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NIAID - National Institute of Allergy and Infectious Diseases

Project summary: Development of Antivirals for Arthritogenic Alphaviruses This NIH grant proposal aims at the optimization of a New World (NW) alphavirus-specific quinazoline scaffold for the development of an anti-Old World (OW) arthritogenic alphavirus therapeutic candidate. With a particular focus on Chikungunya virus and other alphaviruses with a pandemic potential, this project responds to an urgent public health need for effective antiviral treatments for OW alphaviruses, given the recent increase in global prevalence of OW alphavirus infections. Dr. Chung's research group has made significant advancements in developing a quinazolinone-based antiviral specifically targeting NW alphaviruses like Venezuelan Equine Encephalitis Virus, exhibiting remarkable efficacy in preclinical models. This success, coupled with the compound's unique mechanism of action targeting the viral replicase complex, positions the quinazoline scaffold as a valid starting point for developing effective antivirals for other alphaviruses. However, the compound series necessitates significant enhancement in potency against OW alphaviruses. The team has identified a key residue that contributes to the NW alphavirus-specific antiviral spectrum. Here by elucidating the molecular interactions and clashes between the quinazolinone scaffold and OW alphavirus replicase complexes, the team aims to develop next- generation inhibitors with enhanced efficacy toward OW arthritogenic alphaviruses. Recognizing the need to enhance the potency of the quinazoline series against OW alphaviruses, the project proposes three Specific Aims undertaking a comprehensive and systemic approach integrating structural insights, medicinal chemistry, and preclinical evaluations. Dr. Luo's work in Specific Aim 1 focuses on mapping molecular interactions and clashes between the current quinazolinone scaffold and the OW alphavirus replicase complex, providing critical structural insights that will inform the design of new inhibitors in Specific Aim 2 led by Dr. Bannister. Specific Aim 2 efforts are directed towards identifying chemical groups leading to resistance in OW alphaviruses, designing and synthesizing new compounds tailored for OW alphaviruses, and ultimately developing lead candidates with optimized pharmacological profiles. Dr. Chung, in Specific Aim 3, will assess these novel antivirals in vitro and in vivo, supporting the structure-activity study for Aim2. Aim 3 will also study the mechanisms of action and resistance of the series. The overarching goal of the project is to deliver novel, potent therapeutic candidates for OW arthritogenic alphaviruses. With a highly integrated team of experts and a meticulous approach spanning molecular structural biology, medicinal chemistry, and virology, this project holds the promise of advancing preclinical development, ultimately addressing a critical gap in public health preparedness against OW alphavirus infections. Upon successful completion, the project will deliver promising therapeutic candidates ready for advanced preclinical development to address the critical needs for arthritogenic alphaviruses therapeutics.

NIAID - National Institute of Allergy and Infectious Diseases

Project summary: Development of Antivirals for Arthritogenic Alphaviruses This NIH grant proposal aims at the optimization of a New World (NW) alphavirus-specific quinazoline scaffold for the development of an anti-Old World (OW) arthritogenic alphavirus therapeutic candidate. With a particular focus on Chikungunya virus and other alphaviruses with a pandemic potential, this project responds to an urgent public health need for effective antiviral treatments for OW alphaviruses, given the recent increase in global prevalence of OW alphavirus infections. Dr. Chung's research group has made significant advancements in developing a quinazolinone-based antiviral specifically targeting NW alphaviruses like Venezuelan Equine Encephalitis Virus, exhibiting remarkable efficacy in preclinical models. This success, coupled with the compound's unique mechanism of action targeting the viral replicase complex, positions the quinazoline scaffold as a valid starting point for developing effective antivirals for other alphaviruses. However, the compound series necessitates significant enhancement in potency against OW alphaviruses. The team has identified a key residue that contributes to the NW alphavirus-specific antiviral spectrum. Here by elucidating the molecular interactions and clashes between the quinazolinone scaffold and OW alphavirus replicase complexes, the team aims to develop next- generation inhibitors with enhanced efficacy toward OW arthritogenic alphaviruses. Recognizing the need to enhance the potency of the quinazoline series against OW alphaviruses, the project proposes three Specific Aims undertaking a comprehensive and systemic approach integrating structural insights, medicinal chemistry, and preclinical evaluations. Dr. Luo's work in Specific Aim 1 focuses on mapping molecular interactions and clashes between the current quinazolinone scaffold and the OW alphavirus replicase complex, providing critical structural insights that will inform the design of new inhibitors in Specific Aim 2 led by Dr. Bannister. Specific Aim 2 efforts are directed towards identifying chemical groups leading to resistance in OW alphaviruses, designing and synthesizing new compounds tailored for OW alphaviruses, and ultimately developing lead candidates with optimized pharmacological profiles. Dr. Chung, in Specific Aim 3, will assess these novel antivirals in vitro and in vivo, supporting the structure-activity study for Aim2. Aim 3 will also study the mechanisms of action and resistance of the series. The overarching goal of the project is to deliver novel, potent therapeutic candidates for OW arthritogenic alphaviruses. With a highly integrated team of experts and a meticulous approach spanning molecular structural biology, medicinal chemistry, and virology, this project holds the promise of advancing preclinical development, ultimately addressing a critical gap in public health preparedness against OW alphavirus infections. Upon successful completion, the project will deliver promising therapeutic candidates ready for advanced preclinical development to address the critical needs for arthritogenic alphaviruses therapeutics.

