University Of Notre Dame

NIH Research Projects · FY 2025 · 2021-09

Tuberculosis (TB) is a highly contagious airborne pathogen that infects > 2 billion people, of whom an estimated 1.5 million people per year are killed by the disease. The global spread of multi-drug resistant (MDR), extensively-drug resistant (XDR), and totally drug resistant (TDR) strains of tuberculosis emphasizes the great need for new effective treatments. This renewal/Merit Award application capitalizes on the discovery of hits against two critical targets in Mycobacterium tubersuolsis – the imidazo[1,2-a]pyridine-3-carboxamides and the imidazo[2,1-b]pyridine-5- carboxamides that target QcrB and novel scaffolds that target complimentary BD oxidase – and seeks to advance these to potential TB treatments. As the first to patent, prolifically publish, and propose the mechanism of action for the imidazo[1,2-a]pyridine-3-carboxamide (IAPC) series, we are the most experienced group to continue development of this series through primate evaluation in preparation for clinical (human) studies. Additionally, we have disclosed the impressive in vitro properties of imidazo[2,1-b]thiazole 5-carboxamide (IT) series a new promising, rationally designed, scaffold we will continue to develop. This new class has low nanomolar antiTB activity against H37Rv, multidrug resistant (MDR) and extreme drug resistant (XDR) Mtb as well as good in vitro metabolism and in vivo exposure with greater lung to plasma ratios. Most recently, we have discovered a small molecule inhibitor of cytochrome bd oxidase in Mtb. A functional redundancy between the cytochrome bcc:aa3 and the cytochrome bd oxidase protects M. tuberculosis from the preclinical imidazopyridine (Q203)-induced bacterial death, highlighting the attractiveness of the bd- type terminal oxidase for drug development. Combination of our QcrB and bd oxidase inhibitor is bactericidal against replicating, nutrient-starved and hypoxic antibiotic-tolerant mycobacteria and showed increased efficacy in a mouse model of infection. These results indicate that further complementary development of a compound scaffold inhibiting the cytochrome bd oxidase will enhance the value of a drug combination targeting oxidative phosphorylation for treatment of tuberculosis. Furthermore, all of these heterocyclic scaffolds (IAPC, IT and bd oxidase inhibitor) can be prepared in bulk (50 – 100 g) inexpensively and, from these penultimate intermediates, lead compounds with animal efficacy can be prepared in just one synthetic step (amide bond formation or nucleophilic aromatic substitution) and in multi-gram quantities (>15 g). Through our extensive collaborations, we will evaluate all samples and combinations for antiTB activity. We will also perform related studies, including microbe selectivity, gross toxicity particularly looking to avoid mitochondrial toxicity, metabolism, pharmacokinetics (PK), maximum tolerated dose (MTD), mice and/or monkey efficacy and mode of action studies of any new compounds with promising activity and physicochemical attributes including metabolite identification. Our criteria for a clinical candidate are: selective nanomolar potency against H37Rv and drug resistant Mtb, in vivo efficacy comparable to first line drugs isoniazid and rifampicin (at a dose <100 mg/kg), low toxicity (at least 10x over effective dose), minimal drug-drug interactions, good aqueous solubility (>100 g/mL) and synthetic simplicity/cost effectiveness. A highly qualified team of coworkers and collaborators from experienced laboratories has been assembled to accomplish the overarching goal of providing the TB-research and biomedical communities a promising new anti-tb drug treatment as well as validated new drug targtes (respiratory bc1 complex bd oxidase of Mtb). RELEVANCE (See instructions): Tuberculosis (TB) is a serious global health risk that infects more than 2,000,000,000 people worldwide and causes a death every 20 seconds! The objective of this proposal is to develop cost effective anti-TB agents. The focus is on studies of new small molecular weight compounds that are easily synthesized, non-toxic, and yet effective at inhibiting TB growth.

