biomarker-guided personalized immunosuppression in the transplantation setting. The study will also offer valuable insights into the molecular mechanisms underlying immune-mediated allograft injury associated with the tapering of immunosuppression.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Transcranial Magnetic Stimulation (TMS), sometimes in conjunction with Speech-Language Therapy, has been demonstrated to improve language function in subjects with chronic aphasia in a number of small studies. Several lines of evidence suggest that it is more effective when provided in the subacute stage after stroke but its efficacy in this setting has not been adequately assessed. We propose to study the effects of continuous Theta Burst Stimulation (a type of TMS) combined with a modified form of Constraint Induced Language Therapy (mCILT) in 63 subjects with subacute aphasia. Subjects will be randomized in a 2:1 ratio to cTBS with mCILT or sham cTBS with mCILT. After pre-treatment evaluation, subjects will receive 10 days of treatment; post-treatment evaluations will be performed 3-5 days and again at 4 months after the completion of treatment. We will use electrical field modelling to personalize stimulation intensity for each subject. Change from baseline in the Western Aphasia Battery-Revised Aphasia Quotient at 4 months after the end of treatment will serve as the primary outcome measure. A secondary aim is to identify anatomic and behavioral predictors of response to treatment. Finally, a third aim is to identify the mechanism underlying the beneficial effect of the treatment on brain organization using network neuroscience approaches. We will identify changes in the strengths of connections between nodes in the language network to address specific hypotheses regarding the effects of cTBS and mCILT on brain organization that are associated with beneficial response to treatment.

NIH Research Projects · FY 2025 · 2024-08

Populations experiencing health disparities often face limited access to resources, which impact the quality of mental healthcare that these communities receive. Researchers have focused on promoting cultural competency (CC) among therapists to address mental healthcare disparities. Therapist CC is broadly associated with improved clinical outcomes and therapeutic alliance. However, current methods of assessing therapist CC are infeasible to implement in clinical settings. Therapist-client power differentials limit clients from providing specific feedback, and behavioral coding is infeasible to implement in healthcare systems given difficulties with scaling up session evaluations. Natural language processing (NLP) provides a promising alternative to current methods of assessing and providing feedback on therapist CC, and recent applications of NLP based tools to psychotherapy settings indicate that they are viable methods of improving culturally responsive care. The purpose of the current study is to develop a prototype of a NLP-based therapist CC feedback tool (HEAL). We will first create an annotated dataset by coding 300 therapy sessions with a behavioral coding system assessing culturally responsive care (Aim 1). We will then create a prototype of HEAL (Aim 2) by (a) developing a feedback visualizer based on input from community advisory boards (CABs) comprised of supervisors and therapists, and one of individuals from populations experiencing health disparities who have accessed therapy in the past, or are currently in therapy, (b) selecting a speech recognition software, and (c) developing and validating NLP models based on the annotated dataset from Aim 1. We will use rapid cycle prototyping and testing to iteratively revise HEAL, meeting monthly with CAB members. We will assess acceptability, appropriateness, feasibility, and usability through interviews with CAB members, and quantitative measures. Finally, we will pilot the prototype of HEAL with 15 therapists using standardized patients, and assess acceptability, feasibility, appropriateness, and usability through individual feedback interviews and quantitative measures (Aim 3). The results of this study will culminate in the development of a novel therapist CC support tool, and ideally position me to pursue R01 funding to evaluate its effectiveness in a randomized clinical trial. The proposed aims are in line with NIMH’s strategic plan to develop innovative service delivery models to dramatically improve the outcomes of mental health services received in diverse communities and populations. The current study and training plan will allow me to develop expertise in community partnership in user centered design, and understand how cultural contexts and biases impact NLP models, and strongly position me to be a leader in multidisciplinary community-based research developing innovative methods of addressing mental healthcare disparities.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Vertebrate eggs lie dormant until fertilized, when a process called egg activation ensues that triggers the completion of meiosis and other processes that initiate embryonic development. These processes occur within the first several minutes after fertilization and depend on maternal factors supplied by the mother to the egg during oogenesis. Maternal factors exclusively drive early embryonic development in all animals, since the zygotic genome is not activated until at least one and typically several cell division cycles after fertilization. The large maternal contribution to vertebrate embryonic development is evident by the large size of eggs and early embryos compared to somatic cells (~10 to 1000 times greater, depending on the species). Moreover, unique features of this stage of development, such as acentrosomal meiotic divisions and mitotic spindles that cannot scale to the large cell sizes, necessitate unique maternal functions, not found in somatic cells. However, the roles that maternal factors play and how their vast supply is regulated spatiotemporally in the large egg and early embryo is little studied, despite its importance in reproductive success and fertility. The zebrafish provides an excellent model for studying the maternal regulation of development in vertebrates. Many maternally-regulated gene functions are conserved between zebrafish and mammals. Zebrafish eggs are externally fertilized, so are easily accessible, and their large size and translucency is advantageous for live imaging with fluorescent markers, which will be profited from here. A key event in egg activation is the completion of meiosis. At the end of oogenesis in vertebrates, the mature oocyte and egg are arrested in metaphase of meiosis II. The second meiotic division ensues when the egg is activated, thus generating the second polar body and a haploid complement of chromosomes to contribute to the zygote. Meiotic divisions are unique in oocytes and eggs as they lack the centrosome, the primary microtubule organizing center (MTOC) of a cell. While the meiotic acentrosomal MTOC (aMTOC) proteome has recently been described, little is known about how acentrosomal spindles are regulated and what normally restricts their nucleation in time and space. Here a zebrafish maternal- effect mutant named volcán will be studied, which displays multiple ectopic spindle-like MTs in the single cell blastodisc that cause a novel defect, the generation of numerous vesicle-like structures at the animal pole potentially polar body-like structures, but devoid of DNA. The dynamics, etiology in development, and nature of the spindle-like MT and vesicle-like structures abrogated by Volcan will be investigated, as well as the molecular nature of the volcan gene. The results of these Aims are expected to reveal the molecular identity of a key protein acting to restrict formation or organization of aMTOC spindle MTs in the egg to ensure a single polar body-like structure is extruded and that the egg is competent for embryonic development. These studies have direct relevance to reproductive success and women's fertility, including potential targets for infertility treatments.

