University Of Chicago

NIH Research Projects · FY 2025 · 2021-07

Project Summary Cancer is a leading cause of death worldwide. Recent advancements in the use of immune checkpoint blockade have transformed oncology, and immunotherapy has extended the potential application of radiotherapy to systemic disease. More than 200 clinical trials of combined checkpoint blockade and radiotherapy are ongoing or completed. The results of these preliminary trials demonstrate efficacy only in a limited subpopulation of patients. Treatment resistance likely manifested by poor T-cell priming and tumor- mediated immunosuppression continue to constitute significant barriers to optimal patient outcomes; therefore, the opportunity for transformative clinical impact is real in this setting. We propose a new and innovative strategy guided by new findings to improve the interaction of radiotherapy and immunotherapy by incorporating the latest techniques in the emerging field of mRNA modification with well-established radiobiological and immunological approaches. We will leverage collaboration with our Co-Investigator Chuan He, who helped to discover and decipher reversible RNA methylation in post-transcriptional gene expression regulation. We will use the novel techniques to identify the binding sites of Y1 and Y2, and incorporate integrated bioinformatics analysis approaches to investigate the impact of m6A readers in functional pathways of immune cells in irradiated tumors. These techniques are new and, to our knowledge, have yet to be applied in the context of radiotherapy and radioimmunotherapy. We hypothesize that targeting regulation of m6A modifications associated with m6A-binding protein YTHDF1 (for improved antigen presentation and T-cell priming) and YTHDF2 (for alleviation of immunosuppression) will potentiate anti-tumor immunity in the context of both RT alone and RT combined with anti-PD-L1 antibodies. Our proposal focuses on 1) establishing YTHDF1 (Aim 1) and YTHDF2 (Aim 2) as viable targets for RT and radioimmunotherapy, and 2) uncovering underlying pathways. Small molecules from the He Lab will be used to validate our hypothesis. The ultimate goal of this therapeutic approach is to modulate gene expression via targeting m6A methylation related to translation (Y1) or degradation (Y2) of mRNA in order to potentiate immune response. We are uniquely positioned to discover new knowledge and elucidate an unprecedented level of mechanistic understanding of the complex molecular and cellular interplay between radiotherapy and checkpoint inhibition in the context of the immune system. These new findings will provide the mechanistic data required for translational pursuit of superior treatment strategies. Increased local and/or distant control to actualize radio-immunotherapy would be a practice-changing and would broadly enhance cancer care and expand the pool of patients who respond to inhibition of the PD-1/PD-L1 axis.

NIH Research Projects · FY 2025 · 2021-07

Project Abstract This proposal concerns the development of new reagents and strategies for the preparation of basic, aromatic, nitrogenous heterocycles through single-atom insertion reactions. The medicinal importance of such structures (pyridines, pyrimidines, pyridazines) is difficult to overstate – of the thirty new small molecule drugs approved in 2019, ten of them contain one or more of these motifs and their prevalence among medicinally-relevant compounds is a long-standing trend. This privileged status has prompted the development of a variety of synthetic strategies for their preparation, which can largely be subdivided into de novo assembly of the heteroaromatic nucleus (typically condensations), and attachment of preformed heteroarenes via cross-coupling and nucleophilic aromatic substitution approaches. These strategies have enabled the proliferation of such compounds for wide-ranging medicinal applications, but their implementation is nonetheless far from trivial, necessitating the continuing development of novel strategies. A conceptual mid-point exists between de novo synthesis and attachment wherein one heterocyclic structure is converted into another. Such an approach has limited historical precedent but holds substantial promise due to the orthogonal reactivity preferences (e.g. nucleophilic vs. electrophilic) and reaction compatibility of 5- vs. 6- membered heterocycles. We propose herein a set of reagents which will enable such transformations to be realized in a synthetically straightforward manner. Our focus on single-atom changes is calculated: rearrangement reactions, though lauded, are rarely employed in synthesis due to their retrosynthetic complexity. By developing transformations that are easy to recognize in a retrosynthetic sense (e.g. “remove this nitrogen atom”) we hope to provide user-friendly tools for medicinal chemists to employ. Moreover, the ability to transform pyrroles, pyrazoles, and imidazoles into a variety of 6-membered ring heterocycles feeds naturally into late-stage skeletal editing of pharmaceuticals, allowing diversification of bioactive scaffolds for more efficient structure- activity relationship determination and for site-specific isotopic labeling. As such, successful realization of the goals enumerated herein will advance the ability for chemists to interrogate biological function of heteroaromatic compounds by affording a powerful new set of tools for their synthesis.

NIH Research Projects · FY 2025 · 2021-07

Emerging lines of evidence suggest an intimate crosstalk among energy metabolism, metabolites and epigenetics. Post-translational modifications (PTMs) on histones (histone “marks”) (e.g., lysine acetylation (Kac) and methylation (Kme)) are known to be regulated by metabolism, contributing to the epigenetic programs that are associated with cellular physiology and disease. However, we do not yet know if additional histone PTM pathways exist and if they can be modulated by diverse cellular metabolites. Thus, chemistry and biochemistry of metabolites-mediated chromatin changes remain poorly characterized. Lactate, a widely known cellular metabolite, can be dramatically induced under some cellular conditions (e.g. hypoxia) and in the Warburg effect, an observation most commonly shared among diverse cancers and associated with many diseases. Lactate concentration can rise to 20-40 mM in cancer tissues. Although this compound was discovered ~200 years ago, its non-metabolic functions in physiology (e.g., hypoxia, stem cell differentiation and immunoresponse) and disease (e.g., cancer and diabetes) remain unknown, representing a long-standing question in biology. We recently discovered a lactate-derived, new lysine modification, lysine lactylation (Kla). We comprehensively validated this PTM by chemical and biochemical approaches. This PTM can be stimulated by the Warburg effect-derived lactate and has different temporal dynamics from the widely studied lysine acetylation (Kac). Our epigenetic studies suggest that histone Kla represents a new type of metabolism- regulated epigenetic changes and contributes to gene regulation. We hypothesize that the histone Kla pathway is molecularly distinct from Kac pathway and contribute to gene regulation. We therefore propose to characterize the Kla pathway by defining its key regulatory elements: enzymes that can remove the modification (or delactylases), and their targets on histones and non-histone substrate proteins. We will also study their role in epigenetic regulation in cyclic behavior of hair follicle stem cells (HFSCs) in which lactate and its regulatory enzyme play a key role. We will use an integrated strategy involving chemical biology, enzymology, quantitative proteomics, and biochemistry approaches. The knowledge gained from this study will likely have a broad impact on our understanding of epigenetics, and will lay a foundation for studying Kla and the Warburg effect.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract: The natural evolution of aortic dissection is notoriously unpredictable under current methods of evaluation and management. There is an urgent need to more completely elucidate the biomechanical stability of type B aortic dissections and identify signatures in the imaging data allowing for optimal patient classification based on aortic fragility. The long-term goal is the development and validation of image-based analysis algorithms to classify aortic stability and allow a personalized risk stratification for a given patient’s aortic geometry providing the basis for optimizing clinical management. The overall objective of this proposal is to utilize modern approaches in differential geometry, continuum mechanics, and computer vision to discover and characterize high-risk geometric structures hidden within computed tomography angiography (CTA) data of fragile aortas. The central hypothesis of this application is the existence of a fundamental link between aortic shape and aortic stability as it relates to the risk of aortic dissection and fragility. The rationale for this work is development of an easily translatable geometry and mechanics-based algorithm to predict dissection stability and intervention timing by discovering a richer and more nuanced mapping of aortic shapes hidden in existing patient imaging data. The central hypothesis will be tested by pursuing three specific aims: 1) develop a modern geometric classification for aortic shapes, 2) develop a computational model that provides the mechanism underpinning the shape evolution of aortic dissections, and 3) develop a modern successor to the traditional ‘maximum diameter’ measure of aortic dissections that integrates geometric, finite element, and physiologic factors. Utilizing a large pre-identified CTA data set of normal and dissected aortas at various stages of disease and intervention, aim 1 will use tools from computer vision to reduce aortic shape to distributions of shape index and curvedness. Aim 2 will utilize advanced morphoelastic finite element growth models to discover the biomechanical mechanism underpinning aortic shape changes in aortic dissections and validate these models on patient specific geometries over clinically relevant time periods. These novel shape and mechanical stability classifiers will be used in both linear and non-linear dimensionality reduction methods to define aortic shape sub-spaces for different clinical scenarios in aim 3. This proposal is innovative as it challenges the status quo of evaluation and treatment by deploying novel measures and techniques that analyze clinically relevant aortic geometry and the evolution of aortic shape. Every patient is taken to the operating room under the full intent of having a positive clinical outcome. The research outlined is significant because it is expected to provide surgeons and patients a more discriminative framework with which to make better informed management decisions concerning type B aortic dissections and ultimately optimize outcomes.

