Bruce R. Pitt - US grants

The themes of this proposal are: 1) that alterations of growth of the pulmonary microcirculation contribute substantially to the genesis and maintenance of pulmonary vascular disease associated with many cardiorespiratory disorders of the newborn and 2) that quantitative assessment of metabolic functions of pulmonary endothelium will provide a sensitive biochemical probe of microcirculatory growth and integrity during pathologic as well as normal development. To test these hypotheses, we outline a comprehensive quantitative functional and structural assessment of the pulmonary microcirculation in lambs with aberrations of pulmonary vascular development. Two groups will be studied: a) chemically-induced lung injury via delivery of bleomycin with a s.c. osmotic pump during the first week of life; and b) surgically-induced lung changes in the left lung after right pulmonary artery ligation in one day old lambs. Conscious lambs will be studied for one week to six months. Indicator-dilution techniques will be used to quantify the apparent kinetics (maximal velocity: Vmax; concentrataion at Vmax/e2:Km) of Pulmonary angiotensin-converting enzyme (ACE) activity. Pulmonary amine uptake will also be measured in-line with a nuclear detection system and (125I)-meta-iodo-benzyl-guanidine (a photon-emitting synthetic substrate that mimics norepinephrine uptake in biological systems). These biochemical properties of the endothelium will be correlated with physiological assessment of gas exchange surface area (carbon monoxide diffusing capacity) as well as post-mortem morphometric assessment of the microcirculation (stereologic evaluation of the alveolar septum after glutaraldhyde fixation and election microscopy) and arterial circulation (barium sulfate fixation, radiography with light microscopy). We have already shown that Vmax of pulmonary ACE is directly proportional to perfused area during normal development. These new data during pathologic states will provide information regarding: 1) the degree to which alterations in the pulmonary microvascular structure and growth are reflected in lung metabolic function and gas exchange. 2) the diagnostic potential of in vivo metabolic measures to detect abnormalities in microvascular development for clinical as well as experimental use. 3) the role of the pulmonary microcirculation, independent of or in addition to the pulmonary arterial circulation, in the pathogenesis of pulmonary vascular disease.

The theme of this renewal is that strategies involving overexpression of pulmonary endothelial cell metallothionein (MT) will provide a novel approach to augmenting the antioxidant system of the neonatal lung. Metallothioneins, the major intracellular thiol-containing (cysteine: 30 mol%) heavy metal (Cd,Zn,Cu) binding proteins, are inducible by a variety of pharmacological and environmental conditions and participate in cellular defense against partially reduced oxygen species (PROS). We propose to study the effects of overexpression of MT in the sensitivity to hyperoxia of cultured ovine pulmonary artery endothelial cells (OPAEC) as well as intact newborn lambs. In preliminary experiments, we have noted that induction (with cadmium pre-treatment or direct gene transfer) of MT expression in OPAEC is associated with decreased sensitivity to oxidant injury (tert butyl hydroperoxide or 95% O2). The late term fetal lamb is ideal to evaluate a protective role for MT against pulmonary vascular oxidant injury since it can be safely manipulated in utero and unlike many other experimental animals, it remains sensitive to hyperoxia as a neonate. Accordingly, we will determine if overexpression of MT in OPAEC with cadmium pretreatment (AIM I) or direct transfer of mouse MT-I gene (AIM II) affects the sensitivity of these cells to hyperoxia (95% O2; 24-72 hrs). We will then determine the mechanism (AIM III) by which overexpression may protect these cells including assessing whether MT: a) interferes with hyperoxic induced increases in PROS; b) acts as a direct interceptor of these radicals; or c) indirectly affects other antioxidants (CuZn SOD; glutathione peroxidase). We will then (AIM IV) use immunoliposomes targeted to the pulmonary vascular endothelium of 143d fetal lamb as a vehicle for transferring mouse MT-I gene and determine the sensitivity of the full term (147d) neonate to hyperoxia (100%; 1-4d postnatal). Overexpression of MT will be studied at mRNA (Northern blotting and in situ hybridization) and protein (ELISA and immunocytochemistry) levels in OPAEC and lung tissue. Functional determinations of oxidant injury in OPAEC will include 5-hydroxytrypta- mine transport and radioactive chromium release. Structural (capillary stereology) and functional (lung water balance via lymph fistula) studies will be performed in the intact neonate exposed to hyperoxia with or without MT gene transfer. These studies will provide new information regarding the function of MT in vascular endothelium. Furthermore a novel strategy regarding augmentation of antioxidant defense mechanisms of the newborn lung via in utero gene transfer in the late term fetus will be evaluated.