NIH Research Projects · FY 2025 · 2024-08

SUMMARY/ABSTRACT 1 Current estimates indicate that ~30 % of the global adult population is affected by non-alcoholic fatty liver disease 2 (NAFLD). NAFLD is associated with an increased risk of morbidity and mortality from hepatic and extra-hepatic 3 complications, such as cardiovascular disease. Recent research has identified hepatic lipid droplets (LDs) as 4 important players in NAFLD, yet their exact role in the pathogenesis and progression of this disease is not fully 5 understood. One of the key functions of hepatic LDs is to store polyunsaturated fatty acids (PUFAs) and regulate 6 the availability of arachidonic acid (AA). Recent evidence has shown that plasma levels of PUFAs and 7 eicosanoids are associated with NAFLD severity and cardiovascular disease. However, the role of LD proteins 8 in regulating PUFA metabolism and liver inflammation has not been investigated. To address this gap, the 9 principal investigator (PI) will study common genetic coding variants in two LD proteins that may alter PUFA and 10 eicosanoid metabolism and, thus, affect NAFLD progression. The first is PNPLA3-I148M which is well-known as 11 a risk factor for NAFLD and associated comorbidities. The second is PLIN2-Pro251 which has been recently 12 shown to be associated with a reduction in hepatic triglycerides in a NAFLD mouse model. The hypotheses at 13 the heart of this project are that PNPLA3-I148M augments the release of AA from the LD membrane, increasing 14 eicosanoid production, liver inflammation and NAFLD progression, whereas PLIN2-Pro251 decreases the 15 release of AA from the LD membrane, thereby reducing eicosanoid production, liver inflammation and NAFLD 16 progression. Using a genome-first approach, in the K99 phase, the PI will conduct a `recall-by-genotype' study 17 to deep phenotype subjects who are PNPLA3-I148M homozygous without liver disease, and matched controls 18 (Aim 1); in parallel, the PI will use human-derived induced pluripotent stem cells (iPSCs) differentiated to 19 hepatocyte-like cells (HLCs) to determine the role of PNPLA3-I148M on eicosanoids metabolism and LD biology 20 (Aim 2). In the R00 phase, the PI will turn her attention to PLIN2-Pro251. Further embracing a `human phenomic 21 science' approach, she will deep-phenotype subjects homozygous for PLIN2-Pro251 without liver disease, and 22 matched controls (Aim 3) and study the role of this genetic variant in eicosanoid production, VLDL secretion and 23 LD biology in human-derived HLCs (Aim 4). The PI will learn the necessary techniques to accomplish the 24 proposed research under the guidance of her mentors (Dr Rader and Dr FitzGerald) and Advisory Committee. 25 Her training will include 1) becoming proficient in designing and conducting deep phenotyping studies involving 26 genetic variants, and 2) mastering the use of iPSCs as tools to recapitulate metabolic variations observed in the 27 population. The PI will also gain a better understanding of mass spectrometry, bioinformatics, extracellular 28 vesicles, and flow cytometry techniques by working alongside specialist collaborators. Combining these new 29 skills with her prior expertise in LD metabolism, hepatic inflammation and NAFLD, will enable the PI to develop 30 a `big-picture' systems biology perspective, an invaluable asset in her future role leading her own research team.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Specific microbial and immune features of vaginal ecosystems, including a Lactobacillus-deplete microbiota, have been linked to spontaneous preterm birth (PTB). Mechanisms driving associations remain incompletely understood. Premature cervical remodeling, involving cervicovaginal (CV) epithelial barrier dysfunction, is a key biologic process on the pathway to PTB. In other systems, metabolomics has emerged as a lens through which phenotypes of microbial ecosystems can be more clearly elucidated. Microbial and host metabolite- modification of immune responses and epithelial barrier integrity has given rise to novel targeted postbiotic therapeutics. The bioactive potential of the vaginal metabolome remains under investigated. Our group recently identified a role for vaginal polyamines in discerning individuals at greatest risk of PTB in those with similar microbiota-related risk — among individuals with a Lactobacillus-deplete microbiota, those who delivered preterm had a 25-fold reduction in spermine. Polyamines spermidine and spermine play key roles in immunity and oxidative balance in other systems. The overall goal of this proposal is to elucidate their effects on barrier function in the CV space. Our central hypothesis is that vaginal spermidine and spermine maintain immune homeostasis and mitigate oxidative stress to enhance barrier function in the setting of a Lactobacillus-deplete microbiota. We further posit that inadequate host production of spermidine and spermine in response to microbial challenge results in CV barrier dysfunction, ascribing a mechanistic link between vaginal metabolites and cervical remodeling. We propose the following aims: 1) determine how polyamines modify immune responses in the CV space; 2) determine how polyamines regulate oxidative balance in the CV space; and 3) quantify associations of vaginal polyamines with premature cervical remodeling. In the first aim, we will use in vitro methodology to interrogate the role of polyamines in inflammasome activation, NFB-induction of immune mediators, macrophage differentiation, and epithelial-immune cell communication. For the second aim, we will use in vitro methodology to investigate polyamine effects on oxidative stress measured by lipid peroxidation, antioxidant enzyme expression, DNA and protein damage, as well as the ability of polyamine-mediated oxidative balance to modify host immune responses. In the third aim, we will leverage an ongoing pregnancy cohort to quantify associations between polyamines, lipid peroxidation markers, and short cervix to assess the potential impact of polyamines on tissue remodeling and PTB. We propose an innovative approach to define mechanisms by which the vaginal metabolome may modify cervical remodeling. Implications of this work extend beyond pregnancy, as immune perturbations and oxidative stress contribute to myriad adverse reproductive outcomes. Findings may be harnessed to modify aspects of the vaginal ecosystem through novel postbiotic strategies, thereby improving reproductive health outcomes.

