Michael Smith - US grants

DESCRIPTION: (Adapted from the applicant's abstract and Specific Aims.) Obstructive sleep apnea syndrome (OSAS) results in transient periods of hypoxemia, hypercapnia and sleep interruption with associated acute and chronic sympathoexcitation and hypertension. Treatment of the disorder relieves many of the disorders and associated symptoms. The mechanism(s) explaining the acute and chronic states of sympathoexcitation have not been studied prospectively and may be relevant to the hypertension and elevated cardiovascular mortality in OSAS. Abnormalities of chemoreflex and baroreflex function have been speculated to explain the cardiovascular consequences of OSAS, but have not been studied prospectively.The Specific Aims are to: 1) determine the relative contribution of hypoxemia, hypercapnia, and effects of inspiratory effort to acute sympathoexcitation during sleep apnea or simulated apnea during wakefulness in patients with OSAS and healthy subjects; 2) develop a model of these reflex-mediated effects including interactive effects between hypoxemia and hypercapnia in patients with OSAS and healthy subjects; 3) determine the effect of sleep interruption on baseline conditions, chemoreflex and baroreflex function in normotensive and hypertensive patients with OSAS and in control subjects; 4) determine the effect of short-term or long-term treatment of OSAS with nasal CPAP on chronic sympathoexcitation, chemoreflex sensitivity and baroreflex sensitivity during wakefulness; and 5) determine the relation of chronic sympathoexcitation, chemoreflex sensitivity and baroreflex sensitivity during wakefulness to the presence of clinical hypertension in patients with OSAS. Understanding the control of sympathetic nerve activity (SNA) in these patients may lead to improved management of this condition and may lend provide new insight into the importance of chemoreflex control of SNA in healthy and diseased humans.

DESCRIPTION (provided by applicant): How aging and noise exposure interact to produce hearing loss is an important issue for understanding its etiology. The relationship between age-related hearing loss (AHL) and noise-induced hearing loss (NIHL) is not always additive. Studies using mammalian models suggest that sensitivity to acoustic trauma may be greatest during the extremes of ontogeny - that is, in developing animals and in senescent animals. Zebrafish (Danio redo) have become a popular vertebrate model for examining embryogenesis and genetic defects because there is considerable synteny between zebrafish and human genes. Thus zebrafish mutations that affect ear development, or interact with age to affect hearing, may shed light on similar pathologies in humans. Although age-related shifts in hearing has been examined in developing fishes, the effects of degeneration due to senescence have not been examined in zebrafish or any other teleost. In order to fully understand the effects of inner ear genetic mutations and NIHL in the zebrafish model, baseline data of age-related shifts in hearing capabilities in wild type zebrafish are needed. The purposes of the proposed research will be to 1) examine shifts in hearing capabilities with age and 2) determine how aging (AHL) and noise exposure (NIHL) interact to cause hearing loss in zebrafish. To fulfill these goals, zebrafish will be exposed to specific sound exposures at various ages, and then pathology of the inner ear (via SEM) and hearing thresholds (via auditory brainstem response) will be examined.

