Pilot Projects are small research projects funded by the INBRE Developmental Research Project Program via a competitive proposal process. These projects are selected based on their potential to lead to external funding for the researcher, the potential impact on UW research on INBRE thematic foci, and the possibility the project, if lead by a junior investigator, could expand into a thematic research project.
Brian Cherrington, Associate Professor Zoology and Physiology. Estrogen Mediated Epigenetic Regulation of miRNA Biogenesis in Lactotrope Cells. Peptidylarginine deiminase (PAD) enzymes epigenetically regulate gene expression in pituitary lactotrope cells; yet, the physiological consequences of this on lactotrope function during pregnancy are unknown. This gap in knowledge is important because lactotrope remodeling during pregnancy is absolutely required to maximize prolactin synthesis, initiate lactation, and stimulate breastmilk production. This is a highly relevant medical question because breastfeeding has profound health benefits for both the mother and infant. Our long-term goal is to understand hormone mediated epigenetic control of lactation at the molecular level. The objective of this proposal is to show that PAD catalyzed histone citrullination is a novel regulator of miRNA biogenesis that mediates E2 induced lactotrope population changes during pregnancy. Our data demonstrates that PAD expression is highest in lactotropes from late pregnant mice and that these enzymes suppress expression of a riboprotein termed DGCR8, which is critical for miRNA biogenesis. We propose a model in which E2 stimulates PAD expression, and then histone citrullination suppresses miRNA biogenesis. With decreased miRNAs, mRNAs encoding important proliferative, growth factor and gap junction proteins increase to drive lactotrope remodeling during pregnancy. Our central hypothesis is that E2 increases expression of PAD enzymes which then citrullinate histones to suppress miRNA processing in lactotropes during pregnancy. The central hypothesis will be tested with the following specific aims: (1) Determine the mechanism by which E2 regulates PAD expression and histone citrullination in lactotropes; (2) Determine the role of DGCR8 in miRNA biogenesis in lactotropes. The work is significant because it is an important step to characterize a completely novel, unexplored mechanism that is essential for lactotrope population changes during pregnancy and ultimately lactation. The proposed research is innovative because investigating histone citrullination induced miRNA biogenesis represents a new and substantial departure from current studies in the field. The aims of this proposal were submitted as part of a R01 application that was competitively ranked, and additional preliminary data generated with this pilot funding should make this proposal highly competitive for external funding.
Jason Gigley, Associate Professor of Molecular Biology. Dissecting how host available iron impacts pathogenesis of and immunity to chronic Toxoplasma gondii infection. Chronic Toxoplasma gondii (T. gondii) brain infections have severe health impacts, however, there are no effective approaches to eliminate them from the brain. The long-term goal is to define mechanisms by which chronic T. gondii brain infections develop and are controlled to design better therapies to cure this disease. The overall objectives of this proposal are to dissect how host available iron works in development of chronic T. gondii brain infections and immune responses to control them. The rationale is elucidating how host available iron works in development of chronic T. gondii infection and immunity could offer a strong scientific framework to develop new therapies to eliminate this infection. How host available iron affects T. gondii infection is unclear. Preliminary data demonstrates limiting host available iron during in vitro culture of T. gondii prevents parasite growth and replication. Limiting host available iron in vivo results in significantly higher cyst burdens in the brain. This could occur by stimulating parasites to develop cysts early during infection and/or there could also be an immune response defect that results in higher cyst burdens. The central hypothesis is that host available iron is a key factor for parasites cyst formation, parasite dissemination and CD8+ T cell function to control the parasite. Two aims will test the hypothesis: 1) Identify the mechanism(s) of how different host available iron levels affect cyst formation and chronic infection in human tissue and mice; and 2) Dissect how host available iron affects development of short term and long-term CD8+ T cell responses to the parasite. Aim 1 will test how decreasing or increasing host iron in vitro and in vivo affects cyst development (RT-PCR, cyst wall formation), parasite dissemination (quantitative PCR, RFP reporter parasites and confocal microscopy) and chronic infection outcomes in mice. Aim 2 will test how decreasing or increasing host iron in vivo affects CD8+ T cell activation, memory cell differentiation and function (spleen, brain, flow cytometry, confocal microscopy) during acute and chronic T. gondii infection. The research proposed is innovative because it will define a novel process of how available host iron impacts parasite biology in vitro and in vivo and identify novel pathway(s) pathways involved in immunity to T. gondii infection. The work if significant because it is expected to provide strong scientific background to identify novel targets in iron regulated pathways to eliminate chronic T. gondii infection. This knowledge has the potential to lead to more effective therapeutics to treat chronic T. gondii infection and other chronic brain infections.