NIA - National Institute on Aging

PROJECT SUMMARY Despite the growing number of adults that are living with Alzheimer’s disease (AD) worldwide, there still lacks disease modifying treatments. Physical activity and exercise mitigate AD risk and boosts cognitive functions in patients, yet over a quarter of the elderly do not meet these activity guidelines, often due to physical limitations and resource constraints. Current therapeutic approaches focused on AD are restricted to targeting neuropathology within the central nervous system. Thus, there is an imperative need to develop alternative therapies that confer benefits of exercise to AD patients, circumventing their physical limitations. Prior work by our research team identified an exercise-induced platelet-derived factors in blood plasma that mediates the neurogenic and cognitive benefits of exercise in aged mice. Systemically increasing this blood based exercise mimetic conferred beneficial effects of exercise on cognitive function and AD-related neuropathology in aged mice and mouse models of AD pathology. The purpose of this study is to translate the scientific discoveries identifying this platelet-derived exercise mimetic as novel potential therapeutic to treat AD and related dementias. This is a Fast- Track Phase I to Phase II proposal that in Phase I aims to (1) generate various clones of the platelet-derived exercise mimetic with decreased safety risks and retained function, (2) optimization of the expression and purification of this platelet-derived exercise mimetic and (3) demonstrate that the protein engineered platelet-derived exercise mimetic can confer improved cognitive function in aged mice and a mouse model of AD pathology. In Phase II, we aim to (4) perform exploratory toxicity studies to better understand the therapeutic window and (5) begin CMC process development in support of IND-enabling studies.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

Summary – We will establish and validate stable system for CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) in zebrafish, employing attP safe harbor PhiC31 integration sites. The application of CRISPRi and CRISPRa technologies in zebrafish has the potential to expand its capacity for the study of gene function significantly. It will afford the modulation of promoters and regulatory sequences alike, facilitating efficient loss of function and gain of function evaluation of biological and pathological relevance. We recently developed codon-optimized CRISPRi/a constructs for zebrafish, establishing their function in proof-of-principle experiments, using RNA injection of system components to modulate key genes in established phenotypes. We synthesized a zebrafish codon-optimized cas9 gene, harboring mutations D10A and D839A to render the protein catalytically inactive (dCas9). We then cloned codon-optimized Krüppel associated box (KRAB) and methylated CP2 (MeCP2) inactivating domains or VP64 activator domain downstream from dCas9 for CRISPRi and CRISPRa, respectively. To validate the biological function of our initial CRISPRi construct, we targeted the promoter sequences of key genes in melanocyte differentiation (sox10, mitfa, and mitfb); and melanin production (tyrosinase; tyr). Microinjection of CRISPRi mRNA with single guide RNAs (sgRNAs) targeting their promoters resulted in hypopigmented larvae (epidermal melanocytes and retinal pigmented epithelium. In addition, we evaluated CRISPRi/a modulation of mrap2a, which controls energy homeostasis and somatic growth via inhibition of the melanocortin 4 receptor gene (mc4r). Targeting the mrap2a promoter proximal region with CRISPRa or CRISPRi increases and decreases larval body length, respectively. However, RNA-based injections inherently display time-limited effects whose impact is unreliable beyond the development. Thus, here we propose to establish transgenic lines, stably expressing CRISPRi and CRISPRa from constructs integrated into attP safe harbor integration sites in chromosome 14 facilitated by the PhiC31 integrase (Aim 1). We will screen the efficacy of these new lines via Tol2-mediated delivery of sgRNA expression constructs directed at known genes whose modulation yield readily scored phenotypes, as above. Similarly, we will establish and validate constructs for efficient delivery of sgRNA expression to an alternate attP PhiC31 site in chromosome 24 (Aim2). The lines expressing CRISPRi/CRISPRa will be crossed with the empty alternate PhiC31 site, allowing the use of the second site for targeted integration of sgRNA expression constructs. The establishment of lines established in Aims 1 and 2 will be confirmed by the inclusion of discrete reporter cassettes expressing Cerulean [blue, Aim 1] and Venus [yellow, Aim 2] in the pineal and the lens of the eye, respectively. The efficiency of lines generated in aim 2 will be evaluated using guides designed to the promoters of genes employed in Aim 1 validation. Robust CRISPRi and CRISPRa systems in zebrafish will facilitate efficient assay of candidate gene function and disease relevance through bidirectional modulation of gene expression.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