The Graduate School of Biomedical Sciences (GSBS) is collaborating with the Texas College of Osteopathic Medicine (TCOM) at UNTHSC, and the Arizona College of Osteopathic Medicine (AZCOM) to propose a Developmental Center for Research on Osteopathic Manipulative Medicine (DCR-OMM). OMM is a body-based therapy as defined by the NCCAM definitions of complementary and alternative medicine (CAM). The varied principles and practices of OMM are unique among other body-based therapies primarily because they are applied by fully licensed physicians and therefore can be applied to alleviate both musculoskeletal and visceral disease processes and readily integrated with conventional health care. Four key elements of osteopathic principles and practices will be investigated in this DCR-OMM: Study #1) Effects of direct biomechanical strain on the fascial tissues of the musculoskeletal system; Study #2) Effects of OMM (lymphatic pump) on the lymphatic duct lymph flow and the resultant potential beneficial effects on edema and immune function subsequent to an improvement in lymphatic circulation; Study #3) Effects of OMM on sympathetic neural activity either by affecting the sympathetic nervous system directly or by affecting the sympathetic nervous system indirectly by reduction of somatic dysfunction induced pain; and Study #4) Combined synergistic clinical outcome effects that result from applying OMM in patients post -CABG who have a complex combination of fascial restrictions, pathologic fluid shifts, somatic pain and hypersympathotonia. In 2001, the leading national osteopathic professional organizations endorsed and funded these investigators to establish a national Osteopathic Research Center (ORC) housed within the Physical Medicine Institute at the University of North Texas Health Science Center. The OMM research mission of the ORC is perfectly aligned with the goals of the U-19 DCR-OMM and the research priorities of NCCAM. We are all dedicated to the success of this DCR-OMM with the goal of developing into a P-01 Center of Excellence for Research on OMM to enable quality investigation and publication of the mechanisms of OMM.

Osteopathic manipulative medicine (OMM) is a body-based therapeutic approach that has been proven to be efficacious for many clinical conditions. In particular, OMM is commonly used for musculoskeletal conditions and is known to reduce pain. A theoretical foundation of OMM has long been that somato-visceral interactions also can be affected by specific OMM techniques; however, very limited data exist to support or refute this tenet. This proposal will be the first systematic series of studies that address this fundamental hypothesis. OMM techniques include 1) those directed at reducing sympathetic neural activity (SNA), or sympatholytic OMM, and 2) techniques directed at relieving pain and somatic dysfunction at the site of musculoskeletal injury. An initial study, tested the effect of sympatholytic OMM and found that basal sympathetic nerve activity (SNA) can be reduced (20-50%) in healthy subjects free of any pain syndrome. The proposed studies represent an extension of these initial findings. This project #3 will contribute to the overall goal of the proposed developmental center on the mechanisms of OMM by testing the efficacy of the two OMM modalities noted above. Pain accompanies most musculoskeletal injuries and an important part of a pain-mediated stress response is an activation of the sympathetic nervous system; thus, it follows that if OMM can reduce pain in certain conditions, it may also reduce SNA. In Study 1, intermittent cold pressor stimulus will be used to produce an experimental state of sustained elevation of SNA. We will use this condition to determine whether sympatholytic OMM can decrease a sustained elevation of SNA, and whether sustained pain-induced elevations in SNA persist when the stimulus is removed. Although hypothesized, it is not known whether OMM treatment of back pain and somatic dysfunction, can reduce SNA associated with either the pain or the somatic dysfunction. Therefore, in Study 2, it will be determined whether either OMM treatment modality can affect SNA in patients with back pain and somatic dysfunction. In addition, it will be determined whether OMM treatment effects on SNA are direct, or related to pain or somatic dysfunction. Like each of the other Units, these studies will provide critical seminal evidence regarding long-hypothesized, but under-investigated mechanisms of osteopathic manipulative medicine.

