Colorado State University

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ ABSTRACT Family violence, which includes intimate partner violence (IPV) and child maltreatment, is a significant public health problem and disproportionately affects low-income and minoritized women and children under one year of age. Family violence during the perinatal period, the months during and after pregnancy, has been linked to numerous health disparities intergenerationally, including elevated stress in mothers as well as increased risk of poor cardiometabolic health and socioemotional development in infants. IPV during pregnancy and its associations with maternal stress may also place infants at greater risk of child maltreatment and interfere with mother-infant coregulation of physiology and behavior, in turn negatively affecting infant health and development. Moreover, individuals differ in how they respond to adverse environments, with extant models suggesting that maternal and infant resilience factors interact with family violence to confer vulnerability to risk or to promote resilient outcomes. What remains unknown, however, is an advanced understanding of the stress regulation and interpersonal mechanisms that link characteristics of prenatal IPV to subsequent child maltreatment and infant outcomes postpartum. Therefore, in this longitudinal and prospective study, we propose to examine a mechanistic model highlighting biological, behavioral, and social pathways to understand the role of family violence in the progression of infant cardiometabolic health and socioemotional development during the perinatal period. The proposed study will collect self-report, medical record, observational, and biobehavioral data from 330 low-income, racially and ethnically diverse families across 5 time points: 1st (T1), 2nd (T2), and 3rd (T3) trimesters and 6 (T4) and 12 months (T5) postpartum. Our innovative dyadic approach to studying the mechanisms linking family violence to infant outcomes during the sensitive period of perinatal development will address three aims. Aim 1 is to examine how characteristics of prenatal IPV (timing, type, frequency, severity, predictability) relate to maternal perceived and physiologic stress. Aim 2 is to examine the direct and indirect associations among prenatal IPV, maternal stress, child maltreatment risk, mother-infant biobehavioral coregulation, and infant health and development. Aim 3 is to examine how mother and infant resilience factors moderate the impacts of prenatal IPV, maternal stress, child maltreatment risk, and mother-infant biobehavioral coregulation on infant health and development. Our unifying hypothesis is that characteristics of prenatal IPV and maternal stress will carry over into the postnatal period to negatively affect mother-infant relations and regulation but will be differentially associated with infant health and development based on maternal and infant resilience factors. This multi-method study will provide critical insight into sensitive periods whereby family violence that occurs prenatally has a lasting impact on infant outcomes. Identifying modifiable mechanisms of risk and resilience will also greatly inform strategies for prevention and intervention to lessen the intergenerational transmission of stress dysregulation, thus improving the lives of vulnerable mothers and infants.

NIH Research Projects · FY 2024 · 2023-09

Multiple system atrophy (MSA) is a prion-like movement disorder caused by misfolding and self- templating of the protein α-synuclein (α-syn), which spreads throughout the central nervous system to cause progressive degeneration. Similar to many other prion and prion-like neurodegenerative diseases, there are currently no therapeutics available that alter the course of disease for MSA patients. To interfere with α-syn self-templating, several groups have proposed various strategies for knocking down α-syn expression to reduce the amount of protein available as substrate. Unfortunately, these strategies may interfere with normal α-syn function in the brain, leading to loss-of-function deficits for MSA patients. Alternatively, MSA cannot propagate in transgenic (Tg) cells or mice expressing α-syn with the E46K mutation, raising the possibility of using gene therapy to generate conversion-incompetent α-syn to disrupt self-templating. However, to date, this approach has not been tested as a therapeutic intervention for MSA. The objective of the proposed work is to establish proof-of-concept that introducing a single residue change in the α-syn primary sequence can disrupt templated misfolding. We hypothesize that generating conversion-incompetent α-syn using CRISPR prime editing will reduce or prevent MSA propagation. Our approach will capitalize on our recent discovery that non- pathogenic α-syn mutations at residue K80 inhibit MSA propagation in vitro. In Aim 1, we will use CRISPR prime editing to insert our novel K80 mutations into Tg cells and mice expressing wild-type human α-syn prior to challenging the models with MSA patient samples. We have shown that MSA induces α-syn aggregation in unedited cells and mice expressing wild-type protein. We anticipate that successful gene editing will block transmission to these model systems. Cryo-electron microscopy has been used to resolve the structures of α- syn fibrils in MSA patient samples. This work has shown that misfolded α-syn adopts a Greek key conformation that is stabilized by a salt bridge between residues E46 and K80. In Aim 2, we will determine if our non-pathogenic K80 mutations exert their protective effectives by preventing salt bridge formation. We will also quantify the effect of these mutations on lipid binding and protein fibrillization. These orthogonal studies will determine if the K80 mutations are a viable clinical candidate for an MSA gene therapy. This work is innovative because it represents a paradigm-shift in how we approach gene therapies. Rather than focusing on correcting a disease-causing point mutation, we will establish proof-of-concept that gene therapy can be used to interfere with the self-templating disease mechanism underlying prion and prion-like neurodegenerative disorders. This work is significant because it has the potential to serve as a novel treatment strategy for patients with both sporadic and familial prion-like diseases. Through investigating the ability of conversion- incompetent α-syn to prevent MSA propagation, this work has the potential to transform the way we approach therapeutic development for neurodegenerative disease patients.

NIH Research Projects · FY 2025 · 2023-09

Arthropod-borne viruses (arboviruses) adapt to local conditions, maximizing their potential to perpetuate and emerge as health threats. The adaptive potential of arboviruses is driven by error-prone replication, which creates a genetically diverse pool of competing virus genotypes within each host. One of the most important ways that the environment is changing is that temperatures are rising. This proposal examines some of the ways that temperature may impact arbovirus evolutionary biology. Aim 1 will address how a comprehensive temperature gradient that includes both constant and fluctuating temperatures with varying means and amplitudes alters natural selection on WNV populations within mosquitoes and the strength of bottlenecks. Our predictions are that fluctuating temperatures will increase the strength of purifying selection, that diversity will be maximized at optimal constant temperatures, and that bottlenecks will become wider as temperature increases. Flaviviruses infections are most frequently initiated by aggregates of virus particles. Aim 2 will address the extent that this occurs in a host- and temperature- dependent manner, bringing our previous work into a more ecologically relevant, realistic context. In the second phase of Aim 2, we will ask whether these genome aggregates can help to facilitate the maintenance of genetic diversity in the WNV population. This is important because population bottlenecks can significantly impact virus fitness, and aggregation of genomes in individual infections may help viruses escape from them. We have found that birds that generate high WNV viremia and are highly infectious to mosquitoes (crows) have significantly more unique WNV genomes per cell than those that have lower viremias (robins). Aim 3 will assess whether this also may occur in mosquitoes. We also will assess the degree to which this phenomenon may allow for the maintenance of low fitness viral genotypes while preventing those of high fitness from gaining dominance. Preliminary data supporting the feasibility of these studies is provided in the application. The significance of this work is that it will provide novel, comprehensive data on the ways that changing environmental conditions such as those that we are now experiencing may alter the fundamental population biology of arboviruses. Arboviruses are uniquely susceptible to these conditions because they must replicate in mosquitoes. This is inherently significant. Our work is also significant because it will provide mechanistic data on how viruses may maintain genetic diversity in the face of both selective and stochastic reductions in genetic diversity. Finally, the significance of our work is that we have provided technical and analytical tools that are broadly useful and have permitted us to collaborate effectively with a wide array of investigators. The proposed studies are technically and conceptually innovative because of the ways that we can combine realistic transmission systems in the lab with barcoded viruses, single cell approaches, and other new molecular tools.