NIH Research Projects · FY 2025 · 2024-08

Project Description Hearing loss is the most prevalent sensory deficit in humans. Half of all cases of early onset hearing loss in developed countries have a genetic etiology, with single gene mutations in over 100 different loci identified so far. Mutations in the majority of these genes result in nonsyndromic sensorineural hearing loss, where abnormal inner ear function is the only diagnostic feature. In comparison to children, the genetic causes of hearing loss in adults are less well understood. Approximately, 80% of hearing loss cases are diagnosed after the second decade of life and yet, adults are rarely tested for genetic mutations as a possible cause of their condition. To help address this gap in knowledge our research seeks to elucidate the unexplained genetic underpinnings of adult-onset hearing loss, focusing on rare coding variants within established and undiscovered hearing loss genes. Taking advantage of a genome first approach in a hospital-recruited biobank cohort and functional studies in mice we identified ZNF175, and its mouse orthologue Zfp719, as a novel adult onset hearing loss gene. ZNF175/Zfp719 is a member of a large family of Krüppel-associated box zinc-finger proteins that bind DNA and silence target gene expression, including transposable elements and host genes, through the formation of heterochromatin. Our preliminary data indicate that Zfp719 mutant mice exhibit several inner ear pathologies, including synaptopathy, hair cell dysfunction, and hair cell degeneration that progress with age and hearing loss severity in a gene dosage-dependent manner. The overarching goal of experiments in this proposal is to identify the genomic targets of Zfp719 mediated heterochromatin silencing, and determine the pathogenic consequences of their misregulation on ribbon synapses, ion homeostasis, and innate immunity. Importantly, since dysfunction in each of these auditory processes has been implicated in adult onset hearing loss, our studies are likely to provide a better general understanding of this condition. Moreover, the outcome of these efforts may lead to the identification of new drug and gene-based therapies for hearing loss prevention and enable screening of high-risk individuals at an earlier age who might benefit from preventative and restorative treatment options.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY With the global scale-up of antiretroviral therapy (ART), increasing numbers of children with perinatally-acquired HIV (PHIV) are surviving into adolescence and beyond. Adults living with HIV on ART are at an increased risk for chronic age-related illnesses, including neurocognitive impairment, as well as oral disease compared to the general population. Early precursors of these disorders are also highly prevalent in children and adolescents living with HIV (ALHIV). Given that mental and oral health co-morbidities associated with HIV and lifelong ART could be driven by overlapping or distinct biological and immunological mechanisms, a better understanding of these mechanisms is needed to support interventions, particularly among ALHIV. Microbiota- or aging-mediated processes have been shown to contribute directly to HIV comorbidities. Increased incidence and prevalence of dental pathologies observed by our group and others among people living with HIV appears predicated by a differential colonization with pathogenic and commensal microbes. Emerging evidence suggests that controlled HIV infection alters microbial communities, contributing to a chronic low-grade inflammatory state that underlies age-associated conditions in children and youth with PHIV. In parallel, epigenetic age acceleration is observed in adults with HIV on ART when compared to controls and has been linked to neurocognitive deficits. The objective of this proposal is to identify oral microbial taxonomic and functional features, and aging markers associated with neurocognition and oral health in ALHIV. Our multidisciplinary research team is comprised of experts in HIV epidemiology, microbiology, dentistry, medicine, pediatrics, neuropsychology, bioinformatics and statistical modeling. We will leverage an established NIH-funded cohort of approximately 600 children and adolescents perinatally exposed (+/- PHIV) and unexposed (controls) to HIV living in Nigeria. In 50 ALHIV and 50 sex- and age-matched uninfected adolescents (aged 10-13), we will analyze shotgun metagenomic sequences of salivary samples to identify salivary taxonomic and functional profiles and will use the comprehensive Illumina MethylationEPIC BeadChip array to characterize DNA methylation and measure markers of epigenetic age acceleration in blood samples. In Aim 1, we will characterize shotgun metabolic sequences of salivary samples and measures of epigenetic age acceleration in blood samples to examine associations between (a) oral microbial and functional profiles and (b) epigenetic age acceleration with neurocognition outcomes in Nigerian adolescents with and without HIV. In Aim 2, we will test whether epigenetic age acceleration is associated with oral health outcomes in Nigerian adolescents with and without HIV. The research proposed in this R01 is significant because it will generate new insights into how microbiota- or aging- mediated mechanisms contribute to neurocognitive impairments and oral conditions in ALHIV.

NIH Research Projects · FY 2025 · 2024-08

ABSTRACT Cryptosporidium infections are a leading cause of diarrheal disease in young children for which no vaccine or universally effective treatment is available. Apicomplexan parasites such as Cryptosporidium, Toxoplasma, and Plasmodium actively invade their host cells and reside within a specialized membrane-bound compartment, or parasitophorous vacuole (PV). To survive and replicate, intracellular parasites must evade immune detection and extract nutrients from the host cytosol. Toxoplasma and Plasmodium secrete the contents of specialized secretory organelles, such as dense granules, to the PV where they orchestrate vacuole remodeling and immune evasion, while solute transporters at the parasite plasma membrane import small molecules such as amino acids and sugars. In addition, a conserved Kelch13-containing complex in these parasites is responsible for the formation of specialized membrane invaginations in the parasite plasma membrane that facilitate nutrient extraction from the host by bulk endocytosis. Cryptosporidium resides in a unique intracellular but extra-cytoplasmic niche. Transmission electron microscopy of intracellular Cryptosporidium revealed intricate ultrastructural features at the host-parasite interface, including electron dense bands, tight junction-like rings, and a highly invaginated membrane termed the feeder organelle. The three-dimensional architecture, composition, and function of these features are poorly understood. Recent studies and preliminary data have identified a handful of proteins that localize to the C. parvum host-parasite interface, including secreted effectors and solute transporters. The relationship between these components to each other, to the host cytosol, and to the ultrastructural features observed at the interface are unknown. In addition, the machinery responsible for shaping the membrane invaginations at the feeder organelle has not been identified. I hypothesize that transporters and secreted effectors occupy separate compartments at the interface, with transporters assembled in the parasite plasma membrane and effectors delivered to the PV membrane or lumen. I will uncover the three-dimensional ultrastructure, molecular architecture, and composition of the host- parasite interface at unprecedented resolution using cryogenic electron-microscopy-based volume imaging techniques. I will also use high-resolution optical microscopy and fluorescence complementation to determine whether transporters and dense granule effectors are trafficked to the same compartment. I also propose that the feeder organelle is shaped by K13 complex-mediated endocytosis, and I will determine the localization of these candidates in intracellular C. parvum parasites using light and electron microscopy. These studies will leverage innovative structural and molecular parasitology approaches to reveal mechanisms of membrane remodeling and nutrient acquisition fundamental to the biology of intracellular parasitism, yielding insights that will aid the rational design of therapeutics to combat this important disease.