NIH Research Projects · FY 2025 · 2021-06

Project Summary In this application, we examine the molecular mechanisms that instruct neural wiring and axon terminal elaboration. We focus on the Drosophila neuromuscular system due to its invariant connectivity, limited synaptic partners, and accessibility. Given that this ‘simple’ circuit has been studied for over four decades, it is somewhat surprising that fundamental questions still exist as to how motor neurons choose their appropriate muscle targets and how each motor neuron develops a unique, yet stereotyped, axon terminal structure that underlies synaptic function. Conceptually, both of these developmental processes rely on specificity cues to guide synaptic partner matching (role 1) and synaptic elaboration at each axon terminal (role 2). In support of the first role, we previously discovered two interacting cell surface proteins (CSPs), DIP-α and Dpr10, that are required for wiring a motor neuron to a subset of muscles. In support of the second role, these CSPs continue to be expressed after connectivity, implying additional functions in synaptic development. Our central hypothesis is that combinatorial Dpr-DIP interactions, in addition to specifying synaptic connections, also participate in determining the structure and function of specified synapses. Insights into circuit development arose in a prior collaboration where we characterized the ‘Dpr-ome’, the set of interactions between two families of the immunoglobulin superfamily, the Dprs and DIPs. These 32 proteins bind to one another in unique combinations, and our preliminary data reveal unique expression patterns in the Drosophila larval neuromuscular circuit. Additionally, our data support a combinatorial Dpr-DIP interaction model that leverages cis/trans interactions to instruct highly specific synaptic partnerships. We also reveal a novel signaling pathway that underlies local synaptic elaboration. Given our findings and genetic reagents, we are in a unique position not only to compare axon branch-specific identification tags but also to ask if synaptic elaboration of neighboring axon terminals can be independently regulated. In the first aim, we capitalize on the Dpr-ome and the expression of 6 DIPs in multi-innervating motor neurons to perform single-cell genetic manipulations and examine how combinatorial Dpr-DIP codes instruct connectivity. In addition, we generate affinity variants to reveal a coordinated cis/trans interaction model that enhances specificity. In the second aim, we utilize functional and genetic approaches to understand how co-innervating inputs develop unique morphological and functional properties. We identify a novel crosstalk signaling pathway between axon arbors that locally sculpts NMJ size. Overall, these studies will uncover fundamental developmental programs required for neural circuit wiring and axon terminal elaboration, with emphasis on how CSP codes modulate each of these processes.