NIH Research Projects · FY 2025 · 2023-09

Abstract Down syndrome (DS) is associated with an elevated likelihood of co-occurring neurodevelopmental conditions, including autism spectrum disorder (ASD). Although dimensions like sleep and brain activity are considered critical areas of interest in the investigation of nonsyndromic ASD, these physiological dimensions have not been characterized to date in co-occurring DS and ASD. This Administrative Supplement will expand the scope of R01HD110542 (Autism in Young Children with Down Syndrome) to evaluate the feasibility of collecting physiological measures of sleep and neural function in young children with DS, and their potential association with autism-related features. We will leverage the ASD-related phenotyping data collected in the parent R01 and collect both sleep actigraphy and fNIRS data concurrently with ASD assessment visits for 50 participants in R01HD110542 Project Year 2. We will collect child actigraph data across seven continuous days and nights and calculate sleep duration (sum of nighttime and daytime sleep episodes), timing (sleep start and end time), consolidation (ratio of daytime sleep duration/nighttime sleep duration), and fragmentation (number and duration of nighttime awakenings). Caregivers will also complete a standard daily sleep diary and sleep questionnaire to assess contextual factors impacting child sleep environment. Brain activity will be measured during the study visit via fNIRS. We will evaluate the feasibility of collecting usable data from these measures in children with young children with DS and subsequently analyze how physiological dimensions are associated with early social communication and play, two dimensions relevant for early ASD detection. Results may facilitate the discovery of additional indicators of ASD risk in DS, and potentially identify mechanisms that contribute to the onset of ASD in DS in ways that can inform future treatments in this population.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Mesenchymal stromal cells (MSCs) have been tested in nearly one thousand clinical trials, mostly because of their ability to secrete factors that can modify host environments. For instance, MSCs can adapt to their niches and remodel the extracellular matrix. While this property of MSCs can potentially be beneficial to treat fibrosis and promote tissue regeneration, leveraging this property has been challenging because specific signals that enable MSCs to remodel the matrix remain to be defined and leveraged in MSC-based therapeutics. Here, we describe a highly efficient approach to encapsulate individual cells in engineered gel coatings with specifically defined biophysical and biochemical cues. We have developed this approach to show that soft, thin gel coating is an enabling cue that increases the production of soluble interstitial collagenases in response to tumor necrosis factor-α (TNFα). Importantly, our preliminary data show that gel-coated MSCs decrease collagen deposition in a murine muscular dystrophy model. In this K99/R00 proposal, I will build upon these results to test the hypothesis that programming of MSCs using specifically engineered microgels activates the potential of MSCs to inhibit muscle fibrosis. In Aim 1, we will determine the role of engineered gel coating in TNFα- induced activation of MSCs to produce high levels of interstitial collagenases and degrade collagen, and understand mechanisms behind this process. In Aim 2, we will investigate the role of gel-coated MSCs in inhibiting muscle fibrosis and restoring muscle functions. Success of this proposal will lead to an effective strategy to treat muscle fibrosis, which is a major unmet clinical need. During the K99 phase, I will work with my advisory committee to enhance my knowledge in biomaterial design, cellular mechanobiology, pathophysiology of fibrosis, as well as advanced microtechnologies, imaging, and computational approaches to effectively drive this research project. These experiences together with career development activities described in this proposal will ensure my smooth transition to an independent investigator at the interface between mechanobiology and bioengineering to develop novel therapeutic strategies for muscle disorders.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT This proposal describes a five-year mentored research career development project focused on evaluating gene editing delivered via extracellular vesicles (EVs) as a novel approach for contraception. Almost half of United States pregnancies are unintended, and available contraceptives often have undesirable side effects and are effective only when utilized properly. Therefore, new easy-to-use contraceptive products with less side effects are required. A survey of postpartum women indicated about 50% prefer permanent sterilization compared with other contraceptives. The only permanent contraceptive available is surgical sterilization; while routinely performed, there are anesthetic risks, high surgical cost, and requires facilities and personnel that are absent in most low income locations. Non-surgical options for permanent contraception are currently unavailable. By secreting proteins that allow sperm capacitation, fertilization, and early embryo development, the oviduct (fallopian tube) is essential for fertility. Oviductal epithelial cell progesterone receptor (PGR) and oviduct specific glycoprotein (OVGP1) gene knockouts in mice induce infertility and reduce litter sizes, respectively. Gene editing using CRISPR-cas9 has recently gained popularity in a clinical trial for sickle cell anemia and has been used to edit neoplastic genes resulting in tumor shrinkage in rodent models in vivo. However, tissue-specific targeting of CRISPR-cas9 ribonucleoproteins (RNPs) and in vivo transfection are challenging. EVs contain proteins and nucleic acids within a lipid bilayer and are naturally secreted for intercellular communication. Because EVs are produced in vivo for cell transport, they circumvent immune clearance, avoid hypersensitivity reactions, and gain entry to cells that may not be accessible for foreign compounds alone. The overall goal of this proposal is to evaluate the utility of EVs for targeting RNPs to oviductal cells. Specifically, we will evaluate in vivo biodistribution of EVs deposited intrauterine in a mouse model to determine dissemination of EVs (Aim 1). Then, we will determine in vitro gene editing of EVs loaded with RNPs by designing guide RNAs to knock out essential fertility genes (PGR, OVGP1) in oviductal organoids (Aim 2.1) prior to assessing in vivo gene editing in a mouse model followed by a breeding trial to evaluate utility as a permanent contraceptive (Aim 2.2). In addition to generating knowledge on mechanisms influencing oviductal function, this project will lead to optimization of conditions for efficient delivery of CRISPR-cas9 RNPs to oviductal cells. Findings could lay the foundation for development of a non-surgical, permanent contraception for women. The candidate is a postdoctoral fellow at Colorado State University and has assembled a diverse team of experts to serve on her advisory committee. This proposal builds upon the candidate’s previous research background and will augment her expertise in reproductive biology with specialized training in gene editing, EV characterization and engineering, and mouse colony management. Furthermore, the training and development plan is comprehensive and tailored to her needs, which will facilitate her transition to an independent researcher.