NIH Research Projects · FY 2025 · 2021-08

PROJECT ABSTRACT Protein-protein interactions are governed by recognition events between peptide secondary structures (a- helices, b-sheets, loops), which in turn provide design cues for the development of selective chemical probes. However, removal of ordered peptide domains from the context of the surrounding tertiary structure compromises folding and conformational stability. Mimicry and disruption of b-strand/sheet interactions remains a considerable challenge. This is largely due to the inherent flexibility of short peptide sequences, the propensity for b-strands to aggregate, and the large surface areas and diverse modes of b-sheet packing. The early oligomerization of several amyloidogenic proteins involves conformational reorganization into parallel b-sheet structures, followed supramolecular assembly into toxic fibrils. Recent atomic-level structural data using patient-derived extracts has revealed that neurotoxic amyloids may be characterized by unique structural polymorphs, or ‘strains’, depending on the disease. Despite the need for amyloid- and strain-specific ligands, b-rich amyloid assemblies represent particularly challenging targets. We recently established peptide backbone N-amination as a subtle yet remarkably effective approach to b-strand/sheet stabilization. The conformational and non-aggregating characteristics of N-amino peptides (NAPs) render them uniquely suited for capping the growth of sheet fibrils while maintaining the facial packing and sidechain interdigitation important for amyloid recognition. Here, we will further develop soluble mimics of diverse b-sheet-like folds to disrupt amyloid aggregation in a sequence and strain-specific manner. As a proof-of-concept, we will target the assembly and cellular transmission of tau fibrils that characterize numerous sporadic and hereditary neurodegenerative disorders. Our overarching hypothesis is that the structural features of peptide N-amination will enable the development of ligands that selectively target b-rich amyloid folds. In Aim 1 we will expand the utility of NAP modification in pursuit of hyperstable b-strands and amyloid mimics based on parallel b-sheet macrocycles. A library of NAP-based tau mimics will be synthesized in Aim 2. These compounds will be evaluated for their ability to block aggregation and cellular transmission of recombinant tau fibrils as well those extracted from AD patients. In Aim 3, we will synthesize a series of aggregation-resistant NAP macrocycles that mimic the cross-b packing observed in pathogenic tau strains. These will be evaluated for their capacity to specifically inhibit cellular seeding by tau fibrils derived from AD and CBD brains. We anticipate that ligands emerging from this study will enable a robust examination of the the pathogenic strain model of tau transmission. More broadly, these studies will have a significant impact on the design of other selective disruptors of b-sheet and amyloid assemblies that are inherently difficult to target.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT The lymphatic system is an integral part of the circulatory system, where extracellular fluid flows from vascular capillaries into the lymphatic vessels and is returned to the vascular system via the thoracic duct. Additionally, lymphatic vessels regulate homeostasis of tissue fluid, absorption of dietary fat, and trafficking of immune cells. Consequently, dysfunction in lymphatic vessels is associated with development of many diseases, including obesity and metabolic disease, aging and Alzheimer’s disease, chronic wound and cancer, as well as inflammation and cardiovascular diseases. Therefore, controlling lymphatic vascular formation and augmenting its function is postulated as a promising therapeutic target for preventing and treating these debilitating diseases. Unfortunately, therapeutic lymphangiogenesis has not been widely explored partly due to the unavailability of a clinically-relevant cell source and controllable matrix environment. The overall goal of the research program is to derive lymphatic endothelial cells (LECs) and lymphatic muscle cells (LMCs) from human pluripotent stem cells (hPSCs) that can be used as a clinically-relevant cell source for modeling lymphatic function and physiology, as well as therapeutic lymphangiogenesis in a synthetic and controllable matrix environment. To this end, our lab is at the forefront of developing multi-disciplinary approaches to utilize stem cells and synthetic biomaterials for basic understanding of stem cell differentiation and lymphatic vessel morphogenesis, as well as approaches in therapeutic lymphangiogenesis. We have recently established xeno-free, well-defined and controllable differentiation protocols to direct hPSCs differentiation to clinically-relevant vascular progenitor cells with high reproducibility and efficiency, as well as wide clinical applicability. Furthermore, synthetic matrices can be used to provide spatial and temporal control for these progenitor cells to undergo lymphatic vascular morphogenesis, useful for basic understanding of lymphatic vascular biology and a range of therapeutic applications. These results establish a fundamental link between vascular and lymphatic morphogenesis within synthetic matrices. We are currently focused on bridging the large knowledge gap between molecular understanding of vascular and lymphatic differentiation and morphogenesis in a developmental context. Furthermore, we are also testing the impact of lymphatic vasculature to attenuate inflammatory response, prevent edema, and eventually promote tissue regeneration in a wound healing model. Cumulatively, we are combining approaches in stem cell and bioengineering, biomaterials and microfluidics, as well as lymphatic and systems biology to develop the necessary component in therapeutic lymphangiogenesis: reliable human cell sources from hPSCs within a biologically rational synthetic and controllable matrix environment. Collectively, this research has the potential to not only advance our basic understanding of lymphatic vasculatures in health and disease, but also to revolutionize the way we manage and treat a myriad of diseases that will benefit from innovative therapeutic lymphangiogenesis.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY: Modern healthcare has implemented medical devices to help and improve the life quality of people with chronic and lifestyle diseases. Paradoxically, although these devices are successful in achieving their purpose, they make the patient susceptible to infections. Urinary catheters are among the most widely used medical devices, and currently, catheter-associated urinary tract infections (CAUTI) are the most common healthcare-associated infection (HAI) worldwide, accounting for 40% of all HAIs. In addition, the treatment and control of CAUTI is becoming increasingly challenging due to the rise of antibiotic-resistant pathogens. Critically, CAUTIs are very different from uncomplicated urinary tract infections (UTIs), exhibiting unique clinical and pathological manifestations, as well as causative organisms. For example, in uncomplicated UTI, E. coli accounts for >95% of the causative agent, whereas in CAUTI, urinary catheterization allows pathogens such as Enterococcus spp., Staphylococcus aureus, Candida spp., Proteus mirabilis, Pseudomonas aeruginosa, and