PROJECT SUMMARY/ABSTRACT Development of novel contraceptive strategies is central to the mission of the Contraceptive Research Branch of the NICHD. This goal is driven by a global need for effective contraceptive methods to address: 1) the glut of unintended pregnancies (~45% of US pregnancies in 2011); 2) the high rate of elective abortion (1.15M unintended pregnancies ended in abortion in 2011 in the US); and 3) the high risk of maternal mortality (~830 women/day worldwide die due to pregnancy or childbirth complications). In a search for novel male contraceptive drug targets, we identified RBM46, which is a germ cell-specific RNA binding protein expressed by germ cells on the basement membrane of seminiferous tubules (outside the blood-testis-barrier), and is essential for spermatogenesis. Indeed, Rbm46 knockout mice are sterile and have no other phenotype, raising the distinct possibility that targeting RBM46 could lead to safe and effective male contraception by blocking spermatogenesis at the differentiating spermatogonial stage. Thus, we propose to develop drugs that target degradation of RBM46 as a means of oral, non-hormonal male contraception, which will significantly advance additional safe and reversible options for male contraception towards the clinic. Specifically, we will combine: 1) exceptional expertise in drug screening and development at UTSA and UT Health San Antonio; 2) leading expertise in male reproduction, spermatogenesis, and infertility at UTSA and ECU; 3) close proximity to one of two NIH-designated Marmoset Breeding Colonies, maintained at the Southwest National Primate Research Center; 4) growing and ongoing experience collecting and assessing marmoset sperm; 5) published experience in the use of cutting- edge single-cell genomics to assess normality of spermatogenic cell types; and 6) documented expertise with spermatogonial stem cell (SSC) transplantation. In Aim 1, we will identify small molecules that bind RBM46 and could be used to develop PROTACs. In Aim 2, we will produce initial RBM46 PROTACs and validate that they degrade the protein in vitro. In Aim 3, we will use medicinal chemistry to optimize the drug-like characteristics of top validated RBM46 PROTACs. In Aims 4 and 5, we will determine whether optimized RBM46 PROTACs induce reversible contraception in vivo using mice and marmosets, respectively. Together, these Aims are designed to advance RBM46 degradation as a novel strategy to achieve reversible, non-hormonal male contraception and provide key results to justify further preclinical investigation and eventual commercialization.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract: Tumor-associated carbohydrate antigens (TACAs) are aberrantly expressed glycans associated with cancer cells, playing critical roles in cancer progression and immune evasion. Despite their potential as diagnostic and therapeutic targets, the lack of comprehensive tools to study TACA interactions has significantly limited progress in this field. This project addresses these challenges by developing the first comprehensive TACA glycan library and glycan array, integrating 58 existing TACAs with 22 newly synthesized glycans and glycopeptides for MUC16 (CA125), sTRA, and sLeA. These newly synthesized TACAs enable detailed exploration of the glycoform-specific interactions of MUC16, sTRA, and sLeA. The TACA glycan array will feature TACAs displayed at varying densities and valencies, simulating the diverse glycan presentations found on cancer cells. This innovative design allows for the systematic evaluation of TACA interactions with therapeutic antibodies, biologics, and glycan-binding proteins. By providing tools to study these interactions under biologically relevant conditions, the project aims to advance the development of TACA-targeted diagnostics and therapeutics. Phase I focuses on developing and validating the TACA library and associated array, leveraging Z-Biotech’s expertise in glycan synthesis and array fabrication. The amine-tagged TACAs, both pre-existing and newly synthesized in this project, will also support broader applications, such as conjugation to fluorescent beads for autoantibody screening. Phase II will expand the library to include over 300 TACAs and further refine the array for commercial deployment. This initiative will accelerate the discovery of new TACA-targeted diagnostic and therapeutic candidates, establish a transformative research resource, and deepen our understanding of TACA-mediated cancer biology.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT Precision genome editing presents opportunities for treatment of disease and for use in biotechnology. Tyrosine family site-specific DNA recombinases (Y-SSRs), such as Cre, are uniquely positioned as chemical biology tools for genetic engineering because they are capable of excision, integration, and inversion of DNA sequences without requiring exogenous co-factors. A major focus in tyrosine recombinase research is the retargeting of these enzymes to new DNA sites, primarily using substrate-linked directed evolution (SLiDE). Although successful in many regards, SLiDE has two substantial drawbacks that limit its use: limited capacity for negative selection can lead to promiscuous recombinases, and lack of selection tunability limits the ability to select for highly active recombinases. Off-target activities could have serious negative consequences in the human genome, limiting the use of evolved recombinases as probes or eventual therapeutics against human disease. Rigorous characterization of the activity of evolved recombinases towards off-target sites and the development of methods to lower or eliminate these off-target activities is a high-priority enabling technology. Likewise, development of a directed evolution scheme that can select for high recombination efficiency would enable the production of recombinase libraries with higher average efficiency in less time. Here, we aim to address both specificity and efficiency of recombination during evolution by developing novel SLiDE methodology. First, to combat promiscuity in evolved recombinases, we will develop a SLiDE method that leverages simultaneous positive selection at a desired target site and negative selection against a library of off- target sequences. We will use this method to evolve novel recombinases for activity against loxHTLV, the target of the previously-engineered RecHTLV. The activity of novel recombinases with the off-target library will then be quantified and compared to that of wild-type Cre and RecHTLV to observe differences in their promiscuity profiles. Second, to enable selection for higher efficiency recombinases, we will establish a bacterial system by introducing toxic “kill switches” into evolution vectors that are temperature controlled. Recombinase-expressing E. coli that have been transformed with these vectors will select for enzymes that can excise the toxic gene from all plasmid copies before timed activation of the kill switch. We will validate this system with existing recombinases and then evolve a new recombinase for activity on loxHTLV to compare to the activity and evolutionary timeline of the previously evolved RecHTLV. We hypothesize that this novel selection for recombination efficiency will rapidly produce an enzyme with improved efficiency in fewer rounds of evolution. Development of these selection systems will accelerate the development of specific, efficient evolved recombinases, streamlining the timeline for development of novel tools and potential therapeutics.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

