Colorado State University

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY / ABSTRACT No prophylactic or therapeutic regimen is known for any prion disease. The BSE outbreak in cattle with the subsequent emergence of a new and transmissible human prion disease (vCJD) highlights the public health threat from prion diseases. The commonalities among mammalian prion diseases are quite remarkable 1. The ongoing and expanding major threat from prion disease in North America is chronic wasting disease (CWD). CWD is an emergent rapidly spreading disease in cervids 2 with uncertain zoonotic potential 3. Approximately 9 million Americans hunt deer and elk 4 with an estimated 7,000-15,000 CWD- infected cervids consumed annually (increasing 20%/yr) 5. As hunting is a roughly $26 billion (per annum) 4 industry, CWD also represents an enormous potential health and economic threat. We have developed novel vaccine strategies which show promise in animal models 6-8 and have established a carefully-graded CWD challenge 9,10 in tandem with a longitudinal monitoring system in our unique indoor-housed white-tailed deer facility 10-14, permitting CWD vaccine assessment in the native host. We propose 3 aims to determine the ability of homologous or heterologous immunization strategies to protect deer from CWD infection and mitigate prion shedding. In Aim 1 we will determine the efficacy and safety of CWD vaccines that have complementary targets, a highly innovative and rationally designed vaccine which targets PrPSc 8,15 and a second vaccine strategy which aims at eliminating the substrate for prion conversion 7,16, and whether combining the vaccines is more effective. In Aim 2 we will determine if these vaccine strategies, alone or in combination, protect deer from CWD infection. Aim 3 will further determine if vaccination reduces prion shedding in excreta and secreta (urine, feces and saliva) of CWD challenged deer, thereby reducing infection between cervids and environmental contamination. The translational impact of the results of these studies will: a) develop prototype vaccine candidates for prion diseases b) provide management tools for the cervid industry and wildlife management c) mitigate the risks of zoonotic transmission of cervid prions. Thereby, this research will have immediate impacts in both human and animal health.

NIH Research Projects · FY 2025 · 2021-07

Project Summary This proposal aims to train a dual-degree, DVM-PhD, student in preparation for a successful career as a clinician-scientist. The applicant will earn a PhD in Biomedical Sciences while simultaneously earning a DVM. Given that cardiovascular disease and metabolic disorders are an increasingly prevalent global epidemic and these disorders significantly contribute to both increased mortality and increased years lived with disability, it is vital to understand the pathogenesis of these disorders. Epidemiologically, chronic stress has a prominent role in cardiometabolic risk. Neural processes are known to influence physiologic responses to stress. However, the specific mechanisms that underlie sex-dependent changes in endocrine and metabolic physiology after chronic stress are not well understood. Therefore, the research outlined in this proposal aims to determine how specific neural circuitry influences stress reactivity and, consequently, metabolic health in male and female rats. Specifically, testing the hypothesis that signaling from the infralimbic cortex (IL) to the rostral ventrolateral medulla (RVLM) mitigates endocrine stress reactivity after chronic stress in a sex-specific manner. The following specific aims will be addressed: 1) determines if activation of the IL-RVLM circuit mitigates endocrine responses to glycemic challenge and psychological stress in male and female rats. 2) Determines if activation of the IL-RVLM circuit following exposure to chronic stress reduces female susceptibility to endocrine hyper- reactivity. Activation of the IL-RVLM circuit will be achieved using optogenetic stimulation. Acute restraint will be used as a psychological stressor to measure activation of stress hormones, namely glucocorticoids, glucose, glucagon, angiotensin II, and insulin. Glycemic challenge in the form of a glucose tolerance test will be used a metabolic stressor. In aim 2, chronic variable stress (CVS) exposure will consist of 14 days of twice- daily randomized stressors. Following exposure to CVS, animals will undergo acute restraint and glycemic challenge. In addition to stress hormones, glucose, glucagon, angiotensin II and insulin, non-invasive measures of metabolism and autonomic activation such as heart rate, blood pressure, and body temperature will be taken during acute stress in both aims. Corticotropin Releasing Hormone mRNA will be quantified in the hypothalamus. Additionally, basal metabolic measures will be taken in the form of bodyweight and food intake. Taken together, these studies will provide novel insight into how cortical and brainstem processes integrate to influence metabolic health in a sex-specific manner. This will further our understanding of how stress contributes to metabolic and cardiovascular diseases.

NIH Research Projects · FY 2025 · 2021-06

Summary Mycobacterium abscessus (MAB) is a nontuberculous mycobacterium that causes chronic pulmonary infections (pMAB) and patients with pre-existing lung disease (especially cystic fibrosis patients) have a predisposition to pMAB. Due to MAB’s intrinsic antibiotic resistance, treatment is often complex and with low cure rates. Tigecycline, a glycylcycline class antibiotic, demonstrates bactericidal effects against pMAB without eliciting bacterial resistance mechanisms. For pMAB treatment, patients receive twice daily intravenous administration of tigecycline during at least one month resulting in significant side effects and many patients withdraw from treatment. Tigecycline has the potential to qualify as the first-line agent during therapy for pMAB and the backbone for new combination regimens but to achieve its fullest therapeutic potential, we need to improve tigecyclines ratio between efficacy and safety/tolerability, i.e. its therapeutic index. One approach to address this challenge is to develop inhalational formulations of tigecycline that are easy to administer and are well tolerated. In preliminary studies, GM-CSF KO mice with pMAB were treated by intrapulmonary aerosols of tigecycline for 28 days. The pulmonary bacterial burden after full treatment duration showed that inhaled tigecycline has high, dose-dependent efficacy, and is well tolerated. Here we hypothesize that aerosol delivery of tigecycline is a viable therapeutic approach for pMAB. In Aim 1, to avoid the tigecycline requirements for reconstitution, we will develop a dry powder formulation of tigecycline with well characterized aerodynamic properties suitable for inhalation. Aim 2 will study the relationship between dose, dosing regimen and resulting exposure of aerosols of tigecycline in different body fluids, organs and tissues. In particular, we will study the dose-exposure relationship of inhaled versus intravenous tigecycline and its availability in plasma, lung, abscesses and epithelial lining fluid. In Aim 3, we propose to test the efficacy, dose, dosing frequency, and duration of inhaled tigecycline against pMAB using animal models. We propose using first the GM-CSF KO murine model, subsequently, we will test the best regimen in b-ENac Tg mice with pMAB infection, as a representative model for cystic fibrosis patients. The best regimen will be validated in mice infected with selected clinical isolates from MAB clones 1 and 2 and isolates obtained from cystic fibrosis patients. Aim 4 will determine efficacy of inhaled tigecycline in multidrug therapies. Mice with pMAB as in Aim 3 (with strain #21 or clinical isolates) will be treated with binary or ternary combinations of inhaled tigecycline and clarithromycin (oral), clofazimine (oral), bedaquiline (oral). These studies will be performed by a consortium of experts located at Colorado State University, University of Tennessee, Research Triangle Institute and National Jewish Hospital. Working together, we aim to provide an inhalational therapy regimen of tigecycline with well- defined aerodynamic and PK properties and well characterized in vivo efficacy for future preclinical toxicology studies in larger animal models, IND application, and ultimately administration to patients.