Acinetobacter baumanii to colonize the bladder, something that otherwise would not occur. Given that the frequency of catheter usage is only expected to increase due to both an aging population and medical advances, it is imperative to understand the pathophysiology of CAUTI if we are to develop ways to treat and/or prevent it. Recent work has found that urinary catheterization elicits bladder inflammation and mechanically disrupts the host defenses, compromising the host for microbial colonization. Further findings in mice and humans have shown that fibrinogen (Fg) is released and accumulated in the bladder to heal the damaged tissue. Fg is also deposited on catheters, coating them and forming a platform for colonization by CAUTI-associated pathogens. It was found that Fg levels modulate outcome of the infection and, in the absence of Fg, E. faecalis is unable to stick to the catheter and colonize the bladder. On the other hand, high Fg levels enhance enterococcal bladder and catheter colonization. This suggests that protein deposition on urinary catheters is a key factor for microbial infection. This proposal tests the hypothesis that by controlling the amount of protein deposition on the surface using a novel liquid surface coating, we will be able to control the rate and extent of uropathogen biofilm formation, urinary tract colonization, and systemic dissemination, as well as the inflammation response. Through a combination of material modification, proteomics, histological, and immunological approaches with a mouse model of CAUTI, we will: 1) develop liquid-infused catheters that control protein deposition; 2) assess their contribution in reducing protein deposition and biofilm formation in vitro; and 3) characterize in vivo how protein deposition modulation affects biofilm formation, the outcome of infection, and inflammation. Understanding the role of protein deposition in promoting pathogen-material-host interactions will provide new perspective in the establishment and progression of CAUTI, generating key insights into the development of alternative treatments that do not contribute to microbial resistance.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT The majority of breast cancers express estrogen receptor a (ER+), a molecule that typically promotes cell proliferation when activated by its ligand estrogen. Despite successes seen in treating patients with ER+ breast tumors with endocrine therapies, approximately 33% of these treated patients will develop recurrent metastatic tumors after endocrine treatment. These recurrent tumors primarily metastasize to bone and do not respond to current treatment options. Bone is the most common and one of the most dangerous sites for breast cancer metastatic tumor growth. At death, roughly 73% of women with breast cancer have bone metastasis. The bone architecture and the ubiquity of vascular sinusoids provide accessibility to and easy exit from bone to increase the spread through the body from the metastatic bone tumor. The bone microenvironment releases cytokines, chemokines, and growth factors that inhibit colonization of cancer cells in healthy bone or support colonization in cancer. These factors also can create an immunosuppressive environment that prevents a normal immune response or response to immunotherapy. In the proposed research, we will investigate how the chemokine Cxcl5, its decoy receptor Ackr1, and its receptor Cxcr2 contribute to ER+ breast cancer metastatic colonization of bone. We also will examine CXCR2 inhibitors for their efficacy as single agents and as a combination therapy (with bisphosphonates or immunotherapy) to inhibit breast cancer metastasis to bone. CXCR2 inhibitors are attractive therapeutics against metastatic breast cancer that may have efficacy in treating the formation of metastatic bone tumors that are dependent on the CXCL5:CXCR2 signaling axis and resistant to current therapies. Since patients with these tumors currently have few treatment options and often are incurable, this study could have significant translational potential. Significantly, if targeting or inhibiting these factors can reduce bone metastasis in preclinical animal models, then we will be in a position at the end of the grant period to propose that these compounds be used in a clinical trial in breast cancer patients with ER+ tumors metastasized to bone. Since these recurrent tumors typically do not respond well to current therapies, this treatment strategy would be significantly impactful and bring significant hope for patients with this disease. Future development of additional optimized small molecules or peptide inhibitors of CXCL5/CXCR2 will expand the therapeutic options available to clinicians in the care of breast cancer patients with metastatic disease.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY The gut microbiome has repeatedly been linked to major diseases of aging, including frailty, osteoporosis, and diabetes. However, after more than a decade of searching, there is still no consensus on which microbial species or taxonomic features provide reliable hallmarks of aging in adults or the elderly. Different people harbor different collections of microbes with densities and dynamics that vary considerably from one person to the next. This personalization arises, in part, because a given microbe may perform different functions in different people, and even in the same person at different times. This variability constrains the utility of microbiome taxa (e.g. species, phyla, biodiversity) to measure health and healthy aging. Overcoming this hurdle requires a shift in strategy, away from taxonomic data and towards data types that reflect the gut microbiome’s functional capacities, including the microbial genes and metabolic pathways found in the gut microbiome’s metagenome. Developing gut microbiome markers of healthy aging will also require prospective, longitudinal population-based research. However, we lack prospective data sets that track longitudinal changes in individual gut microbiome function and health outcomes across adulthood and old age. Our objectives in this proposal are to use a prospective, full life course, nonhuman primate model to: (i) identify changes in the microbiome’s functional capacities across the life course; (ii) test how social and environmental factors affect the nature and pace of microbiome aging; (iii) test how taxa-function relationships change at different life stages; and (iv) learn which microbiome features predict physical/behavioral aging and all-cause mortality. Our system, the well-studied Amboseli baboon population in Kenya, captures the complexity of human behavioral and social conditions better than other animal models. We have already profiled gut microbial taxonomic composition in 17,277 fecal samples collected over 14 years from 501 baboons. These data reveal personalized microbiome dynamics and aging trajectories that are shaped by individual social and environmental conditions. We propose to expand this data set for 10 more years to include 800 total individuals and analyze microbiome functional capacity in 12,000 samples. By identifying drivers and patterns of microbiome functional aging, we will identify targets for interventions aimed at building and sustaining healthy aging. Our results will help harness the promise of the gut microbiome to predict and improve human health.