NIH Research Projects · FY 2025 · 2023-09

Project Abstract The goal of this project is to develop new understanding and predictive models for the formation, reactivity, and selectivity of organic radical and diradical intermediates. Triplet diradicals can undergo a variety of transformations that are not accessible in the singlet ground state. However, developing efficient photocatalytic triplet energy transfer processes, particularly in an enantioselective fashion, remains an enduring goal. We will show that computational approaches can be leveraged to develop general principles for substrate and sensitizer design to harness triplet-state reactivity. We will use computation to target the mechanism-guided discovery of unexplored reactivity in the triplet state, such as homolytic aromatic substitution, and the design of chiral Lewis acids to promote asymmetric photocatalytic cyclizations. We will also develop a qualitative and quantitative understanding of the factors controlling the reaction rates and site selectivities of radical homolytic substitution, such as hydrogen and halogen atom transfer reactions. These conceptual insights will be used to rationalize experimental observations and underpin the development of new radical reagents for site-selective C(sp3)-H chlorination. The development of quantitative models, aided by new physical-organic parameters, can be used to accelerate this process. Machine learning models grounded in mechanistic understanding will provide new tools to parametrize substrates and reagents to accelerate reaction discovery and optimization. We will employ this strategy to predict the site-selectivity of P450 oxidation small molecules and to establish general workflows to predict the metabolic degradation pathways.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Current drug resistance surveillance for leprosy is solely based on the monitoring of clinical symptoms and the molecular identification of specific mutations in known drug resistance genes, which are few and fail to encompass the range of molecular mechanisms responsible for treatment failure. We used comparative genomics of drug-susceptible and drug-resistant Mycobacterium leprae strains to identify novel molecular markers of antibiotic resistance in leprosy. Top candidate genes whose polymorphism potentially associated with drug resistance were characterized using a surrogate Mycobacterium (Mycobacterium tuberculosis) since M. leprae cannot be cultured in vitro. Our preliminary results show that the deletion of one candidate gene in particular, fadD9, in M. tuberculosis significantly enhances dapsone resistance. Analysis of the potential function of this gene combined with the results of an earlier metabolomics study on the effects of antifolates on M. tuberculosis metabolism point to the existence of a previously unknown target of dapsone, independent of the FolP1 enzyme from the folate pathway, the deleterious pharmacological inhibition of which is mitigated by mutations reducing or inhibiting the activity of FadD9. This exploratory project aims to characterize these new mechanisms of susceptibility and resistance to dapsone in mycobacteria. Specifically, we hypothesize that dapsone inhibits the g-aminobutyrate (GABA) aminotransferase, GabT, responsible for the production of succinate semialdehyde (SSA) from GABA, thereby limiting the amount of succinate entering the TCA cycle through the GABA shunt. We further hypothesize that FadD9 converts the product of GabT, SSA, to succinaldehyde and that loss of/reduced function mutations in FadD9 thus prevent the limited amounts of SSA produced by the dapsone-inhibited GabT from being diverted away from the TCA cycle. Aim 1 will use genetic and enzymatic approaches to test the hypothesis that dapsone inhibits GabT. Aim 2 will similarly use a combination of cell-free and whole cell-based approaches to test the hypotheses that (i) FadD9 converts SSA to succinaldehyde and that (ii) clinically-relevant mutations lead to reduced or loss of FadD9 activity. Aim 3 will finally attempt to correlate different levels of dapsone resistance in a collection of well-defined M. leprae isolates to the presence of mutations in folP1, fadD9, and/or potentially gabT, in the same isolates.

NIH Research Projects · FY 2025 · 2023-08

Project Summary: The Rocky Mountain Regional Biocontainment Laboratory (RMRBL) at Colorado State University (CSU) has been responsive to the national RBL mission to: 1) “Conduct research on biodefense and emerging infectious disease agents”; 2) “Be available and prepared to assist national, state, and local public health efforts in the event of a bioterrorism or infectious disease emergency” since its opening and full commissioning in 2008. Researchers at the RMRBL and their collaborators rapidly pivoted in response to the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), working on host-derived therapeutics, vaccines, and identifying other potential zoonotic reservoirs as spillover opportunities in wildlife and domestic animals. Our researchers similarly pivoted this past year to work on countermeasures in response to the re-emergence of Mpox, while also continuing to address high consequence pathogens that have chronically plagued public health systems, such as Mycobacterium tuberculosis, the causative agent of Tuberculosis. Beyond our demonstrated ability to rapidly address research and research service needs on pathogens with pandemic potential, our team contributes to training, outreach, and access of our facility via sponsored fellowships, visiting scholars’ programs, workshops, conferences, and through collaborations. Despite these gains, our RMRBL BSL3 suites are aged, requiring constant investments to maintain safe, secure and compliant BSL3 facilities. RMRBL BSL3 researchers, support staff, and biosafety professionals are vulnerable to the strain of the work environment, limited resources, funding gaps, and opportunities to engage in less risky fields equipped with cutting edge technologies. In this application, we respond to the challenges facing the RMRBL BSL3 laboratories with 3 Cores. Core 1 includes an improved management structure, systematic replacement of deprecating scientific instruments, and comprehensive and proactive maintenance of existing facilities needs to ensure compliance and continuous functioning. Operation of the RMRBL BSL3 is additionally enhanced to improve the working environment and increase consistency for operations research support and animal husbandry staff. Core 2’s initiatives seek to develop training programs responsive to adult learning and education best practices, ensuring improved safety and safety compliance in persons working in the BSL3. Biosecurity upgrades will improve the safety and security climate in anticipation of new national standards, and integration between research teams and the office of Biosafety in constructing and training in technical standard operating procedures will accelerate safe performance and technical competence. Finally, Core 3 synthesizes our research strengths to develop a uniquely qualified Biocontainment Research Resources Core, bringing together opportunities to exploit team talent and perform innovative research. Combined, our three Cores ensure that the RMRBL BSL3 facilities are always ‘warm ready’ – to face and combat the next pathogenic pandemic threat.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY The purpose of this F31 proposal is to provide support for Cali McEntee, a doctoral student at Colorado State University (CSU), while she trains to become an independent scientist conducting research on age-related neurodegeneration. With support from this award, Ms. McEntee will determine if reductions in adenosine deaminase acting on RNA 1 (ADAR1) lead to the accumulation of double-stranded RNA (dsRNA), a pathogen-associated molecular pattern that causes innate immune/inflammatory signaling, in the context of brain aging and Alzheimer’s disease (AD). Numerous reports show that ADAR1 suppresses dsRNA-related inflammation and may modulate lifespan, and that innate immune activation/neuroinflammation driven by glial cells, like astrocytes, is a central mechanism of brain aging/AD. The applicant’s preliminary data show that brain aging/AD are associated with reductions in ADAR1 levels, and that reducing ADAR1 expression in astrocytes leads to an increase in transcripts from genomic repetitive elements (RE), which are increasingly implicated in aging and neurodegeneration and predisposed to form dsRNA. As such, ADAR1 may be an important regulator of neuroinflammation with brain aging and/or AD, perhaps by reducing RE-associated dsRNA accumulation. However, this idea has not been investigated. In this project, Ms. McEntee proposes to gain valuable translational research training by using multiple models to test the hypothesis that ADAR1 reduces the deleterious effects of RE-derived dsRNA and thereby protects against age/AD-related neuroinflammation by: Aim 1) using directly reprogramed human astrocytes from healthy young/older adults and AD patients to determine if ADAR1 and dsRNA levels are related to neuroinflammation, performing total RNA-seq and RNA immunoprecipitation sequencing (RIP-seq) to identify RE-derived dsRNAs involved in neuroinflammation, and intersecting her findings with existing clinical datasets; and Aim 2) increasing expression of ADAR1 in reprogrammed human astrocytes and a pre-clinical mouse model of aging to determine if ADAR1 protects against age/AD-related neuroinflammation and cognitive dysfunction. Ms. McEntee’s immediate goal is to gain the fundamental experience and professional skills necessary to perform independent research in the field of age-related neurodegenerative diseases. Her long-term goal is to become an academic, translational researcher investigating mechanisms of aging that contribute to neurodegenerative disease. Ms. McEntee will train in a state-of-the-art environment with an exceptional mentoring team at CSU. The sponsor, Dr. LaRocca, has an extensive background studying aging and neurodegenerative diseases and directs the NIH-funded Healthspan Biology Laboratory at CSU. Under the guidance of Dr. LaRocca and consulting mentors Drs. Chris Link, Ron Tjalkens, Julie Moreno, and Dan Lark, Ms. McEntee will be able to successfully complete the training plan, supporting her development into an independent scientist.