? DESCRIPTION (provided by applicant): The overarching goal is to find platinum(II) complex drugs that show toxicity against cancer cells, while avoiding ototoxicity and hearing loss. This research will be conducted both in vitro, and in vivo in the zebrafish model. Current platinum-based drugs like cisplatin have diamine ligands and are highly ototoxic, i.e., produce sensory hair cell and hearing loss. Some novel platinum(II) complexes have triamine or heterocyclic ligands that exhibit unique DNA and protein binding properties that differ from that of complexes with diamine ligands. Dr. Kevin Williams (Biochemistry) at Western Kentucky University (WKU) has recently found differences in platinum(II) diamine and triamine complexes in their reactions with amino acid and nucleotide targets. These properties may result in reduced drug side effects such as ototoxicity. Dr. Michael Smith (Neurobiology) recently showed that ototoxicity could be examined in the zebrafish inner ear using two ototoxic chemicals- the aminoglycoside antibiotic gentamicin, and the platinum-based anti- cancer drug cisplatin. The proposed project is an interdisciplinary collaboration between these two laboratories at WKU. This proposal has two primary aims: 1) to determine whether novel platinum(II) complexes cause cytotoxicity to cancer cells, and 2) to determine whether the novel platinum(II) complexes are ototoxic (i.e., damage hair cells and cause hearing loss). In aim 1, we will test six novel compounds in six different cancer cell lines for anticancer activity using cytotoxicity assays, inductively coupled plasma-emission spectroscopy (ICP-ES) to measure uptake of platinum into cancer cell nuclei, and DNA binding assays. In aim 2, those compounds that are found to be cytotoxic to cancer cells in aim 1 will be tested for ototoxicity in a zebrafish inner ear model. Following microinjection with either a platinum-compound or buffer control, zebrafish hearing thresholds will be measured using auditory evoked potential (AEP) recording. Then the inner ears will be dissected and cell death and hair cell loss will be quantified in the sensory epithelia. The proposed study establishes a framework for understanding the effects of these novel platinum(II) complexes on cancer cells and hair cells, with the ultimate goal of providing the foundation for future investigations developing new chemotherapy drugs that avoid the side effect of hearing loss. This proposal accomplishes the AREA program objectives of: 1) supporting meritorious research; 2) exposing undergraduate and graduate students to research; and 3) strengthening the research environment in non- research intensive universities. Western Kentucky University aspires to be the best comprehensive public institution in Kentucky. This project supports this mission by enhancing the research environment and by providing undergraduate and graduate students with opportunities to be engaged in biomedical research, and supports both P.I.s' career goals of leading independently-funded research laboratories.

Honey bees, live as a group of organisms that form a cooperative unit: a colony. Colonies, like individuals, have a cycle of growth and development. When a colony transitions between developmental stages, the workers must coordinate the transition. The goal of this project is to understand the cues that non-reproductive worker honey bees use to detect the developmental stage of their colony and coordinate the switch between producing more workers and producing reproductive males and females (drones and queens). This research will encourage collaboration between researchers studying animal behavior, chemical ecology, and electrical engineering. Current methods used by beekeepers to assess the strength and developmental stage of a honey bee colony are based on metrics that humans can easily determine, but are almost certainly not those used by the bees. Honey bees are the primary pollinator of agricultural crops worldwide, providing billions of dollars of pollination services. By identifying the metrics that bees use to detect their own colony's development, this research will help beekeepers determine which colonies need to be managed, and when. Better colony management can in turn improve crop production. The outcomes of this research will be shared through beekeeping classes. This project will also train graduate and undergraduate researchers in behavioral ecology, chemical ecology and engineering.

Descriptive work has shown that social insect colonies invest first in workers (for growth) and then switch to producing reproductive individuals. Theoretical work has shown why colonies invest as they do, but it is unknown how developmental transitions are coordinated. The researchers recently discovered that a threshold number of workers is the trigger that induces honey bee colonies to invest in reproduction, but how workers detect this reproductive threshold is unknown. The goal of this research is to test two hypotheses of the mechanism by which individual honey bees assess their colony?s size: (1) beeswax comb vibrations, and (2) volatile chemical compounds. Using accelerometers to measure comb vibrations, and gas chromatography to analyze chemical compounds, the researchers will determine whether these cues reliably change with colony size. The cues will then be experimentally manipulated, to determine which one(s) the workers use to detect that their colony is above the reproductive threshold. Understanding the cues that superorganisms use to coordinate their developmental transitions will give insights into how evolution has solved similar problems at different levels of biological organization such as individuals and societies.