NSF Awards · FY 2024 · 2024-08

Pathogens are a devastating stressor that result in hundreds of billions of dollars in lost crops every year. Thus, plants have evolved mechanisms to recognize and respond to these pests by turning specific sets of genes on and off in response to infectiontion. Part of this gene regulation includes regulating RNA, which are intermediary molecules made from reading the DNA of genes. Traditionally, scientists believed that the activity of RNA was dictated only by the sequence of the DNA from which it is transcribed, but recent discoveries have shown that regulation of RNA molecules is also highly controlled after it is transcribed. Recently, it was discovered that RNA stability can be controlled through the addition or removal of small molecules, such as methyl groups, to individual bases in RNA molecules. This new form of regulation, called epitranscriptomics, is still largely understudied in plants and has the potential to be used to help crops better deal with their pathogens. This project aims to characterize the addition and removal of one of these modifications (N6-methyladenosine, or m6A) to RNA at a global level and identify how these processes may improve plant tolerance to various pathogens. To accomplish this goal, the project will leverage and pioneer advances in RNA isolation and sequencing as well as new computational tools to analyze large amounts of sequencing data. These methods and tools will be made available for other science groups to rapidly apply the same techniques to their research questions. Overall, these findings will produce important new mechanistic insights and new resources for further studies focusing on functions of covalent RNA modifications, as well as studies developing more pathogen resistant crops. In addition to the broader impacts that our new findings will have on the fields of epitranscriptomics and crop improvement, we will also apply a novel training program that will prepare the next generation of epitranscriptomics researchers for the future of biology as a data-driven science. Plants have developed numerous regulatory mechanisms to recognize and, subsequently, direct precise transcriptome regulation in response to pathogen infection. However, the mechanisms responsible for directing transcriptome reprogramming during eukaryotic pathogen response are still quite unclear. Significant recent attention has revealed that post-transcriptional processes are just as important to regulating plant gene expression as transcriptional regulation. One feature of RNA molecules found to have significant post-transcriptional regulatory effects are covalent chemical modifications (e.g. methylation) of nucleotides; these are both widespread and physiologically relevant. This is especially true of m6A, currently the most abundant known internal messenger RNA (mRNA) modification. However, little is understood about the addition and removal of m6A or the role of these processes in post-transcriptional regulation of the plant transcriptome, either in normal development or during pathogen response. The project will address this significant knowledge gap using a combination of genomic, proteomic, and bioinformatics approaches, together with analytic software packages and web-based tools developed in our laboratory. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY Imagine having to choose between an apple or an orange for a snack. To decide between these famously incomparable options, you may think back to previous experiences you have had with either fruit and choose the one you enjoyed the most. To make a choice in this manner, it is crucial that you remember your previous snacks with a relatively high level of detail, specifically when it comes to the identity of the fruit. Conversely, imagine a decision-maker who is asked to choose between two novel options: a new brand of chocolate, and a fruit they have never tasted before. In this case, choosing the right snack relies on abstracting over the details of past experiences to infer the high-level value of chocolate versus fruit – the specifics of each experience are no longer relevant. Thus, less precise memory can facilitate the generalization of value to novel exemplars of a particular kind, while hindering people’s ability to choose between similar options. Here, we propose to test this hypothesis by leveraging the decreases in memory precision that occur naturally in healthy aging. We will collect fMRI data while participants take part in two variants of a decision task that relies on estimating value from past experience in service of choice. In Aim 1, participants will choose between items that are semantically and perceptually similar, meaning that precise memory is necessary for accurate choice. We predict that older adults will perform poorly at this task because of decreases in the precision with which they can remember stimulus identities. Furthermore, we predict that decreased pattern separation in the posterior hippocampus – a phenomenon by which similar items are kept separate in memory via enhanced neural differentiation – will be at the root of this effect. In Aim 2, participants will be asked to make choices between novel exemplars of specific categories whose value they can learn over time. We hypothesize that older adults, because of this same decrease in memory precision, will actually generalize category value faster than their younger counterparts, who may be more biased by noise from individual exemplars. Neurally, we expect to find greater pattern completion (the opposite of pattern separation) in the hippocampus of older adults, as well as increased connectivity between the hippocampus and other brain regions known to track category information (ex: medial prefrontal cortex). We will also characterize these effects computationally by comparing the performance of an “object only” reinforcement learning (RL) model in which each exemplar is assigned a separate value to a model that understands categories by separating them into discrete states towards which value can be assigned.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Despite antiretroviral therapy, people living with HIV (PLWH) have an increased risk of acquiring oral HPV and ultimately developing HPV-associated oral and oropharyngeal cancers compared to uninfected groups. While efforts are being made to understand the epidemiology of HPV-at-risk adult populations, the natural history of oral HPV in children and adolescents is largely uninvestigated. Determining the natural history of oral HPV infection in childhood and adolescence is critical in mitigating long-term risk and targeting vaccine intervention programs. Furthermore, the mechanisms underlying the persistence of oral HPV infection in PLWH are unclear, yet crucial to preventing HPV-associated cancers and diseases. Recent studies of the impact of HIV infection on the oral microbiome have shown differences in abundance of certain bacteria as compared HIV+ and HIV- children. Yet, there is limited understanding regarding the key microbial functional pathways that could facilitate oral HPV infection. The overall objectives of this project are to investigate the natural history of oral HPV infection and determine the taxonomic and functional roles of the oral microbiome in HPV persistence in the context of perinatal HIV exposure, infection, and treatment. To accomplish this, we will take advantage of a unique and large cohort of children/adolescents born to HIV-infected mothers vs. those born to HIV uninfected mothers. The proposed prospective observational study will recruit three groups of children and their respective mothers in Nigeria. Namely: 1) HIV-infected (HI), 2) HIV exposed-and-uninfected (HEU) and 3) HIV-unexposed-and- uninfected (HUU) children. The central hypothesis is that HI and HEU children, compared to HUU children, will have higher rates of oral HPV type-specific persistence, which is associated with a distinct microbial community and functional pathways. We will test this hypothesis via three specific aims: 1) Characterize and compare the natural history of oral HPV infection among youth born to HIV-infected women and uninfected women; 2) Identify inflammatory-mediated functional pathways associated with oral HPV persistence in children infected with HIV; and 3) Develop and test a deep learning algorithm based on clinical and oral taxonomic/functional microbiome profiles to predict oral HPV persistence. For the first aim, oral rinse samples from child-mother pairs will be used to characterize the dynamics of oral HPV subtype distribution over 2 years of follow-up. The second aim involves functional characterization of the oral microbiome using whole genome (shotgun sequencing) to investigate its relationship with HPV-type-specific persistence. For the third aim, we will train neural networks based on longitudinal microbiome features, cytokine levels, and other risk factors to determine which features strongly predict oral HPV. This project will lead to a more complete understanding of how impaired immunological states increase susceptibility to oral HPV infection and persistence. Knowledge gained regarding the contribution of oral microbiota, will lay a foundation for future prevention and therapeutic interventions.