NIH Research Projects · FY 2024 · 2021-06

Epistatic interactions within proteins can, in principle, make the paths and outcomes of evolution contingent on chance events; they can also entrench proteins with residues that appear to be optimal but are accidents of history. The extent to which epistasis actually affected the trajectory and outcomes of molecular evolution depends on the fitness effects of substitutions when they occurred in history compared to their potential effects earlier or later in time and on the temporal order in which interacting substitutions occurred. Deep mutational scanning studies have revealed pervasive epistasis among the huge number of possible mutations, but no studies have directly assessed how the fitness effects of substitutions that happened during history changed over time as the protein evolved. We will perform the first comprehensive experimental analysis of the fitness effects of all amino acid states that evolved in a protein during a long-term phylogenetic trajectory, both at the time they occurred and if they had occurred at other points in history. These data will be analyzed in the ordered temporal context of the protein's phylogeny and supplemented with biochemical experiments, enabling a deep characterization of the causes and consequences of epistasis, contingency and entrenchment across the billion-year history of an essential protein. Our model system is ideal for this purpose. Hsp90, the essential molecular chaperone in all eukary- otic cells, plays key roles in protein folding and maturation, cell signaling, and a wide range of diseases. Strong phylogenetic signal allows confident reconstruction of the billion-year evolutionary history of Hsp90's protein sequence from the last common ancestor of animals, fungi and related protists to present-day Saccharomyces cerevisiae. We will generate targeted protein libraries containing every ancestral and derived state that occurred during this phylogenetic trajectory, singly and in every possible pair, in the background of all 30 reconstructed ancestral proteins along the trajectory. Using a high- resolution bulk competition assay in yeast, we will precisely measure selection coefficients and epistatic interactions and quantify how these properties changed over time. This will reveal the fitness effects and interactions of every substitution at the approximate time it occurred, as well as the effects and interactions it would have had if it happened (or reverted to the ancestral state) at any point earlier or later during the trajectory. We will also apply biophysical and structural techniques to elucidate the underlying biochemical mechanisms that drove these genetic and evolutionary phenomena. This work will provide deep new insight into the ways in which proteins' genetic and physical architecture influences, and is influenced by, the processes by which they evolve; it will also strengthen our understanding of sequence-structure-function relationships in a biologically essential protein.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Infancy is increasingly being recognized as a key time point of microbiome establishment that impacts neonatal health as well as later outcomes. The intestinal microbiome has specifically been implicated in neurologic outcomes via the gut-brain axis. However, means by which the intestinal microbiome can have influence on the brain are poorly understood. The preterm infant is at the nexus of these unknowns. Preterm infants are a vulnerable patient population at risk for significant poor long-term neurodevelopmental outcomes. Preterm infant brain development occurs in parallel with intestinal microbiome development, thus modification of the intestinal microbiome is a potential means of improving neurodevelopmental outcomes. In this proposal, we will test the hypothesis that distinct gut microbiome taxa and metabolites at key time points improve preterm infant neurodevelopmental outcomes at school age. Our preliminary and published data in gnotobiotic mouse models demonstrates that different early preterm infant microbiota impact neuron number, myelination, and behavior. This proposal will use our ongoing MIND (Microbiome In Neonatal Development) preterm infant cohort to determine how the gut microbiome impacts neurodevelopmental potential in the NICU, and how it may alter neurodevelopmental trajectories post- NICU discharge. We will conduct longitudinal sampling of participant fecal and blood samples to monitor gut microbiome as well as fecal and serum metabolites. We will also perform neurodevelopmental testing during the NICU course and up until preschool/school age (3.5-5 years old). School readiness, which describes children's strengths, challenges, and needs for supports when learning in the classroom, is a functional outcome that differs from single summary measures of intelligence (IQ) and will be the outcome measure. A combination of 16S rRNA gene sequencing, metagenomics and metabonomics will be applied to the collected fecal samples. Sophisticated machine learning strategies will be used to develop novel models of preterm infant gut microbiome succession with time as a critical element. Serum cytokine analysis and metabonomics will provide mechanistic insight into how the gut microbiome may be impacting neurodevelopment. We have established complementary in vivo gnotobiotic mouse models, in which germ-free mice are transfaunated with preterm infant microbiota. This state-of-the-art experimental model will allow specific investigation of the impact of clinically relevant microbiota on brain development that is not possible in human infants. The goal of this proposal is to discover intestinal microbiome patterns associated with school readiness, identify the key time points that represent windows of opportunity for microbiome optimization, and identify mechanisms by which the intestinal microbiome impacts brain development and behavior. This new knowledge will enhance our understanding of the gut-brain axis and lay the foundation for microbial based therapeutics to improve infant neurodevelopmental trajectories.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Treatment advances in psychosis are limited by the use of phenomenology-defined diagnoses based on symptomatic outcomes, rather than by neurobiological constructs monitored by quantitative characteristics. The Bipolar-Schizophrenia Network for Intermediate Phenotypes (B-SNIP) uses biomarkers to define psychosis subgroups with the goal of testing the advantages of B-SNIP biomarkers for diagnostic and therapeutic decisions, consistent with principles in the NIMH Strategic Plan (NSP). With >3000 phenotyped psychosis probands, relatives and healthy controls (HC), B-SNIP has a multilevel biomarker library for psychosis and used that library to re-conceptualize psychosis subgroups as biomarker-defined Biotypes (B1, B2, B3), where B1 and B2 are the low cognition/high symptom groups and B3 shows lower symptoms and relatively normal cognition. We replicated Biotypes in a new sample, “forging a future where measures of an individual's … neural and physiological state will form the basis of an increasingly specific and informative diagnosis” (NSP). In this grant we propose that B1, with its low cognition and low cortical activity, will respond uniquely to clozapine, a drug which will generate active cortical attractor networks in B1 to support symptomatic improvement. Clozapine is the most effective antipsychotic drug (APD) with unique clinical efficacy. It is the least used APD because its side effects are serious (neutropenia, myocarditis, seizures) and its administration complex. A predictive biomarker would allow targeting of cases most likely to respond and improve prognosis in psychosis. B-SNIP has shown that clozapine is associated with increases in EEG measures of alpha/theta power, and we identify this increase in time periods without stimulus processing requirements as intrinsic EEG activity (IEA), across all Biotypes. Because B1 cases express low IEA, clozapine's action to increase EEG power will be normalizing for this psychosis subgroup, with increased cortical attractor states. Because B2 express accentuated IEA, clozapine is associated with more deviant IEA in B2. We propose to test B1 psychosis cases with clozapine vs. risperidone (n=40/group clinical trial completers), over a 6 week cross-titration (to therapeutic plasma levels) and a 9 week stable dose extension, predicting that the B1/clozapine group will respond significantly better, as measured with total PANSS, than the B1/risperidone group and also better than either B2 group. It is our hypothesis that the cortical attractor networks will be normalized and their function increased by the increase in intrinsic EEG activity.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT A fundamental question in immunology lies in understanding how the immune system can mount robust T cell responses to foreign pathogens, while restricting collateral damage to endogenous tissues, a state often referred to as "self vs. non-self discrimination". Although many self-reactive conventional T (Tconv) cells are removed from the body by clonal deletion, considerable evidence demonstrates that this process is imperfect. The control of remaining self-reactive Tconv cells requires suppression by CD4+Foxp3+ regulatory T (Treg) cells, which function throughout life to prevent autoimmunity. Efforts to define the mechanisms by which Treg cells suppress Tconv cells have revealed numerous potential mechanisms, including the masking of co- stimulatory ligands, the local production of suppressive cytokines, or the hoarding of key accessory factors. However, these antigen non-specific "bystander" mechanisms are not sufficient to explain self vs. non-self discrimination, especially in the context of innate immune activation during infection, highlighting the importance of new research examining the mechanisms of Treg-mediated suppression. Previously, we identified two self-peptides ("C4" and "F1" peptides) that are recognized by naturally occurring Treg cell populations and are derived from a single prostate-specific protein, Tcaf3. Here, we demonstrate that selection on the C4 peptide during repertoire formation is critical for the prevention of prostatitis, and that polyclonal Treg cells of other specificities can not compensate for the shift in the C4-specific T cell pool. This reveals a key role for Treg-mediated suppression of Tconv cells of matched peptide/MHC-II (pMHC-II) specificity, as opposed to broad antigen non-specific mechanisms. The objectives of this application are to elucidate mechanisms by which Treg cells coordinate pMHC-II-specific immune suppression at steady state and during infection. We will achieve our objectives in close collaboration with Dr. Ron Germain, an expert in advanced imaging techniques, and Dr. Nancy Freitag, an expert in the genetics of the bacterium Listeria monocytogenes (Lm). In Aim 1, we will use functional experiments and advanced confocal imaging to define the mechanistic basis of pMHC-II- specific Treg cell suppression at steady state, testing the hypothesis Treg cells do not prevent the initial activation of pMHC-II-matched Tconv cells, but instead rheostatically respond to activated Tconv cells to restrict their subsequent differentiation and expansion. In Aim 2, we will define the role of Treg cell pMHC-II specificity in coordinating self vs. non-self discrimination during Lm infection, testing the hypothesis that robust pMHC-II-specific suppression by self-selected Treg cells is imparted by both quantitative (numerical) advantages and qualitative properties induced by the recognition of peripheral self-ligand prior to infection. In all, our work is expected to elucidate key mechanisms by which the immune system orchestrates host defense while limiting collateral damage to self tissues.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Here, we propose to thoroughly characterize the origins of pairwise correlations in cortex using a synergistic mix of experimental methodologies, behavior, and computation in mice and macaques. We will elucidate the mechanistic underpinnings of normalization and test our hypothesis that changes in cortical pairwise correlations and other signature arise from ongoing cortical computations. In Aim 1 we will record from populations of neurons in the middle temporal visual area of trained, behaving monkeys to test the hypothesis that pairwise spike correlations, gamma oscillation and transient responses at the onset of visual stimuli arise in part from the dynamics of the circuits that normalize neuronal responses. These tests require measurements with a precision that is not feasible in mice. Conversely, the experiments in Aim 2 and 3 address questions that are not feasible in monkeys. In Aim 2 we will exploit the accessibility of mouse visual cortex by using both two-photon laser scanning microscopy and multielectrode arrays to comprehensively measure the relationship between normalization and pairwise correlations in populations of V1 neurons and measure how spatial separation within cerebral cortex affects that relationship. Finally, in Aim 3 we will establish the contributions of specific cell classes to normalization and pairwise correlations in mouse V1. We record the activity of pyramidal neurons and the three most thoroughly characterized classes of cortical interneuron (VIP, SST and PV) during normalization. We will then separately manipulate the activity of these cells classes to revealing the role that changes to the ratio of excitation and inhibition play in driving normalization. In this way, we will establish the role these neurons play in changing pairwise correlations within the excitatory pool of neurons. Results from all three Aims will be tied together using a new family of dynamic, recurrent circuit models of normalization to formalize the hypothesis that normalization imposes pairwise correlations and other activity signatures, and will use experimental data to constrain and refine these models.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/ Abstract How cells dynamically control their proteome in response to stress is a fundamental aspect of understanding how organisms are able to react to changing environments. Two representative features of the cellular stress response, which is universally conserved across eukarya and occurs in response to a variety of different noxious environmental conditions, are 1) the upregulation of the cytoprotective heat shock proteins and 2) biomolecular condensation of RNA and protein into assemblies. Most translation is shut down, while proteins involved in the stress response are efficiently produced. How translation is reprogrammed to favor heat shock protein production post-transcriptionally is poorly understood, but biomolecular condensation has been linked to translational control. Basic questions are incompletely answered: 1) Which mRNAs condense in response to stress? 2) What cellular mechanisms are responsible for mRNA condensation? And 3) What is the functional relevance of mRNA condensation to translational control? Herein, we present unpublished work measuring mRNA solubility of >5,000 genes during temperature stress in S. cerevisiae by biochemical sedimentation followed by RNA-Sequencing. These data inform our hypothesis that blocking translation initiation triggers condensation of an mRNA through specific binding by unknown protein factor(s). We also predict that mRNA condensation during stress is an adaptive process contributing to the preferential translation of stress response messages. To test these hypotheses, we aim to confirm that blocking translation initiation triggers mRNA condensation both on a transcriptome-wide and individual message level, to determine protein factors required for mRNA condensation, and to test the role of mRNA condensation in translational reprogramming during stress. Preliminary data measuring the solubility of both native and reporter mRNAs support that blocking translation initiation triggers condensation. We have identified and will interrogate a set of the translation initiation factors as candidates putatively required for mRNA condensation. We will test whether the candidates are required for mRNA condensation and measure the translational effect of perturbing mRNA condensation during stress. Biomolecular condensates are intimately related to cellular RNA homeostasis, and their dysfunction has been linked to the pathogenesis of several neurodegenerative diseases including Alzheimer's and Parkinson's. Knowledge of how mRNAs condense and the functional role of condensation informs disease pathogenesis and may inform future treatments for those affected.