NIH Research Projects · FY 2025 · 2021-06

Abstract Phosphatidylinositol mannosides (PIM) and their multiglycosylated counterparts, lipomannan (LM) and lipoarabinomannan (LAM), are complex glycolipids and lipoglycans found in the cell envelopes of all mycobacterial species. They play various essential although poorly defined roles in mycobacterial physiology and are important immunomodulatory molecules in the course of tuberculosis and leprosy as well as key ligands promoting the entry of mycobacteria and their survival within phagocytic and non-phagocytic cells. Although much progress has been made over the last 25 years in elucidating the structures and biosynthesis of these molecules, fundamental questions remain about the pathways leading to their biosynthesis and translocation to the cell surface. Furthermore, while the pleiotropic biological activities displayed by purified PIM, LM and LAM in cellular models suggest that they play important roles in pathogenesis, studies aimed at validating this assumption and precisely delineating their contribution to host-pathogen interactions when carried by intact bacilli are still limited by the paucity of mutants deficient in well-defined aspects of the biosynthesis and export of these molecules that are available. We propose to pursue structural, genetic and biochemical studies toward a complete definition of the structure (Aim 1), biosynthesis (Aim 2) and export (Aim 3) of PIM, LM and LAM. Completing our understanding of PIM, LM and LAM biogenesis, in addition to providing fundamental knowledge about the biochemistry of Mycobacterium tuberculosis (Mtb), is expected to lead to the discovery of essential enzymes and transporters which, much like the arabinosyltransferases of the Emb family and the epimerase DprE1, could provide new opportunities for anti-tuberculosis drug development. The availability of recombinant strains accumulating structurally defined biosynthetic precursors will facilitate structure-function relationship studies, and that of defined Mtb mutants deficient in various aspects of PIM, LM and LAM synthesis will allow a precise assessment of the contribution of these molecules to the immunopathogenesis of tuberculosis. Abbreviations: AG, arabinogalactan; AM, arabinomannan; AcylT, acyltransferase; Araf, arabinofuranosyl; AraT, arabinosyltransferase; CZE, capillary zone electrophoresis; DOC, deoxycholate; GT, glycosyltransferase; Ino, myo-Inositol; LAM, lipoarabinomannan; LM, lipomannan; LPS, lipopolysaccharide; MALDI-TOF, Matrix-Assisted Laser desorption/ionization time of flight; Manp, mannopyranosyl; ManT, mannosyltransferase; MPI, mannosylated phosphatidylinositol; MS, mass spectrometry; MTX, methyl-thio-xylose; ORF, open reading frame; OM, outer membrane; PIM, phosphatidyl-myo-inositol mannosides; TLC, thin-layer chromatography. Nomenclature: PIM is used to describe the global family of phosphatidylinositol mannosides that carries one to four fatty acids (attached to the glycerol, inositol and/or mannose) and one to six mannose residues. In AcXPIMY, x refers to the number of acyl groups esterified to available hydroxyls on the mannose or myo-inositol residues, y refers to the number of mannose residues; e.g. Ac1PIM1 corresponds to the phosphatidylinositol mono-mannoside PIM1 carrying two acyl groups attached to the glycerol (the diacylglycerol substituent) and one acyl group esterified to the mannose residue.

NIH Research Projects · FY 2025 · 2021-04

Innate defensive behaviors promote survival by allowing animals to detect and respond to threats within their environment, such as predators. Although innate, these defensive behaviors show a remarkable amount of flexibility and are subject to robust habituation, suggesting that the circuitry underlying defensive behaviors is subject to modulatory control. Our recent work suggests that the cerebellum may provide one source of afferent modulatory influence within the periaqueductal gray. We find that cerebellar afferents to the freezing-related circuitry in ventrolateral periaqueductal gray (vlPAG) predominantly activate local dopaminergic interneurons, which subsequently alter the relative strength of synaptically evoked inhibition and excitation onto freezing premotor neurons. More specifically, cerebellar activation increases IPSC amplitudes and decreases EPSC amplitudes, which is predicted to increase spike threshold and alter the input-output relationship, altering the integrative properties of freezing premotor neurons. These results motivate testing the hypothesis that the cerebellum modulates the expression of innate defensive behaviors through activation of local vlPAG dopamine neurons. To test this hypothesis, a combination of in vivo behavioral and systems-level approaches will be used to manipulate cerebellar input and record cerebellar activity during innate freezing behaviors to determine the nature of the cerebellar signals produced, and their resulting effects on expression of freezing behavior. To study the role of vlPAG dopamine neurons in modulating innate freezing behaviors, I will similarly record and manipulate vlPAG dopamine neuron activity using fiber photometry and optogenetic actuators. Finally, because selective lesions of vlPAG dopamine neurons disrupt both fear memory formation in mice (as do disruptions in cerebellar plasticity), I will test the hypothesis that cerebellar associative plasticity contributes to fear memory formation by modulating vlPAG dopamine activity. Together, these experiments provide a framework for understanding how the cerebellum modulates both innate freezing and more broadly how cerebellar plasticity contributes to learned defensive behaviors.