NIH Research Projects · FY 2025 · 2021-04

Acinetobacter baumannii is listed by the CDC as a clinical pathogen that poses a serious antibiotic resistance threat in the United States, due to its resistance to the last resort carbapenem antibiotics (carbapenem-resistant A. baumannii or CRAb), which were the drugs of choice for treatment of infections caused by this microorganism. In addition, CRAb is often resistant to antimicrobial agents of different classes (multi-drug-resistant A. baumannii or MDRAb), which severely limits available therapeutic options. The major mechanism of resistance of A. baumannii to carbapenems is production of antibiotic-inactivating enzymes, carbapenem-hydrolyzing class D β- lactamases or CHDLs. In addition, carbapenemases of classes A and B, sensitivity of carbapenem targets (bacterial penicillin-binding proteins or PBPs), rates of antibiotic penetration into the bacterial cell and their expulsion by efflux pumps can also contribute to resistance. Levels of resistance to carbapenems reach up to 90% in some parts of the world, and mortality rates from infections caused by such bacteria are staggeringly high, up to 50%. Our long-term goal is to develop novel antibiotics for treatment of deadly MDRAb infections. Over the last decade, the Vakulenko group has performed in-depth characterization of clinically important CHDLs, which provides guidance for development of a new generation of carbapenems capable of inhibiting these enzymes. Concurrently, Dr. John Buynak’s (co-PI) group developed dozens of novel atypically-modified carbapenem antibiotics. We evaluated these antibiotics for their activity against MDRAb and demonstrated that three of them possess superior activity (when compared to commercial carbapenems) against MDRAb. All three inhibited the most prevalent A. baumannii CHDL, OXA-23, and had varying spectra of inhibitory activity against other CHDLs and carbapenemases of other classes. One of these compounds had an unprecedented wide spectrum of activity and resisted hydrolysis by a wide range of clinically important carbapenemases of all molecular classes. In this grant application, we propose to perform detailed characterization of our novel carbapenem antibiotics. We will determine activity of our compounds against A. baumannii strains expressing major CHDLs and other carbapenemases and unveil kinetic and structural features responsible for their ability to inhibit these enzymes (Aim 1). We will study interaction of our novel carbapenems with their targets, PBPs, and determine to what extent efflux pumps and porins influence bacterial resistance to these antibiotics (Aim 2). We will design and characterize several dozen novel carbapenem antibiotics to further improve their antimicrobial activity by enhancing their inhibitory potency against various carbapenemases, improving affinity for PBPs and increasing penetration rates and resistance to efflux (Aim 3). We will perform in vitro characterization of our best novel carbapenems to assess their solubility, stability and toxicity. Finally, our best compounds will be evaluated in animal studies to appreciate their potential as novel therapeutic agents against MDRAb (Aim 4).