NIH Research Projects · FY 2025 · 2021-04

PROJECT ABSTRACT An attractive modality for bone and soft tissue regeneration involves the use of pluripotent mesenchymal stem cells that are induced by osteogenic and dermogenic cues. Furthermore, the delivery of engineered cells within 3D-printed, “smart” scaffolds tailored to any shape would make an ideal approach to rapidly repair battlefield injuries. Our overall goal is to investigate capacity of BMP-9-induced, Notch pathway-synergized human adult- derived urine stem cells seeded onto unique, polydiocitrate-graphene hybrid scaffolds, to heal critical-sized multi- tissue defects in the rat scalp/cranium. This project is based on the hypothesis that the combination of BMP-9- Notch-induced stem cell osteogenesis and a three-dimensional scaffold capable of hosting stem cell differentiation along osteogenic and dermogenic lineages will lead to adequate reconstruction of critical-sized multi-tissue craniofacial defects. To test this hypothesis, the following specific aims are proposed: 1) To investigate the mechanisms by which BMP9 induced human urine progenitor stem cells (HUPs) repair trauma- induced cranial defects in vivo; 2) To develop structural composite scaffolds that can be customized to fit and regenerate a critical-sized skin and bone calvarial defect in a rodent model. These specific aims will be addressed by the following experimental design: 1) Treatment of critical-sized rat cranial defects with iHUPs transduced with BMP9 and abrogation of defect healing with Notch inhibition and; 2) Design and incorporation of 3D-printable mPOC-graphene/A5G81-PPCN hybrid scaffolds using micro-CLIP technology and testing of scaffold permutations in a novel multi-tissue rat scalp-cranial defect.

NIH Research Projects · FY 2025 · 2021-03

We will conduct a Hybrid Type I effectiveness-implementation randomized controlled trial of an evidence-based, flexible, and tailored intervention that harnesses social support to promote retention in care and viral suppression among people living with HIV aged 18-49. The study will take place in Cook County (Chicago, Illinois) and Alabama, two high-burden areas prioritized in the national Ending the HIV Epidemic Plan. Existing efforts to improve Continuum of Care outcomes for people living with HIV often rely on newly created network members, e.g., peer navigators, support groups, case managers. Often missing from these approaches is a focused attempt to harness organic social network supports, i.e., those people who already offer critical forms of emotional, informational, and instrumental support. In contrast, the intervention was developed to identify, activate, and harness organic social network support for people living with HIV. The intervention uses (1) social network visualization and theory to help men identify a support confidant to engage in care; (2) the Information-Motivation-Behavioral Skills Model targeted at the support confidant to activate and maintain dyadic social support; (3) a linked social support model to target the drivers of retention in care and viral suppression. Content is delivered via a single face-to-face session and mini-boosters. The intervention’s flexibility ensures that support confidants are selected based on their supportive function rather than their role. In a pilot randomized controlled trial in Chicago, we demonstrated feasibility, acceptability, and efficacy. To now test effectiveness, N=600 people living with HIV in Chicago and Alabama will be randomized to receive the intervention (n=300) or treatment as usual (n=300). We also will enroll 300 support confidants. At 12-month post-intervention, we will re-randomize dyads to continue receiving quarterly mini-boosters (sustained support: n=150) or return to treatment as usual (n=150). Data collection at baseline, 12 months, and 18 months will include surveys and electronic medical record data. To study implementation, we will use the Consolidated Framework for Implementation Research as the determinants framework and Reach, Evaluation, Adoption, Implementation, and Maintenance framework as the evaluation framework. The specific aims are to: (Aim 1) Evaluate the (a) effectiveness of a social support intervention versus treatment as usual over 12 months with 600 people living with HIV ages 18-49 and (b) value of continuing to offer social support over another 12 months. The primary outcomes are retention in care and Viral Suppression, as measured by electronic medical record data on missed visit proportion and viral load; (Aim 2) Examine if intervention effects (a) vary between Chicago and Alabama, (b) are mediated by changes in the Index’s level of motivational readiness, self-efficacy, and stigma expectancies, and (c) are moderated by mental health and substance use at the Index level; and (Aim 3) Evaluate the implementation of the intervention using the Consolidated Framework for Implementation Research and the Reach, Evaluation, Adoption, Implementation and Maintenance frameworks. We will conduct surveys and focus groups with key stakeholders to assess the inner and outer settings, implementer and intervention characteristics, and multi-level process factors within the Consolidated Framework for Implementation Research. We will assess the following implementation outcomes for the study in each clinical setting and geographic context: Reach, Adoption, Implementation, and Maintenance.