NIH Research Projects · FY 2025 · 2021-02

The overall objective of this project is to establish how infant diet with different protein-rich foods regulate growth trajectories and gut microbiota development. Both NIH and USDA are now addressing the urgent need for evidence-based dietary guidance early in life, particularly regarding protein intake, but a significant knowledge gap exists in the effects of protein-rich foods on growth and development during early complementary feeding. Early complementary feeding (~5 to 12 months of age), when infants start to consume foods beyond breastmilk or formula, is a critical transition period of developmental plasticity. Growth trajectories and shifts of the gut microbiota during this critical period have the potential to program long-term body weight, composition and disease risks and are greatly influenced by diet. Preliminary data from our pilot study in formula-fed infants demonstrated that consuming diets with two protein-rich foods: meat and dairy at a high-intake level, resulted in distinctive growth patterns from 5 to 12 months of age. An important new preliminary finding was that meat- and dairy-based foods directly affected gut microbiota diversity, composition and short-chain fatty acid production at 12 months. Changes in gut microbiota composition were also associated with infant linear growth (length gain). Here we propose three specific aims to determine how the introduction of common protein-rich foods impact infant growth (Aim 1), the development of gut microbiota (Aim 2) and the relationship between gut microbiota and infant growth (Aim 3), in a randomized controlled trial. Healthy, term infants (n=300) will be recruited and randomized to meat-, dairy-, plant-based diet groups or the reference group (standard of care), from 5 to 12 months. We will use controlled feeding (all foods provided, and formula if needed) with longitudinal assessments of gut microbiota, infant growth, blood biomarkers (IGF-1, IGFBP3, insulin, amino acids, lipids, etc.), dietary intakes, body composition, and total energy expenditure. Our multi-disciplinary team is ideally positioned to conduct this project, with collective expertise in pediatric nutrition, human clinical trials, microbiome, biostatistics, and a long-standing record of collaboration. Findings are expected to have significant scientific and health implications for determining dietary patterns that promote optimal infant growth and identifying gut microbial changes that are beneficial to the host metabolism and growth during early complementary feeding. The results of the study will also support evidence-based dietary recommendations in infants to prevent the risk of overweight and later obesity.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY/ABSTRACT Dr. Neha Lodha is an Assistant Professor in the Department of Health and Exercise Science at Colorado State University (CSU). Dr. Lodha's long-term career goal is to become an independent investigator advancing the science of functional mobility to improve the quality of life in aging and age-related neurological disorders and dementias. Up to this point, Dr. Lodha has acquired strong scientific background in neurophysiology and motor control. To accomplish her career goal, a key additional element required is the training in cognitive sciences. Understanding how cognitive and motor systems interact to enable independent mobility is important for identifying the precise mechanisms and developing interventions to promote functional mobility. The short-term training goals of the current K01 application are to train in 1) conducting cognitive assessments with specific emphasis on the intra-individual variability (IIV) approach, 2) clinical trials design and analysis, 3) multivariate statistics, 4) driving technology, and 5) transition to independence. To achieve these goals, Dr. Lodha has assembled a mentoring team that consists of 1) an eminent gerontologist, Dr. Manfred Diehl (primary mentor) with expertise in aging, IIV and cognitive development across the lifespan, 2) an experimental psychologist, Dr. Karlene Ball (co-mentor) who is internationally renowned for her work in cognitive correlates of driving mobility in aging and dementia, 3) a vascular neurologist, Dr. Sharon Poisson (advisor) with extensive experience in clinical trials in stroke survivors, and 4) a renowned biostatistician, Dr. Haonan Wang (co-advisor) with experience in statistical inference and modeling for clinical data. The proposed research plan focuses on identifying the motor and cognitive impairments underlying driving deficits in older stroke survivors with mild cognitive impairment as they are most likely to resume on-road driving after stroke. Conventionally, driving impairments are shown to be independently influenced by mean-level performance in specific motor and cognitive domains. However, this mean-level approach neglects the well-established fact that performance becomes highly variable and unstable in older adults with neurological disorders, such as those at preclinical stages of Alzheimer's disease and mild cognitive imapirment. The proposed research will apply the novel intra- individual variability (IIV) approach to investigate driving mobility in older stroke survivors. The goal of the research plan is to identify the impact of stroke on IIV in motor and cognitive performance (Aim 1); determine the contribution of IIV measures to predict driving performance (Aim 2); and use IIV measures to predict change in driving performance over a two-year period (Aim 3). The training plan will accelerate Dr. Lodha's career to pursue a unique and independent line of research that combines cognitive and motor processes to promote the functional mobility of older adults and is directly aligned with NIA's AD/ADRD goal to identify sensitive behavioral measures for earliest manifestations of functional impairments in older adults.

NIH Research Projects · FY 2023 · 2020-12

Unmanaged reward seeking is a shared central feature of eating and substance use disorders that expose patients to lifelong relapse vulnerability. Recent research shows that rewarding experiences induce synchronous activation of a discrete number of neurons in the nucleus accumbens core (NAcore) that are causally linked to reward-related contexts. This proposal intends to characterize the neuronal ensembles that are built through reward experience and code for reward seeking and extinction in the NAcore and adjacent circuitry. Throughout the proposal, special emphasis is given to comparing ensemble-specific changes between drugs (cocaine) and natural (sucrose) rewards to address whether or not addictive drugs usurp circuitry used by biological rewards or involve distinct circuitry mechanisms. I will use targeted recombination in active populations (TRAP) strategy, specifically the FosCreERxAi14 mouse line that allows tdTomato tagging of the neurons specifically activated during distinct behaviors. The K99 aims address the characterization of the cocaine- and sucrose-seeking ensembles and their colocalization within the same animal using an innovative version of the well-described model of reward self-administration and cue-induced reinstatement of seeking (Aim1A). The newly generated FosCreERxAi14xD2GFP reporter mice and RNAscope strategy (training component #1) will allow rigorous identification of the cell types comprising each ensemble (Aim 1B). Functional measurements of AMPA/NMDA ratios, a marker of plasticity (training component #2), will be performed within tagged cells during reward seeking (Aim 2). The R00 portion of the proposal evaluates necessity and sufficiency (Aims 3A-B) of the ensembles during cued-reinstatement using inhibitory and excitatory designer receptors exclusively activated by a designer drug (DREADD) specifically expressed in the ensemble cells, as well as the functional connection between reward-specific ensembles (Aim 3C). Characterization of the extinction ensemble will be achieved using FosCreERxAi14xD2GFP reporter mice and RNAscope (Aim 4A). Finally, ensemble-specific projections profiles of seeking and extinction will be completed using ensemble-dependent retroviral expression (Aim3B) followed by ensemble and projection specific plasticity measurements (Aim 4C). Preliminary data demonstrate that cocaine, sucrose and extinction ensembles form largely distinct but partly overlapping ensembles in the NAcore, and that AAV-targeted DREADD expression is restricted to the ensembles. The data obtained will shed new light on the mechanisms sustaining maladaptive reward-oriented seeking and extinction behaviors towards drugs and natural rewards. The Medical University of South Carolina provides an excellent research setting. Under the mentorship of Dr. Kalivas and my mentoring committee, I will experience advanced technical training and career development guidance to successfully complete this project while transitioning to an independent faculty position and further study of the intrinsic properties of ensembles underlying reward seeking.