NIH Research Projects · FY 2026 · 2024-08

Nearly half of all mammalian genomes originate from mobile sequences and ancient retroviruses called RetroElements (REs). REs consists of tens-of-thousands of nearly identical repetitive copies per RE-family and have been historically considered “Molecular Parasites” or “Junk DNA” as most are immobilized by mutations and epigenetic silencing. However, rare instances of gene disruption following RE integration have led to an “US versus THEM” model that fails to reflect symbiotic relationships developed over millions of years of co- evolution. This is best demonstrated by the well-known but poorly understood phenomenon of “RE-reactivation” in mammalian preimplantation embryos, in which thousands of related REs express simultaneously at distinct stages. REs are essential for development, as their disruption is embryonic lethal. Some REs have maintained regulatory features that influence expression, structure, and function of nearby genes and RE-signatures tightly overlap with events that are unique to preimplantation embryos, including zygotic genome activation, totipotency, reprogramming and pluripotency, supporting the hypothesis that RE-reactivation was perhaps the innovative catalysts for promoting conserved embryo specific programs through domestication of viral functions and rewired gene networks. In support, our single cell technologies and preimplantation RNA-Seq analysis to assess RE function revealed highly conserved expression patterns of RE families across 8 mammalian species. To study these, we developed an embryo editing technology (CRISPR-EZ) to delete 5 specific RE insertions in mice, each resulting in unique developmental defects. These findings revealed the first essential retrotransposon in mammalian development and established reactivation as part of “Normal Development” and shifting our view on “Junk DNA”. While silenced in most tissues, unintentional RT reactivation is commonly observed (and largely ignored) during epigenetic breakdown occurring in aging, autoimmunity, neurodegeneration, and cancer. Therefore, the goal of this proposal is to characterize RE regulatory networks and mechanisms that ensure proper development and leverage this information to explore the therapeutic potential of “synthetic RE- reactivation” to re-engage beneficial “emergent properties” in epigenetically compromised cells or block RE- Signatures in compromised cells to ameliorate or even reverse pathologies. This proposal pioneers a combination of comparative biology, genome editing and parallel in vivo / in vitro strategies using CRISPR/CAS9 variants and consists of three interconnected but independent aims. We use a human centric criterion to select RE candidates to 1) characterize conserved RE-mechanisms in development through humanized mouse models, 2) define RE regulation through a combined in vitro/in vivo CRISPR strategy and 3) assess the therapeutic potential of RE modulation in adult and diseased cells. Together, our findings will provide an atlas of REs that have been domesticated by host genomes as a reservoir of genetic innovation and how they can be further harnessed to study human embryo development and used as a therapeutic tool to improve human health.

NIH Research Projects · FY 2025 · 2024-08

Despite aggressive medical therapy, over one-third of the world’s 70 million epilepsy patients suffer from uncontrolled seizures. Surgery and implantable devices can control seizures in many patients, but these treatments are only effective when targeted accurately. Currently, targeting is manual due to the lack of rigorous methods to quantify epileptic networks, and when these targets are identified, there is no rigorous way to select the best surgical approach. Consequently, therapy varies dramatically across centers and patients. There is a critical need for standardized, quantitative methods to map epileptic networks and to target and optimize therapy. My long-term goal is to develop these quantitative methods, create a scalable infrastructure to implement them at scale, and rigorously validate and translate these methods into clinical practice. My overall objective is to integrate non-invasive structural imaging, which provides a comprehensive anatomical view of the brain, with invasive IEEG that aims to pinpoint seizure origin and spread. I will develop rigorous, quantitative methods to map epileptic networks that cause seizures to guide epilepsy surgery. My central hypothesis is that patient outcome after epilepsy surgery depends on what percentage of abnormal regions quantified on neuroimaging and IEEG are removed. With this central hypothesis, I will develop tools for clinical translation by (1) developing standardized quantitative methods that generalize across epilepsy centers, (2) developing and validating new methods to integrate structural imaging and IEEG, (3) implementing methods to run at scale on a large number of patients, representing the diversity of epilepsy, across centers. In Aim 1, I will develop scalable methods to automate aggregation and multimodal analysis of structural imaging, IEEG, and clinical data from multiple epilepsy centers. In Aim 2, I will develop normative methods that merge structural imaging and IEEG data, to identify abnormal epileptic networks by comparing individual patient’s data with the norm. Undertaking Aims 1 and 2 during the K99 phase will enhance my proficiency in cloud computing, scalable analysis, multicenter biostatistics, and clinical translation. In Aim 3, I intend to implement quantitative methods to run at scale to predict surgical outcomes in two specific populations: patients with temporal and extratemporal lobe epilepsy. Multiple conceptual and technical innovations are embedded in this proposal to overcome translational barriers that limit generalization, rigorous validation, and scalability. These include innovative tools to scale analysis, novel personalized localization methods, collaborative validation, and data sharing across 15 US epilepsy centers. This work is significant because it merges state-of-the-art engineering, neurology, and neurosurgery to make practical tools to improve and standardize patient care by quantitatively guiding epilepsy surgery.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Cortical mechanisms for contrast gain control in auditory perception in noise. Everyday natural auditory environments are variable, ranging from quiet beaches to raging waterfalls. In order to identify and robustly encode important sounds like speech from the background noise, the auditory system needs to flexibly adapt to the current environment. One way the auditory system does this is by modifying neuron response properties according to the statistics of the auditory environment. Neurons in the primary auditory cortex (A1) adapt response function slope (gain) to account for changes in auditory variability (contrast). This gain adaptation process is associated with corresponding changes in perceptual sensitivity. The mechanisms driving this process are still unknown. The goal of this proposal is to identify the circuit mechanisms responsible for contrast-dependent perceptual changes and neuronal gain adaptation in A1. In addition to excitatory neurons, A1 comprises many types of inhibitory interneurons. Two subtypes, parvalbumin-positive (PV) and somatostatin-positive (SST) interneurons, are capable of shaping the gain of their surrounding excitatory neurons. We hypothesize that PV and SST neurons work together to differentially adapt neuron gain to auditory contrast. We will first test this hypothesis by establishing whether PV and SST neurons are sensitive to the environment’s contrast. Using electrophysiological recordings with optotagging, we will record PV and SST neuron activity in mouse A1. Next, we will test whether SST and PV activity drives the perceptual sensitivity changes associated with gain adaptation. By optogenetically perturbing PV and SST activity in A1 of mice performing target-in-noise detection tasks with variable stimulus contrasts, we will quantify the relative contributions of PV and SST neurons to perceptual sensitivity changes. Together, these results will characterize the roles of inhibitory cortical circuits in adaptation to the current auditory environment, deepening our understanding of effective auditory processing in noisy environments.