NIH Research Projects · FY 2025 · 2021-03

Abstract: Molecular Mechanisms of Adjuvant Triplet Combinations The immune system makes decisions in response to complex combinations of microbial inputs. Live vaccines that are empirically attenuated from pathogens have been a powerful means to yield life-long immunity against many deadly pathogens because they mimic immune responses to combinations of microbial signals. However, the rational design of non-live vaccines using immunomodulatory agents such as adjuvants has remained an elusive task in many cases where live vaccination is not efficacious or feasible, in part because identifying potent adjuvant combinations and associated molecular mechanisms that explain cross talk remains a major challenge. Based on our recent findings and extensive preliminary data, we propose to define the molecular mechanisms through which two adjuvant triplets – containing agonists for Toll-like receptor (TLR), C-type lection receptor (CLR), RIG-I-like receptor (RLR), and cytosolic dsDNA sensor (CDS) pathways – induce protective CD4+ and CD8+ T cell responses in mice. We will use an innovative approach which is (i) comparative – by contrasting the quantitative effects of adjuvant triplets and matching singles and pairs as means to accurately pinpoint molecular mechanisms explaining cross talk; and (ii) multiscale – by studying molecular mechanisms at play in cells, tissues, and the whole body. First, we will determine the molecular mechanisms of intra-cellular signaling cross talk by adjuvant triplets by testing hypotheses at the level of protein complexes proximal to adjuvant receptors, phosphorylation cascades and kinase-substrate relationships, and gene regulatory networks. Second, we will identify the molecular mechanisms through which adjuvant combinations impact inter-cellular signaling between dendritic cells (DCs) and T cells by testing hypotheses on the regulatory mechanisms shaping the cellular, surface, and secreted proteome of DCs. Third, we will test hypotheses on the effects of adjuvant triplets on the organism-wide spreading and seeding of effector and memory T cells, and the underlying cell circuits of the skin (vaccination site) and draining lymph node that explain the induction of protective, long-term T cell immunity. Results from this work will produce critical insights at the forefront of adjuvant combination research by characterizing higher-order combinations of adjuvants that can mimic the effects of well-established, potent live attenuated vaccines and inform future vaccine designs against infection.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY In B lymphopoiesis there are alternating and mutually exclusive state of stochastic immunoglobulin gene recombination and cell proliferation with selection. Following successful rearrangement of the Ig heavy chain gene, Igµ pairs with surrogate light chain (SLC) to form the pre-BCR, expression of which is associated with clonal large pre-B cell expansion. However, at the subsequent developmental stage, small pre-B cells must fully exit cell cycle before initiating Ig light chain (IgL) gene recombination. Failure to do so risks genomic instability and leukemic transformation. Work from our lab and others has demonstrated that the IL-7R drives proliferation while the pre-BCR primarily appears tasked with IgL recombination. However, there is an apparent paradox in that IgL rearrangement occurs in small pre-B cells in which there is concurrent strong repression of SLC. There are two possibilities. The pre-BCR could initiate a complex developmental program in large pre-B cells that is executed in small pre-B cells. Alternatively, there could be other receptors or mechanisms that orchestrate IgL chain recombination. We now demonstrate that CXCR4, which is upregulated in small pre-B cells, directly transmits signals that open Igk to recombination. Indeed, it is CXCR4-mediated ERK activation, and not escape from IL-7, nor expression of the pre-BCR, that mediates late B lymphopoiesis. These and other data suggest a new model of B lymphopoiesis in which sequential signaling through three receptors, the IL-7R, pre-BCR and CXCR4, orchestrate critical cell fate decisions. We propose to test this mode in the following Specific Aims: Aim 1. Identify the signaling pathways specifically downstream of the pre-BCR. Aim 2: Determine how CXCR4 signals integrate with pre-BCR/IL-7Resc to drive Igk recombination. Aim 3. Determine how CXCR4 regulates receptor editing.

NIH Research Projects · FY 2025 · 2021-03

ABSTRACT Tumor-associated macrophages (TAMs) are the most prevalent immune cell in the tumor microenvironment. TAMs adopt an M2-like phenotype that supports angiogenesis, attenuates anti-cancer immune responses, and facilitates metastatic dissemination. Studies in humans and experimental animal models support targeting TAMs for anti-cancer therapy. However, the environmental conditions triggering M2 polarization and molecular mechanisms mediating this process are poorly understood. This knowledge is required to target TAMs therapeutically and to identify patients that would benefit from such therapies. One potential pathway for TAM polarization is via metabolic reprogramming. Previous studies showed that glycolysis supports a pro-inflammatory, anti-tumor M1 phenotype in macrophages, while mitochondrial respiration is required for the M2 phenotype. Our recently published work challenged this paradigm. We showed that treating macrophages with LPS or bacteria (M1 stimuli) induce lactate production which in turn, promotes a late phase switch to an M2-like phenotype. The mechanism underlying this surprising observation involves a novel lactate-induced epigenetic modification (H3 lysine lactylation (Kla)) at promoters of genes associated with the M2-phenotype that directly promotes transcription. Hypoxic conditions, such as those found in tumors, also induce lactate production by macrophages. In preliminary work, we show that hypoxia induces expression of M2-like macrophage genes, and these genes are marked by Kla at their promoters. We further show that TAMs isolated from tumors with high hypoxia have elevated levels of histone Kla and M2-like proteins in comparison to TAMs tumors with low hypoxia. Finally, we show that inhibiting endogenous lactate production by TAMs (via myeloid cell specific deletion of Ldha) attenuates the M2-like phenotype of TAMs, lessens tumor growth, and increases CD8+ effector T cells in tumors with high hypoxia, but not in tumors with low hypoxia. Based on these studies, we hypothesize that a lactate-Kla pathway induces the M2-like phenotype of TAMs during hypoxia and promotes tumor growth by suppressing adaptive immunity. To test this hypothesis, we propose three aims: (1) determine the contribution of TAM lactate production to its M2-like phenotype during hypoxia, (2) dissect mechanisms by which lactate production by TAMs promotes tumorigenesis, and (3) determine the contribution of histone lactyltransferases to histone Kla and M2-like phenotype and function of macrophages. By integrating human studies with mechanistic animal, cell-based, epigenetic, and biochemical studies, our proposed work seeks to delineate the mechanisms that promote M2- like TAMs and their effects on tumor development. Delineating these mechanisms may identify potential molecular targets for TAM-based therapeutics that improve anti-tumor immunity and reduce tumorigenesis.