NIH Research Projects · FY 2025 · 2021-01

Project Summary/Abstract Cost-effective, brief programs to support family communication and improve mental health in youth are a pressing need; yet few evidence-based programs exist. Our group has developed and rigorously tested an empirically-supported family-systems approach to improving communication and conflict in families, thereby improving mental health in youth. Beneficial effects for youth mental health and other indices of adjustment associated with the Happy Families Curriculum have been supported in several efficacy trials with families from a variety risk contexts. However, the value of efficacy research is limited unless it is subsequently tested in the context of an effectiveness trial. Given the potential large-scale benefits of broad implementation of the Happy Families Curriculum, a critical need exists for an effectiveness trial to evaluate the program when it is implemented in community settings by facilitators who would provide the program in “real world” settings. Our objective in this proposal is to test the effectiveness for a large sample, in different contexts of risk, of the brief (i.e. 4 session) psycho-educational and communication training approach used in our efficacy trials, and to examine the mechanisms associated with change processes that occur as a result of the program, including emotional security as a mediator of program effects and moderators of effects associated with participants’ socioeconomic and contextual risks as well as organizational factors that may impact program effectiveness. Our central hypothesis is that participation in the program will improve patterns of communication and conflict in families, thereby improving youth mental health. This hypothesis is supported by extensive efficacy research on the Happy Families Curriculum and conclusions based on a recently conducted feasibility study of the proposed effectiveness trial. Our rationale is that providing a family-systems approach to improving the family environment will support youth mental health over time. The specific aims are: (1) evaluating program effectiveness for improving communication, reducing destructive conflict in families and enhancing mental health in youth, (2) testing process models, guided by the Emotional Security Theory (EST; Davies & Cummings, 1994), to explain how, why, for whom and when, changes occur as a result of the program, and (3) evaluating organizational factors associated with program effectiveness, including the impact of organization structure and facilitator type, and organizations’ subjective evaluation of the program. This approach is innovative because it utilizes an RCT design to test the effectiveness of a proven family-systems approach that represents a brief, inexpensive and readily scalable approach to foster change in families’ communication patterns and improve mental health. The program is based on a well-established theoretical model for “mechanisms of effect” and backed by evidence for program efficacy. This research is significant because it will result in an inexpensive model program for family-system-level interventions that is sustainable in the organizations it is tested in, and readily adjusted to other contexts.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY: Even with an ever-expanding arsenal of active drug molecules validated in vitro, ensuring these reach their desired target in the body, while at the same time limiting toxic exposure in healthy tissue, remains a challenge. Routes for targeting drugs using antibodies or targeted carriers still result in less than 1% of drug arriving at the site of need. Molecular-scale targeting may have inherent advantages relative to these approaches due to more extensive tissue distribution and more rapid clearance of unbound attenuated therapeutic agents, leading to more drug arriving at the site of need or clearing prior to onset of systemic toxicity. Routes using `click' chemistry and related covalent ligations have been explored for homing drugs to pre-targeted sites. Here, we describe our progress and plans in developing a versatile and modular molecular-scale approach that uses synthetic non- covalent affinity to home drugs to desired sites in the body. Relative to covalent molecular-scale approaches, the chemistry we use has faster kinetics of association and also enables future reuse of the targeted site. Through prodrug methodology, we have shown that drugs of interest can be modified with affinity motifs through labile linkers, to be recognized at desired tissue sites by the presence of a corresponding binding partner. Serial re- dosing of these sites, or the possibility to temporally change the drug delivered, adds further benefit to our modular non-covalent approach. With this proposal, we seek to further define this research program and more fully capture the benefits of non-covalent recognition relative to `click'-based alternatives. Specifically, we will elucidate the importance of prodrug design and pharmacokinetic properties. So as to enable serial re-targeting of a drug site – a distinct benefit of non-covalent recognition – we will explore new chemistry for in situ immolation to lower-binding variants. We will also explore this approach in overcoming common physiologic barriers to the administration of protein and small molecule therapeutics, using the systemic administration of innocuous agents to trigger the release of therapeutic compounds bearing affinity tags from locally applied depots. Finally, to expand the therapeutic scenarios wherein this targeting route may be useful, we will explore this affinity axis for integration with metabolically engineered cells. In summary, we are optimistic that the new targeting technology we are developing will unlock the vast therapeutic potential of active agents which are presently limited by systemic toxicity or poor target localization. A platform such as that we are pursuing would have broad application in therapeutic delivery for the treatment of a variety of diseases or for remote intervention in implanted biomedical device practice.