NIH Research Projects · FY 2025 · 2024-08

Myocardial infarction (MI) from coronary artery obstruction affects more than 1 million people annually in the United States resulting in symptoms, reduced quality of life, and substantially increased risk of heart failure and sudden death. Rapid diagnosis on electrocardiography and treatment with percutaneous intervention (PCI) are essential to minimize the risk and size of permanent myocardial injury as well as the risks of ischemia or reperfusion triggered malignant arrhythmia. Ironically, restoration of blood flow to infarcting myocardium can result in reperfusion injury and expand infarct size. Recent data from animal studies suggest that reperfusion injury may promote lipomatous metaplasia (LM) or intra-myocardial fat deposition. LM has been shown to associate with negative cardiac remodeling, leading to worsening heart function and higher chances for congestive heart failure. We have shown that LM is prevalent but highly variable in distribution among patients with prior MI and ubiquitous among those presenting with ventricular tachycardia (VT). We have also shown that corridors critical to VT circuitry traverse infarcted tissue through or near LM. The latter association appears to be mediated by prolonged local action potential duration, reduced conduction velocity, as well as increased regional resistance and reduced current loss as impulses traverse corridors adjacent to LM. In prior studies, we have also shown that reperfusion injury, the apparent precursor to LM, can be quantified by cardiac magnetic resonance (CMR) as pathologic iron deposition, and is present in most patients despite nominally successful reperfusion. However, the association of reperfusion injury with LM incidence and progression in humans have not yet been defined. Additionally, no prospective study has evaluated the longitudinal evolution of scar, LM, and viable tissue architecture with the incidence of VT. Thus, we propose a prospective observational study of 175 patients <2 months since PCI for acute ST elevation MI (STEMI) that undergo CMR at baseline, and at 1- and 2-years’ follow-up. Using data from this cohort we will test the following premises: 1. That LM incidence occurs early post STEMI and that its volumetric progression is associated with time since reperfusion; 2. That the extent of LM is associated with reperfusion injury following PCI for STEMI; and 3. That LM precedes and predicts the incidence of sustained VT following MI. Our group has extensive experience with STEMI and VT management, image acquisition and analysis, epidemiology, and biostatistics. The findings of this study will have wide applicability to our mechanistic understanding and management of cardiac remodeling and VT.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/ Abstract Osteoarthritis (OA), one of the leading causes of disability in the US, is characterized by cartilage degradation, synovitis, and subchondral bone remodeling. While there are currently no treatments for OA, there is increasing evidence that chronic low-level inflammation plays a role in driving disease progression. Inflammation during OA manifests as the infiltration of joint tissues (especially the synovium and subchondral bone) by immune cells, including macrophages, T cells, and NK cells. Although mechanisms driving synovial inflammation are currently an intense area of study, little is known regarding how inflammation in the subchondral bone (SCB) occurs and its consequences on remodeling and joint homeostasis. Recently, Toll-like receptor (TLR)-mediated inflammatory pathways have been implicated in OAassociated synovitis, cartilage erosion, and bone remodelling. Our previous work demonstrated that mice deficient in CD14, a TLR co-receptor, are partially protected from disease in a post-traumatic OA model. Specifically, CD14- deficiency resulted in a significant reduction in SCB bone thickening and cartilage damage after joint-injury compared to wild type mice, with the most substantial amelioration of the SCB pathology. OA bone pathology extends beyond signs of remodeling, and also includes an increase in inflammatory cells in the bone marrow. 1bis inflammation may contribute to pain in OA, but the mechanisms driving SCB inflammation are poorly studied. Thus, I propose that TLR signalling in osteoclasts modulates the osteoclast secretome, resulting in osteoblastogenesis as well as T cell migration. To explore this, I will first show in vitro that TLR signaling leads to the release of factors that are both proinflammatory and osteoblastogenic (Aim 1 ). Next, I will use IMC to characterize the T cell landscape in the subchondral bone of osteoarthritic mice and determine anatomic associations between T cells and osteoclasts 1 (Aim 2a). Lastly, I plan to develop a novel osteoclast T cell migration assay to show that TLR signaling in osteoclasts increases proinflammatory T cell migration (Aim 2b). Successful completion of these Aims will provide a better understanding of the cellular and tissue-level changes that occur in the subchondral bone in OA, as well as new targets for therapeutic intervention. I have collected a significant amount of preliminary data, motivating the direction of these aims and showcasing the skillset and tools I have already become proficient in. Additionally, with the help of my mentors as well as my collaborators Dr. Su and Dr. Liu, I have all the necessary expertise to ensure the successful completion of this project.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT Hyperphosphorylation to the microtubule (MT) associated protein Tau is the primary driving force of Tau aggregation, a pathological hallmark of Alzheimer’s disease. Tau binds to and stabilizes MTs, especially in neuronal axons. Phosphorylation causes Tau to dissociate from MTs, thus destabilizing them while also increasing the vulnerability of Tau to further phosphorylation and toxic aggregation. Yet, clinical trials aimed at either stabilizing MTs or degrading phospho-Tau (pTau) have yet to succeed. A major caveat of these trials is that they did not stabilize the interactions of specific Tau species to MTs, which is a feasible strategy to stabilizing Tau function and preventing its toxic aggregation. It has been challenging to achieve this clinical goal because there are critical gaps in the Alzheimer’s disease field in distinguishing pathological from functional pTau species, and mapping dysregulation of the pathways that drive Tau toward its hyperphosphorylated state. Most Tau research to date has focused exclusively on the longest Tau isoform even though it is the least abundant isoform in the brain. However, there are a total of 6 Tau isoforms in the brain, expressed at varying protein levels. I have made several discoveries connecting phosphorylation of Tau to its function and dysfunction in an isoform-dependent manner. I found that i) Tau isoforms have different phosphorylation levels in human neurons, ii) pTau species that are associated with Alzheimer’s disease are also abundant and soluble in normal developing brains, iii) phosphorylation regulates Tau-MT interactions and aggregation levels in an isoform- dependent manner. These findings support my central hypothesis that phosphorylation impacts Tau-MT interactions in an isoform-dependent manner (AIM 1), modulated by isoform-specific regulation of Tau-MT interactions (AIM 2). While the study of Tau hyperphosphorylation is a rich field, many studies have been limited by Tau overexpression systems and/or exclusive focus on pathology. Furthermore, high-throughput tools to test for regulators of Tau-MT interactions were not available until recently. I will address these concerns by using new technologies to contrast Tau function and pathology. I will receive training to apply CRISPR prime editing, a new and highly efficient gene editing technique with reduced off-target effects, to endogenously edit phosphorylation sites of interest to Tau to test for isoform-specific effects on its binding to MTs in neurons (AIM 1). Second, I will apply a powerful new technology, pooled optical CRISPR screening, to identify trans-regulators that increase dissociation of Tau from MTs. Optical screening allows for genotype-phenotype matching of virtually any phenotype that can be imaged under a microscope. This aim will uncover both known and novel genetic risk factors for Alzheimer’s disease, thus opening several avenues for my future studies beyond this proposal.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY AND ABSTRACT Macromolecular signaling complexes play fundamental roles in biology, ranging from the control of development to regulation of the immune system. The inflammasome is an example of a macromolecular signaling platform that activates the innate immune system in response to invading pathogens or endogenous host-derived danger signals. As the innate immune system regulates inflammation and has crosstalk with the adaptive immune system, dysregulation of inflammasome signaling is associated with autoinflammatory and autoimmune disorders. Activation of the inflammasome involves the recognition of intracellular danger signals by germline- encoded pattern recognition receptors that then recruit an adaptor protein known as ASC (although in some instances ASC is dispensable) to form the inflammasome. ASC then recruits a cysteine protease known as caspase-1 to the inflammasome where it undergoes autoproteolytic maturation. Active caspase-1 then cleaves substrates that are important for restoring cellular homeostasis, including the interleukin family of cytokines, IL- 1 and IL-18, and the pore forming protein GSDMD to induce a lytic type of cell death known as pyroptosis. The mechanism of how inflammasomes are assembled, in particular, how the different proteins are organized in space and time to ensure proper function, is not well understood. Of note, the incorporation of ASC on the inflammasome is required for cytokine processing, but the mechanism of how ASC induces cytokine activation has remained unclear for over a decade. Additionally, some inflammasomes are ASC-independent but can form ASC-containing inflammasomes. How these inflammasomes decide when to form an ASC-independent or ASC containing inflammasome is a mystery. The goal of this proposal is to address these fundamental questions in innate immunity. We will elucidate the mechanism of cytokine activation and delineate the spatiotemporal regulation of inflammasomes. To accomplish this, we have developed new tools that will allow us to build a spatiotemporal map of inflammasome formation and signaling, which will provide key mechanistic insights to aid therapeutic development for immune related disorders. Furthermore, we anticipate these tools will become important biomedical research tools.