NIH Research Projects · FY 2024 · 2021-01

PROJECT SUMMARY / ABSTRACT The transition from drug use to abuse and, eventually, to dependence may be mediated by biological factors that are present prior to drug use. Although chronic exposure to drugs of abuse is known to disrupt many signaling pathways, little is known about the molecular mechanisms that mediate addiction susceptibility. There is considerable interest in identifying early biomarkers for addiction susceptibility to improve addiction prevention strategies. Most studies have been conducted in substance-dependent individuals where the dissociation between ‘susceptibility’ and ‘consequence’ is ambiguous. I have recently identified a behavioral phenotype in rats that reliably predicts future drug-taking behaviors and identified three proteins as potential mediators of addiction susceptibility: sorting nexin 1 (SNX1), ryanodine receptor 2 (RYR2), and ataxin 2-like (ATXN2L). These proteins are involved in intracellular trafficking, calcium signaling and cytoskeleton reorganization, and have been previously linked to addiction. Precisely how differences in expression of these proteins impact the signaling cascades underlying addiction susceptibility is not known. The overarching goal of this proposal is to determine the functional and molecular role of proteins that mediate addiction susceptibility and investigate how these factors are mechanistically linked to genetic and/or environmental components of risk for addiction. To accomplish this goal, the proposed research will combine sophisticated behavioral and computational assessments with viral, proteomic and bioinformatic approaches. In Aim 1 I will use an inducible and reversible viral construct to bi-directionally manipulate expression of SNX1, RYR2, and ATXN2L and also determine how changes in expression of SNX1, RYR2, and ATXN2L alter methamphetamine self-administration and protein signaling mechanisms. In Aim 2 I will determine if variation in the expression of SNX1, RYR2, and ATXN2L is altered in a model of genetic addiction susceptibility and is associated with increased addiction risk. In Aim 3 I will determine if variation in expression of SNX1, RYR2, and ATXN2L is altered in a model of environmental addiction susceptibility and is associated with increased addiction risk. Completion of these aims will generate new insights into the signaling mechanisms of addiction susceptibility that could identify early biological markers of risk for addiction and improve current strategies for addiction prevention. The Principal Investigator will receive mentorship and technical training in viral and proteomic technologies by experts in cell signaling and cellular mechanisms, and viral technologies in motivated behaviors. Yale University and the Department Psychiatry provide exceptional facilities and resources for completing the proposed experiments, as well as having an exceptional reputation and track record for mentoring and transitioning early-stage investigators in to independent investigators. The proposed training, education and research will provide the PI with the technical and professional training to become a successful, independent addiction investigator.

NIH Research Projects · FY 2025 · 2021-01

Project Summary/Abstract Amyotrophic lateral sclerosis (ALS) is a uniformly fatal neurodegenerative disease caused by neuronal death in the motor system, both in the brain and spinal cord. It results in progressive weakness throughout the body, with death typically from respiratory failure within 3 years of symptom onset. Therapy initiation and drug development are hindered, in part, by the lack of quantitative biomarkers for the disease. In the proposed project a multi-center study will be carried out to validate and further characterize a potential biomarker for ALS, known as intermuscular coherence (IMC). IMC measures the correlation of activity between two muscles and represents the shared input to the muscles from motor neurons in the brain and spinal cord. In vivo studies in both non-human primates and humans suggest that IMC in the range of 15-40 Hz (β-to-γ frequencies) represents input to muscle pairs from upper motor neurons. When motor neurons in the brain are damaged, as happens in ALS, IMC decreases in the βγ frequency range. In a preliminary report we showed that patients with ALS have lower IMC in the βγ range than do age- and sex-matched control subjects. Because the measurement of IMC is quick, non-invasive, painless, and requires only equipment found in standard clinical neurophysiology labs, the method, if validated, would be an important biomarker for ALS. Proposed is a multi-center validation study of IMC in the clinical environment. First, the accuracy, sensitivity, and specificity of the biomarker will be determined in patients who present to neurology clinic for an initial evaluation when ALS is suspected. In order to provide the most specificity, the distribution of IMC values will be characterized in neurotypical subjects across several demographic subgroups. Finally, IMC will be monitored over time in patients with ALS to determine how IMC changes with ALS disease progression. Preliminary data suggest that IMC could be a useful biomarker for diagnosing ALS, allowing differentiation of ALS from ALS-mimic disorders, and that it can be used to objectively monitor the progression of ALS over time. A multi-center study to test the validity of these preliminary findings is important before this method can be implemented to speed diagnosis and provide faster access to treatments of ALS for patients.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY / ABSTRACT Colorectal cancer (CRC) is a major health concern with nearly 2-million new cases of CRC diagnosed worldwide in 2019. While surgical resection of the primary tumor offers a cure for some, up to half of patients undergoing colorectal surgery will develop a postoperative recurrence. With a median survival of only 24 months, almost all patients whom develop a postoperative recurrence will die from their disease with current therapies; there is thus an immediate need to develop new strategies to understand and prevent CRC recurrence. Despite increasing evidence that intestinal bacteria plays a major role in the pathogenesis of primary CRC, how gut microbes influence the development of CRC recurrence has never been addressed. To address this gap in knowledge, Benjamin Shogan MD has developed exciting data demonstrating that CRC recurrence is a microbial driven process. For reasons that remain poorly understood, a high-fat Western diet is the major risk factor for the development of both primary and recurrent CRC. He has discovered that when mice fed a high-fat diet undergo intestinal resection (mimicking the surgery patients undergo for CRC cure) collagenase producing organisms, especially Enterococcus faecalis preferentially colonizes the site of reconnection. He has found that E. faecalis can over-activate critical extracellular matrix proteases, including the urokinase(uPA)-plasminogen system, creating an environment abundant in signals (i.e. uPA, MMP9, plasminogen) well-known to promote tumor progression. Strikingly, when CT26 mouse carcinoma cells are present intraluminally at the time of surgery (mimicking exfoliated viable tumor cells that exist in human patients), they can migrate through healing intestinal tissue to develop tumors identical to human CRC recurrence only when mice are fed a high-fat diet and colonized with collagenolytic organisms. Recent in vitro experiments have found that E. faecalis promotes enhanced invasion and migration of CT26 cells, suggesting that at the intersection of CRC recurrence is bacterial induced metastasis of tumor cells through permeable intestinal tissue. In this K08 application, Dr. Shogan creates a career development plan to acquire his long-term goal of becoming a principal investigator examining how modulation of the intestinal microbiome can improve survival outcomes in patients with CRC. With the guidance of his mentors Ralph Weichselbaum MD and Eugene Chang MD, he will test the hypothesis that the perioperative proliferation of collagenolytic organisms by a high-fat diet creates an intestinal microenvironment that promotes the extraluminal migration of cancer cells, driving CRC recurrence. Using in vivo and in vitro approaches, and samples from his human patients, he will explicate the mechanisms by which collagenolytic organisms, via its interaction with the extracellular matrix, drives the transluminal migration of CT26 cells to form extraluminal tumors. Completion of this work will inform the interaction between host-microbe-cancer cells, and force a complete rethinking and development of novel strategies to prevent and treat colorectal cancer.