Daniel Levy, Associate Professor Molecular Biology. Organelle size scaling in living sea urchin embryos. Cell sizes vary dramatically throughout biology, in different cell types and organisms as well as during early development when reductive cell divisions occur without growth. A fundamental question in cell biology is how organelle size is regulated relative to cell size, referred to as organelle size scaling. My lab focuses on size regulation of the nucleus. Nuclear size is physiologically and pathologically relevant, changing during development, cell differentiation, and cancer progression. For example, cancer cells with enlarged nuclei almost always represent more aggressive metastatic disease. The long-term goals of my lab are to elucidate mechanisms of nuclear size regulation and to use that information to test how nuclear size impacts cell and nuclear function. The primary experimental model system that my lab utilizes is Xenopus frogs. While we have discovered mechanisms that regulate nuclear size in Xenopus, one difficulty we have encountered is in visualizing nuclei in vivo in living Xenopus embryos, due to yolk and pigment granules that interfere with imaging. For this reason, we are limited in our ability to extrapolate our in vitro findings to living cells. Sea urchin (P. lividus and L. pictus) embryos offer an ideal complementary system to address this limitation, as the embryos are optically clear and it is routine to perform live imaging of nuclei in dividing embryonic sea urchin cells. Two years ago, I spent 5 months in the lab of Dr. Nicolas Minc at the Institut Jacques Monod (Paris, France) for a sabbatical, where I learned the sea urchin system and collected data on how nuclear size and import change during early development. Through different chemical and mechanical perturbations we showed that nuclear growth and size can be uncoupled from cell size, a surprising finding that differs from many other studies and was made possible by live imaging (manuscript in revision at Developmental Cell). The goal of this INBRE Pilot Grant is to establish the sea urchin system in my own lab. We will use live imaging in sea urchin embryos to validate and further explore our Xenopus findings related to mechanisms of nuclear size regulation. Perhaps more excitingly, this new system will allow us to address a wide array of new questions that are difficult or impossible to address using Xenopus. For example, we will use microfabrication approaches to examine if nuclear size is sensitive to the shape and size of the embryo and test how laser ablation of various cytoskeletal and membrane compartments impacts scaling. Furthermore, we can investigate developmental scaling of other organelles, such as the ER and mitochondria. We will also provide sea urchin embryos and necessary equipment to others on campus as well as to community colleges that could use the system to teach various aspects of developmental biology. In the long-term, introducing this new model system will expand my lab’s breadth and productivity as well as the resources available to the University.
Grant Bowman, Assistant Professor Molecular Biology. Genetic, Biophysical, and Structural Analysis of Network Connectivity at an ntrinsically Disordered Hub Protein Interface. Several of the most prominent proteins in cancer biology, including BRCA1 and p53, are key components in complex signaling networks that regulate cell growth. BRCA1 and p53 help to simplify network complexity by acting as centralized “hubs” that bind to many different types of proteins from several different signaling pathways. p53 can interact with almost 100 different proteins, and most of these interactions occur through special protein binding domains that are characterized by intrinsic disorder. Intrinsically disordered binding domains have no 3D structure by themselves, but contact with a binding partner triggers a disorder-to-order transition and the subsequent formation of a context-specific binding interface. Because of the inherent ambiguities in structure and the context-dependent shifts in the amino acids that are critical for binding, these types of protein interaction interfaces have been difficult to study. My lab is interested in the fundamental molecular mechanisms underlying the specificity and binding affinity between intrinsically disordered hub proteins and their many binding partners. To understand these interactions, we propose to study an intrinsically disordered hub protein in bacteria, a system that offers robust forward genetics, biochemistry, and cell biology in a single package.
Rebecca Carron, Assistant Professor Nursing. A Pilot Management Plan for American Indian Women with Polycystic Ovary Syndrome. The mission of the NIH is to develop new knowledge about the state and behavior of living systems that can be used to improve the health of living systems and decrease illness and disability. Polycystic ovary syndrome (PCOS) is a known risk factor for type 2 diabetes (T2DM), metabolic syndrome (MBS), and cardiovascular disease (CVD). Insulin signaling defects found in women with PCOS contribute to inherent insulin resistance, even in normal weight women. PCOS occurs in 6-15% of reproductive-aged women. Symptoms and problems associated with PCOS are known to vary by culture. Therefore, the 3rd PCOS Consensus Workshop Group identified a need for determining prevalence and best management practices in different ethnic groups. Based on results from our previous Wyoming INBRE 3 Thematic Research Project, our project purpose is to develop and pilot test a community-based, best practice management program for American Indian (AI) women with PCOS. The pilot management program has 3 aims: Aim 1: Develop and pilot a patient-centered, culturally appropriate, self-management program for AI women with PCOS that includes pathophysiology of PCOS, diet, exercise, fertility, mental health, weight, chronic disease risk, and appearance. Method: Focus groups with previous AI participants with PCOS. Outcome: A best practice self-management program that can be pilot-tested tested on Wind River Indian Reservation (WRIR) and then in other AI communities to improve health and decrease comorbidity risk. Aim 2: Explore strategies for developing a community-wide public awareness program about PCOS. Method: Focus groups of AI women with PCOS from previous study. Outcome: A program that can be pilot tested and refined on the WRIR and then used in other tribal communities. Program will include strategies to increase PCOS knowledge and decrease the stigmatizing effects of PCOS described by AI women. Aim 3. Explore the feasibility of implementing a PCOS screening tool in a WRIR clinic. Method: Plan-Do- Study-Act (PDSA) cycles in a WRIR clinic. Outcome: Determination of whether a PCOS screening tool provides early identification of AI women with PCOS. A pre- and post-test PCOS search in clinic health records will provide information on the success of tool and PCOS prevalence. To our knowledge, this is the first project to develop a management plan that includes self-management, community awareness, and screening for AI women with PCOS.