NIH Research Projects · FY 2025 · 2023-08

Project Summary / Abstract The bunyavirus order (Bunyavirales) includes significant human, animal, and plant pathogens. As with all segmented viruses, reassortment is a major driver of bunyavirus evolution. Reassortment can produce viruses with undesirable phenotypes, including the ability to infect new hosts. As the world becomes increasingly interconnected and disrupted by climate change, the opportunity for previously isolated bunyaviruses to meet and reassort is increasing. Compatibility between the proteins and RNAs of two viruses is a key determinant of whether they can produce viable reassortant progeny. And, because reassortment joins proteins and RNAs that have not adapted to work together, new reassortants face an uphill evolutionary battle when competing with their parents and other viruses in the population. This proposal will generate an improved mechanistic understanding of molecular barriers to reassortment and investigate evolutionary pathways that permit reassortants to emerge despite initial fitness disadvantages. We have devised a new system that uses libraries of competing minigenomes to quantify reassortment potential between large numbers of viruses simultaneously, and to define the molecular breakdown in cases when they can’t. Using these “minigenome melees” in concert with traditional techniques, we propose to answer targeted questions about the molecular compatibility between bunyaviruses. Our team combines expertise in viral genomics, molecular virology, mosquito infection, and virus evolution. In aim 1, we identify steps in the viral lifecycle that break down when bunyavirus replication proteins are mismatched and perform directed evolution experiments that force mismatched proteins to adapt to work together. This will explain a key constraint on bunyavirus reassortment and detail molecular interactions at the heart of the bunyavirus life cycle. In aim 2, we use our minigenome melee system to test the hypothesis that orthobunyavirus reassortment is relatively unconstrained by packaging. In aim 3, we test the hypothesis that bottlenecks associated with replication in mosquitoes enable less fit reassortant genotypes to gain a foothold in populations via the stochastic effect of genetic drift. At the conclusion of this project, we expect to have a substantially improved understanding of the molecular rules that determine whether two bunyaviruses can reassort, the fitness consequences of reassortment, and the evolutionary pathways by which reassortant viruses emerge. Our results could be used to parameterize models that predict bunyavirus emergence risk and will shed light on conserved interactions that could be targeted by antiviral drugs. Minigenome melees could in principle be used to study the biology and evolution of all kinds of RNA viruses, and we expect this work to establish this as a broadly useful platform.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Human tuberculosis (TB) is caused mainly by Mycobacterium tuberculosis (Mtb) and represents an enormous challenge to global health because of the inadequacy of currently available drugs and vaccines. The most common clinical manifestation is pulmonary TB, and Bacille Calmette Guerin (BCG) is the only licensed vaccine for protection against TB; however, its efficacy is highly variable. Today at least 52 countries have reported multidrug-resistant (MDR) and extensive drug-resistant (XDR) TB cases which cannot be cured or contained by current TB therapy. Thus, there is an urgent need to develop new therapies/vaccines that effectively prevent or cure TB. It is well established that the generation of an adaptive immune response against Mtb occurs inside germinal centers (GCs) in secondary lymphoid organs (SLOs), such as spleen and lesion draining lymph nodes, where antigen‐presenting cells (APCs) and antigen-specific circulating T and B lymphocytes interact, clonally expand, and are disseminated to sites of infection. We found that mice vaccinated with BCG and exposed to Mycobacterium avium [a non-tuberculous mycobacterium (NTM)] via drinking water provide more robust and longer-term protection than BCG alone as determined by reduced Mtb bacterial burden and inflammatory progression of infection. Interestingly, these mice also developed ectopic germinal centers (eGC) in the lungs and have an increased number of B-cells and higher levels of anti-Mtb cell lysate-specific IgA and IgG antibodies. These findings suggest that NTM and B-cells play a critical role in generating protective immunity against pulmonary Mtb infection, and the formation of eGC in these mice is a crucial factor in this improved immunity. Thus, investigating the mechanism of eGC formation and the role of NTM and B-cells in its stimulation is an important question to understand TB pathogenesis and develop effective vaccines and therapies. In this K99/R00 application, we propose three aims: 1) investigating the key differences between eGC in lungs and conventional GCs in lymph nodes, 2) evaluating the role of NTM and B-cells in eGC stimulation, and 3) characterizing the antigen-specificity and affinity of eGC B-cells against Mtb antigens. The results of this proposal will bring us one step closer to understanding the B-cell and antibody-mediated mechanisms of protection from TB.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ABSTRACT There are many diseases (e.g. arrhythmias, coronary artery disease, and infections) and situations (e.g. medical devices, trauma, and surgery) that require rapid and accurate point-of-care hemostatic tests, especially when regulating antithrombotics or after a blood transfusion. However, there are few options in resource-limited environments like low- and middle-income countries (LMICs), rural clinics, combat environments, and many others. This is because current hemostatic assays require electrical power, capital expense, skilled technicians, large equipment, and can be slow. As cardiovascular disease increases in LMICs, there is an urgent need to provide innovative tools for these lower resource environments. As an alternative, microfluidic-paper based analytical devices (PAD)s have been developed for low resource settings as a point-of-care technology since these are rapid, cost-effective, portable, disposable, eas-to-use, and typically require electricity. Such devices include the rapid antigen tests developed for COVID-19, which provide critical rapid feedback for making educated health decisions. These can be used almost anywhere with relatively little skill. Our goal is to develop a similar point-of-care tool that we call the Paper-based Clotting Analysis Test (P-CAT). Current PADs are limited for hemostatic analysis since they typically produce low flow conditions that can’t test many of the processes/pathways in primary hemostasis. We have developed a fast-flow PAD that will be incorporated into the P-CAT, enabling us to investigate the spectrum of pathways involved in hemostasis with only a drop (microliters) of blood. P-CATs can be created rapidly and at very little cost, thereby producing high throughput data. Here, we will develop the P-CAT to test blood samples with high specificity and and sensitivity to 1) pathways in primary (platelet-dependent) hemostasis, and 2) intrinsic and extrinsic coagulation cascades in secondary hemostasis. If successful, we will expand upon the P-CAT to incorporate additional biomarkers in an automated point-of-care device that could have a major impact on enabling portable, rapid, and inexpensive tests for hemostasis that can be widely distributed.