The ability of living cells to maintain their internal mechanical stress in response to external disturbances is essential for normal physiological functions of cells and tissues and for a protection against various diseases. This is known as tensional homeostasis. Breakdown of tensional homeostasis is the hallmark of most advanced solid cancers (carcinomas), as well as of stiffening of the arteries (atherosclerosis) and clot formation (thrombosis). While physical interactions between cells appear to be fundamental to tensional homeostasis, important questions remain. First, it is not known how the transmission of forces between cells impacts tensional homeostasis. Second, there is little understanding of how cells respond to external changes in their mechanical environment (e.g., mechanical stretch) after having initially achieved a steady state. In order to address these questions, this project will advance and apply a biomechanical imaging computational platform. The fundamental questions that can be answered using this technique will improve basic understanding of how cells respond to their mechanical environment. The biomechanical imaging methods and software will be distributed to any interested researcher in order to broadly disseminate this technique within the cellular mechanobiology and tissue engineering communities. In addition, graduate students and undergraduate students will be trained in a highly multidisciplinary environment and will mentor high school students for a six-week research experience.

The biomechanical imaging computational platform maps intracellular stress distribution through simultaneous measurements of (1) traction forces that are transmitted by cells to the substrate and (2) intracellular displacements generated by externally applied stretch. In this project, the researchers will first improve the ability of the biomechanical imaging technique by using mitochondria as fiduciary markers in order to obtain more accurate maps of intracellular stress. The improved technique will then be used to address two fundamental questions. First, the impact of external stretch on tensional homeostasis of multicellular clusters will be investigated. This will be done using bovine aortic endothelial cells and primary human umbilical vein endothelial cells. Second, the system will be used to unravel the relevance of intercellular stress transmission on tensional homeostasis. This will include the investigation of the role of cell-cell adherens junctions and the impact of substrate stiffness on both cell-cell vs cell-matrix force transmission.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Promoting progress in science and building a globally competitive workforce, a core part of the National Science Foundation's mission, requires increasing diversity in science, technology, engineering and mathematics (STEM). This project is supported by the Divisions of Chemistry and Physics in the Mathematical and Physical Sciences Directorate, and the Division of Chemical, Bioengineering, Environmental, and Transport Systems in the Engineering Directorate. The GEM Consortium aims to improve diversity in the STEM workforce through a two-pronged approach. First, GEM encourages selected underrepresented minority (URM) students to pursue graduate degrees in STEM fields by ensuring that the students receive a living stipend and tuition while enrolled in a GEM member-university program. Next, GEM provides access to paid internships for the selected students, providing the students with work experience and networking opportunities. Ultimately this project works to illustrate and provide pathways for advanced STEM education and careers for URM students.

This project provides training pathways for both Ph.D. and Master's level students in STEM fields, particularly in Chemistry, Chemical and Environmental Engineering, and Physics. GEM consortium-selected URM students apply to the university-member programs, and upon acceptance, GEM provides a stipend towards living expenses and tuition. The universities cover the remainder of the tuition, and in the case of Ph.D. level students, a living stipend (through teaching or research assistantships or other methods) for the duration of the degree. This encourages member universities to accept URM students, while assuring students of the economic feasibility of an advanced degree. Students also participate in a paid internship that GEM helps to arrange, which enables students to build a career network and provides them with work experience.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

An award is made to Western Kentucky University for the acquisition of a Laser Scanning Confocal Microscope for research and instruction at a primarily undergraduate institution. The microscope will build imaging capacity at WKU and in the Southern Kentucky region. It will not only impact the research projects of several faculty members who train Graduate (Masters) and Undergraduate students in their laboratories but also enhance the student learning and research experience. The students will present their findings at the local Student Research Conference and at regional and national meetings. They will also disseminate their findings in the form of refereed publications. The exposure of students to this routine yet state-of-the-art technique and the dissemination of acquired data will benefit their research careers by making them competitive for graduate programs. An existing undergraduate Developmental Genetics, Cell Biology and Microscopy course will incorporate this technique and expose students to this state-of-the-art imaging. Additional courses at graduate and undergraduate level will develop modules for this technique as well. To engage and attract young scientists into choosing careers in the sciences, we will offer teachers and students from local high schools opportunities to visit the facility and learn firsthand the power of confocal microscopy. Additionally, rising high school junior and senior students from the Gatton Academy of Mathematics and Science at WKU will also benefit from the availability and technical capabilities of this microscope as several of these students conduct research in the laboratories of the PIs who spearhead a variety of research projects. The microscope will be made available to other regional academic institutions on a fees per use basis and will be advertised at the regional conference organized by the Kentucky Academy of Science. Furthermore, professors and their students within the Kentucky Community and Technical College System will have opportunities to visit the facility for a demonstration of the Confocal Microscope and its capabilities.