NIH Research Projects · FY 2024 · 2020-09

Project Summary West Nile virus (WNV) is a mosquito-borne flavivirus, and the leading cause of domestically-acquired arboviral disease in the United States, resulting in significant disease and death every year among humans and other animals. Efficacious, vector-targeted interventions are critically needed, but current vector control methods (primarily insecticides sprayed into the environment) have been inadequate for WNV control. We have been developing and testing stations that contain endectocide-treated bird feed as a way to control WNV transmission to humans in the western U.S., the region which suffers from the highest incidence WNV disease in North America, by targeting the primary bridge vector mosquito, Culex tarsalis. This project is designed to develop endectocide-treated birdfeed as a novel, safe and scientifically-validated way to control the risk of West Nile virus transmission to humans in the western U.S. The concept is that birds will self-medicate by birdfeed stations containing the treated feed, which would be placed as arrays on homeowners' properties, in neighborhoods or in parks. The blood of treated birds would then kill the primary mosquito vectors of West Nile virus that blood feed on them, which would lower the risk of West Nile virus transmission to people who live, work and recreate around the birdfeed stations.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract – Overall The overarching aim of this project is to support food defense efforts in the State of Colorado by establishing a radioanalytical measurements laboratory capable of monitoring human and animal food products for radioactive contaminants. Currently none of the analytical laboratories maintained by the State of Colorado have radioanalytical measurement capabilities that could be utilized to perform measurements in support of food defense in either routine or emergency situations. The Health Physics program at Colorado State University has a long-standing tradition of carrying out radioanalytical measurements and radiochemical separations for training and research. The initial efforts will focus on providing a laboratory capable of monitoring food samples for gamma-emitting radionuclides in support of food defense (Track 1). Simultaneously the capability to measure alpha-emitting radionuclides (Track 2) will be developed by drawing on the facilities available at CSU and the expertise and the background of the faculty in the Health Physics program.

NIH Research Projects · FY 2024 · 2020-08

Project Summary As part of the Research Domain Criteria (RDoC) initiative, the NIMH seeks to improve measures of neuronal and psychological targets for use in intervention research. RDoC tools must precisely measure cognitive and neuronal systems to produce reliable findings. Unfortunately, many RDoC tasks have not been psychometrically evaluated, nor refined, and are liable to confounds that can lead to inaccurate claims of differential deficit, weakened effect size, and contradictory brain imaging findings. As highlighted by FOA PAR- 18-930, there is a critical need for modern psychometric methods and tools designed to support cognitive and clinical neuroscience research. This project responds to this FOA by evaluating and refining a methodology designed to administer statistically robust variants of RDoC tasks, especially in the context of brain imaging. We have created a quantitative methodology designed to administer computerized adaptive tests (CATs) in the context of cognitive and clinical neuroscience research. CATs manipulate stimulus properties in real time in order to improve measurement precision, avoid ceiling and floor effects, and maximize effect size, even for individuals and groups with highly discrepant levels of cognitive functioning. CATs also perform psychometric adjustments to cognitive tasks so that brain functioning abnormalities can be interpreted independent of performance deficits. In pilot work, we have used this approach with an RDoC working memory task, the N- back, to show that the methodology improves the reliability of both cognitive and brain imaging data. We will evaluate the generalizability and impact of adaptive testing, beyond the N-back task, for use in translational and experimental testing. Patients with schizophrenia and controls will be administered both adaptive and non- adaptive versions of four RDoC paradigms used to assess working memory: delayed match-to-sample, Sternberg, self-ordered pointing, and N-back. Additionally, participants will be administered the 5-Choice Continuous Performance Task and Probabilistic Learning Task, translational measures of control and learning respectively. Both groups will undergo functional neuroimaging and respond to adaptive versions of these tasks. The specific aims are to: (1) Determine whether adaptive testing improves the precision and effect size estimates of performance differences produced by RDoC tasks; and (2) Determine whether adaptive testing improves the reliability and effect size estimates of brain activation differences produced by RDoC tasks. While these aims are designed to evaluate a methodology, and to address critical concerns related to the use of RDoC working memory tasks as neural probes, mediators, and outcomes in psychosis research, results also have broad implications across populations, brain regions and networks, and cognitive domains. By addressing concerns of poor reliability, weak effect size, and brain activation confounds, this project will show that adaptive testing broadly improves cognitive neuroscience tasks. To facilitate rapid deployment of adaptive RDoC tasks, we will develop freely available versions of the paradigms used and a companion R package ‘catCog’.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY/ABSTRACT People who work evening, night or rotating shifts (i.e. “nonstandard” work hours) represent one in five U.S. employees and are alarmingly 44% more likely to develop Type 2 diabetes (T2D) compared to people who work standard day shifts. Circadian misalignment is one mechanism suggested to increases the risk of obesity and diabetes in people who work non-standard hours, and is highly prevalent and often unavoidable in modern, 24-hour society (e.g. shift work, long work hours, jet lag, medical residency, emergency responders, military personnel, Daylight Savings Time changes, etc). Disruptions in sleep and circadian rhythms have been linked to insulin resistance, increased energy intake, weight gain, and increased total body, abdominal and intrahepatic fat content, yet there have been limited attempts at identifying strategies or countermeasures to prevent the impact of such disruption on T2D risk in a sizeable proportion of the population. Therefore, our long-term goal is to identify and develop effective, behavioral countermeasures to combat the increased risk for metabolic diseases associated with sleep and circadian disruption when these behaviors are unavoidable. The overall objective for this project is to test the impact of time-restricted feeding to a 7h period in the day as a noninvasive countermeasure to the metabolic impairments associated with circadian misalignment. Our central hypothesis is that time-restricted feeding to the daytime period will prevent metabolic impairments during circadian misalignment compared to a condition where energy is consumed throughout the day and night. The rationale for the proposed project is that defining a non-invasive, scalable and feasible countermeasure to circadian misalignment could mitigate the risk of obesity and T2D. To test our overall hypothesis, will use a randomized crossover study with a rigorous inpatient diet-, activity and light-controlled protocol in 32 healthy men and women. We will determine the impact of time- restricted feeding during circadian misalignment on 1) muscle tissue insulin sensitivity and gene expression; and 2) muscle tissue lipid accumulation and circulating nocturnal FFA and glucose concentrations. Findings from this study represent a critical advancement in the fields of translational circadian and metabolic physiology by identifying and testing a countermeasure to circadian misalignment. Achievement of our proposed aims could lead to the development of new intervention strategies for chronic disease prevention and management. The knowledge to be gained offers the potential to support cost-effective programs that may inform our healthcare approach to metabolic disease prevention in populations at risk for these diseases such as shift workers, individuals with sleep disorders and anyone who eats outside of daytime hours.