Kyle De Young, Assistant Professor Psychology. A pilot test of mood and circadian rhythm mechanisms driving binge eating. Mood and circadian rhythm disruptions are associated with binge eating (BE), which is a discrete, episodic behavior characterized by: 1) eating an objectively large amount of food while 2) experiencing a subjective sense of loss of control. BE episodes are often preceded by negative mood states, and a reliable subtype of individuals with BE has high levels of negative moods. This group has more comorbid psychopathology and a poorer response to treatment. Thus, understanding the role of negative mood is a critical area for research on BE. Individuals with BE demonstrate disruptions in several circadian rhythms, including the diurnal timing of meals, hormone patterns (e.g., daily cortisol rhythms), and mood variations. The most potent synchronizer of circadian rhythms is light. Exposure to light varies over both minutes and months, by location, weather, and season, and humans can volitionally or unwittingly modify the light in their environments. Thus, exposure to light may explain other phenomena that fluctuate similarly, such as mood and the occurrence of BE. Mood is subject to the influence of light, and BE is also influenced by exposure to bright light. However, it is unknown whether regulating circadian rhythms via regular exposure to light improves BE through its effects on mood or via changes in other biological or behavioral rhythms. This knowledge can inform the development of treatments that target biobehavioral mechanisms that maintain BE and indicate for whom this may be most effective. This project aims to test the roles of negative mood and circadian rhythms in the relationship between light exposure and BE and identify subtypes of individuals in whom this effect is strong.
Jay Gatlin, Associate Professor Molecular Biology. Engineered Approaches to Study Aster Positioning Biomechanics. The microtubule cytoskeleton is critical for organization the insides of cells. At times during the cell cycle, this network adopts the form of an astral array with microtubule fibers emanating from a central focus and branching outward throughout the cell. Importantly, the center of this array is typically found near the geometric center of the cell where it is anchored to the surface of the nucleus. Forces generated by the microtubules themselves move the internal organs of the cell during interphase, and because the network also serves as a scaffold upon which these organs attach, its position dictates their spatial arrangement within the cell. During mitosis similar forces positioning the cell division machinery and establish the eventual location of the cell division plane. Though aster centering is likely critical in all cells, it is particularly relevant in large cells immediately after fertilization. Here, the male pronucleus (from the sperm) must transverse large distances to reach the cell center and establish the location of mitotic spindle formation and division plane positioning during the subsequent mitosis. Thus, errors in this process can lead to erroneous cell division and have deleterious effects on development. Precisely how the microtubule aster generates and responds to forces to move to the cell center remains unanswered. In this work, we describe the application and continued development of a new approach engineered to overcome existing limitations inherent to other approaches used to study aster positioning. This approach provides exquisite control of cytoplasmic shape and volume in a platform that is amenable to visualization using standard wide-field and confocal light microscopy.
Cynthia Hartung, Associate Professor Psychology. Acute Effects of Exercise and Stimulant Medication in College Students with ADHD. There is an urgent need for developing novel treatments to address the limitations of existing interventions and improve ADHD-related functioning among college students. Physical exercise is as a promising option because it has been shown in preliminary studies to improve executive functioning (EF) in individuals with ADHD. Additionally, physical exercise has the ancillary benefits of improving physical health and reducing the risk for obesity for college students with ADHD, who are more likely to be in poor health. Our overall objective is to examine exercise as an acute intervention for ADHD. Our central hypothesis is that college students with ADHD will exhibit greater degrees of improvement in EF immediately following sprint interval training relative to non-ADHD peers. This hypothesis was formulated based on preliminary studies demonstrating improved EF following exercise. The rationale of our proposed study is that exercise could serve as an effective treatment for college students with ADHD that has low costs, low risks, ancillary health benefits, and may address limitations of existing treatments. First, we will examine the immediate effects of exercise on EF. Our working hypothesis is that college students with ADHD will show greater improvement on measures of EF following exercise than controls. Next, we will examine the individual and combined effects of exercise and stimulant medication on EF. Our working hypothesis is that improvement in sustained attention and working memory will be greater when college students with ADHD take stimulants than when they exercise; but, the combination of stimulants and exercise will be more tolerable than taking stimulants alone. Our expected outcomes are to confirm these hypotheses and demonstrate the need for further study into the efficacy of exercise for college students with ADHD. This outcome is expected to have an important positive impact because exercise may serve as an effective treatment for college students with ADHD that is less risky than stimulants, less time-consuming than therapy, and provides ancillary health benefits. Our proposed research is innovative because it represents a substantive departure from the status quo of managing mental health with psychotherapy and/or medication. Specifically, we seek to shift current research and clinical practice paradigms by testing the efficacy of a novel treatment approach for college students with ADHD. The proposed research is a significant departure from previous treatment approaches. Overall, an examination of the acute effects of exercise on the EF of college students with and without ADHD will allow us to determine whether continued work in this area is justified. Even if acute effects of exercise do not exceed those of stimulants, the combination of exercise and stimulants may alleviate some of the negative side effects of stimulants. If exercise has a significant impact on EF or if it significantly reduces stimulant side effects, continued research into the chronic effects of exercise on college students with ADHD will be warranted. That is, studies examining the prolonged effects of regular exercise in college students with ADHD may demonstrate continued positive effects on attention and memory, as well as sustained effects on mood, sleep and physical health.
Caleb Hill, Assistant Professor Chemistry. In the proposed work, analytical methods will be developed and applied to simultaneously monitor the cellular uptake of individual nanoparticles (NPs) and resulting effects on cell viability in situ. This will be accomplished through a systematic approach combining (a) super-resolution optical imaging to visualize the route of entry and subsequent intracellular trafficking with nm-level precision, (b) optical spectroscopy to probe the structure of individual NPs, and (c) local electrochemical measurements via scanning electrochemical microscopy to quantify the generation and release of reactive oxygen species, which will provide insights into oxidative stress resulting from NP uptake. The model system employed in these studies will be the infiltration of Au nanoparticles into HeLa cells, which is ideal due the numerous geometries in which Au NPs can be synthesized (spheres, rods, tetrahedrons, cubes, decahedrons, etc.), providing a powerful model for studying the effects of geometry in the uptake process without interfering chemical effects. The developed methodology will applied to answer the following 3 general questions, examining the role of NP geometry in each: 1. Is NP uptake uniformly distributed across the cellular membrane or is it highly localized? 2. How quickly does a NP cross the cellular membrane? 3. How many NPs are required to adversely affect cell health?