NIH Research Projects · FY 2025 · 2021-01

ABSTRACT Preeclampsia (PE) is a disease of late pregnancy characterized by hypertension and organ damage, which not only increases peripartum morbidity but is associated with the postpartum development of heart failure, including myocardial fibrosis and systolic heart failure. Despite two decades of research demonstrating this association and a large public health burden, the molecular mechanisms mediating PE-induced postpartum heart failure remain poorly understood and therapies to prevent this outcome are lacking. One potential trigger may be the profibrotic growth factor Activin A, a member of the transforming growth factor beta family produced by the placenta and inflammatory cells. Using a randomized mechanistic clinical trial of aspirin vs. placebo for patients with preeclampsia, integrated with studies of the mechanisms by which aspirin and Activin A might affect the heart, we will dramatically advance knowledge of cardiac dysfunction in preeclampsia and its possible treatments. We pursue two specific aims to answer these questions. AIM 1 is a randomized trial to test whether aspirin improves cardiac function and decreases Activin A in women with preeclampsia. AIM 2 Identifies how increased plasma Activin A during pregnancy causes postpartum cardiac dysfunction and how aspirin prevents it. This project innovates methodologically and at the bench in order to dramatically advance our understanding of a major cause of morbidity and mortality in women globally.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Post-transcriptional modifications of mRNA have emerged as a central regulatory mechanism in genetic information flow. N6-methyladenosine (m6A) is the most abundant post-transcriptional modification in eukaryotic mRNAs. m6A mRNA methylation is reversible and dynamically regulated by writers, erasers and readers. Writers are methyltransferases that install the methyl group on adenosine residues, erasers are demethylases that remove the methyl group, and readers are proteins that recognize and interact with the m6A site. m6A methylation influences all fundamental aspects of mRNA metabolism, including mRNA processing, stability and translation. Despite tremendous progresses, the in vivo roles of m6A mRNA methylation in macrophage biology remains unclear. Sepsis is a major clinical problem and leading cause of death in patients in intensive care units. Sepsis is usually caused by Gram-negative bacterial infection that triggers a fast cytokine storm. Macrophages as the first line of defense are the predominant producer of pro-inflammatory cytokines during infection. Proper resolution of the cytokine response is essential for the host's well-being. The intensity and duration of cytokine storm is delicately regulated by negative feedback regulatory loops, and the SOCS family of proteins are the central players of this feedback regulatory mechanism. We have sought to understand the role of m6A methylation in macrophage biology by genetically targeting METTL14, a core subunit of the m6A methyltransferase (a writer). We found that mice carrying METTL14 deletion in myeloid cells are hypersensitive in both cecal ligation puncture (CLP)- and lipopolysaccharide (LPS)-induced sepsis models. These tissue-specific METTL14-mutant mice produced and maintained much higher levels of serum pro-inflammatory cytokines and suffered much higher mortality than control mice. METTL14-depleted macrophages produced and sustained much higher levels of pro-inflammatory cytokines than the control macrophages, and the underlying cause is that METTL14 deletion impairs SOCS1 induction in macrophages following bacterial infection or LPS challenge. Our data support the hypothesis that m6A methylation plays a critical role in controlling the intensity and resolution of cytokine storm in sepsis by increasing Socs1 mRNA stability and translation. Our data strongly suggest that LPS or bacterial infection activates the NF-κB pathway that stimulates Socs1 mRNA transcription; LPS/bacterial infection further increases Socs1 m6A methylation by promoting FTO (an eraser) mRNA degradation, and then YTHDF1 (a reader) binds to the Socs1 m6A sites to promote Socs1 mRNA stability and increase its translation. In this proposal we will validate that SOCS1 is an essential METTL14 target to control macrophage activation in septic response using in vivo and in vitro models (Aim 1), validate that YTHDF1 is a critical reader to promote Socs1 mRNA stability and translation in septic response (Aim 2), and validate that FTO is a critical eraser whose mRNA degradation promotes Socs1 m6A methylation and greatly contributes to negative feedback control of macrophage activation (Aim 3).

NIH Research Projects · FY 2024 · 2020-09

γδ T cells constitute an important component of the immune response against infectious agents and cancerous transformations, yet the biochemical mechanisms by which they detect antigen through their somatically recombined T cell receptor (TCR) remain unclear. Unlike αβTCRs, which are restricted to recognizing antigens in the context of Major Histocompatibility Complex (MHC) molecules, γδTCRs can recognize a diversity of ligands ranging from self MHC to intact, unprocessed, viral glycoproteins. Our recent work has established CD1 molecules as ligands for a subpopulation of human Vδ1 γδ T cells, producing robust functional, biochemical and structural evidence. We seek to extend our studies to the human gut, where γδ T cells, and in particular, Vδ1+ T cells, predominate. Our preliminary data suggests that CD1 recognition is robust and present in all individuals examined, and that there exist important functional differences between CD1 reactive γδ T cells in tumors versus healthy adjoining tissue. Thus, the long-term goal of this proposal is to fully characterize this CD1 reactive population in tumors versus healthy tissue, examining their functional effector phenotypes, TCR repertoire and immunomodulatory signals, in addition to the TCR, that shape the recruitment, activation and potential expansion of these cells in the context of a highly relevant human disease, colorectal cancer. Our first aim, “Characterization of CD1-specific γδ T cells in normal and diseased tissue.”, seeks to use classical cellular expansions complemented by direct ex vivo functional and transcript analysis to profile CD1 reactive and non- reactive T cell populations derived from tumor and adjoining healthy tissue. These data will provide insight into the signals that regulate γδ T cells within the tumor microenvironment compared to healthy tissue. Our second aim, “Elucidation of the molecular mechanisms by which γδ TCRs bind to CD1/lipid complexes.”, will focus on characterizing the interaction between the γδ TCRs expressed by these cells and CD1/lipid antigen. We will use protein biochemistry, biophysics and x-ray crystallography to elucide the molecular mechanisms by which the γδ TCR recognizes CD1/lipid. Our effort will significantly expand our understanding of the specific signals that regulate γδ T cell activity in human health and disease. Our third aim, “Determine the presence and role of ligand, co-stimulatory and/or co-receptor molecules in CD1 specific γδ T cell activation and phenotype in the colon” will characterize the ligand and immunomodulatory signals that may regulate the activity of CD1 reactive γδ T cells in the context of human colorectal cancer. We will combine RNAseq and differentiation assays using cord blood derived, naïve Vδ1 cells to test the relevance of candidate signals. This will be complemented by in vitro derived native Vδ1 T cells through the OP9/DL1 system. γδ T cells can be either pro-inflammatory or regulatory, therefore we seek to understand which role these cells play, if any, in this disease state. Together, these aims will begin to unravel the mystery of γδ T cells in human immunobiology, both at the cellular and molecular levels.