NIH Research Projects · FY 2024 · 2020-08

Summary: Northern Colorado Bridge to Baccalaureate (NOCO B2B) traineeship program aims at increasing participation of underrepresented (UR) students in research careers in behavioral and biosciences. Each year, a cohort of 10 new trainees will be recruited from Front Range Community College (FRCC) underrepresented student population who are pursuing transfer to behavioral and bioscience research programs at Colorado State University (CSU). Trainees will receive a monthly stipend as they engage in research and scholarship support for two full years spanning their enrollment at FRCC and CSU. The NOCO B2B traineeship program will provide a scaffold of academic support services, co-curricular and extra-curricular programming and mentored-research opportunities to underrepresented Front Range Community College (FRCC) through a B2B learning community. Trainees will be coached to follow an optimal enrollment pattern to advance toward completion of a bachelor’s degree in a timely manner via an articulated pathway for transfer to their major at CSU. Trainees will begin their traineeship at FRCC the spring semester prior to transfer to CSU and participate in summer research experiences for undergraduates (REU) at CSU. Scholarships will provide financial support to allow students to enroll full-time. Stipends will alleviate students’ need for low-wage employment unrelated to their career path and allow them to focus on their research training. The program creates continuity between the FRCC and CSU experience through the use of articulated MAPs, co-enrollment opportunities at CSU prior to transfer, and a transfer learning community at CSU with social and academic events, and an upper division, 1-credit Transfer Student Seminar, “Becoming a Scientist.” The cohort-based program allows students to make strong connections amongst faculty, mentors and peers contributing to retention and completion. Sixty FRCC UR students will participate in the traineeship program and receive two years of scholarships and stipend support. 100% of trainees will complete a Summer REU; 100% of the trainee cohort who transfer to CSU will join the Transfer Student Learning Community and complete the Transfer Student Seminar “Becoming a Scientist;” 75% of trainees will complete a B.S. degree within three years of transferring; 80% of completers will obtain employment in a field related to their major or apply to a graduate program in behavioral or bioscience.

NIH Research Projects · FY 2023 · 2020-07

PROJECT SUMMARY Obesity is one of the defining public health problems of our time. At its root, increases in fat storage is caused by an imbalance in energy homeostasis, favoring energy intake over expenditure. Physiological mechanisms are in place to prevent excess caloric intake, yet these defense mechanisms fail in the face a modern food environment that promotes food intake. This is underscored by the lack of efficacy of non-invasive strategies, such as caloric restriction or medications, to sustain long-term weight loss. Thus, there is a critical need to understand the pathophysiology leading to food overconsumption and develop novel strategies to promote weight loss. The vagus nerve provides direct communication about nutrient intake from the gut to the brain. Removing of the vagus in lean animals results in significant overeating when presented with palatable calorie dense diets, suggesting a protective role of the vagus nerve to prevent overconsumption of calories. In obesity, vagal communication of gut metabolic cues to the brain is impaired, and preventing vagal signaling results in weight loss in animals fed high fat diet. The mechanisms for the switch from protection against, towards promoting obesity are unclear, but we have recently demonstrated that chronic consumption of high fat diet results in anatomical restructuring of vagal fibers in the brain. Therefore, we propose a new hypothesis that vagal gut-brain axis is reprogramed in response to high fat diets to drive obesity. We use a combination of molecular and genetic approaches to deconstruct the sensory vagus into cellular components based on their site of innervation to fully elucidate the role of high fat feeding on vagal remodeling. In aim 1 we assess the impact of diet on vagal fiber anatomy, synaptic function, and the behavioral consequences, including meal termination and motivation for food. In aims 2 and 3 we consider the mechanisms by which diet causes vagal remodeling. We hypothesize that a gut microbiota-driven immune response triggers the rewiring of the gut-brain axis. This is supported by our previous work and preliminary data showing abnormal microbiota composition is necessary and sufficient to alter vagal innervation in the NTS. In aim 2, we will use germ free rats and microbiota transplant to determine 1) if microbiota dysbiosis is sufficient for vagal remodeling, and 2) if restoring a symbiotic microbiota in obesity can normalize vagal signaling, feeding behavior and body weight. In aim 3 we will combine genetic and molecular tools to investigate the recruitment of immune cells with the vagal afferent pathway as mediators of diet-driven vagal maladaptation. Completion of these studies will identify vagal rewiring as a novel pathway in the etiology of obesity, and establish microbiota and microglia as potential tools for the development of weight loss strategies. .

NIH Research Projects · FY 2025 · 2020-07

This Medical Scientist Training Program renewal application from Colorado State University requests funds for continued support of a DVM-PhD dual-degree program with a 20-year history of translational clinician-scientist training. The program recruits three students per year from a pool of ~40 applicants and has a 97% retention rate across 49 total matriculants. Thirty trainees have graduated; 63% are employed in a variety of academic, agency, and nonprofit research positions. Seven students have received NIH F30/F31 fellowships since the first year DVM-PhD students were eligible applicants in 2015. Trainee research projects span broad disciplines across the College of Veterinary Medicine and Biomedical Sciences, which has provided nearly $4.2M in direct support of this DVM-PhD program since 2004. Forty-six participating mentor faculty represent a variety of career stages and expertise and hold 187 active research awards totaling over $34.6M. These mentors have trained, or are training, 209 predoctoral and 171 postdoctoral trainees, 33% of which are DVM-PhD or post-DVM/PhD trainees. Twelve MSTP students were supported by this T32 during our first 4 years of NIGMS funding. These 12 trainees have collectively published 22 total manuscripts, secured two NIH F30/F31 fellowship awards, and two completed their PhD degrees while participating in a rigorous program of biomedical research training (3-4 years) integrated with DVM didactic and clinical training (4 years). Current NIGMS funding also facilitated expansion of dual-degree program specific clinician-scientist training and career development activities which have reinforced the technical, operational, and professional skills trainees acquire from laboratory research and DVM clinical training. These activities have included ‘Translational Medicine’, a dual-degree specific rotating topics course focused on scientific communication, careers in biomedicine, grant and manuscript writing, and animal models of human disease. Trainees also completed annual workshops in experimental rigor and data reproducibility, while preceptors participated in mentor training through the CSU Graduate School. This renewal application focuses on more robust program evaluation and outcomes assessments, expanded national recruitment efforts, and enhanced engagement of our External Advisory Committee for guidance in directing this next phase of programmatic success and growth. Over the five-year award period, we request funds to support 28 training slots to be distributed in years one and two of DVM training. This funding will leverage institutional support to continue program expansion by enrolling 5 additional trainees over the award period, maximizing the impact of this MSTP T32 in fulfilling the national need for translational biomedical scientists.

NIH Research Projects · FY 2026 · 2020-07

Project Summary There is a critical need to develop synthetic methods that improve access to chemicals required for the discovery and production of new medicines. The long-term goal of this research program is to invent generalizable base-promoted reactions that address major limitations in organic synthesis. In contrast to widely used traditional base-promoted reactions that combine a pronucleophile with an electrophile, this proposal introduces general strategies for oxidative and reductive coupling reactions that combine two nucleophiles or two electrophiles, respectively. These protocols unlock a wide array of new base-promoted reactions that employ common functional groups to provide previously difficult-to-access products. Specifically, haloarenes are used as a new type of oxidant to transfer halogens to transiently formed carbanion species that enable oxidative coupling processes of mildly acidic C–H bonds. This process greatly streamlines the synthesis of ether and amine-containing arenes, alkenes and alkanes. Additionally, base-activation of disilanes generates silyl anions that transform electrophiles into nucleophilic species for C–C bond-forming reactions. This approach is used for the monoselective substitution of trifluoromethylaryl C–F bonds to access new difluoroalkyl chemical space and for the combination of alternative electrophile classes to form complex Csp2-Csp3 and Csp3-Csp3 carbon backbones. The methods in this proposal rely on base-activation strategies and thus have distinct scope, selectivity and utility over alternative means of reaction promotion such as the use of transition metals, electrochemistry or photochemistry. These impacts are demonstrated through single-step access to compounds that were either previously inaccessible or required tedious multistep syntheses and through shortened and modular routes to specific drug substructures. The reactions can also be used on complex drug-like structures to modify and diversify bioactive molecules. Upon completion of this work, chemists will have at their disposal a set of powerful reactions that increase the efficiency, practicality and range of access to molecules desired in the pharmaceutical industry.