PROJECT SUMMARY Epilepsy affects over 50 million people worldwide, with developmental and epileptic encephalopathies (DEEs) representing the most severe and treatment-resistant forms. DEEs are characterized by early-onset seizures and developmental delays, often caused by pathogenic variants in genes critical for brain function. Understanding these genetic underpinnings has revolutionized epilepsy research, paving the way for precision medicine—therapeutic strategies tailored to specific genetic and molecular mechanisms. Among these disorders, EEF1A2-related neurodevelopmental disorder (EEF1A2 syndrome) stands out as a rare yet compelling example, highlighting the need for targeted research to understand and treat epilepsy. Mutations in the EEF1A2 gene, which encodes the eukaryotic elongation factor 1A2 (eEF1A2), have been identified as a cause of severe neurodevelopmental disorders, including DEEs. eEF1A2 facilitates protein synthesis by delivering aminoacyl-tRNAs to ribosomes in a GTP-dependent manner. Approximately 50 distinct missense mutations have been reported, with recurrent variants such as G70S, E122K, and D252H associated with severe intellectual disability. These mutations map to key functional domains of eEF1A2—GTP binding (G70S), tRNA binding (E122K), and actin binding (D252H)—and exhibit distinct clinical phenotypes. While G70S and E122K are linked to early-onset epilepsy, D252H is associated with milder or no epilepsy. The role of eEF1A2 in actin dynamics through its third functional domain is also unclear, suggesting potential additional roles in neurons. This project aims to address these gaps through two specific aims: 1. Determining how EEF1A2 mutations alter protein synthesis in human neurons. 2. Determining how EEF1A2 mutations alter actin dynamics, neuronal morphology, and development. Previous studies have been limited by the species-specific isoform switch between eEF1A1 and eEF1A2 that occur in humans but are not fully replicated in mouse models. By utilizing human-induced pluripotent stem cells (hiPSCs) differentiated into cortical neurons, this research will overcome these limitations by determining the alterations due to these mutations on protein synthesis, elongation rate, translatome profile, and translational efficiency (Aim 1). Additionally, alterations in actin mobility and subcellular structure, as well as neuronal morphology, synapse number, and neuronal electrophysiological properties will be determined (Aim 2). The findings will advance our understanding of eEF1A2's neuron-specific roles and inform precision medicine strategies for EEF1A2 syndrome treatment.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract Atopic dermatitis (AD) is a chronic skin condition characterized by T cell-driven Type 2 inflammation. The MC903- induced dermatitis model has enabled dissection of pathways driving acute AD-like disease such as keratinocyte TSLP production and Th2 signaling; however, its ability to model persistent disease states remains unexplored. Using a repeated-challenge protocol with MC903, we found that adult mice undergoing a primary bout of AD-like inflammation develop persistent, tissue-specific inflammatory memories in skin that manifest as exaggerated pathologic responses during secondary MC903 challenge. This aligns with the emerging idea that AD chronicity stems from local memories of inflammation that persist in healed lesions and exacerbate future disease flares. However, AD in childhood follows a distinct course, with a unique immune composition and lower incidence of chronic disease. We thus tested our model in neonatal mice, and despite observing similar primary response kinetics to MC903-treated adults, we strikingly saw no signs of aggravated pathology during secondary MC903 challenge. This suggests that AD-like inflammatory memory fails in early life. Characterizing the cellular and molecular mediators of these divergent skin phenotypes will be the focus of our proposed work. Our data suggests that inflammatory memory in adults is driven by the emergence of Type 1 (T1) immune features in skin such as T1 tissue-resident memory T cells (Trms), mirroring recent findings in human AD. Transcriptional profiling suggests fibroblasts help to organize these networks by supporting T cell positioning and Trm development within inflamed adult skin. Given emerging data to suggest that neonatal T cells and fibroblasts exhibit distinct inflammatory behaviors from their adult counterparts, we hypothesize that inflammatory memory is impaired in neonatal skin due to age-related differences in T cell and fibroblast function. To test this, we will first use lineage tracing to define the fates and phenotypes of adult and neonatal T cells during AD-like inflammation in developing and adult skin (Aim 1). Subsequently, we will interrogate fibroblast-T cell interactions in the atopic skin of adult and neonatal mice using in vivo profiling via scRNA-seq and immunofluorescence staining as well as in vitro functional assays involving fibroblast-T cell co-cultures. Our work is conceptually innovative and clinically relevant, especially given the growing incidence of chronic AD in adults. Completion of the proposed work will clarify the basic mechanisms by which neonatal skin escapes inflammatory memory, potentially aiding in the identification of new therapeutic interventions for chronic AD.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Early life exposures play a significant role in shaping health and disease susceptibility. Yet, our understanding of the developmental programming of adult stem cells that maintain tissue homeostasis is limited. Mature adult intestinal stem cells (ISCs) sustain continuous life-long adaptations to diet and the intestinal microbial milieu to preserve tissue stability and prevent sequelae leading to diseases such as inflammatory bowel disease (IBD) or sporadic cancer. ISC’s developmental trajectory is influenced early in life by a maternal environment that provides extrinsic patterning cues and nutrients to influence cell, tissue, and system maturation. Despite the fundamental nature of these developmental phases, we do not understand how durable programmable molecular mechanisms are established or how adverse maternal environmental exposure is maintained in a stable and homeostatic manner within ISCs. There is a critical need to establish how the environment contributes to intestinal homeostasis and long-term disease risk. Using genetically engineered mouse and organoid models, this project aims to distinguish ISC developmental programming and determine both early intrinsic adaptations and extrinsic environmental interactions that promote establishment of a stable pre- pathological ISC ground state. Our central hypothesis is that offspring exposure to an obesogenic maternal environment during pre- and postnatal development establishes a maladaptive pre-pathological ground state ab initio. In Aim1 we will characterize the extent of altered ISC changes in offspring exposed to maternal pro- obesity high-fat Western diet (HFD) during pre- and postnatal phases of maternal dependence, and test the duration that these features exist as offspring age. We will further challenge offspring with the reintroduction of the HFD and test for pathological states. In Aim2, we will investigate how lipid metabolism drives ISC- programming by testing for necessity of lipid regulators, Ppar-d and Ppar-a, and for sufficiency with increased PPAR-d activity. In Aim3, we will explore the role that external signaling from the immune system contributes to developmental programming and later risk of disease. We will test the necessity of pro-inflammatory cytokine IL-17 and the influence in tumorigenesis and inflammatory states. In summary, these efforts will enhance our understanding of how developmental programming in early life leads to health and disease disparities as we age.