NIH Research Projects · FY 2025 · 2023-06

Project Summary Mycobacterium tuberculosis, the causative agent of Tuberculosis (TB), is the leading cause of human mortality due to an infectious disease, outside of the COVID-19 pandemic. The limited knowledge on risk factors and comorbidities for TB progression and the mechanisms by which these promote susceptibility limits the ability to develop new prevention and treatment approaches. Recently, we published data showing a causal relationship between vitamin A deficiency and progression to clinical active TB disease, carrying up to a 10-fold higher risk for human TB progression. The substantial TB risk associated with vitamin A deficiency highlights the need to understand the mechanisms by which this molecule contributes to TB pathogenesis, particularly in the context of malnutrition among TB-affected communities, which are often the same communities affected by vitamin A deficiency. Vitamin A has been shown to have an impact on both innate and cell-mediated immunity, where diverse roles in immunity convolute the potential contributions of this molecule to TB immunity. We hypothesize that vitamin A is required for effective cell-mediated immunity to control infection after exposure, and respond properly to vaccination, requiring production within the lung for development of effective immunity. The goals of this research are to better understand the contribution of vitamin A bioavailability to the development of the immune response, the metabolic perturbations of vitamin A during infection that may limit bioavailability, and how vitamin A status impacts efficacies of BCG vaccination practices. These goals will be achieved through three Aims using a guinea pig model of vitamin A deficiency developed in our laboratory. We will first determine the contribution of vitamin A bioavailability to the development of the coordinated granuloma immune response using single cell and spatial transcriptomic approaches on infected guinea pig lung tissues. Next, we will evaluate cellular, organ-level, and systemic vitamin A metabolic patterns during infection using stable heavy isotope tracing methods in the guinea pig model throughout the course of infection. Finally, the impact of vitamin A on protective efficacy of BCG vaccination, the only vaccine available for TB, will be assessed under conditions of physiologic and pathologic neonatal vitamin A deficiencies. Upon completing these experiments, we will have determined the role of vitamin A in the development of the granuloma and cell mediated immunity, the requirements for, and availability of, vitamin A at the site of infection in the lung, and the impact of existing and proposed human vitamin A supplementation programs on the efficacy of BCG vaccination. These results will elucidate mechanisms of TB disease progression, identify the role of vitamin A in TB immunity and propose informed options for preventive or therapeutic intervention on vitamin A deficiency.

NIH Research Projects · FY 2026 · 2023-06

Project Summary Parkinson’s disease (PD) is a debilitating movement disorder affecting the central nervous system (CNS) and is the second most common neurodegenerative disease worldwide after Alzheimer’s disease (AD). In addition to age and genetic background, environmental exposures are strongly associated with the development of PD. Agents implicated as risk factors for PD include pesticides and heavy metals, as well as infectious agents such as bacteria and viruses. The question of how cumulative environmental exposure to these agents throughout lifespan can promote the development of PD and related disorders remains largely unanswered. Inflammatory activation of glial cells in the brain is recognized as a critical early event in the prodromal phase of PD. Still, the molecular signals regulating conversion to this damaging inflammatory phenotype are only now being elucidated. This R35/RIVER application addresses the critical question of how the neuro-immune axis responds to environmental exposures encountered across the lifespan and how the combination of such exposures can trigger the onset of neurological disease through phenotypic conversion of glial cells to a neurotoxic state. To develop a deeper understanding of the molecular regulation of phenotypic changes in glial cells that underlie the progression from prodromal to symptomatic disease, the proposed studies will employ sophisticated transgenic models and molecular tools to address these scientific questions. The key scientific questions raised in this application coalesce around three well defined and interrelated goals: 1) Determine how developmental exposure to environmental neurotoxins modulates innate immune signaling in glial cells to alter the inflammatory response of the neuro-immune axis to subsequent toxic exposures during aging, 2) Identify key molecular pathways in glial cells altered by exposure to environmental neurotoxicants that regulate inflammatory responses of the brain to viral infection, and 3) Elucidate molecular signatures in regional populations of glial cells corresponding to various states of activation across the progression of neurological disease triggered by exposure to environmental neurotoxicants and viruses. The scientific goals addressed by this R35/RIVER application concerning multiple exposures across lifespan and risk for neurological disease will use both long-term studies involving exposure to multiple neurotoxic and infectious agents, as well as powerful approaches such as high-content imaging, neural network-based informatic analysis, single-cell and spatial transcriptomics and generation of new transgenic models. The R35 mechanism is ideal for integrating these complex resources and approaches with the personnel expertise necessary to address fundamental questions about how environmental exposures alter innate immune responses of the brain that promote the development of neurological disorders like PD and AD.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY High mutation rates in mitochondrial DNA (mtDNA) are a major cause of inherited and age-related diseases. There is a longstanding view that the mutation-prone nature of mitochondrial genomes in humans and animal models is a byproduct of the intense metabolic activity associated with oxidative phosphorylation and energy conversion. However, recent evidence has challenged this idea, and there is growing recognition that differences in the enzymatic machinery responsible for mtDNA replication and repair are a key cause of high mutation rates in human mtDNA. Variation in mtDNA replication and repair pathways may also explain why some eukaryotic lineages (e.g., plants) are able to suppress mutation rates in their mitochondrial genomes to exceptionally low levels. An overarching theme of our research program is to identify the mechanisms responsible for variation in mitochondrial mutation rates across eukaryotes and thereby help resolve the long- term uncertainties about why rates are so high in humans. To overcome the technical challenges associated with investigating rare events like de novo mutations, we have applied multiple innovative sequencing technologies that can detect new DNA sequence variants present at ultra-low frequencies, essentially capturing mutations and damaged bases as they occur and mapping them to nucleotide-level resolution. Our future work will address two major questions. First, how do plant mitochondria achieve some of the lowest rates of point mutations ever observed (less than one substitution per site per billion years)? This line of investigation will build off our recent discovery that plant-MSH1 is necessary for maintaining low mutation rates in plant organelle genomes. MSH1 is enigmatic member of the MutS gene family which was likely acquired by horizontal transfer from giant viruses. We will test the hypothesis that it is part of a novel mechanism of mismatch repair that induces double-stranded breaks followed by template-based recombinational repair. Second, how do mitochondria repair bulky DNA damage introduced by UV? We will test the hypothesis that mitochondria contain a previously unrecognized repair pathway with similarities to classic nucleotide excision repair (NER). This hypothesis is motivated by our recent observation that exposure of divergent eukaryotic model systems to UV light results in the release of mtDNA-derived oligonucleotides that carry damaged bases at consistent positions and exhibit characteristic length profiles, a hallmark of NER. Overall, this research program will help determine why mitochondrial genomes exhibit such extreme mutation rate variation across the eukaryotic tree of life.