Confocal microscopy is routinely utilized by biologists to understand the localization of gene products (proteins and RNA) within a cell in space and time. It also provides valuable insights into the cellular and tissue architecture of developing living systems at resolutions far superior to conventional fluorescence microscopy. Acquisition of this state-of-the-art microscope will impact WKU faculty projects in diverse fields ranging from the Cell and Developmental Biology of fruit flies, Microbiology, Neurobiology of Zebrafish, Endocrinology, Plant Biology, Immunolocalization of proteins in Chlamydomonas to Nanotechnology. The confocal microscope will expand capability and capacity in the Southern Kentucky region and enable students to be part of high caliber research projects. Cellular localization of proteins, generation of high resolution cellular images and understanding mechanisms involved in normal development will all be possible with this microscope, thereby exposing students to sophisticated research.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary Elucidating the Mechanism of B-Raf Dimerization Inhibition Using (+)-Griffipavixanthone Derivatives Ras, Raf, MEK, and ERK are common hotspots for oncogenic expression as they are proteins involved in a highly-regulated pathway controlling cell proliferation. A single mutation can result in a hijacked cell exponentially replicating without regulation. Developing tools to probe, understand, and prevent the effects of these mutations are of great interest as there are currently no FDA-approved drugs that inhibit mutant Ras, an oncogene accounting for approximately 1/3 of all human cancers. The FDA has approved few inhibitors of mutant B-Raf; however, life expectancy is extended for only 3-8 months as resistance is acquired. (+)-Griffipavixanthone (GPX), a dimeric xanthone natural product that we can readily access by asymmetric synthesis, has demonstrated anticancer activity in various cell lines. In particular, we have found maximal potency in cancer cells with K- RasG12 and B-RafV600E mutations. When compared to the FDA-approved drug, sorafenib, GPX demonstrated a lower GI50 and similar or higher LC50. We have found that B-Raf dimerization, an important event mediated by Ras in cell proliferation, is inhibited upon treatment with (+)-GPX. Interestingly, the response is greater in (+)-GPX than its unnatural (-) enantiomer with a long response time for inhibition of B-Raf dimerization (18 h). Objectives: The proposed study will (1) expand on the key asymmetric reaction and employ late-stage functionalization to generate a compound library to understand the delayed response time of B-Raf dimerization inhibition, as we believe that GPX is likely a prodrug that undergoes intracellular oxidation to a reactive p- quinone methide. These advancements would have considerable impact on drug development and inhibitor design, and in the chemistry community; (2) ascertain the mechanism of action as it attenuates a deregulated, hijacked pathway. This will have high broader impact on the community and those studying this oncogenic pathway; (3) elaborate GPX to be more potent, drug-like, and target specific. Three aims are proposed to address the aforementioned objectives. Aim 1: A library of analogues with varying functionality, GPX-derived prodrugs with increased lipophilicity, and oxidized variants will be synthesized and evaluated in a time-dependent manner for B-Raf dimerization inhibition. Aim 2: A complete characterization of intracellular effects and downstream signaling by GPX and its derivatives will be performed in WT and Raf/Ras mutant cells. We will also study direct GPX-Ras/Raf surface binding in vitro and in vivo. Additionally, an unbiased pull-down experiment will be used to confirm intracellular targets. Aim 3: We will develop structural models of GPX derivatives and candidate receptors, including K-Ras mutants, and use this information to design higher affinity analogues. Overall, this research will impact chemical synthesis, prodrug development and metabolomics, and will provide important information on and tools for study of Ras/Raf/MEK/ERK, the most deregulated oncogenic pathway.