NIGMS - National Institute of General Medical Sciences

Biological oscillators, the rhythmic and regular increases and decreases of gene expression or protein activity, drive many dynamic molecular and morphological processes. Probably the most familiar is the circadian clock, but there are also ultradian clocks that operate on the order of minutes and/or hours. Examples of ultradian clocks are those that regulate periodic root branching in Arabidopsis, molting cycle in C. elegans, timing of mitosis to tightly coordinate cell proliferation and tissue morphogenesis, stem cell maintenance, bulk protein degradation and re-synthesis in proliferating mammalian cells, and tissue patterning during somitogenesis, a fundamental vertebrate developmental process that we have studied for many years. Somitogenesis is the anterior to posterior sequential segmentation of the vertebrate embryonic mesoderm into blocks of tissue called somites, which later give rise to axial skeletal muscle, vertebrae, and dermis This rhythmic and regular process is controlled by a molecular oscillator called the segmentation clock which operates in the unsegmented presomitic mesoderm (PSM) and is characterized by rapid cycles of mRNA and protein expression with a species-specific periodicity. Like many cell-autonomous oscillatory networks, the segmentation clock is regulated by a self-sustaining negative feedback loop, where a core oscillatory factor, a transcriptional repressor, inhibits expression of downstream oscillatory genes, including itself. For segmentation clock oscillations to persist, the transcript and protein molecules of clock genes must be short- lived. In all vertebrates examined to date, transcriptional repressors of the Hes/her gene family are considered the evolutionarily conserved pacemakers of the segmentation clock. For proper somite formation, rates of each oscillatory step, from transcription to translation to decay, must be regulated to ensure correct somite size and number. Computational modeling and experimental perturbations have shown that transcriptional and translational time delays (the amount of time from transcription or translation initiation to the emergence of a mature mRNA or protein, respectively) and degradation rates of both transcript and protein are parameters having the largest influence on clock period and evidence from in vivo studies assessing the impact of timing of mRNA and protein processing and decay mirrors modeling predictions. Transcriptional regulation alone is not sufficient to produce genetic oscillations and we are focusing on understanding the post-transcriptional regulatory mechanisms that promote proper oscillatory expression. The two key questions that we address in this proposal are: (1) What are the key targets of the core Hes/Her oscillators that carry out oscillatory output? and (2) What are the post-transcriptional regulatory mechanisms driving rapid decay of oscillatory gene transcripts. Answering these questions will address how oscillatory gene expression regulates pattern and fate.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY The objective of this proposal is to elucidate the complex genetic, transcriptional, and histological landscape underpinning palate morphogenesis by leveraging a systems genomics approach in genetically diverse mice. Craniofacial morphogenesis involves the outgrowth and fusion of facial prominences, which must be coordinated with skeletal specification and differentiation to generate a functional craniofacial complex. Underscoring the sensitivity of this intricate choreography, orofacial clefts (OFCs) are the most common facial anomaly in humans, affecting 1/700 births worldwide. Mouse models provide a key platform for human disease allele discovery owing to recent developments in gene editing that enable rapid validation of novel variants. However, multiple published examples demonstrate the profound effect that mouse genetic background can have on the penetrance and expressivity of craniofacial phenotypes. Thus, our limited understanding of the effect of natural strain variation on developmental processes presents a major challenge to validation of disease variants and our ability to model human congenital malformations. While individual genes are critically required for normal development, it is the collective function of genes and their interactions, modularly organized into gene regulatory networks (GRNs), that control the transcriptional dynamics and timing of morphogenesis and differentiation. Variation in these dynamics likely accounts for normal variation in facial shape but can also underpin craniofacial birth defects. However, feasibility limits studies of genetic variation in developing embryos to only a few genes at a time and experimental models that accurately relate genomic sequence variation to variation in transcriptional dynamics are critically lacking. These challenges complicate a systematic address of genome-scale mechanisms that drive morphogenesis and the potential for pathological outcomes. The complementary Collaborative Cross (CC) recombinant inbred strains and Diversity Outbred (DO) heterogeneous stock combine the genetic and phenotypic diversity of 8 common inbred founder strains of mice in a design optimized for systems-level genetic dissection of complex traits. It has been demonstrated that QTLs for facial shape in adult DO mice are enriched for genes with established function in craniofacial and skeletal development. However, these studies do not address mechanisms of where, when, or how QTL candidate genes influence facial shape. The proposed aims will apply a developmental systems genomics approach to map the influence of genetic variation within a model of palate morphogenesis. Aim 1 will define the temporal dynamics of morphogenetic networks within the segmentally organized upper jaw and identify QTL/eQTL underlying strain differences in developmental timing and palate morphogenesis. Aim 2 will employ single-cell and spatial genomics to genetically dissect the molecular signatures of QTL/eQTL with cellular and histological resolution. Aim 3 will leverage quantitative phenotyping to relate differences in morphogenetic dynamics and resulting morphology to variation in underlying genotype and thereby derive mechanistic insight into the integration of genomic regulatory systems.

NCATS - National Center for Advancing Translational Sciences

ABSTRACT There is a need in the market for a true platform technology that automates the tissue processing experience. Syntr Health Technologies has dedicated the last nine years to developing the technology that can streamline tissue processing. Our FDA-cleared SyntrFuge System and SyntrFPU360, is a platform technology that can process a variety of different tissue types such as cancer tumors of kidney, liver, brain, and breast tissue by dissociating them into single cells to better understand tumor tissue heterogeneity, of which the single cell analysis market is valued at $3.3 Billion and expected to grow to $6.3 Billion by 2026. Additionally, our platform can be used to microsize autologous adipose tissue to be used for reinjection back into the patient to treat medical conditions of the aging population such as diabetic foot ulcers, knee osteoarthritis, and breast cancer reconstruction surgery. Syntr Health successfully completed an SBIR Phase I grant where we utilized microsized adipose tissue to heal diabetic ulcers in an animal model. This study had an important role in developing a treatment modality that translated successfully into humans. Preliminary human clinical data shows significant diabetic foot ulcer wound closure within 28 ± 4 days using autologous adipose tissue processed in our platform. The diabetic foot ulcer market is currently valued at $1.98 Billion with a CAGR of 8.2%. This high growth potential is attributed to the increase of the diabetic population. Moreover, the use of microsized adipose tissue is showing great promise in intra-articular injections to knee joints for the treatment of osteoarthritis (OA). The knee OA market in North America is currently valued at $5.9 Billion and growing at a CAGR of 8.8%. Over 88% of individuals with OA are 45 or older. The high growth potential of this market is largely attributed to the increase of the aging population along with the increase in obesity rates. Our platform technology for the rapid processing and/or microsizing of tissues can disrupt these markets in a major way. The full automation and market readiness of this technology in this Phase II SBIR is paramount to increase favorability in the adoption of this much needed technology in various medical fields..