NIH Research Projects · FY 2026 · 2020-04

PROJECT SUMMARY The objective of this proposal is to address the contrast problem in cellular electron microscopy. The cloneable fluorophore, Green Fluorescent Protein (GFP), and related fluorescent proteins complement small molecule stains and dyes to essentially solve the contrast problem in optical imaging. For cellular electron microscopy, however, contrast options are limited and there are no widely used cloneable contrast agents. Cloneable contrast in electron microscope imaging of biology can arise from a `cloneable inorganic nanoparticle (cNP).' A cNP is an NP made by a protein. The protein determines the properties of the nanoparticle such as elemental composition and shape. Because protein sequence, structure and function are encoded in DNA, the properties of the nanoparticle are also encoded in DNA. Modifications to DNA encoding a cNP may modify the resulting cNP. Our cNPs are based on inorganic ion oxidoreductase enzymes. Such enzymes select for and reduce inorganic biocoordination complexes, creating metal(loid) nanoprecipitates. Additional proteins/peptides (fused to the enzyme) act as ligands, influencing size, morphology, et cetera of the nanoparticle. DNA encoding the cNP can be concatenated to DNA encoding any protein of interest. Resulting protein chimeras contain an integral inorganic nanoparticle. The nanoparticle contrast allows the protein of interest to be identified against cellular background in electron micrographs. We have developed a cloneable selenium nanoparticle (cSeNP). We demonstrated cSeNP molecular labeling of FtsZ filaments in E. coli. The goal of this proposal period is to continue evaluation of this cSeNP and produce additional distinguishable cNPs. The proposal proceeds in 3 aims. Aim 1 is to evaluate the cSeNP in Drosophila, as a multicellular model organism, more complex than E. coli. Aim 1 also proposes to label ribosomal protein L1 in Caulobacter with the cSeNP and other cNPs. Because ribosomes can be unambiguously identified in electron cryo-tomograms, they provide an internal-standard for evaluation of cNPs. We will determine efficiency of cNP constructs by assessing what percentage of ribosomes have an associated cNP label. This assessment identifies shortcomings in cNPs, informing their subsequent refinement. Ideally, cNPs will emerge that label quantitatively. Aims 2 and 3 engineer different aspects of cNPs. Aim 2 uses protein design tools to minimize cNP protein size. Aim 2 also proposes directed evolution of cNP enzymes to produce cNPs of novel elemental composition. Aim 3 is to expand the palette of biomolecules that ligate cNPs, influencing their resulting size and morphology. Aim 3 will identify proteins within the protein coronas that form around cNPs, followed by identification of any serendipitous nanoparticle binding domains. Aim 3 also proposes to isolate nanoparticle binding peptide ligands through ribosome display approaches. Overall, this proposal is to develop and evaluate a pipeline of many size/shape/morphology distinguishable cNPs, facilitating multiplexed protein imaging in cellular electron microscopy.

NIH Research Projects · FY 2025 · 2019-09

Abstract Opiates are the cornerstone of analgesic therapy, but also produce numerous side effects. Besides their high propensity for addiction, repeated opioid administration leads to progressive sleep disorders, expressed as worsening insomnia and daytime sleepiness/sleeping. Opioid- induced sleep disorders (OISDs) are strong predictors of multi-substance use disorders, and psychiatric comorbidities including suicidal ideation. The primary target of opioid analgesic drugs is the µ-opioid receptor (MOR). MORs are widely expressed within the sleep/wake circuitry, therefore systemically delivered opioids might interfere with sleep at multiple sites of action. Importantly, no consensus has been reached on which specific CNS sites therapeutic or abused opioids act upon to trigger OISD, nor do current therapies specifically address OISD. Melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) synchronize the sleep/wake schedule to environmental cycles of light/darkness (“photoentrainment”) by sending light-evoked spike trains to the brain in humans and most mammals. Our recent work demonstrated that ipRGCs express MORs by which MOR-selective agonists directly inhibit the light responses of ipRGCs. Furthermore, we have shown that upon chronic, systemic delivery, morphine accumulates in the eye, and acts on MORs expressed by ipRGCs to alter the regular rhythm of sleep/wake while contributing to the chronic morphine-triggered behavioral sensitization. The objectives of the current proposal are to analyze whether kinetics of opioid deposition in the eye along with specifics of MOR signaling in ipRGCs directly contribute to the gradually worsening OISD. We will also test if antagonist of MORs delivered in engineered nanoparticles into the eye can reduce OISDs. The results will provide a mechanistic description of a novel neural pathway by which systemically administered opioids alter sleep/wake cycle. Additionally, the results will show the feasibility of using intravitreal MOR selective antagonists to reduce the severity and inherent comorbidities of sleep disorders in patients receiving long-term opioid therapies.

NIH Research Projects · FY 2025 · 2019-07

The Quantitative Cell and Molecular Biology (qCMB) Training Program provides comprehensive and flexible training in quantitative approaches in order to facilitate cross-disciplinary and collaborative research in the broad field of Cell & Molecular Biology. The Program produces outstanding graduates with the skills to meet the computational and professional demands of modern life science research. The objectives include to produce more graduates able to apply computational approaches to solve biological problems, provide a robust curriculum for training in this area, encourage and support cross-disciplinary collaborations, and offer training and professional development opportunities to better prepare students for the workforce. The qCMB program is designed to support students engaging in collaborative research projects requiring the generation and analysis of large biological datasets, particularly those generated through sequencing, imaging and flow cytometry. We introduce our early‐stage trainees to coding and its applications through a gateway course led by a supportive group of preceptors and more experienced trainees. As students build confidence, a plethora of elective computational courses support their individual research interests by expanding their quantitative skillsets. Symposia, workshops and group meetings will enhance and support collaboration between program members and between the qCMB program and other campus academic groups. Preceptors will hone their mentoring skills and learn strategies to ensure mentees engage in rigorous, reproducible and responsible research through qCMB resources and trainings. qCMB graduates are prepared to enter the workforce by engaging in career management and leadership training and through opportunities such as internships and guided mentor training. Throughout their PhD studies, qCMB trainees experience a supportive scientific environment that values their contributions, celebrates their successes and guides them through the challenges of graduate school. The effectiveness of training will be demonstrated through evaluations and by following the career trajectories of our graduates. Trainees (6 per year) are selected through a comprehensive process from among the pool of applicants to the CMB program and funded as teaching assistants for the first semester while rotating through preceptor labs. Each will be appointed to fellowship after one semester and will continue with rotations until joining a preceptor lab in Apr/May of the first year. After two years on the award, trainees will be funded through the preceptor's research grant or other fellowship for stipend. The award will support a total of 40 trainees who will then take on positions in academia or industry that require data analysis, collaboration and research experience.