NIH Research Projects · FY 2026 · 2023-05

Project Summary / Abstract The broad, long-term goal of my research project is to understand the parameters controlling prion transmission and evolution within and between species, and ultimately to prevent recurrent epidemics in humans and animals. Chronic wasting disease (CWD), a burgeoning epidemic in cervids of increasingly uncertain zoonotic potential, is a particular focus within this general framework. My research group is one of only a handful with the resources and expertise in transgenic, cell biological, biochemical, molecular genetic and in vitro approaches to study prion diseases. Our output has exerted a powerful and sustained influence on the field. This application leverages a longstanding relationship with NINDS which is a feature of my uninterrupted record of NIH funding as an independent investigator for a period covering 26 years. Since prion studies require long-term experimental commitments requiring sustained and highly coordinated approaches, this proposal explores the feasibility of an alternate funding mechanism with improved stability and flexibility leading to improved efficiency which will enhance our already significant capacity to innovate, conduct transformative research, and capitalize on new developments. This application is designed to build on the advancing trajectory of our research by addressing key questions relating to naturally-occurring prion diseases with a particular focus on CWD. We will address the prevalence, properties and origins of emergent and established CWD strains; explore how strain conformations and species-specific PrP primary structural differences regulate interspecies prion transmission; investigate the parameters which stabilize strain phenotypes or promote prion adaptation/evolution; address the roles played by peripheral compartments and the central nervous system in strain selection/adaptation by the host; ascertain the risks posed by established and emergent strains to humans; and determine the structural properties of CWD prion strains at high resolution. The proposed mechanism also provides enhanced opportunities for dedicated mentoring and supervision of trainees and senior scientists, and to optimize my ability to generate a legacy for the next generation of independent investigators.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Multi-Track Post-Baccalaureate Research Education (MT PREP) The College of Veterinary Medicine & Biomedical Sciences (CVMBS) at Colorado State University (CSU) proposes to create the first national Postbaccalaureate Research Education Program (PREP) (R25) that will provide a unique training opportunity in veterinary and translational medicine research that will enable recent baccalaureate science (BS) graduates from underrepresented minority (URM), disabled and/or socioeconomically disadvantaged backgrounds to enter either a PhD or DVM/Ph.D. degree program. Data over the last 20 years from National Academy of Sciences, National Institutes of Health, and other biomedical research organizations reveal an insufficient number of individuals are being trained in translational medical science, despite the significant increase in job opportunities currently and in the future. In particular, the lack of training opportunities for Under-represented individuals interested in STEM severely limits the capacity to fill this national need. The proposed MT PREP will incorporate existing pre-doctorate DVM research training program activities as well as Ph.D. training program elements with specific postbaccalaureate activities to achieve the goals. Former postbaccs currently enrolled in graduate programs in CVMBS, trainees appointed to our CSU IMSD T32 and DVM/PhD students will serve as peer mentors. Well-qualified faculty mentors with diverse backgrounds and research disciplines, with over $15M in extramural funding, and demonstrated success in undergraduate and graduate training, have been recruited to provide experiential research opportunities for trainees. The goals of this program are to 1) recruit and support postbaccs from under-represented backgrounds with interest and aptitude for foundational, translational and/or clinical research in our MT PREP , 2) develop MT postbaccs’ independent critical reasoning, creative ideation, observation and rigorous experimental design and analytical skills, 3) provide faculty and peer mentorship for these candidates to practice responsible and ethical conduct of research and develop and implement their own mentoring philosophy and plan and 4) provide support to leverage postbacc’s PREP experience in writing federal GRFP applications, PhD and DVM/PhD program applications and presentation of results. Our long-term goal is to increase competitiveness of URMs for nationally recognized graduate programs and contribute to the talent pool of well-trained biomedical URM scientists.

NIH Research Projects · FY 2025 · 2023-01

PROJECT SUMMARY The long-term goal of the proposed research is to determine the neural pathways by which physiological signals regulate gonadotropin secretion, which ultimately determines reproductive function. Modulation of luteinizing hormone (LH) secretion determines fertility and controls gonadal steroid concentrations, which has profound effects on cardiovascular, musculoskeletal, and mental health. Pulses of luteinizing hormone secretion are organized by neurons in the arcuate nucleus of the hypothalamus that contain kisspeptin, neurokinin B and dynorphin (KNDy neurons) in males and females. In females, the preovulatory LH surge is induced by estradiol and is dependent upon kisspeptin cells in the anteroventral periventricular region (AVPVKiss1). However, the higher order neural circuitry that governs these populations of kisspeptin cells remains a significant outstanding question. The nucleus of the solitary tract (NTS) is located in the brainstem and consists of a heterogenous population of neurons that receive rich interoceptive and central inputs and projects widely thought the brain. Interestingly, these neurons are implicated in both the inhibition of pulsatile LH secretion during stress, and the facilitation of enhanced LH secretion during the preovulatory LH surge. To address this apparent paradox, this K99/R00 proposal will test the central hypothesis that distinct subpopulations of neurons in the NTS suppress pulsatile LH secretion via inhibition of KNDy neurons and enhance LH secretion via activation of AVPVKiss1 cells. During the mentored phase, we will employ viral-mediated cell activation labeling techniques and light sheet microscopy of optically cleared tissue to determine if the same neurons are activated during stress and the LH surge, as well as single-cell RNA sequencing to identify the subpopulations of NTS neurons that are activated during stress and the LH surge (Aim 1). The mentored phase will consist of critical training in advanced neuroanatomical and neuroimaging techniques, next generation sequencing technologies, bioinformatic analysis, as well as career development experiences that are necessary for transitioning to an independent academic research position. In the independent phase, I propose to use chemogenetic and cell- specific viral-mediated neural ablation techniques to determine whether subpopulations of neurons identified in Aim 1 are sufficient and necessary for stress-induced suppression of LH secretion and KNDy cell activation (Aim 2) or for the preovulatory LH surge and AVPVKiss1 cell activation (Aim 3) and determine the locations in the brain these subpopulations project (Aims 2 & 3). These studies will launch my independent research program and will provide a neural framework that may influence the development of therapies to treat disorders of altered LH secretion, including amenorrhea, infertility, and polycystic ovary syndrome. Collectively, the commitment of the sponsoring/co-sponsoring team to my scientific and professional development, coupled with the stimulating academic environment and impressive resources at UC San Diego available to me will ensure achievement of the aims of this Career Development proposal and the training mission of UC San Diego and the NIH.