NIH Research Projects · FY 2025 · 2024-08

The Penn Injury Science Center (PISC) will build on its past successes as an injury control research center (ICRC) at the University of Pennsylvania to advance our mission of reducing injuries and violence across the lifespan through equity-centered, actionable science, outreach, and education. The PISC is evolving and progressing to specifically focus on the integrated application of research, training and education, and outreach to improve the lives of the populations disproportionately affected by violence and injury. We recognize that the injuries we study are not distributed equally across populations; historic disinvestment and other forms of structural racism have led to marked disparities that catalyze, moderate, or mitigate the incidence of injury, access to prevention and treatment, and ability to recover. In this next cycle as an ICRC, we will continue partnering with Black and Brown communities in which the PISC is situated to conduct local, actionable injury science that prioritizes public health equity and addresses the social and structural conditions that contribute to the burden of violence and injury these communities experience. The PISC’s outward-facing slogan of Stop It, Fix It, Live On aligns with our mission of empowering community-research partnerships to address primary (Stop the event), secondary (Fix the causes of the event), and tertiary (Live On in a context of safety rather than risk) injury and violence prevention goals. The PISC is organized around three Cores (Administrative, Outreach, Training & Education), essential Special Advisors (Equity and Academic-City partnerships), integration with the Children’s Hospital of Philadelphia (CHOP), and three advisory boards (Institutional Advisory Board, External Advisory Board, and Community Action Board) as well as four embedded Research Projects and new research development efforts. Our four Research Projects address some of the most salient injury problems: cross-cutting violence prevention, drug overdose and adverse childhood experiences. Across the PISC we have prioritized diversity in leadership from underrepresented groups in our proposed Cores and Projects, as well as mentoring of developing injury researchers from diverse backgrounds and disciplines to become scientific and programmatic leaders. By being the hub for injury science in our University and City, our innovative approaches in the bidirectional research-practice-policy exchange will enhance the highest caliber research, disseminate findings, implement new practices, promote sustainable outreach activities, and train the next generation of injury scientists to maximize the impact of our work for equitable outcomes in disproportionately affected populations.