NIH Research Projects · FY 2026 · 2019-03

Project Summary The goal of mitotic cell division is to produce two cells from one and to ensure that each daughter cell inherits an exact copy of the original genetic material. When mitosis malfunctions, a common result is aneuploidy, in which cells contain the incorrect number of chromosomes. This impacts human health, since aneuploidy is a leading cause of birth defects and is implicated in cancer initiation and progression. The fidelity of mitosis depends on kinetochores, which are structures built at defined regions of chromosomes called centromeres. Kinetochores have several essential functions, including the following: (1) they connect chromosomes to spindle microtubules, (2) they regulate the strength of these connections, and (3) they ensure that cells do not exit mitosis if chromosomes are incorrectly attached to microtubules (via the spindle assembly checkpoint). Our lab focuses on understanding kinetochores, and specifically how they establish and regulate attachments to microtubules. Our lab also investigates how tumorigenesis results in kinetochore defects, and how these defects lead to cancer cell-specific vulnerabilities that can be exploited for cancer therapies. The first two projects in the proposal aim to uncover the mechanisms cells use to establish and regulate kinetochore-microtubule attachments in human cells. Based on our recent findings, we have generated new hypotheses for how the Aurora family of kinases (i.e., Aurora A, and Aurora B) impact kinetochore-microtubule attachment stability. A significant obstacle that has hindered progress in understanding how kinetochore kinases precisely regulate kinetochore-microtubule attachments is the lack of tools that permit unobtrusive tracking of the dynamics of these kinases and their activities at kinetochores with high spatial and temporal resolution. We have overcome this by developing methods to generate genetically-encoded, fluorescently-tagged, antibody-based probes that can be used to track these phenomena in living cells. We will use this approach to generate phosphorylation “sensors” that recognize active, phosphorylated forms of Aurora A and Aurora B kinases, as well probes directed to their target substate sites at kinetochores. We will use these tools – and generate new ones – to discover how kinetochore kinases regulate kinetochore-microtubule attachment stability throughout mitosis to ensure successful chromosome segregation. A related project will address how kinetochore-microtubule attachment status is communicated to the checkpoint. For this, we will employ our phospho-sensors, super-resolution imaging, and a newly-developed in vitro chromosome capture assay. Finally, we made the recent discovery that hyperactive signaling by the RAS and MAPK pathways over-stimulates kinetochore kinases to induce kinetochore defects and cancer cell-specific vulnerabilities in laboratory-transformed cells and glioblastoma tumor isolates. We aim to identify the targets of MAPK in mitosis and define new roles for RAS/MAPK signaling in dictating cancer vulnerabilities, with the long- term goal of devising strategies to target kinetochore dysregulation in cancer cells for therapeutic intervention.

NIH Research Projects · FY 2026 · 2019-02

PROJECT SUMMARY Hundreds of human proteins contain prion-like domains (PrLDs), defined as protein domains that are compositionally similar to yeast prion domains. In recent years, a growing number of PrLDs in various organisms have been shown to form functional assemblies that regulate various cellular activities. These assemblies vary in both complexity and material states, from highly stable amyloid aggregates that can act as a form of cellular memory to complex, reversible biomolecular condensates such as stress granules. Additionally, mutations in PrLDs have been linked to various degenerative disorders, including amyotrophic lateral sclerosis and frontotemporal dementia. Disease-associated mutations tend to increase the aggregation propensity of the PrLDs. This observation has led to the hypothesis that many PrLDs are designed to mediate dynamic reversible interactions involved in cellular regulation, and that mutations alter the dynamics of these assemblies, promote the conversion to more stable structures, or cause aberrant aggregation of the PrLDs. However, while PrLDs are highly over-represented in eukaryotic genomes, the functions of only a small fraction of PrLDs have been characterized. Our long-term goal is to develop a comprehensive understanding of the roles of PrLDs in normal physiology and in disease. Towards this end, we are examining the basis for recruitment of PrLDs to complex assemblies such as stress granules, examining how specificity of targeting is achieved between different assemblies, and characterizing novel functions of these domains. Collectively, these studies will provide a framework for understanding the diverse functions of PrLDs.

NIH Research Projects · FY 2024 · 2018-01

Project Abstract. The goal of this project is to introduce a new synthetic strategy to functionalize pyridine and diazine heterocycles. Pyridines are the second most common nitrogen heterocycle found in FDA approved drugs, and there are numerous examples of diazines in these structures. The widespread occurrence arises because of a combined effect of the heterocycle and its substituents. The key drug-receptor interaction is often comprised of a hydrogen bond between the heterocycles N-lone pairs and the biological target. These heterocycles are also polar, can engage in p-stacking interactions and are resistant to oxidative metabolism. The substituents enable tuning of the steric and electronic environment of the heterocycle as well as serving as additional binding sites. As such, medicinal chemists require chemical process that can directly and selectively install a range of substituents at various stages of drug discovery from C–H precursors. In this proposal we will develop three different approaches for azine functionalization. First, we will install heterocyclic phosphonium salts and exploit their unique reactivity to develop coupling reactions with amines, thiophenols, cysteine containing molecules and alkynes. Using phosphines with pendant functional groups will enable coupling with water and ammonia. Second, direct coupling reactions between NTf-pyridinium salts and nucleophiles will be exploited for C–Heteroatom bond formation. additionsThis platform will enable direct coupling with aliphatic amines, anilines, amides and sulfonamides. Third, we will use sulfur nucleophile to change the regioselectivity of nucleophilic addition from the 4-position of pyridines to the 2-position of the scaffold. Once embedded in the substrate, these sulfur nucleophiles also serve as versatile functional group the enable other transformations to make C–N, C–O and C–F bonds.