NIH Research Projects · FY 2025 · 2022-12

Project Summary/Abstract Cannabis is the most commonly used substance among individuals who consume alcohol, but there is conflicting evidence regarding the effects of cannabis on alcohol use and specific health outcomes. Furthermore, physiological responses to long-term and regular use of these substances differ substantially. The present study aims to investigate physiological and psychological differences across individuals with different patterns of alcohol and cannabis use (individuals who exclusively and regularly use cannabis-only, alcohol-only, and individuals who regularly use both alcohol and cannabis). The specific aims are to (1) investigate the effects of regular cannabis and alcohol consumption on the gut microbiome and intestinal permeability in the three groups, (2) examine baseline circulating levels of endocannabinoids and differences in relation to substance use patterns, and (3) explore trait depression and anxiety symptoms and diagnoses among the three groups with validated indices such as Beck’s depression and anxiety inventories. These aims will be tested through collection of fecal and blood samples, as well as administration of several psychological assessments and substance use questionnaires to individuals between the ages of 21-40 who regularly use alcohol or cannabis or a combination of both. The current proposal is innovative in its multi-faceted approach to examining the effects of alcohol and cannabis, and novel in its consideration of an endocannabinoid-microbiota-gut-brain axis. Structural equation modeling will be used to investigate the aims, with data from an advanced biostatistics pipeline for microbiome analysis. This proposal is designed to advance the candidate’s long-term goal of becoming an independent scientist with advanced methodological and statistical knowledge that can be applied to clinical treatment of substance use and mental health disorders. The training objectives are to (a) develop skills and knowledge to bridge physiological processes and human psychopathology/behavior (alcohol and substance use, depression and anxiety), (b) develop proficiency and applied expertise in human gut microbiome research and analysis, and (c) develop competency and applied expertise in cannabinoid and endocannabinoid system research (methodology and analysis). Foundational knowledge generated from this proposal will inform the candidate’s future research directions and grant applications. The training will enable the candidate to become an innovative, skilled researcher capable of utilizing advanced methods and statistics to conduct rigorous research on integrative psycho-physiological processes and how they are impacted by alcohol and cannabis.

NIH Research Projects · FY 2025 · 2022-09

Imaging the full lifecycle of viral proteins in vivo is essential for understanding the molecular processes underlying viral infection. Live-cell imaging has long been performed using fluorescent protein fusion tags such as GFP. However, these tags can alter the size and function of targeted proteins. Furthermore, slow maturation, degradation, and photobleaching of tags results in the loss of signal, making it difficult to track the early life and ultimate fate of many proteins. Viral polyproteins, in particular, remain refractory to imaging in vivo due to their hypersensitivity to tags and the extensive processing and assembly they undergo during viral biogenesis. The use of linear epitope tags reversibly labeled by genetically encoded live-cell probes can solve many of these issues. Unfortunately, engineering functional probes for live-cell imaging of epitopes has been costly and time-consuming. In the proposed research, we combine expertise in protein engineering, single-molecule microscopy, and biochemistry to refine and accelerate the rational design of orthogonal epitope/probe pairs for highly multiplexed imaging of full viral protein lifecycles in living cells. We demonstrate the power of our strategy in our Preliminary Data by creating novel scFvs that bind the commonly used HA and Flag epitopes with high affinity in a variety of demanding live-cell imaging scenarios. In Aim 1, we will use our tested strategy to develop scFv against additional viral epitope tags and validate their utility in imaging experiments. To identify chimeric scFv that are both soluble and active within the cellular milieu, we will graft known epitope-specific CDR loops onto a unique panel of stable scFv scaffolds. In Aim 2, we will use state-of-the-art machine learning protein modeling and design methods to develop predictive binding models for scFv:viral-epitope complexes, validate a scFv design pipeline, engineer scFv libraries encoding multiple new peptide-binding solutions, and screen using innovative high-throughput, high-content in vivo methods. In Aim 3, we will demonstrate the utility of our newly developed scFv in live-cell imaging experiments by probing several critical aspects of viral biology. Specifically, we will use our engineered scFv to visualize and quantify the translation dynamics of flavivirus transmembrane polyproteins, and to monitor alphavirus particle assembly kinetics. Overall, this project will provide a powerful new pipeline for generating scFv proteins that can track viral proteins in living cells. The reagents we generate will provide the virus molecular biology community with new, versatile imaging tools to better illuminate many important biological processes.

NIH Research Projects · FY 2025 · 2022-09

Project Summary This project aims to determine the chemical mechanisms of lipid peroxidation in membranes. The accumulation of lipid peroxides on cellular membranes is a process that accompanies aging, as well as a host of age-related diseases, such as cancer, Alzheimer’s disease, stroke, and heart diseases. More recently, it was discovered that lipid peroxidation leads to a programmed cell death, called ferroptosis, which may provide the connection between lipid peroxidation and diseases. Despite their fundamental role in aging and health, the mechanistic nature of lipid peroxidation and its connection to the diseased state in cells remain elusive. This may be because lipid peroxidation studies are usually carried out in live cell context, where quantitative measurements are difficult, or with lipids in organic solvents, where it lacks biological relevance. There are two important questions with respect to lipid peroxidation and ferroptosis: one is which of the two fundamentally different pathways, enzymatic peroxidation or non-enzymatic autoxidation involving free iron, is responsible for the membrane lipid peroxidation in cells. The other is how lipid peroxidation leads to ferroptosis and other adverse cellular outcomes. To investigate these problems, we will develop a membrane-based assay for quantitative measurement of lipid peroxidation. By using surface-selective fluorescence microscopy to detect the generation of lipid peroxides, we will elucidate chemical mechanism of lipid peroxidation in the membrane context. We hypothesize that both the enzymatic and non-enzymatic pathways are important in efficient lipid peroxidation, as they play different roles in the process: the enzyme, lipoxygenase, initiates peroxidation with the selective binding for lipid substrates. Then, after a critical amount of lipids have been oxidized, membrane structural disintegration allows iron- catalyzed propagation of further peroxidation. We will test this hypothesis by directly measuring the rate of lipid peroxidation in membranes under enzyme-driven and autoxidative conditions. We further hypothesize that lipid peroxidation inflicts a chemical damage to membrane proteins, and the impairment of their function leads to cellular death. To test this hypothesis, we will evaluate the structural and functional impact of lipid peroxidation on Ras, an important signaling protein that is known to be sensitive to oxidative damage. The results of this study will provide a better understanding of lipid peroxidation, which underlies many age-related health concerns and therapeutic strategies to combat them.