NEI - National Eye Institute

Project Summary The proposed Diabetes Endothelial Keratoplasty Study (DEKS) Renewal will follow up from the initial findings of the DEKS, driving evidence-based decisions regarding use of corneal tissue from donors with diabetes for Descemet membrane endothelial keratoplasty (DMEK), the leading form of endothelial keratoplasty in the US. There remains the need to definitively resolve the discrepant findings regarding the use of diabetic donor tissue to guide the corneal transplant community. While we hypothesize that overall non-diabetic corneas are superior, it is unlikely that all donors with diabetes are inferior. Rather, based on preclinical and DMEK eye bank stripping data, we presume that the extremes of diabetes severity in donors drive the adverse effects on DSAEK outcomes that our group noted in the Cornea Preservation Time Study (CPTS) where diabetes in donors and recipients was not collected systematically. In the current DEKS, we seek to determine graft success varies between donor subgroups predefined by presence or absence of diabetes as well as various diabetes severity scales after 1 year of follow-up which we expect will be welcomed findings by eye banks and corneal surgeons guiding them to avoid inefficient distribution of inferior tissue for DMEK. However, the current DEKS, with follow-up limited to 1 year, does not address long term endothelial cell loss (ECL) and accompanying graft failure of diabetic vs non-diabetic donors as well as the recipient diabetes effect. Nor does it examine long term effects of diabetes severity and other predictors (genetic) on ECL and graft failure. The renewal of the DEKS would address this by a powered analysis of long-term ECL out to 5 years as a marker for graft survival in the first specific aim while exploring impact on graft failures in the second specific aim. The third specific aim in the renewal of DEKS will be continuation of the third specific aim in DEKS, exploring the relationship of severity of diabetes in the donor (as measured by eye bank-determined diabetes risk categorization scores, post-mortem HbA1c, and skin AGEs and oxidation markers), and in the recipient (as measured by diabetes risk categorization scores, and HbA1c). In addition, in the most novel aspect of this renewal, we will explore donor and recipient genetics (mutations in TCF4, other Fuchs dystrophy genes, and diabetes polygenic risk scores) on ECL and graft failure 5 years following DMEK. These long-term insights on the diabetic donor and associated graft outcomes as well as recipient diabetic status and Fuchs genetics will provide further guidance to the eye banking and corneal transplant surgeon community on the use of an increasing number of diabetics in the corneal donor pool and increasing number of diabetics in recipients also affected by Fuchs Dystrophy.

NIGMS - National Institute of General Medical Sciences

Project Summary Piperidines are the most common nitrogen-containing, privileged scaffold, present in numerous FDA- approved drugs. However, to derive desired physiochemical properties, biological activities, and target selectivity, chiral substituents are added to the piperidine core in enantio- and diastereoselective manners. Accordingly, medicinal chemists prepare various stereoisomers of the drug candidate to test and compare their properties, but the traditional preparation of chiral piperidines drugs are a target-oriented synthesis which that relies on chiral ligands, catalysts, and auxiliaries for every derivative. Therefore, chiral piperidine synthesis remains a bottleneck in drug optimization campaigns. To address this challenge, I propose that a hydrogen-bond donor (HBD) catalyst can generate a common chiral environment that enables a general, diastereoselective strategy for selectively accessing highly substituted diastereomeric piperidine products bearing an epoxide moiety. Once the common HBD/iminium ion intermediate is created upon the addition of a Lewis acid, two subsequent synthetic pathways emerge: the “oxidation-first” pathway and the “nucleophile-first” pathway. Hence, the proposed diastereoselectivity will be controlled by order of addition of nucleophiles and oxidants; since the positive charge of the common intermediate persists when the oxidant is added first, the HBD catalyst is still bound to the substrate which presents an opportunity to set different chiral centers. If nucleophile is added first, then the positive charge of the iminium ion is quenched, and the stereochemistry of the subsequent epoxidation will be controlled by the d.r. of the substitution at the C2 position. This proposed system will be realized by two aims. First, the prochiral piperidine substrate will be rapidly diversified by adding amine-, alcohol-, and TMS-based nucleophiles to form new C-N, C-O, and C-C bonds at the C2 position. To ensure success of this aim, the structure of HBD catalyst will be rigorously optimized by tuning its pyrrolidine arm, anion-binding bridgehead, and electron-withdrawing substitutions. Once optimal catalysts and conditions are found for each type of nucleophile, mechanistic studies will be performed to understand molecularity, rate-, and selectivity-determining steps. Then, the resulting epoxide moiety in the chiral piperidine product will be further functionalized through nucleophilic ring-opening strategies. In the second aim, a concurrent, reigodivergent reduction of the epoxide moiety of the HBD/iminium ion intermediate will be carried out to achieve net reductive resolution and 1,2- transposition of the 4-OH group of the piperidine substrates. This goal will be achieved by using magnesium catalysts with butyl and bistriflimide ligands that facilitate regioselective hydride attack at the epoxide. Overall, the transformations outlined in this proposal closely mirror the crucial principles of drug design, lowering the barrier to accessing valuable chiral piperidine building blocks.

NIDCR - National Institute of Dental and Craniofacial Research

Phosphate (PO43-/Pi) is a natural component of animal and plant food sources and is widely used as food additive in the modern Western diet. With the high consumption of processed foods, the average dietary Pi intake greatly exceeds the body requirements and recommended dietary allowance in the United States. The pathological consequences of the dietary Pi overload include inflammation and disrupted bone metabolism. On the other hand, Pi insufficiency, either systemic or at the cellular level, results in impaired formation of skeletal and dental tissues. Pi is a signaling molecule that changes cellular physiology. This function of Pi depends on levels of available Pi, but outcomes depend on a type of a cell exposed to Pi, that is on its molecular makeup and transcriptional program. In vitro studies from our and other labs implicate GATA-type transcription factors in regulation of gene expression in response to Pi. We have shown that Trps1 (a GATA-type transcription factor) is critical for recognizing and responding to available Pi in cells producing mineralized extracellular matrix. Importantly, our published and preliminary data also revealed that Pi regulates the activity of this transcription factor suggesting that Pi and Trps1 form a regulatory loop controlling gene expression in skeletal and dental tissues. The goal of the proposed study is to determine the interplay between dietary Pi availability, its pro-osteogenic and pro-inflammatory effects and the Trps1-regulated formation and homeostasis of the periodontium. Periodontium is a multi-tissue structure that anchors the tooth to the jaw bone and provides a barrier protecting the underlying tissues from the oral microbiota. At least two of the periodontal tissues (cementum and bone) depend on adequate Pi availability, which is highlighted by increased susceptibility and severity of periodontal disease in individuals with Pi deficiency. Periodontal disease is a highly prevalent inflammatory condition affecting at least 2 in 5 adults aged 45−64 years in the United States. Our preliminary data from Trps1Col1a1 cKO mice detected periodontal defects characteristic for Pi deficiency, further supporting the involvement of Trps1 in cellular responses to Pi. Hence, we hypothesize that the interplay between the Pi availability and Trps1-regulated bone and cementum development maintains the homeostasis of periodontium. We will address this hypothesis through a set of in vivo experiments that will: 1) determine the effects of dietary Pi intake on Trps1 activity in osteoblasts and cementoblasts; 2) define the relationship between the dietary Pi intake and the Trps1-dependent formation of the cementum and alveolar bone; 3) determine the consequences of the dietary Pi overload on sound and genetically compromised periodontium in the context of disrupted periodontal homeostasis. Results of these studies will provide the understanding of the differences between healthy and impaired alveolar bone responses to Pi availability and inflammatory challenge, which will facilitate development of preventive and therapeutic strategies aimed at improving periodontal health in individuals with genetically impaired periodontal structure.