Domen Novak, Assistant Professor Electrical and Computer Engineering. Pilot evaluation of a spinal exoskeleton for prevention and relief of low back pain. Although spinal orthoses are often used in the management of chronic low back pain, their efficacy is highly contested. Such orthoses have shown limited evidence of benefit, and there is no consensus on how they should be designed in order to successfully manage back pain. This is an enormous missed opportunity, as low back pain is the leading cause of disability in most of the 195 countries examined by the 2015 Global Burden of Disease Study. The costs of low back pain in the United States alone are estimated to be above 100 billion dollars annually, with an associated 149 million work days lost per year. There is thus a great need for improved spinal orthosis designs that could more effectively manage back pain. This project will perform a pilot evaluation of a novel type of spinal orthosis: a reconfigurable exoskeleton that can be manually adjusted by the wearer in order to provide personalized support during different activities. Such a device has several potential advantages over state-of-the-art orthoses; however, these advantages must be validated in short-term studies before the device can be deployed in long-term pain management research. The device will thus be evaluated in two settings: brief activities of daily life performed in a lab and regular office work performed over the course of an 8-hour workday. This evaluation will be done both with participants who have not experienced chronic back pain and with participants suffering from chronic low back pain. During the evaluation, we will measure key neurophysiological (electromyogram of postural muscles) and biomechanical (posture, pressure on different areas of the trunk) parameters that are known to be correlated with long-term development of low back pain. Measurements obtained with the reconfigurable exoskeleton will be compared to those obtained with a state-of-the-art spinal orthosis. By quantifying the short-term advantages of the proposed exoskeleton compared to state-of-the-art designs, we will thus obtain preliminary data for future long-term efficacy evaluations of this technology.
Karen Wawrousek, Assistant Professor Chemical Engineering. Bacterial Magnetic Nanoparticles for Diagnostic Assays. Bacteria will be used to produce doped magnetic nanoparticles, with dopings selected to alter the magnetic properties of the nanoparticles. This project focuses on characterization of nanoparticles simultaneously doped with two metals, which has not been reported in the literature. Potential applications for these particles in biomedical applications include use in diagnostic assays, embedding the magnetic nanoparticles in another material to create an artificial muscle, use as contract agents for imaging, as well as targeted drug delivery and hyperthermia treatment of cancer. The bacterial magnetic nanoparticles will then be incorporated into raman spectroscopy-‐based diagnostic assays. As a proof-‐of-‐concept, bacterial magnetic nanoparticles will be incorporated into an assay to detect West Nile virus in two ways. First, antibodies to West Nile virus will be conjugated to magnetic nanoparticles to detect West Nile virus itself. This should give a positive result in the diagnostic assay if the organism has an active West Nile infection. Second, the bacteria producing the magnetic particles will be genetically engineered to display West Nile antigens on the surface of the magnetic nanoparticles. These particles will then be used to detect the presence of antibodies to West Nile virus, with a positive result in the diagnostic assay indicating that the organism has been exposed to the West Nile virus. Success of these proof-‐of-‐concept studies will validate this model for use to detect and diagnose other infectious diseases, including those for which current diagnostic assays are not sensitive, are not selective, and cannot be conducted at the point-‐of-‐care.
Jared Bushman, Assistant Professor Pharmacy. Localized immunosuppression for peripheral nerve allografts. Using allografts to repair segmental peripheral nerve defects is extremely effective but rarely used due to the risks and complications of systemic immunosuppression. This is despite the fact that peripheral nerve allografts only require temporary immunosuppression. We hypothesize that it will be possible to provide mmunosuppressants local to the allograft instead of systemically to delay the immune response against the graft for a sufficient amount of time for regeneration to occur. To test this hypothesis we will build upon our preliminary data and determine the concentrations of immune suppressants that can be tolerated in combination by peripheral nerve cells in vitro. We will also develop procedure to harvest anti-T-cell antibodies from hybridomas, which will form part of our multi-factorial approach to locally suppress the immune response. The project will culminate in the testing of this strategy in the rat peripheral nerve injury where nerves from GFP Sprague-Dawley rats will be allografted into Lewis hosts, using the sciatic nerve model.
Carl Frick, Professor Electrical Engineering. Removable and Replaceable Glaucoma Treatment Device Pilot Study. The proposed research will challenge the standard approach to lowering intraocular pressure (IOP) in patients suffering from glaucoma. Glaucoma is the second leading cause of blindness worldwide and it is estimated that 70 million patients worldwide suffer from the disease. The underlying cause is related to elevated IOP, which leads to damage of the optic nerve and results in vision loss. Treatment of glaucoma focuses on lowering IOP; however, a successful long-term solution remains elusive. Topical medications have limited efficacy and suffer from poor patient compliance, while surgical strategies suffer from high morbidity and limited long-term success. For example, drainage devices lower IOP by shunting aqueous humor from the anterior chamber to another compartment outside of the eye; however, these devices are prone to eventual scarring and closure of the artifical drainage pathway.We propose a transcorneal drainage device that utilizes a novel material platform of antimicrobial liquidcrystalline elastomers (A-LCEs) to overcome the existing challenges and barriers in drainage devices. Thistreatment strategy would reduce IOP by directly draining aqueous humor to the tear film, bypassing the conjunctiva and tissues that commonly scar and prevent outflow. This would promote ease-of-implantation, allowing surgeons to perform the procedure with minimal training. The drainage device would consist of an A-LCE porous filter that has reversible shape-changing properties, which enables removal and replacement to tailor outflow and ensure long-term efficacy if the filter becomes clogged. Furthermore, the antimicrobial properties would prevent bacteria from entering the eye. If successful, this research could significantly alter how IOP is treated with drainage devices as well as introduce a novel, unexplored ophthalmic materials platform for future devices.