Multiple organ dysfunction is a life threatening syndrome that affects a considerable proportion of patients after resuscitation from hemorrhagic shock (HS). Lung is a critical target in this insidious process. Histopathologic and physiologic studies have shown that structural and functional changes in the pulmonary endothelium contribute significantly to the pathogenesis of post-HS lung injury. Injury to lung appears to be secondary to systemic events including delivery to the pulmonary circulation of activated neutrophils, cytokines and bacteria (and their toxins) via translocation from ischemic gut and/or liver. The molecular mechanisms responsible for pulmonary damage in post HS lung injury remain unclear but a role for an imbalance between free radicals and cellular defense mechanisms is apparent. We propose that the molecular determinants for enhanced sensitivity of the lung to pro-inflammatory stimuli during resuscitation arise early in the course of HS within resident cells of the alveolar capillary barrier. We theorize that adaptive responses to HS, including increased intrapulmonary synthesis of NO, result in endothelial cell metabolic changes thereby initiating a sequence of events including apoptosis. These compensatory responses may initially be critical components of pulmonary defense, but in the transition to more severe HS, they become maladaptive. The molecular events leading to apoptosis become unbalanced due to excessive production of iNOS-derived NO in the presence of oxidative stress (enhanced superoxide anion production and decreased intracellular thiols). Accordingly, SPECIFIC AIMS are to determine: I. a protective role for metallothionein (MT) in modifying pulmonary endothelial cell injury after resuscitation from HS or hepatic ischemia/reperfusion (I/R) in MT knockout and transgenic mice; II. a contributory role for iNOS-derived NO in post-HS and IR lung injury in iNOS knockout mice; III. the molecular mechanism and intracellular signaling pathways in which NO causes endothelial cell damage and apoptosis during oxidative stress in cultured murine lung endothelial cells(MLEC); and IV. effects of NO on novel gene expression in lung tissue and MLEC during oxidative stress by RT-PCR-differential display.

DESCRIPTION (provided by applicant): Pulmonary endothelium is the locus of early structural and functional changes in hyperoxic lung injury. An imbalance between the production of partially reduced oxygen species and their elimination appears to account for the genesis and/or maintenance of such pathology and evidence exists that such injury may be exacerbated by partially reduced nitrogen species. Nonetheless, it is apparent that nitric oxide (NO) may actually limit endothelial cell injury. The molecular mechanism underlying the protective role of NO, especially in pulmonary endothelium, remains unknown. From recent reports and preliminary data, we hypothesize that iNOS derived NO may be protective to lung endothelium by its potential antiapoptotic effects mediated by posttranslational S-nitrosylation of proteins. Furthermore, we have recently shown that S-nitrosylation of zinc thiolate clusters in metallothoinein (MT) is a critical component of cellular redox sensitivity linking NO to zinc homeostasis in pulmonary endothelial cells. The well known contributions of zinc to transcriptional activity and the inhibitory effects of zinc on apoptosis underscore the importance of SNO-MT in the signaling pathway underlying NO mediated cytoprotection. Accordingly we will determine the: Aim I: role of iNOS derived NO in affecting pulmonary endothelial cell structure and function in hyperoxia. We hypothesize that iNOS derived NO is cytoprotective in lung. iNOS-/- mice, endothelial cell transgenic mice and anti-PECAM targeted somatic gene transfer of iNOS to pulmonary endothelium will be used to test this hypothesis in pulmonary circulation of intact mice exposed to hyperoxia. Aim II: mechanism by which NO is antiapoptotic in cultured murine lung endothelial cells (MLEC). We hypothesize that NO mediated release of zinc underlies antiapoptotic effects of iNOS derived NO in LPS induced apoptosis in MLEC. Full spectral confocal and multiphoton laser scanning microscopy (MPSLM) of GFP-labeled metal regulatory factor-1 and fluorescence resonance energy transfer (FRET) will be done. Aim III: role of zinc in NO induced protection of hyperoxic injury to pulmonary endothelium in intact mouse lung. We hypothesize that release of zinc is an important contributing factor to NO mediated resistance to hyperoxia in intact mice. NO induced changes in labile zinc via MPSLM and FRET in perfused lung and zinc depletion or targeted disruption of MT on sensitivity of intact mice to hyperoxia will be studied.