NIH Research Projects · FY 2025 · 2017-09

Project Summary Single-cell imaging can quantify intricate spatial and temporal dynamics of gene regulation that underly important biomedical process ranging from bacterial infections to cancer. This gene regulation is subject to complexities and randomness of biological processes, and its observation is further subject to measurement artifacts due to inefficiencies in biochemical labels and distortions in microscope imaging. Yet, despite these complications, preliminary work shows that it is possible to integrate data and computational models to predict gene regulation in myriad environmental and genetic conditions provided that: (1) models must be constrained by informative and reproducible data, (2) models must be rigorously verified to account for biological and technical variations, and (3) models must be systematically explored to quantify uncertainties. The overarching hypothesis of this project is that spatial and temporal fluctuations observed in subcellular dynamics contain unique information that can be unlocked with improved computational methods and model-guided experiments. To test this hypothesis, this project will create a new research platform to be known as the single-cell Graphical Utility to Interpret and Design Experiments. scGUIDE will combine experimental analysis (e.g., image processing and single-particle tracking to extract quantitative data from fluorescence microscopy experiments), spatial stochastic simulation (e.g., reaction-diffusion models to generate realistic videos to mimic cellular experiments), model abstraction and identification (e.g., parameter inference and uncertainty quantification to translate quantitative observations into predictive insight), and experiment design (e.g., statistical methods to pinpoint which specific experimental conditions are most likely to reveal new biological insight). To demonstrate its broad capabilities, scGUIDE will be used to analyze and design single-cell experiments for four different health-related processes. In yeast, the project will examine the coordination between stress-activated MAPK dynamics and Spt-Ada-Gcn5 Acetyltransferase (SAGA) subunits that control chromatin and RNA transcription/transport dynamics, and which have been implicated in carcinoma, skeletal dysplasia, and retinal degeneration. In human cells, the project will examine the spatiotemporal clustering and phosphorylation of RNAP Polymerase II as it engages in single-gene transcription under CDK-inhibitor cancer treatments. In osteosarcoma cells, the project will explore how competition for local tRNA resources affects translation of single-mRNA molecules in different sub-cellular regions and in human and viral contexts. Finally, the project will explore the effects that epigenetic memory and molecular competition have on the multi- generational activation or repression of the pap operon that allows E. coli to establish uropathogenic infections. Each project will build mechanistic and quantitatively predictive models for how spatial and temporal interactions of transcription or translation factors, enzymes, and complex molecular machines combine with environmental influences to regulate expression at the single-gene, single-mRNA, single-cell, and population levels.

NIH Research Projects · FY 2025 · 2017-08

PROJECT SUMMARY/ABSTRACT: Cells of the Caenorhabditis elegans early embryo diversify their mRNA content even in the absence of de novo transcription. This remarkable feat has given my lab a unique vantage from which to study post- transcriptional regulation. Surprisingly, we found that many mRNA transcripts which exhibit cell-specific patterning also localize to discrete subcellular structures such as biomolecular condensates or membranes. My lab aims to understand how subcellular mRNA patterning arises mechanistically, how it functionally links to protein production, and how it impacts development. Recent advances have identified 190,000 localized mRNA transcripts found at 44 subcellular locales across 65 species (RNALocate Database). These transcripts include many that impact human neurobiology and whose mislocalization is associated with disease. Many more represent mRNAs that accumulate at cellular regions through unknown mechanisms and for undefined purposes, underscoring the potential for new discoveries that we aim to make. In the first funding phase, my group determined mechanisms that localize mRNAs to P granules (cytoplasmic condensates important for germline development and fertility). Transcripts that associate with P granules undergo either temporary sequestration or permanent decay. In the next phase, we will address the signals, mechanisms, and dynamics that distinguish P granule-associated mRNA sequestration from decay. Specifically, we will differentiate between competing models explaining how the conserved transcript nos-2 (nanos) exits P granules and initiates its translation to ensure fertility. Previously, we identified several mRNA transcripts that localize to membranes along with the proteins they encode, a finding echoed in other organisms. Among these were erm-1 a member of a conserved family of cytoskeletal membrane linker proteins that impacts cell shape and cancer. We found that erm-1’s mRNA localization is translation-dependent. Next, we will address the mechanisms and principles explaining how and why complexes of translating erm-1 move to membranes. We will also use genomics to characterize the membrane-enriched transcriptome to better understand localized translation at membranes. Maternal mRNA transcripts undergo decay in early embryos often creating cell-specific patterns that direct cell differentiation. In the first funding cycle, we demonstrated a requirement for the RNA binding protein SPN-4 in the clearance and cell-specificity of some transcripts. In the next phase, we will determine how SPN-4 shapes the transcriptome and works in concert with other mechanisms of mRNA clearance. The sum of these projects will be to create an overarching research program aimed at describing how mRNA transcripts organize spatially within the cell and how that organization can impact gene expression and embryogenesis.

NIH Research Projects · FY 2025 · 2017-08

PROJECT SUMMARY Colorado State University Veterinary Diagnostic Laboratory Participation in the FDA Veterinary Laboratory Investigation and Response Network Project Director: Kristy Pabilonia Institution: Colorado State University Co-Project Director: Josh Daniels Institution: Colorado State University Co-Investigator: Gary Mason Institution: Colorado State University The Colorado State University Veterinary Diagnostic Laboratory (CSUVDL) is accredited by the American Association of Veterinary Laboratory Diagnosticians (AAVLD) as a full-service, all species laboratory. The CSUVDL has a robust Quality System that meets and in most places, exceeds, AAVLD guidelines, which are based on ISO17025 standards. The CSUVDL has strong capabilities for veterinary diagnostic service regionally, nationally and internationally and performs more than 500,000 tests per year. The CSUVDL currently serves as a Tier 1 Laboratory for the United States Department of Agriculture (USDA) National Animal Health Laboratory Network (NAHLN) and as a Sentinel Laboratory for the Centers for Disease Control and Prevention (CDC) Laboratory Response Network (LRN). Colorado State University maintains CDC/USDA Select Agent Registration, which includes the Veterinary Diagnostic Laboratory Biosafety Level 3 facility. The CSUVDL is well poised to serve as an active member of the Food and Drug Administration (FDA) Veterinary Laboratory Investigation and Response Network (Vet-LIRN) program. The CSUVDL can provide full veterinary diagnostic testing services, including bacteriology, chemistry, toxicology, virology, parasitology and pathology testing and results interpretation. The CSUVDL is housed in a new, state-of-the-art building with more than 20,000 square feet of laboratory space, including a large necropsy floor, numerous BSL-2 laboratories and a 2,000 square foot USDA/CDC Select Agent approved BSL-3 laboratory. The faculty and staff of the CSUVDL are well trained to analyze veterinary cases and diagnostic samples using more than 250 diagnostic tests offered by our laboratory. In addition, the CSUVDL has an advanced information technology system, including an advanced Laboratory Information Managements System with the capabilities for sending HL7 messages. The CSUVDL routinely contributes to outreach and service activities for animal owners, livestock producers and government agencies. Since its acceptance into the Vet-LIRN in 2011, the CSUVDL has been active in all aspects of Vet-LIRN program, including participation in diagnostic investigations, participation in proficiency tests and attendance at meetings. For this project, the CSUVDL agrees to continue these activities and assist the Vet-LIRN with animal food and drug emergency outbreak testing, provide surge capacities for testing and use standardized Vet-LIRN methodologies.