Karen Gaudreault, Assistant Professor, Kinesiology and Health and Stacy Carling, Research Scientist Kinesiology and Health. After School Engagement in Physical Activity and Associated Attitudes. Our long-term goal is to implement an after-school wellness program for at-risk children that aids in their development of healthy and sustainable behaviors and lifestyles. Physical activity (PA) engagement can prevent several chronic diseases, including obesity. As 17% of children in the United States are obese, childhood obesity is a public health issue affecting child health and wellness. Obesity is a risk factor for many cardio-metabolic disorders, and according to the Centers for Disease Control and Prevention, efforts to combat obesity and its co-morbidities at the community level should address healthy eating and active living. Research indicates positive impacts of child-obesity intervention programs that address healthy lifestyles, however, evidence regarding the long-term effects of such programs on the adoption of active lifestyles into adulthood is limited and further study is needed. Our objective with this proposal is to initiate and evaluate our protocol for assessing PA engagement and associated attitudes regarding PA in the context of a Wyoming-based after-school wellness program called Healthy Pokes (HP). Developing effective protocols for assessing PA and associated attitudes is the next step toward investigating how HP programming impacts PA engagement over time and healthy lifestyle development. Our aims include: Aim 1: To assess changes in PA in three settings: 1) during HP program sessions, 2) during standard recess sessions at school, and 3) during physical education classes at school, following exposure to HP program. Aim 2: To assess changes in attitudes toward PA following exposure to HP program. Aim 3: To evaluate and modify implementation of protocols for assessing PA and associated attitudes. Aim 4: To compare changes in physical activity across settings.
Jason Gigley, Assistant Professor Molecular Biology. Transition Metal Chelators as Novel Therapeutics Against Toxoplasma gondii. The obligate intracellular parasite Toxoplasma gondii can attack and invade any nucleated cell of warm-blooded animals and can never be cleared. All nutrients sequestered by the parasite are derived from the host, including essential ions such as heavy metals copper, iron, and magnesium. In both the host and the parasite cell biology, these metals are primarily used as cofactors in evolutionarily conserved processes such as, respiration, oxidative stress responses, and protein-protein interactions. In addition, the metals are involved in higher eukaryote processes such as signal peptide processing and angiogenesis. Limiting the availability of these ions could alter the course of the infection in the host environment, parasite biology, or host-parasite interactions. Currently, heavy metal chelators such as D-Penicillamine. Trientine and Tetraethylene pentamine (TEPA) are used either as therapeutic agents or in biomedical research, but the effects of these drugs on infectious disease is largely unknown. We hypothesized that a drug treatment of the above chelators for a T. gondii infection will alter the kinetic of infection. So far, our research shows that these drugs used to treat a T. gondii infection slow the growth and replication of the parasite both in vitro and in vivo without showing significant toxicity in drug treatment alone. Our future studies aim to dissect the mechanism of chelation on T. gondii infection and will identify chelation feasibility for therapeutic use as well as reveal off-target effects of these drugs currently in circulation.
Dongmei (Katie) Li, Assistant Professor Chemical Engineering. Microfluidic Production
of Multimodal Therapeutic PEG Hydrogel Nanoparticles. Many classes of nanoparticles have been designed with cancer-specific therapeutic
applications in mind.
The function of these particles can include, for instance, localized delivery of an
imaging dye or contrast agent, targeted delivery of a chemotherapeutic molecule, or
a metallic nanoparticle for hyperthermic treatment. Synthetic polymer-based hydrogels
such as polyethylene glycol (PEG) are ideal candidates for therapeutic drug delivery
because they provide biopassivitiy, programmed drug release, and ease of functionalization
with antibodies, nucleic acids, and peptide sequences. Despite the broad diversity
of techniques by which polymer hydrogels may be customized, there exist no reliable
methods to prepare PEG particles on nanometer length scales with narrow size distributions
and well-defined loading. Recently, we have successfully developed a platform to address
the above challenges by fabricating nano-scale, biodegradable polyethylene glycol
(PEG) particles with narrow size distribution. Here we propose to apply this platform
to produce nanoparticle carriers for applications in cancer therapeutics. The proposed microfluidic
processing approach is superior to existing techniques due to its exquisite control
over particle size and uniformity and facile tunability of the macromolecular network.
The Specific Aims of this two year INBRE Pilot Project are: Specific Aim #1: Utilize
the PEG nanoparticle fabrication device to encapsulate molecular cargo for cancer
diagnostics and therapeutics. Functional molecules to be encapsulated include dyes,
small molecules, and monoclonal antibodies for imaging, and targeted therapies, respectively. Specific
Aim #2: Customize PEG macromolecular architecture to enable targeted delivery and
release through enzymatic degradation. This aim will focus on two goals: 1) conjugating
PEG nanoparticle surfaces with target antibodies for localizing delivery to tumors,
and 2) creating enzymatically degradable PEG nanoparticles for release in response
to matrix metalloprotease secretion by target cells.