NIH Research Projects · FY 2024 · 2020-07

Project Summary/Abstract Gram-negative bacteria have become broadly resistant to known classes of antibiotics. Treatment of infections by these pathogens has become increasingly challenging and efforts in the past two decades in discoveries of new classes of antibacterial agents have failed. In this grant application, we have turned our attention to bulgecins, a group of three natural products (bulgecins A, B and C) discovered in the 1980s, which potentiate the activities of b-lactam antibiotics to Gram-negative bacteria. The three natural products were prepared by total synthesis and we documented the potentiation activity in microbiological experiments. Furthermore, we documented by both fluorescence microscopy and by scanning-electron microscopy that the combination of bulgecin A and a b-lactam antibiotic (ceftazidime or meropenem) cause bulges in the bacterial envelope, which are points of structural instability that burst and lead to bactericidal effect. In addition, we documented that merely two lytic transglycosylases out of 11 in Pseudomonas aeruginosa—Slt and MltD (with ceftazidime) and Slt and MltG (with meropenem)—are the targets of bulgecin A. We also report the X-ray structure for the complex of Slt with bulgecin A. We disclose the next phase of this research in two Specific Aims. Specific Aim 1 addresses our planned analysis of the bulgecin-biosynthetic cluster, which we discovered recently. The eight-gene cluster converts L-serine and L-aspartic acid to bulgecinine, a key structural component of bulgecins, and then in turn, to bulgecins A, B and C. We propose to study these genes both for their enzymological reactions and for their structures. A proposal is outlined to prepare the high-value bulgecinine using a host bacterium as a “one-pot” reaction vessel. We already have reported a chemical synthesis for bulgecinine and a second (shorter) synthetic approach is also proposed. A detailed plan is outlined in Specific Aim 2 to optimize the bulgecin template. The process takes advantage of our X-ray structure for the complex of Slt and bulgecin A in a computational analysis to identify analogs that will bind more potently to lytic transglycosynases and achieve penetration into Gram-negatives more avidly. The proposed targets will be synthesized and fully analyzed in a series of both in vitro and in vivo experiments in identification of a suitable combination of a bulgecin analog with a b-lactam antibiotic in fighting Gram-negative bacterial infections.

NIH Research Projects · FY 2023 · 2019-10

ABSTRACT Approximately 30-50% of the US population experiences acute sleep continuity disturbance (i.e., insomnia) per annum, and approximately 10% of the population report chronic levels of insomnia. Chronic insomnia (CI) is associated with significant daytime impairment and is a substantial risk factor for multiple psychiatric and medical disorders. Given CI’s prevalence and consequences, it is essential to identify factors that perpetuate this disorder. One of the leading candidates for the neurobiological basis of CI is hypothalamic-pituitary-adrenal (HPA) axis dysregulation, specifically, alterations in cortisolinergic tone. Cortisol secretory patterns exhibit both a circadian and an ultradian rhythm. Ultradian pulses (i.e., every 60-120 minutes) are hypothesized to be involved in the maintenance of wakefulness during the day and may be related to the inhibition of wakefulness at night (i.e., the inhibition of pulses promotes the consolidation of sleep). While cortisol pulses naturally occur with transient awakenings, we hypothesize that these pulses can become a conditioned phenomenon in CI that predisposes the individual to awaken and/or experience prolonged nocturnal awakenings. Increased cortisol pulses during the day may also be expected because of the increased effort required to maintain wakefulness, and in turn, these increased pulses may further condition the aberrant occurrence of cortisol pulses at night. The scientific aims are (1) to evaluate whether subjects with CI, as compared to good sleepers, exhibit greater ultradian cortisol pulsatility during the day and/or at night, and (2) to quantify the association between ultradian cortisol secretion and metrics related to spontaneous awakenings from sleep (i.e., timing, frequency, duration, and EEG spectral profile of the awakenings). The proposed study will be conducted as a between-subjects design, examining 20 individuals with CI and 20 good sleepers during two consecutive nights in the laboratory (Night 1 is a screening night). While in the lab, blood will be sampled every 10 minutes for 24 hours and sleep will be polysomographically recorded. A refined delineation of both the circadian and ultradian aspects of cortisol secretion may allow for a better understanding of the etiology of chronic insomnia, the efficacy of established treatments, and potentially the development of new therapeutic approaches. The training plan includes educational activities that encompass three broad topic areas: (1) general skills (i.e., professional, ethics, and research training activities), (2) principles and practice and methodology issues related to neuroendocrinology, and (3) principles and practice and methodology issues related to behavioral sleep medicine and sleep medicine. The training plan builds upon the applicant's background in depression-related sleep research and stress physiology and provides the necessary training in neuroendocrinology, behavioral sleep medicine, and sleep medicine to further explore and document any association of HPA-axis abnormalities with persistent wakefulness at night. The pedagogic approach includes routine one-on-one mentorship, directed readings, course work, mini-fellowships, lab-based trainings, and conferences/workshops.