NIAID - National Institute of Allergy and Infectious Diseases

Asymptomatic carriage of MDROs increases the risk of infection for the carrier and members of the community to whom they may transmit the organism. Due to the increased incidence of community-acquired MDRO infections, there is a pressing need to identify factors influencing MDRO colonization. While diet has been shown to modulate the dynamics and metabolite profiles of human gut microbiota, we lack a detailed and quantitative understanding of how specific dietary factors and human gut microbiome impact MDRO carriage. By integrating a longitudinal human study coupled to a novel sequencing-based approach to elucidate dietary species, advanced computational modeling, high-throughput construction of human gut communities and germ-free mouse experiments, will revolutionize our understanding of the multi-scale fiber-dependent interaction networks shaping MDRO (Clostridioides difficile, vancomycin-resistant Enterococci, third generation cephalosporin- resistant Enterobacteriaceae, and carbapenem-resistant Enterobacterales) fitness and colonization of the mammalian gut. In Aim 1, we will perform a prospective, longitudinal study of community-based participants to elucidate the mappings between diet, human gut microbiome taxa, microbial pathways and MDRO carriage. We will go beyond food intake self-reporting which is limited by systematic biases, to track human diet using a DNA sequencing methodology referred to as FoodSeq. Leveraging the longitudinal data, dynamic computational modeling will reveal microbe-microbe interaction networks across individuals. To identify the key dietary fibers and human gut species shaping MDRO colonization, we will use a high-throughput and automated human gut community culturing pipeline. The species identities as well as the specific combinations of these species will be selected using a novel microbial genome-to-function deep machine learning model (data-driven Community Genotype-Function or dCGF) that predicts MDRO abundance as a function of dietary fibers and the genetic features of constituent community members. Using this expanded design-test-learn (E-DTL) approach, we will identify combinations of species and dietary fibers that significantly influence MDROs fitness in human gut communities. Analysis of the model using explainable artificial intelligence techniques will reveal genes/pathways within constituent community members that impact MDRO fitness, providing mechanistic insights beyond the taxonomic level. In Aim 3, we will use dCGF to design robust and maximally inhibitory or enhancing species- fiber combinations for characterization in a murine C. difficile model. We will evaluate the ability of these designed species-fiber combinations to decolonize C. difficile from the murine gut. Overall, this proposed research will provide critical data and models across multiple scales to understand the interplay between diet, the gut microbiome, and MDRO carriage. Our results will inform future interventions aimed at reducing MDRO carriage, transmission, and infections in the community. Finally, this systems biology framework will be generalizable to study other bacterial pathogens, environmental factors, and microbiome functions.

NIAID - National Institute of Allergy and Infectious Diseases

Proposal Summary Neural regulation of sleep, appetite and energy homeostasis is critical to an animal’s survival and under stringent evolutionary pressure. Despite the prevalence of disorders associated with metabolism and sleep, the neural and genetic processes that regulate interactions between these two systems is unclear. This proposal will investigate how sleep is regulated by blood feeding in the disease vector mosquito Aedes aegypti. We will systematically define sleep in mosquitos, and determine the dietary amino acids that promote sleep. We will then screen neuropeptides for regulators of sleep to test for neuromodulators required for sleep following a blood meal. These experiments leverage decades of sleep analysis in fruit flies, to define sleep in a blood feeding insect. The findings have potential to identify conserved regulators of sleep-feeding interactions, as well as providing the first investigation of how macronutrients regulate sleep in a blood-feeding insect. Further, the methodology used to define sleep can be readily applied to other mosquito species. Therefore, the completion of this work will establish Ae. aegypti as a model for studying dietary regulation of sleep.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY The bacterial family that is particularly associated with infections of the gut are Enterobacteriaceae. One significant but understudied family member, Providencia alcalifaciens, has been linked to sporadic foodborne outbreaks of diarrhea and isolated from stool samples from persons with diarrhea. P. alcalifaciens is also part of the human oral, sputum and gut microbiomes of healthy individuals. What distinguishes commensal from enteropathogenic P. alcalifaciens? Most diarrheal isolates harbor a large plasmid ranging in size from 128 kb - 181 kb that encodes a type III secretion system (T3SS) that is closely related to, and functionally interchangeable with, the invasion-associated T3SS (T3SS1) of Salmonella enterica serovar Typhimurium. Of the plasmid- carrying isolates, a subset invades non-phagocytic host cells in cellulo. The primary objective of this application is to define what role this genomic and phenotypic heterogeneity plays in P. alcalifaciens pathogenesis. A comparative genomic analysis identified nine genes in the 40 kb type III gene cluster on the plasmid as statistically associated with the invasion phenotype. In Specific Aim 1, we will use in silico, in vitro and in cellulo analysis to determine if these predicted genes are the molecular drivers of invasiveness in P. alcalifaciens. In Specific Aim 2, we will establish an oral-challenge murine model to assess bacterial colonization and host response in the gut upon infection with invasive and non-invasive P. alcalifaciens isolates. These results will define the contribution of intestinal epithelial cell invasion to P. alcalifaciens enteropathogenesis via the natural route of infection. The potential impact of our proposed work is high because it will be the first to decipher the genetic and molecular basis of pathogenicity of P. alcalifaciens, and the first systematic assessment of P. alcalifaciens virulence determinants in a small animal model.