Colloidal-based SERS Detection of AMI-associated miroRNAs, Dr. Patrick A. Johnson, Department of Chemical and Petroleum Engineering
MicroRNAs (miRNA) are a class of small (~22-24 nucleotides in length) non-coding RNAs that have been implicated in the control of cellular processes associated with both normal physiological states and disease. Recent demonstrations that unique miRNA expression profiles are highly correlated with a wide range of chronic and acute human pathologies (e.g. sepsis, diabetes, liver and heart disease, arthritis, mental illness) suggest that they could serve as next-generation biomarkers for clinical diagnosis, and perhaps prognosis as well. Currently, efforts to develop assays for disease profiling based on the recognition of miRNA expression signatures in clinical specimens have largely focused on microarray-based platforms that are restricted to testing applications in large clinical research centers.To determine the feasibility of developing a miRNA detection assay, which could be performed using cost-effective reagents and portable instrumentation, we will develop nucleic acid hybridization assays for the capture and detection of the acute myocardial infarction (AMI)-associated miRNAs miRNA-1, miRNA-208s and miRNA-499. Proof-of-concept hybridization assays will include the miRNA targets, Au-coated paramagnetic nanoparticles (Au@PMPs) conjugated with oligonucleotides complementary to the miRNAs, and miRNA-complementary reporter oligonucleotides which are conjugated with Raman reporter dyes. Upon formation of Au@PMP/miRNA/reporter ternary complexes, an external magnetic source will be applied for surface-enhanced Raman scattering (SERS) detection of miRNA capture by concentration and excitation of the Raman reporter within the field of an interrogating laser beam. Once assays have been optimized for the detection of individual AMI-associated miRNAs, we will then develop multiplexed assays in order to establish feasibility for the eventual goal of integrating this assay platform within the detection capabilities of a low-cost, portable Raman instrument reader. The proposed activities are based on technological capabilities currently available in our laboratory, and it is our intention to partner with a local Raman spectroscopic instrumentation company (DeltaNu, Inc., Laramie WY) in seeking follow-up R&D funding from federal agencies once the benchmarks outlined in the proposal have been achieved.
Metabolic Syndrome in PCOS: Understanding the Role of Pituitary Gonadotropes. Amy M. Navratil, Dept. of Zoology and Physiology
It has long been established that the physiological mechanisms controlling energy balance are integrated with those that control reproduction. In humans, insulin resistance is a component of polycystic ovary syndrome (PCOS), a reproductive ovarian disorder characterized by anovulation, polycystic ovaries, high androgen levels, hyperinsulinemia, and predisposition for Type 2 diabetes. It is the most common endocrine disorder among women of fertile age, with upwards of 10% of women being affected. PCOS is complex reproductive disorder with an unclear pathophysiology that often leads to infertility. It has been well documented that altered gonadotropin secretion is associated with the typical form of PCOS. Compared with the follicular phase of the normal menstrual cycle, women with PCOS exhibit a disproportionately high luteinizing hormone (LH) secretion with relatively constant low follicle stimulating hormone (FSH) secretion from anterior pituitary gonadotropes. Previous data from our group suggests that gonadotrope cells in pituitary slices alter their movements in response to gonadotropin releasing hormone (GnRH) by sending out long cellular processes. Preliminary data suggests that these cyto-architectural changes are related to directed movement toward vascular elements in the anterior pituitary presumably to facilitate hormone release into peripheral circulation. Thus, one of the central goals of this proposal is to use a live cell confocal imaging approaches to address how the organization, morphology, and movement of gonadotropes within a living pituitary may change under a PCOS/metabolically dysregulated state to affect LH secretion. At issue is whether the spatial and temporal attributes of gonadotropes are altered with metabolic syndrome to disproportionately increase circulating concentrations of LH seen in PCOS. To directly test this, we propose to utilize a PCOS mouse model to begin our imaging analyses in a complex and physiologically relevant system. Beyond these more descriptive studies, we will also use our PCOS mice to elucidate the molecular alterations in the synthesis of the gonadotropin subunit genes in vivo. Specifically, we will determine if dysregulation of the metabolic state results in an alteration of the chromatin structure surrounding the gonadotropin subunits genes. Taken together, our pilot proposal seeks to advance our understanding between the relationship of energy homeostasis/PCOS, the gonadotrope, and LH secretion. We are hopeful that the experiments outlined will help in identifying the underlying mechanisms of increased LH secretion in the pituitary and provide critical insight in to pathophysiology of PCOS and impaired reproductive function. Presently, our project is in very preliminary stages but following our two year projected research plan, it is our long term goal to submit a NIH research proposal (either R21 or R01) to the National Institute of Child Health and Disease (NICHD) funding agency.
Variability in long-term body weight trajectories among older adults, health, and mortality: implications for public health recommendations, Anna Zajacova, Dept. of Sociology
INTRODUCTION AND BACKGROUND- After decades of research, studies on health consequences of obesity have failed to yield consensus on many key questions. In particular, excess body weight is known to be related to increased health risks but population studies suggest it is also linked to lower mortality risks. We posit that the contradiction may be solved if we understand how body weight changes over the long term in older adults. Special attention should be paid to population variability in the body mass trajectories because some types of weight patterns may be associated with particularly poor health outcomes.
SIGNIFICANCE / WHY IS THE RESEARCH QUESTION IMPORTANT-Rates of adult obesity have nearly tripled in the preceding five decades, from 13% in 1960 to 35% today. This trend has motivated extensive research on the health consequences of excess body mass index (BMI). Even after decades of study, however, major gaps remain in the BMI-health literature. This project is motivated by the paradoxical contradiction between findings from population research that finds excess BMI associated with decreased mortality among older adults versus the predominant understanding that excess body weight is det-rimental to health.