NIH Research Projects · FY 2026 · 2017-08

ABSTRACT Genetic crosses coupled with linkage mapping have provided an outstandingly successful approach for locating the genetic determinants of biomedically important traits such as drug resistance and host specificity in P. falciparum malaria. In the initial funding period of this Program Project (P01), we made great strides in transitioning Plasmodium crosses from the original model using chimpanzees to the human-liver chimeric mouse infused with human red blood cells (the FRG huHep/huRBC mouse) to generate more than 30 crosses in a span of 5 years. This included many replicate crosses of 6 different parental combinations that produced nearly a thousand new cloned progeny. This capacity allowed for optimizations that drove our development of the bulk segregant analysis (BSA) methodology and put within reach the ability to use experimental crosses of new clinical isolates to test specific hypotheses about emerging drug resistance. The combination of routine and replicated experimental crosses with BSA has shifted the challenge from making crosses and identifying genetic loci to instead prioritizing the rapid identification of genes and mechanisms underling parasite drug resistance and fitness. Our capstone discovery identified the role of pfaat1 as an epistatic partner with pfcrt in the evolution of chloroquine resistance that influences the balance between resistance and compensatory fitness, a crucial determinant of how new drug resistances emerge and spread. The overall goal of this P01 renewal proposal is to continue to advance this technology to track in real-time the alarming emergence of artemisinin resistance (ART-R) and its partner drugs used in artemisinin combination therapies (ACTs) in East Africa, an imminent threat to reverse decades of progress against morbidity and mortality due to malaria on the continent where more than 90% of malaria deaths occur. Coding mutations in the kelch13 gene that are strongly associated with ART-R and serve as the only markers for surveillance. However, mutations in kelch13 generate a wide range of resistance levels and fitness effects, and how these effects are compensated by other structural or regulatory changes in the genome remains unknown. Relying on 3 Research Projects, supported by 2 Scientific Cores, we will use targeted experimental genetic crosses to (i) dissect the genetic complexity of ART-R, (ii) clarify the role of kelch13, (iii) define the regulators and partner genes that control ART-R and emerging resistance to lumefantrine, the predominant ACT partner drug in Africa and (iv) establish a sustainable and user-centric valuable community resource including our methodological pipeline, data and biological materials. In the process, we will expand our BSA drug selection protocols to include cutting edge single cell RNAseq as the next step in building this powerful community resource for real-time solutions to clinically urgent questions.

NIH Research Projects · FY 2025 · 2016-05

Abstract Our NIGMS-funded research emphasizes the interface between structural biology, molecular biophysics, and immunology. Broadly speaking, we aim to connect the fundamental physical principles that govern protein behavior with function in the immune system, relying on a wide variety of approaches in biophysics, structural biology, computational biochemistry, and molecular immunology. In addition to providing mechanistic insight into immunology, our work in this interface has been instructional for addressing basic rules of biomolecular recognition and other protein behavior, as well as in the modeling and design of complex systems. In this renewal, we propose to continue this interdisciplinary focus. Our studies emphasize T cell receptors (TCRs) and their ligands, short peptides bound and “presented” by major histocompatibility complex proteins (peptide/MHC complexes). TCR recognition of peptide/MHC complexes is the cornerstone of cellular immunity, as it defines specificity and initiates the signaling that leads to T cell immune responses. Owing to the high diversity in both receptor and ligand, as well as the myriad of processes in which these molecules participate, the TCRs- peptide/MHC interaction is recognized as one of the most complex in biology. Deconstructing how specificity emerges in the face of this extraordinary complexity, learning how to predict and manipulate TCR recognition properties, and understanding the biophysics of T cell signaling processes remains at the core of our studies. We are motivated not only by the desire to gain further mechanistic insight, but also by the growth of new therapeutic approaches such as gene-engineered T cells and peptide-based vaccines. While there have been immunotherapy successes, there have also been significant complications and confounding outcomes. It is widely understood that an improved understanding of the fundamentals of immune recognition is needed for such therapies to reach their potential. Our goals for the next five years include improving our understanding of the mechanisms of TCR cross-reactivity and specificity, with an eventual goal of using structural information and modeling to identify cross-reactive ligands. Advances here will require concomitant improvements in our ability to model and score suboptimal (or as we call them, “sloppy”) protein-protein interfaces, which is a major part of our focus. We also plan to assess the mechanism of enigmatic “catch bonds” in TCR-peptide/MHC interfaces through the lens of physical chemistry, a view which has been largely absent from the discussion of catch bonds in immunology. We also aim to bring elements of physical chemistry and structural biology into predictions of immunogenicity, tackling this by considering the biophysics of protein-protein molecular recognition. Lastly, we aim to continue our work on dynamic allostery, studying how protein dynamics contribute to immune recognition and the still poorly-understood mechanism of T cell triggering. Our work remains highly collaborative and interdisciplinary, allowing it to impact multiple fields in molecular and cellular immunology and protein biophysics.