NIH Research Projects · FY 2026 · 2022-09

PROJECT SUMMARY Growing evidence links transposable/repetitive element (RE) transcripts with Alzheimer’s disease (AD), but the underlying mechanisms and disease relevance are unclear. In this project, we will test the hypothesis that the mechanism linking RE with AD is an age-dependent, global RE transcript increase that causes age/AD- related neuroinflammation. Our rationale is that: 1) aging is the key risk factor for AD; 2) RE transcripts derived from structural/retroviral sequences in the genome accumulate progressively with aging; 3) RE transcripts are prone to form double-stranded RNA (dsRNA) and/or complementary DNA (cDNA), both of which can cause neuroinflammation (a major driver of brain aging/AD that precedes pathology); 4) aging and AD are also linked with impairments in epigenetic control (e.g., hypo-methylation, which could facilitate RE transcription) and quality control systems like autophagy that degrade RE transcripts (which could potentiate RE-derived cDNA/dsRNA accumulation). Emerging data even suggest that RE-derived dsRNAs/cDNAs could spread via extracellular vesicles (EVs), a potential non-cell-autonomous explanation for the pathology observed in AD. These observations suggest a model that is highly consistent with the age-dependence of AD, in which global, age-related RE transcript increases lead to RE-derived dsRNAs/cDNAs that drive neuroinflammation and AD. In support of this idea, our preliminary data show that RE transcripts are hypomethylated in AD patients and correlate with neuroinflammation prior to pathology, and that inhibiting RE transcript buildup in human astrocytes/neurons may reduce inflammation. We also find evidence of RE in circulating EVs from AD patients. Based on these observations, we propose to (Aim 1) determine if age- rather than pathology-related RE transcript dysregulation links RE with AD by performing a large bioinformatics analysis of existing RNA-seq data, probing for RE in human/AD brains, and profiling global methylation (whole-genome bisulfite sequencing) in the same brains, as well as in samples from >100 subjects from a longitudinal study on brain aging, neuroinflammation and AD. In parallel (Aim 2), we will use patient-derived astrocytes and neurons to test the efficacy of clinically translatable treatments (e.g., methylation/autophagy activators) that reduce RE-derived dsRNAs and cDNAs for inhibiting age/AD-related neuroinflammation, and we will identify RE that may cause neuroinflammation by binding to cellular dsRNA and cDNA sensors (PKR and cGAS). Finally (Aim 3), we will determine if RE transcripts induce aged/AD-like neuroinflammation and accumulate in EVs in young astrocytes and neurons, and whether these EV-borne RE transcripts cause inflammation/toxicity in other cells. We also will use existing samples (as described above) to determine if RE in EVs are related to markers of systemic inflammation, neuroinflammation and AD in humans. These studies are specifically designed to extend on our ongoing, NIA-funded pilot projects, generate multi-omics data on RE in aging/AD, and to provide a platform for future diagnostics or therapeutics in this context.

NIH Research Projects · FY 2025 · 2022-09

SUMMARY The success of the High Plains Intermountain Center for Agricultural Health and Safety (HICAHS) is grounded in engaging and listening to stakeholders and responding to regional needs with sound and relevant programs and partnerships. Building on a substantial record of accomplishments in research, intervention, education and outreach, we propose an innovative transdisciplinary Center that leverages expertise and resources among partners to promote a healthy agricultural and forestry/logging population in PHS Region VIII (Colorado, Montana, North Dakota, South Dakota, Utah, and Wyoming) and beyond. Over the past 6 years we have fostered a One Health approach with the rapidly expanding dairy industry in our region, responded to the disproportionate COVID-19 impacts on agricultural workers, and strengthened partnerships in occupational safety with the logging industry in Montana. This proposal brings together an internationally recognized leadership team, outstanding new investigators, and a well-developed regional partnership to address the new and unique needs of this region. Building on the Regional Needs Assessment and following diversity, equity, and inclusion principles, we will continue to advance health and safety in the region. The Mission of HICAHS is to improve the health and well-being of workers in food, fiber, and timber production by engaging partners in transdisciplinary research, education, and prevention programs across the region. The Center’s long-term goal is to reduce the risk of work-related injuries, fatalities, and illnesses primarily in the agriculture and forestry subsectors in the region. We propose integrating new and existing knowledge and using multiple routes of dissemination to translate research into improved agricultural health and safety practices through our three Cores: 1. Evaluation & Planning Core; 2. Research Core including our Pilot/Feasibility Program, two Basic/Etiological Research projects, one Intervention Research project, and one Translation Research project; 3. Outreach Core including our Community-Initiated Grant Program. By achieving our aims, we expect an effective Center infrastructure, cohesive and complementary research and outreach efforts, and strengthened partnerships among researchers and practitioners working together to support the agricultural and forestry workforce. Ultimately, we expect our work to contribute to the reduction of risks of work-related injuries, fatalities, and illnesses and usher in our vision of a healthy and safe agriculture and forestry workforce in the HICAHS region and beyond.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY The open availability of authenticated, well-documented research materials is essential for scientific progress. Plasmids have become essential research tools to address almost any question in biology, and together with other forms of engineered DNA molecules, are essential for clinical research applications including gene therapy, vaccine development, and the production of recombinant drugs. Currently, the two common links between a plasmid and its documentation are the plasmid names and the plasmid sequence. Despite the central role that plasmids play in biomedical research and development, there is no guaranteed way to connect a physical plasmid in a tube to its documentation. A pipetting error, a labelling error, a spontaneous mutation, or an undocumented modification of the plasmid are some of the events that could result in a tube containing a different plasmid than what is indicated on the label. In addition, there is no standardized, secure approach to documenting the sequence, function, and lineage of a plasmid. As a result, there are widespread discrepancies between the physical sequences of the plasmids in circulation in the life science community and their supposed reference sequence. This situation creates reproducibility issues, slows down R&D efforts and raises significant security and safety issues for biotechnology applications. We are proposing to develop a new digital certificate technology, enabled by a web-based resource called MyPlasmid.org, that will provide a robust, physical link between engineered DNA molecules, their electronic documentation, and their authors. This technology will produce unique DNA sequences generated by cryptographic algorithms that can be inserted into an engineered DNA molecule. MyPlasmid.org will allow users to document their genetic designs by aggregating the documentation of individual genetic elements as well as combinations of elements. In addition, it will link the computer records of the engineered DNA sequence directly to the molecule itself and provide a method to retrieve documentation without a priori knowledge of the plasmid's identity. Short unique DNA sequences called certificates will be inserted between the functional blocks of engineered sequences. Unlike DNA barcodes, certificates will be computed by cryptographic algorithms using the DNA sequence itself and the author's identity as input so that users of engineered DNA molecules can verify the origin and integrity of certified DNA molecules. The technology described in this proposal is expected to foster a transition similar what has been observed in the semi-conductor industry where different stakeholders invest in the development of circuits that can be easily combined in larger designs that can then be manufactured by foundries not involved in the chip design. By ensuring that the sequence, origin, function, and lineage of engineered DNA molecules is accurately tracked and conveyed to all users, the proposed technology will improve the reproducibility, utility, and potential application of engineered DNA molecules in the life sciences.