This request is for funds to purchase a multimode live cell inverted fluorescence microscope. This instrument will serve as the primary light microscopic image collection system within the Department of Environmental and Occupational Health (EOH) of the Graduate School Public Health (GSPH). This new need for EOH is the result of: a) immediate and future recruitment of molecular toxicologists with live cell imaging needs; b) the expansion of existing programs within EOH to examine issues of environmental stress on the physiology of live cells and fluorescence in situ hybridization studies for chromosomal aberrations after environmental mutagens. These investigators are currently located in the Center for Environmental and Occupational Health and Toxicology (CEOHT) in Blawnox, PA that is 9 miles from the University of Pittsburgh GSPH and School of Medicine. There are no microscopic facilities that support live cell imaging nor is there an ability to collect images from low light samples such as in situ fluorescent hybridization. There is, however, a highly contemporary Center for Biological Imaging (CBI) directed by Simon Watkins, Ph.D. in the School of Medicine. Although plans are underway to move CEOHT onto the University of Pittsburgh campus in Oakland, PA, the likely new site will remain at a considerable distance from CBI. It is the intent of this request to establish a broad based platform of fluorescence imaging within CEOHT to: a) facilitate studies that otherwise could not tolerate travel between CEOHT and CBI; b) allow developmental work in imaging prior to execution of more sophisticated applications in heavily subscribed CBI; and c) enhance training of pre- and post- doctoral students by introducing routine fluorescence imaging in environmental health science. Accordingly a request is made to purchase a high quality, expandable optical platform able to sample several different fluorochromes with exquisite signal separation between chromes, a sensitive camera system able to perform quantitative analysis of extremely low light signals, the ability to collect images from live cells with minimal cell killing, and a computer system containing modules specifically designed for applications in environmental health science.

Pulmonary endothelium is an early locus of functional and structural changes that contribute to the genesis and/or maintenance of acute lung injury(ALl). We will define the mechanisms of transport and the vascular actions of key inflammatory-modulatory macromolecules [e.g. S-nitroso-albumin (SNO-AIb) and myeloperoxidase (MPO)] that are elevated in the circulation after resuscitation from hemorrhagic shock (HS). Accordingly specific aims are to: Aim #1. Define the physiological mechanism of transcytosis of SNO-AIb in cultured pulmonary endothelium. We will use real time multimode imaging (confocal and multiphoton scanning laser microscopy and total internal reflection fluorescence microscopy, FRET) to define molecular events associated with vascular cell binding, transcytosis and SNO-AIb mediatied signaling via S-nitrosation and/or activation of guanylyl cyclase. Aim #2. Reveal the physiological mechanisms of MPO transcytosis in live intact pulmonary endothelium Aim #3. Explore the biochemical mechanisms by which MPO affects SNO-albumin signaling in pulmonary vascular cells and intact lung. To reveal the central pathways for intra- and extracellular regulatory MPO-dependent modulation of vascular NO signaling, we will examine the actions of MPO on SNO-albumin transfer and decomposition kinetics in increasingly integrated biological systems including: a) cell free models in vitro; b) rat lung microvacular endothelial cells (RLMVEC); and c) rat lung microvasccular smooth muscle cell and RLMVEC co-culture system. Specific Aim #4. Place the pathophysiological contributions of SNO-albumin transport, redox status and MPO into the context of ALl after resuscitation from HS. We will contrast the extent of ALl (permeability index, neutrophil accumulation, morphological changes,pulmonary vasoregulation) in isolated perfused lungs and intact mice of wildtype and caveolin-1 and MPO homozygous null mutants.