NIH Research Projects · FY 2025 · 2024-08

Abstract We propose to investigate the role of the chromatin methyltransferase DOT1L in neuronal function and determine how its disruption leads to neurodevelopmental disorders (NDD). Although the genetic causes of NDDs are heterogeneous, a high proportion of causative mutations are within the genes that encode chromatin regulators. Chromatin is the complex of DNA and the histone proteins that organize the genome and control gene expression. Chromatin regulating enzymes deposit a wide range of posttranslational modifications on histones such as methylation, acetylation, and many others. Interestingly, recent advances have identified mutations in the histone methyltransferase DOT1L in NDD patients with intellectual disability and developmental delays. However, the mechanisms through which DOT1L functions in the brain remain largely unknown. DOT1L is the sole methyltransferase of histone 3 lysine 79 where it deposits methylation marks (H3K79me). Patient mutations are de novo, monoallelic, and cluster in the catalytic domain. Our preliminary data indicate that they likely act as loss-of-function mutations and decrease methylation of H3K79. In addition, we found that DOT1L and H3K79me increase during neuronal development and that DOT1L depletion affects transcription of critical neuronal synaptic genes. Together, this work suggests that DOT1L plays a critical role in neuronal development and function. We hypothesize that partial loss of DOT1L and H3K79me disrupt transcription leading to cognitive deficits and changes in neuronal maturation and synaptic gene expression. To test this, we will bring together biochemical studies, genome-wide sequencing, and new cell and mouse lines to generate a model of the patient disorder and define the function of H3K79me in neurons. Merging new systems with a wide range of approaches has the potential to define how DOT1L affects cognition. We will first employ a heterozygous Dot1l knockout mouse model to examine how partial loss of DOT1L affects chromatin, transcription, neurogenesis, neuronal maturation, and behavior to provide insights into the disorder. Next, we will focus on H3K79me using a new mutant embryonic stem cell line that allows us to specifically examine the effects of H3K79me in differentiated neurons without perturbing other functions of DOT1L. We will use this stem cell model to measure H3K79me genomic localization during neuronal development and determine how H3K79me loss affects transcription and neuronal differentiation. By merging these diverse approaches, we will expand our understanding of both an emerging disorder and the role and regulation of H3K79me in neurons. In addition, these experiments will contribute to the broader understanding of how epigenetic regulators play a role in brain function and how their disruption leads to neurodevelopmental disorders.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract Computations reveal valuable information about organic reactions including those catalyzed by transition metals. Organic chemists can map out potential energy surfaces using density functional theory (DFT) and gain insight into the mechanism of a transformation. Using transition state theory, the reactivity and selectivity of a reaction can be explained by these studies. However, reactions with selectivity that cannot be explained by energy calculations performed with DFT may require the use of quasi-classical molecular dynamics simulations. One transformation where this type of modeling is vital is in the Michael addition and nickel cross-couplings using Watson's pyridinium salts. Despite the best experimental efforts, the Michael addition forms many byproducts in addition to product and the nickel cross-coupling gives only byproduct. The aim of this project is to study the dynamic behavior of alkyl radicals from a series of pyridinium salts and use this knowledge to enable reaction design. While the barrier computed by DFT for the alkyl radical to undergo Michael addition is lower in energy than recombination with the pyridine, a greater amount of byproduct is observed experimentally. This lack of agreement between DFT calculations and experiments points to a need for modeling of dynamic effects. I propose that the generation of the alkyl radical via C–N bond breaking of the pyridinium salt is an ambimodal transition state, which does not follow the intrinsic reaction coordinate (IRC) pathway to the alkyl radical but rather recombines with the pyridine, forming byproduct instead of the statistically favored product. I propose that the nature of the pyridine substitution pattern will affect the distribution of products, with more electron-withdrawn pyridines favoring radical addition. After verifying that more electron-rich, sterically hindered pyridines will favor productive reaction, the escape of the alkyl radical from the solvent cage will be modeled. A detailed understanding of the lifetime of the alkyl radical in solvents of varying polarity is needed, with the hypothesis being that more polar solvent leads to a more stable radical which is more likely to undergo further chemistry. This study is accessible solely through molecular dynamics simulations, an underexplored area that is vital to reaction design in systems with ambimodal transition states. Using the training and information gained in this study, in the R00 phase, I will develop chemistry to expand on existing methods of three-component coupling processes of C-aryl glycosides in which molecular complexity can rapidly be generated from a simple scaffold. These methods are vital to future drug design with the goal of lowering drug cost by simplifying the synthetic pathway to access certain drugs as well as using earth-abundant metals like nickel and iron in the R00 phase.

NIH Research Projects · FY 2025 · 2024-08

Project Summary The cellular and molecular mechanisms controlling cell cycle entry and plasma cell differentiation remain poorly understood. One key facet to this problem is how naïve B cells achieve a state of readiness for the plasma cell (PC) fate. This project strives to understand the molecular mechanisms whereby activation of the Notch pathway in naïve B cells fosters biochemical events that accelerate both mitosis and PC differentiation. Further, this project addresses whether these processes will amplify B cell responses to SARS-CoV-2 immunogens. One major hurdle to vaccine development for complex pathogens are facilitating diverse and durable antibody responses that can keep up with rapid mutations and stand the test of time. As such, understanding the mechanisms that underlie optimal B cell responses are vital to improve vaccine design. One distinctive B cell subset, marginal zone (MZ) B cells, exhibit a selective advantage at generating effector responses. Residing in the marginal sinus of the spleen at the interface between incoming blood supply and lymphoid follicles, this innate-like subset responds to blood-borne antigens, serving as a first line of defense to generate antibody- secreting PCs in a matter of hours. Unlike conventional follicular (FO) B cells, MZ B cells have a distinctive requirement for the signal Notch2, an evolutionarily conserved transmembrane receptor family member that dictates cell fate decisions. Notch2 is known to drive lineage commitment of the MZ B cell pool during development, but how this signal is used continuously to maintain mature MZ B cells is poorly understood. As such, it is reasonable to speculate that Notch2 signaling instructs a constitutively poised state in resting B cells by modifying activation requirements and differentiative events. Indeed, preliminary data demonstrate the induction of Notch2 signaling in non-poised FO B cells enhances their responsiveness to antigen receptor or TLR signals to promote their proliferation and differentiation into PCs. The central hypothesis of this proposal is that Notch2 independently augments PC differentiation and cell division, both features which hold potential to amplify vaccine responses. Herein, this proposal will independently interrogate the mechanism(s) by which Notch2 modifies proliferative and differentiative potentials in aim 1, and the potential for Notch2 signals to improve SARS-CoV-2 vaccine responses in aim 2. The significance of investigating how Notch2 regulates B cell responses is twofold. For one, this proposal will challenge the current understanding of Notch2 as a determinant of cell fate decisions, elucidating how this signal is tied to activation and effector programs. Additionally, this proposal can better inform vaccination strategies using Notch2 signaling as a tool to enhance the frequency and diversity of a given antibody response.