RATIONALE FOR APPROACH- We propose to determine the shape and variability of BMI trajectories among older Americans, using a large, nationally representative longitudinal dataset. We then link specific BMI trajectory patterns to health outcomes. This project presents two major improvements over the previous research. First, we employ cutting-edge ana-lytic methods to model long-term changes in body weight among older adults. Second, we shift attention from analyzing mean BMI trajectories to variability in the trajectories. Based on preliminary analyses, we hypothesize that there is substantiation heterogeneity in the older population in body weight trajectories. De-scribing the shapes and variability can help us identify how excess body weight impacts health at older ages.
PROJECT IMPACT- The current inconsistencies in the literature on body weight and health need to be resolved if health providers and public health officials are to provide a coherent set of recommendations to the public regarding achieving and maintaining body weight. Our results will help resolve a long-standing puzzle of inconsistent findings be-tween population and clinical research. By identifying population subgroups with body weight patterns that are particularly detrimental to health, targeted therapies and interventions can be designed. Additionally, the inno-vative methodology we employ here has potential to be useful in many other health-related research areas. By disseminating results using functional data analysis for longitudinal health research, we will add value to di-verse areas of epidemiological, biomedical, and population-level health and aging research.
CURRENT PROJECT STATUS- We have conducted extensive preparatory analyses to determine the feasibility of a successful completion of both project aims. For Aim 1, we have estimated growth mixture models of BMI trajectories. For Aim 2, we have estimated unadjusted joint growth mixture-survival models. We have also written the statistical code for FDA estimation used in Aim 2. The next step is to master the PACE modeling to examine the heterogeneity in BMI trajectories within the FDA framework.
PLANS FOR EXTERNAL FUNDING- The NIH has identified obesity as one of its main funding priorities. This project fits the major themes of the Strategic Plan for NIH Obesity Research: health consequences of obesity and the application of innovative an-alytic methods. With results obtained in this pilot, we will submit a competitive application. We will consider the R15 mechanism; alternatively, the NIH has an R21 call for applications specifically for secondary analyses in obesity, diabetes, and digestive and kidney diseases.
Engineering red-light activated nucleotide cyclases. Dr. Mark Gomelsky, Department of Molecular Biology
Rapid Diagnosis of Invasive Aspergillosis with Fountain Flow™ Cell Sorting of Bronchoalveolar Lavage fluid followed by Molecular Species Identification. Dr. Paul E. Johnson, Dept. of Physics & Astronomy
Antibiotic Drug Discovery from Myxobacteria. Dr. Daniel Wall, Department of Molecular Biology
Maternal Obesity and Development of Type I Diabetes in NOD Mice Offspring
Dr. Meijun Zhu, Department of Animal Sciences
Engineering red-light activated nucleotide cyclases
Dr. Mark Gomelsky, Department of Molecular Biology
Abstract: Engineered photoregulated proteins have the potential to revolutionize biomedical research. In a photoregulated protein, a photon absorbed by a chromophore bound to a photoreceptor protein domain affects activity of an output domain. Recently, remarkable progress has been achieved in engineering of artifical photoreceptors, which have already made a dramatic impact on the field of neurobiology. However, it is clear that we are just at the dawn of the era of photoregulated proteins as tools for biomedical research and therapy. Visible and far-red light is harmless to mammalian cells, therefore, it can work as a highly specific, and cheap way to regulate protein activities. The spatiotemporal resolution that can be achieved by using photoregulated proteins is unprecedented as a laser beam can be focused not only on an individual cell but on a particular region of the cell. Engineered photoregulated proteins can be broadly used for activation (or inactivation) of proteins of interest in cell cultures, tissues and animal models. Thus far only blue-light photoreceptors have been used for protein engineering. Because of the short wavelengths of light they have low tissue penetration, which drastically limits their utility in animal models of disease. Bacteriophytochromes absorb red/far-red light. It has much higher tissue penetration capacity than blue light and is currently used in deep-tissue phototherapies. The objective of this application is to provide the proof of principle that a chromophore-binding module of bacteriophytochromes can be used for engineering of red/ farred light regulated proteins. The goal of this pilot project is to engineer a red-light activated adenylate cyclase (cAMP synthase). The critical role of cAMP in controlling glucose and lipid metabolism as well as neuronal activity makes adenylate cyclase a highly desired target. Photoactivated adenylate cyclase can be used in various model systems to study neuronal plasticity, progression of diabetes and obesity. Some of these diseases are of particular interest to INBRE. The design of photoregulated enzymes relies heavily on computationally-intensive bioinformatics approaches that involve analysis and modeling of protein structures and dynamics. Bioinformatics is identified as one of the focus areas in INBRE. The assembled research team has complementary expertise in structural protein bioinformatics, genetic engineering and protein-ligand photo- and biochemistry required for the success of this pilot, high-risk/ high-return project.