NIH Research Projects · FY 2024 · 2020-09

PROJECT ABSTRACT The bacterial communities (microbiota) residing on the human body have been linked to a variety of acute and chronic diseases and conditions, such as obesity, inflammatory bowel disorders, Type 2 diabetes, depression, and urinary tract infections (UTIs), as well as to the host’s response to a variety of treatments and health interventions for these diseases and conditions. As the critical role played by the microbiota has become increasingly recognized, microbiome sequencing data sets are now routinely generated under ever more sophisticated experimental designs and survey strategies. While such data share many common features and challenges of modern big data, such as high-dimensionality and sparsity, they also possess characteristics peculiar to the microbiota, including (i) the explicit and latent contextual relationships among the bacterial species, such as their evolutionary and functional relationships; and (ii) the substantial heterogeneity across samples and complex structure in the sample-to-sample variation. Effective analysis of modern microbiome studies calls for new statistical methodology that incorporates these important characteristics in the data generative mechanism. This project’s objective is to develop a suite of statistical models, methods, algorithms, and software that meet this urgent need. An initial aim is to develop a multi-scale probabilistic framework for modeling microbiome compositions that effectively characterizes the high dimensionality, sparsity, and substantial cross-sample variation in microbiome sequencing data, and incorporates a variety of common experimental designs, such as covariates, batch effects, and multiple time points, while striking a balance in flexibility, analytical parsimony, and computational tractability. An additional focus is to develop latent variable models for microbiome compositional data for the purpose of identifying latent structures such as sample clusters and species subcommunities. A final aim is to produce user-friendly, open-source software that implements all of the proposed methods for the analysis of microbiome sequencing data. All of the models and methods developed are informed by two on- going collaborative projects of PI Ma and his team. One is on the identification of microbial communities associated with UTIs in aging women, and the other on the study of longitudinal changes in the microbiome of cancer patients undergoing hematopoietic stem cell transplantation. These studies will serve as testbeds for all development. The models, methods, and software developed will not only result in better prediction of the health outcomes in these and other microbiome studies but also help decipher the roles of microbiome in various diseases and biomedical processes, with the ultimate goal of personalized interventions on the microbiome compositions of patients to lead to improved health.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT The purpose of this Mentored Clinical Scientist Research Career Development Award (K08) is to provide Dr. Loren Saulsberry with a mentored training experience designed to lead to an independent academic career in genomic medicine. Dr. Saulsberry has a PhD in Health Policy. She is currently an Assistant Professor in the Department of Health Sciences at The University of Chicago. Her long-term career goal is to lead a research program that evaluates and guides the implementation of pharmacogenomics (PGx) into clinical practice in a manner that advances health equity in genomic medicine. The proposed work leverages skills she developed through prior training and research and will form the basis for a successful R01 proposal. The career development plan includes didactic and experiential learning in pharmacogenomics; developing research skills in implementation science; acquiring leadership skills for directing interdisciplinary, implementation research teams; and maximizing professional development through a series of objectives. Dr. Saulsberry will also attend seminars, journal clubs, laboratory and research meetings, grant-writing workshops, and present at national meetings, which will all provide additional opportunities to develop and strengthen skills. The mentorship team for the proposed interdisciplinary project covers the full translational cycle of PGx research to ensure training and preparation for independence is comprehensive, incorporating knowledge of each stage of translation from research to practice. The proposed studies build on Dr. Saulsberry's prior work in health disparities and chronic illness; it investigates communication processes essential to PGx implementation, providing a foundation for future studies that evolve approaches for delivering PGx care to patients at risk for health disparities. Aim 1 investigates the inter-ethnic disparities in the use of prescription drugs with evidence-based PGx guidelines and assesses the potential impact of PGx testing on minority populations based on a nationally representative dataset on health care utilization. Aim 2 determines the views of patient-provider pairs on PGx risk communication through interviews/focus groups and examines inter-ethnic variation between patients' risk communication preferences. Aim 3 uses a survey experiment to evaluate the influence of PGx risk information on inter-ethnic patient beliefs and preferences for the clinical use of PGx. This proposal responds to the urgent need to discover methods of tailoring PGx implementation to the expectations and needs of ethnically diverse populations so as to not widen health disparities. The K08 award will lead to Dr. Saulsberry's transition to an independent career in genomic medicine focused on designing and adapting PGx implementation that is minority-centered.

NIH Research Projects · FY 2025 · 2020-09

ABSTRACT Type 1 diabetes (T1D) is characterized by a complex interplay among various cellular constituents within the islet microenvironment, including immune cells, endocrine cells, endothelial cells, and acinar cells. Our team has previously advanced the understanding of stress-responsive signaling cascades within β cells, revealing their role in triggering or exacerbating autoimmunity. Building on these findings, this renewal HIRN application aims to identify pivotal intracellular signaling pathways and develop targeted interventions to modulate early disease processes that shape human islet biology in T1D. Our approach aligns with the objectives of RFA-DK- 23-007, leveraging a synergistic Team Science framework to explore interferon signaling in β cells. Interferons, released by islet-invading immune cells, play a crucial role in T1D. Genes related to interferon response (PTPN2, IFIH1, TYK2) are linked to T1D susceptibility. While the acute β cell response to interferons is adaptive, a sustained response in genetically susceptible individuals may initiate or propagate insulitis, leading to T1D. Our data indicate that an interferon transcriptional signature is present early in T1D, with IFN-α influencing β cells initially, followed by IFN-γ during advanced insulitis. Additionally, post-transcriptional mechanisms, including mRNA translation and posttranslational modifications (particularly S-palmitoylation), finely tune interferon responses to balance pro- and anti-T1D effects. We hypothesize that the interferon response in β cells is a critical early cellular cascade, balancing β cell survival and autoimmune susceptibility in T1D. We will test this hypothesis through the following aims: Aim 1: Define the impact of post-translational S-palmitoylation on interferon signaling in β cells. Aim 2: Define the contribution of mRNA translation to the interferon response in β cells. Aim 3: Investigate the regulatory mechanisms of interferon signaling on β cell PD-L1 production. With the demonstration that T1D onset can be delayed in humans, there is a compelling need to identify stress response pathways in β cells as potential targets for disease prevention. This HIRN proposal builds on a successful prior HIRN team collaboration, employs innovative methods to investigate interferon responses in human β cells, and promises to provide new insights into manipulating this early signaling pathway to modify T1D progression.