NIH Research Projects · FY 2024 · 2013-05

PROJECT SUMMARY: N-terminal (Nt) acetylation is an understudied aspect of bacteriology. Nt-acetylation is the addition of an acetyl group to the amino group on the α-carbon of the first amino acid of a protein. The fundamental mechanisms promoting and regulating Nt-acetylation, and the consequences of this modification in bacteria remain undefined. The objective of this renewal application is to define the fundamental mechanisms underlying Nt-acetylation in mycobacteria. The central hypothesis is that protein Nt-acetylation is a dynamic, regulated process that directly impacts mycobacterial virulence. The central hypothesis will be tested by following these specific aims: 1) Define the enzymes promoting Nt-acetylation in mycobacteria. 2) Establish the mechanisms and consequences of vir- ulence factor Nt-acetylation in mycobacteria. 3) Determine the link between Nt-acetylation and mycobacterial metabolism. Under the first aim, the applicant proposes to use enrichment strategies combined with quantitative proteomics to determine the function and substrate specificity of conserved mycobacterial NATs. Under the sec- ond aim, in vitro biochemical assays will be combined with targeted and quantitative proteomics to identify the NATs that modify essential mycobacterial virulence factors. Genetic and molecular approaches will be used to define functional relationships between predicted NATs in mycobacteria. Under the third aim, the applicant will combine enrichment and proteomics approaches to investigate differential Nt-acetylation following growth of mycobacteria on host-relevant carbon sources. The applicant will use proximity-dependent labeling to identify potential regulators of NAT activity. The successful completion of this proposal will contribute a fundamental understanding of the mechanisms promoting Nt-acetylation and establish a link between NATs, Nt-acetylation and essential mycobacterial virulence pathways. These contributions will be significant because they will ad- vance our understanding of an understudied protein modification important for mycobacterial virulence, which may be applicable to other bacterial species. The topic of this proposal is conceptually innovative because Nt acetylation is an under-investigated protein modification in both areas of tuberculosis research and bacteriology. Furthermore, studying the regulation of Nt-acetylation by metabolism to identify Nt-acetylation events essential for mycobacterial virulence is an innovative idea. The proposal is technically innovative because the applicant combines biochemical screens, enrichment protocols with bioanalytical chemistry, and expertise in molecular and genetic manipulation of pathogenic mycobacteria. The applicant leverages both M. tuberculosis and M. marinum strains to optimize productivity. These studies in bacteria will lay a foundation for focused and informed studies in animal virulence models in the future. By rigorously studying the mechanisms and regulation of Nt- acetylation in mycobacteria, the applicant may establish new therapeutic targets for treating mycobacterial dis- ease.

NIH Research Projects · FY 2026 · 2013-01

Project Summary/Abstract Staphylococcus aureus is a problematic human bacterial pathogen, which is broadly resistant to b-lactam antibiotics. This resistance is inducible and is conferred by a set of genes that encode a b-lactam antibiotic sensor/signal transducer protein, a gene repressor and two resistance determinants (the BlaZ b-lactamase and a unique penicillin-binding protein designated as PBP2a). A key feature of these processes is the recognition of the antibiotic by the b-lactam sensor/signal transduce BlaR, an integral membrane protein. Recognition of the antibiotic by the sensor domain of BlaR unleashes conformational changes through the membrane, which lead to the activation of the protease domain on the cytoplasmic side. This process culminates in expression of the genes for the antibiotic-resistance determinants. In Specific Aim 1, we describe the use of a fluorescent tool in live S. aureus for discovery of agents that shut down the BlaR recognition of the b-lactam antibiotics, which would reverse the resistance phenotype. A discovery funnel is outlined for analysis of structure-activity relationship for these compounds. In Specific Aim 2 we communicate a discovery that the BlaZ b-lactamase is incorporated to the surface of the cytoplasmic membrane in a lipidation- and phosphorylation-dependent manner. We outline a method for purification of the membrane-anchored BlaZ for the purpose of the identification of the sites of phosphorylation. The protein will be used in kinetic studies to compare to the non- membrane-anchored BlaZ, which is not phosphorylated. Plans are detailed for X-ray crystallography for characterization of the structural issues. The generality of the lipidation- and phosphorylation-dependent anchoring of proteins to the membrane surface in S. aureus will be explored for 10 distinct proteins. In each case, plans are outlined to identify the sites of phosphorylation to elucidate rules for phosphorylation and membrane sequestration of these proteins. These studies will shed definitive light on the complex machinery that S. aureus strains have evolved for resistance to b-lactam antibiotics.

Other NSERC · FY 2024

Multiphase flows, Fluid Dynamics, Atmospheric Physics, Dust, Lagrangian particles, Numerical simulation, Transport, Mathematical modeling, Turbulence