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Pilot Research Projects

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.

Current Pilot Project Summaries

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. 

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.

Past Pilot Project Summaries

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.

Past Pilot Projects

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.


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