DESCRIPTION This application is the second re-submission of the University of Pittsburgh's graduate training program in computational toxicology. This program is a joint effort of the Department of Environmental and Occupational Health (DEOH) within the Graduate School of Public Health and the Department of Computer Sciences within the School of Arts and Sciences. The program is administratively housed within the DEOH. The program seeks to offer pre- and postdoctoral training that will allow graduates to apply computational techniques to model and develop: (1) toxicological dose-response relationships, (2) three-dimensional ligand-receptor interactions, (3) expert systems to generate hypotheses regarding toxicological phenomena, and (4) mechanistically informative structure activity relationship (SAR) models. SAR-based approaches are a unifying theme of this program. Since the last re- submission physiologically based pharmacokinetic (PBPK) model development has been dropped as a modeling area. The program is highly interdisciplinary in both the makeup of the faculty and the required curriculum. Dr. Herbert S. Rosenkranz, the Program Director, is chair of DEOH and the co-Director, Dr. Bruce Buchanan is a Professor in Department of Computer Sciences. The involved faculty members have a broad range of primary affiliations including the DEOH, Department of Computer Sciences, Biostatistics, Pittsburgh Supercomputer Center, Carnegie Mellon University, and private industry. Two additional faculty have been added since the last re-submission, Dr. Mazdumar (Biostatistics) and Dr. Cunningham (DEOH). The predoctoral curriculum is designed to be integrated and interdisciplinary. Required courses span the disciplines of toxicology (6 credits), exposure assessment (6 credits), biostatistics (9 credits), epidemiology (2 credits), and computational sciences (15 credits). Six credit hours of electives from among these disciplines are required as is participation in the Survival Skills and Ethics Program. Total course credits required are 46 plus 30 research credits for a total of 76 credits. Attendance at departmental seminars and journal club is also required.

DESCRIPTION (provided by applicant): This request is for funds to purchase an analytical bench top research two-laser flow cytometer. The new instrument, a BD FACSCalibur, will serve as the only analytical cytometer in Department of Environmental and Occupational Health (EOH) of the Graduate School Public Health (GSPH) and for users in nearby Magee Women's Research Institute (MWRI) who share common interests in environmental health science and additional work in maternal/child health. The rationale for this request includes: a) The Department of Environmental and Occupational Health (EOH) is in an extensive recruitment phase with an emphasis on contemporary environmental health science that greatly increases our utilization of flow cytometry. This expansion coincides with the recent recruitment of Dr. Jerry Schatten and colleagues at our Magee Women's Research Institute (MWRI) thereby greatly further expanding our user base; b) EOH is in a newly renovated stand alone facility 0.2 to 0.4 miles from collaborating investigators (with the noted exception of much closer proximity to MWRI) requiring transit through unacclimatized outdoor routes minimizing the opportunity to share flow cytometers located in several other departments in our allied health system; c) Our major Flow Cytometry Core facility, directed by Dr. Donnenberg, relocated (September 2002) to the Hillman Cancer Center that is 1.5 miles away adding, to our need to establish an independent ability to perform analytical flow cytometry; d) The instrument is vital for training of pre- and postdoctoral students; and e) An analytical flow cytometer (e.g., FACSCalibur) will provide valuable quantitative approaches to support more complex research protocols in environmental health science with the aid of our colleagues on our advisory committee from Flow Cytometry Core (Dr. Donnenberg) and Center for Biological Imaging (Dr. Watkins). Applications will include multicolor immunophenotyping, physiological studies on transfected cells, signaling and apoptosis, stem cell biology and microbiology.