Rapid Diagnosis of Invasive Aspergillosis with Fountain Flow™ Cell Sorting of Bronchoalveolar
Lavage fluid followed by Molecular Species Identification
Dr. Paul E. Johnson, Dept. of Physics & Astronomy
Abstract: This project will prove the concept for a low-cost, point-of-care system for real-time diagnosis of invasive aspergillosis. Invasive aspergillosis occurs in 8-15% of allogeneic stem cell transplant patients and 5-15% of solid-organ transplant patients. Mortality of infected transplant patients ranges from 30% to 70% and is nearly 100% if left untreated. Diagnostics are unreliable; too often conventional diagnostics fail to identify fungal infection and a confirmed detection is made only post mortem. This proposal will explore a novel method which has the potential to detect Aspergillus down to the level of a single microorganism in bronchoalveolar lavage (BAL) fluid in minutes and to diagnose the species in 2-4 hours, using fluorescent in-situ hybridization (FISH) RNA probes and/or PCR. The objective of this effort is to build and test a proof-of-concept cell-sorting method, Fountain Flow™ Sorting, for the detection of fungi in BAL fluid. A stream of BAL fluid containing an inexpensive fluorescent, fungal dye is illuminated with an LED, and fluorescent fungal cells are detected with a digital camera. After each detection, a fungal cell is sorted into a smaller volume, which can be then stained with more-expensive, immunolabel or FISH probe for Aspergillus confirmation. Rapid PCR or FISH probes can then be used for species identification, allowing for an early diagnosis of invasive aspergillosis, particularly in immunocompromised patients. This research will be performed by a team of scientists from the University of Wyoming, as well as an infectious disease specialist (Cleveland Clinic) and a pediatric oncologist (Associate Professor of Pediatrics at the University of Colorado Denver Health Sciences Center).
Antibiotic Drug Discovery from Myxobacteria
Dr. Daniel Wall, Department of Molecular Biology
Abstract: To address the growing problem of antibiotic resistance there is an important medical need to develop new antibacterials that work by novel mechanisms. This pilot proposal seeks to exploit myxobacteria as prolific producers of natural product antibiotics. Recent genomic and bioinformatic findings have highlighted this point as a stunning ~10 percent of myxobacterial genomes contain secondary metabolite biosynthetic gene clusters. However, many of these gene clusters are cryptic and the natures of their metabolites are unknown. Here, we will use molecular genetic and microbiology methods to study secondary metabolite encoding genes and their corresponding metabolites. Additionally, this proposal seeks to tackle a central enigma of antibiotic drug discoverynamely, that natural products are the leading source of antibiotics, yet their development is hindered by low fermentation yields and complex chemical structures that make synthesis and optimization difficult. A goal of this project is to test the feasibility of our hypothesis that the predatory behavior of myxobacteria can be exploited to genetically select optimized producer strains. This approach offers innovations over traditional laborious screens because large numbers of mutagenized cells can be processed to identify rare mutants with improved yields or potencies. A focus of these studies is on the natural product antibiotic TA; a promising hybrid polyketide-peptide antibiotic that inhibits cell wall biosynthesis, has broad-spectrum activity and is safe in animals and humans. Optimization and development of TA has been hampered by difficult synthesis and poor fermentation yields. The aims are designed to improve strain yields and test our hypothesis that predation can be exploited for strain optimization. A long term goal is to construct a commercially viable TA producer strain to allow TA use that exploits its adhesive properties for treatment of periodontal disease and as a prophylactic to coat medical indwelling devices. Optimized TA variants will also be sought for possible use as a systemic broad-spectrum antibiotic. This project builds on the expertise of the PI in myxobacteria biology and antibiotic drug discovery.
Maternal Obesity and Development of Type I Diabetes in NOD Mice Offspring
Dr. Meijun Zhu, Department of Animal Sciences
ABSTRACT: SIGNIFICANCE: The obesity rate has increased more than two fold in recent decades. According to the latest NHANES survey (1999-2002), 29% of women at childbearing age (20-39 years old) are obese. At the same time, autoimmune diseases including Type I diabetes are also increasing, indicating a likely link between maternal obesity (MO) and altered immune system development. Major components of immune system development are accomplished during the fetal and neonatal stages. Our preliminary data show that MO led to systemic inflammation in fetuses and the expression of toll like receptor (TLR) 4 was elevated. CENTRAL HYPOTHESIS: MO induces systemic inflammation in fetus, which promotes survival of thymocytes specific to host-derived antigens, increasing the incidence of autoimmune diseases including type I diabetes in offspring. SPECIFIC AIMS: 1) MO increases the incidence of type I diabetes in offspring. 2) MO induces an inflammatory response, which promotes dendritic cell maturation, resulting in the evasion of apoptosis for those thymocytes recognizing self antigen via TLR4 signaling. APPROACH: We will use our well-established obese mouse model non-obese diabetic (NOD) mice fed control (Con) or obesogenic (OB) diet to study the effect of MO on the incidences of offspring type I diabetes. NOD mice are the most commonly used animal model for type I diabetes studies and other autoimmune diseases. In addition, we will utilize the unique advantage of mouse studies, TLR4 knockout mice, to study the role of TLR4 in the fetal immune system development. INNOVATION: The proposed work is novel, because this is the first study designed to determine the development of the fetal immune system is affected by MO, which may result in an increased incidence of type I diabetes and other autoimmune diseases. ENVIRONMENT: All methodologies required are already established in our laboratories. The Center for the Study of Fetal Programming provides excellent animal and laboratory facilities. FUTURE PLAN: Based on the data obtained from this study, the PI will further explore mechanisms associated with the fetal immune system development and immune tolerance, and develop specific strategies to cope with autoimmune diseases. IMPACT: The immune system is responsible for the defense against tremendous numbers of bacteria and opportunistic pathogens and its proper development is crucial for lifelong health. Knowledge obtained in this study will provide targets for interventions to ensure the proper development of the immune system, improving the quality of life for the offspring of the increasing number of obese pregnant women in this country.