NIGMS - National Institute of General Medical Sciences

Eukaryotic cells can rapidly adjust the abundance, size, and shape of their membrane-bound organelles in response to physiological needs. We recently found that fission yeast cells employ different mechanisms to counteract various types of stress: nutritional deprivation sequentially activates organelle degradation, while ER stress, caused by the accumulation of misfolded proteins, induces morphological changes in multiple organelles, such as the ER, Golgi, and mitochondria, without triggering their degradation. Our current research aims to understand the molecular framework underlying these differential adaptive responses in fission yeast and to investigate their conservation in mammalian systems. We propose that organelles communicate and coordinate sequential degradation through selective autophagy. To explore this, we will integrate structural prediction with genetic and cellular assays to systematically identify selective autophagy receptors and use multi-omics approaches to investigate potential crosstalk between organelles during starvation. Given that ER stress is increasingly recognized as a contributing factor in a number of human diseases including neurodegenerative disorders and cancer, we will also use fission yeast and human cell cultures to examine how these cells reorganize multiple organelles to mitigate ER stress. These insights could advance our understanding of disease pathology and suggest new diagnostic and therapeutic strategies.

NIA - National Institute on Aging

Project Summary. Accumulating evidence suggests that Alzheimer’s disease (AD) has a long preclinical phase, lasting years to decades during which the accumulation of pathological proteins such as amyloid beta (Aβ) and tau can occur prior to the appearance of overt cognitive symptoms. Obstructive sleep apnea (OSA) is one of the most common sleep disorders and is composed of both sleep disruption (SD) and intermittent hypoxia (IH). Disentangling the separate contributions of chronic SD and chronic IH may reveal how OSA impacts risk for AD. While the effects of SD and IH on Aβ have been studied in both humans and animal models, less is known about the effects of SD and IH on tau spread and propagation throughout the brain, a crucial step in the formation of neurofibrillary tangles (NFT) throughout the brain. Our rationale for targeting tau stems from our own findings demonstrating significant associations between cerebrospinal fluid (CSF) concentrations of the wake-promoting neuropeptide orexin and both total and hyperphosphorylated tau, and that individuals with OSA might show signs of MCI and AD at a younger age. In addition, the treatment of OSA has been shown to delay the age of onset of MCI and to improve cognitive function in AD. Although these observations and others implicate OSA in the regulation of tau in the brain they do not establish directionality. A causal role for OSA in accelerating NFT formation would be strengthened by both chronic SD and chronic IH in mouse models of tauopathy and forms the basis for Aim 1. Given the association between tau and orexin, effective treatment of OSA holds particular promise in slowing the spread of tau throughout the brain. While hyperphosphorylation of tau is important in the formation of NFT’s, the development of AD may be separately accelerated by the spread of tau from neuron to neuron by particular aggregate conformations, a phenomenon that depends on activity of the seeding neuron. The locus coeruleus (LC) is a key brain region that expresses some of the earliest tau pathology, and importantly, the LC promotes wakefulness through rostral noradrenergic projections and is silent during sleep. Therefore, the increased neural activity of the LC during SD or IH represents a potential mechanism by which chronic SD affects the spread of tau pathology to rostral targets. Identifying the precise brain targets to which tau spreads from the LC and the time course over which this occurs is addressed in Aim 2. Addressing the hyperphosphorylation and spread of tau in models with and without LC specificity and the associated behavioral consequences on spatial, motor and fear learning as a function of chronic sleep disruption or chronic intermittent hypoxia serves as the basis for Aims 3.

NIAID - National Institute of Allergy and Infectious Diseases

Summary Regulation of immune responses is critical to provide beneficial immunity against pathogens and cancer, while avoiding autoimmune diseases and immunopathology. A population of memory-phenotype CD8+ T cells, expressing the transcription factor Helios” has been proposed to play a key role in preventing excessive and autoimmune responses following vaccination or infection. However, the precise identity of these Helios+ “regulatory” CD8+ T cells, the factors controlling their differentiation and maintenance, and the exact ways in which they contribute to immune homeostasis are highly controversial. In this exploratory proposal, we develop model systems intended to dissect the development, homeostasis and specificity of these CD8+ T cells, position ourselves for future studies on the physiological role and therapeutic potential of Helios+ CD8+ T cells.

AXON ENTERPRISE INC

Engineering and Research and Technology Based Services-Computer services-System and system component administration services-Technical support or help desk services

NHLBI - National Heart Lung and Blood Institute

ABSTRACT Lung complications occur in 1 in 20 people that receive a surgery that requires an overnight stay in the hospital, even if their surgery is not near the lungs. Anesthesia during surgery and inactivity during recovery cause air sacs in the lungs to deflate, which puts patients at risk of complications like pneumonia that increase mortality five-fold, increase lengths of stay and recovery periods, and are both costly and non-reimbursable. The current standard of care is to prevent lung complications with use of incentive, or therapeutic, spirometry, where patients must breathe in from an incentive spirometer device 10x each hour to inflate their alveoli. Proper incentive spirometry can prevent 1 in 3 post-surgery lung complications. However, current incentive spirometers are entirely mechanical, making it difficult for nurses to track patient adherence to their exercises beyond labor-intensive bedside monitoring. The majority of incentive spirometry is currently self-monitored and self-reported by patients, which leads to poor adherence and overreporting. This makes incentive spirometry largely inefficient and ineffective in the clinic today. Airalux seeks to solve this problem with a redesigned incentive spirometer that can automatically collect user metrics and display them on a mobile dashboard for clinician monitoring. The Airalux device is also designed to increase incentive spirometry adherence through behavioral nudges and goal setting for patients. We have developed a unique combination of sensors, mechanical design, and a software suite that can directly measure patient airflow and does not rely on the mechanical piston systems of current incentive spirometers, which are clunky, difficult to digitize, and introduce large opportunities for user error. The goal of this technology is to make incentive spirometry easy for patients to use and easy for clinicians to monitor. The aims of this project are to develop a prototype with reduced materials costs that make it viable and affordable for clinical use. Because this technology relies on environmentally sensitive sensor configurations, we must also verify that our reduced-cost device can measure inhalation volumes accurately in a broader range of environmental settings. This will enable advancement of our technology to Phase II, when we will conduct a randomized clinical trial with patients using our lower cost prototype and assess clinical outcomes from increased adherence, such as improved oxygenation and reduced complication rates. Commercially, this device seeks to improve patient outcomes after surgery, while also saving hospitals non-reimbursable costs associated with lung complication treatment and readmissions.