Voltage-gated K+ (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs) have been implicated in the initiation of pulmonary hypertension: inhibition of these channels results in membrane depolarization and an increase in intracellular Ca2+ concentration, leading to vasoconstriction and cell growth / remodeling. The use of anorexic agents (phentermine, fenfluramine and their related drugs) is associated with an increased incidence of pulmonary hypertension. These drugs also decrease the activity and expression of Kv channels in PASMCs. Thus, understanding the mechanisms by which these identified stimuli produce alterations in the function and level of these channels may provide clues for prevention and treatment of primary pulmonary hypertension. The anorexic agents reduce Kv channel activity at multiple steps. They acutely inhibit 4-aminopyridine (4-AP)-sensitive Kv current in PASMCs. Furthermore, long-term treatment of PASMCs with fenfluramine leads to decreases in Kv current density and the expression of Kv1.5 mRNA. Lung tissues from patients with primary, but not secondary, pulmonary hypertension also exhibit reduced expression of Kv1.5 mRNA. These findings suggest acute and long-term exposures to these drugs influence 4-AP-sensitive Kv channels at plasma membrane and transcription of Kv channel subunit genes, respectively. Using Xenopus oocyte expression system, we found that fenfluramine and phentermine inhibit Kv1.5, Kv2.1 and Kv4.2, but not Kv3.1b, current. Using cultured rat PASMCs and heterologous expression systems, we have analyzed molecular mechanisms underlying the anorexigen-induced changes in the activity and expression of Kv channels. First, exposure to fenfluramine decreased endogenous Kv2.1 proteins in PASMCs. The drug also reduced heterologously expressed Kv2.1, but not Kv1.5 or Kv4.3, proteins in a mammalian cell line. In addition, the non-selective kinase inhibitor staurosporin mimicked and occluded the fenfluramine-induced decrease in the channel protein level in PASMCs. Second, the anorexic drugs caused significant decreases in the level of endogenous Kv1.5 mRNA and reporter gene expression driven by the Kv1.5 promoter in PASMCs. Reductions in the channel promoter activity were also seen in A7r5 smooth muscle cells, but not in CHO or HEK293 cells. Finally, fenfluramine and phentermine rapidly and reversibly inhibited Kv1.5, Kv2.1 and Kv4.2, but not Kv3.1b, currents in Xenopus oocytes. Thus, the anorexigen-induced pulmonary hypertension may be mediated by their multitude of actions to produce acute and long-term inhibition of PASMC Kv channels. Hence, this proposal is to identify molecular mechanisms for anorexigen-induced inhibition of Kv channels at the three levels: a slow decrease in Kv2.1 proteins, inhibition of Kv1.5 gene transcription and blockade of Kv currents at plasma membrane.

[unreadable] DESCRIPTION (provided by applicant): This amended proposal is for a high performance gel and blot imager. The new instrument, General Electric-Amersham Biosciences Typhoon 9400, has three methods of detection (storage phosphor, fluorescence and chemiluminescence) and will serve as the only high sensitivity quantitative instrument for low abundance targets for multiple users in the Department of Environmental and Occupational Health (EOH), Center for Clinical Pharmacology, McGowan Institute Regenerative Medicine (MIRM) and Molecular Medicine Institute (MMI). The rationale for this request includes: a) EOH relocated to a new research (Cellomics) building at our Bridgeside Point site 1.5 miles from the main medical campus where the two current phosphoimagers are located; b) Center Clinical Pharmacology, MIRM and MMI are current occupants of the Cellomics Building and Biotechnology Center at the Bridgeside Point site and along with EOH have increased their emphasis on the utilization of molecular biological approaches that routinely require access to a broad based platform of high sensitivity and significant dynamic range; c) Typhoon 9400 will be a valuable platform for the training of pre- and postdoctoral students; d) subtle features of this instrument including 3 laser fluorescence should assure its utility in the future; and e) the short scanning time of the instrument enhances its multiuser capability by allowing more time consuming analysis to be done in individual laboratories. Applications with Typhoon 9400 include fluorescent DNA gel stain, 2-color fluorescent differential display, fluorescent protein gel stain, fluorescent multiplex PCR and Western blot analyses, imaging 2-D gels and chemiluminescence detection of proteins, phosphor imaging of radioisotopes for Northern blot and in situ hybridization. This enabling technology will advance fundamental research in environmental health sciences, regenerative medicine, clinical pharmacology and gene therapy and thereby facilitate broad based efforts in many aspects of medicine and public health. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant) [unreadable] [unreadable] The overall goal of the University of Pittsburgh Short-Term Educational Experience for Research (PITT-STEER) in the Environmental Health Sciences for High School and Undergraduate Students is to encourage entry of students into careers in biomedical research and environmental health sciences. The PITT-STEER will exploit a mentored focus on respiratory biology, metal toxicology, health disparities and other contemporary aspects of environmental health science. During the 10-week program, students will conduct original research in these areas under the mentored supervision of Environmental and Occupational Health (EOH) and Behavioral Community Health Sciences (BCHS) faculty in the Graduate School Public Health (GSPH). Students will work on problems at the frontier of biomedical science using state-of-the art techniques. Students will also follow a dedicated series of lectures and participate in problem based learning to introduce basic fundamentals of environmental health science and toxicology. Additionally, they will participate in a seminar program designed to further their understanding of the scientific process and critical analysis, and workshop forums on career paths within biomedical research. The long-term goal is to promote scientific training of new investigators that exploits interdisciplinary approaches to address significant research problems in environmental health science. Prospective students will be selected from a large and outstanding pool of candidates of emerging junior and senior high school students in the Pittsburgh Public School system. Access to this group of students will be facilitated by ongoing efforts in a separate research project (Healthy Class of 2010) in which the Center of Minority Health in the GSPH has established firm interactive ties with the 9 public high schools in Pittsburgh. Six students per summer will be selected by an administrative committee based on high school transcripts, personal statement and letters of recommendation. The Pittsburgh Public School system has significant underrepresented minority (70%) and disadvantaged youth representation facilitating goals of diversity recruitment. Opportunities will be provided to participate in a second summer experience for outstanding students during their own college undergraduate experience. A core of NIEHS and NIH-HLBI funded, experienced investigators has been chosen as mentors. Ancillary activities will include laboratory safety and scientific ethics. At the end of the program, each student will present the results of his/her work at a colloquium of participating students and mentors. A certificate will be awarded to all students successfully completing the program. Longitudinal follow-up will monitor graduate training and career choices elected by student participants in this program and will be compared with that of entering underrepresented minority biomedical graduate students who did not participate in the PITT-STEER or similar programs. This tracking will provide an assessment of the ability of the program to achieve its goals. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We are requesting funds for a Bio-Rad Laboratories Bio-Plex Workstation and Software (Suspension Array) system to provide a multiplex system to facilitate simultaneous analysis of up to 100 different biomolecules (proteins, peptides or nucleic acids) in a single microplate reader. The multiplex platform integrates xMAP(R) technology and Luminex200" from Luminex Corporation to enable discrete bioassays on the surface of color coded beads (either polystyrene or magnetic). Reagents may include antigens, antibodies, oligonucleotides, enzyme substrates or receptors and thus can provide quantitative information of multiple analytes in small volumes for experimental (animal and human) or cell culture analyte profiling, signal transduction profiling and transcriptional profiling. The multiplex analysis system (Bio-Plex) will be used to support a group of 13 investigators in the Department of Environmental and Occupational Health (EOH). The rationale for this request includes: a) EOH is an extensive recruitment phase with an emphasis on contemporary environmental health science that greatly has increased the utilization of molecular biological approaches in experimental models in genetically modified mice including quantitative trait locus approaches towards gene-environment interactions, mammalian cell culture and human subjects;b) EOH recently relocated to a new contemporary research building (Thermo-Fisher Building) in the Bridgeside Point Site (100 Technology Drive) that is >3.0 miles from the major cluster of Bio-Plex instruments (n=5) at University Pittsburgh Cancer Institute;and our current collaborators at Childrens Hospital of Pittsburgh;c) there is only one Bio-Plex instrument nearby (Clinical Pharmacology) and this is dedicated to a large pharmacogenomic effort and was not configured to accommodate the entire needs of our department;d) Bio-Plex will be a valuable platform for the training of pre- and postdoctoral students in environmental health sciences;e) the versatility (proteins and nucleic acids) of Bio-Plex will assure that it conforms to predicted future needs of serving as a critical interface with other discrete high throughput screening;f) the short scanning time of the instrument enhances its multiuser capability by allowing more time consuming analysis to be done in individual laboratories and the multiuser software components will facilitate off site data analysis further simplifying multiuser access. The Bio-Plex platform is capable of assaying a wide range of biomolecules including proteins and nucleic acids and as such applications can include immunoassays, enzyme assays, receptor ligand interactions and signal transduction assays as well as transcription factor profiling. We anticipate extensive initial use in animal model serum and cell culture model analyte profiling and expanding use in signal transduction network profiling and transcription factor profiling. PUBLIC HEALTH RELEVANCE: The acquisition of Bio-Plex for 13 sponsored investigators in EOH will greatly facilitate existing funded research in environmental health related to respiratory, cardiovascular and central nervous system function. The ability to make multiple measurements of protein and nucleic acids in a small volume will greatly enhance the integrated nature of virtually all experiments and allow for considerably more meaningful results to emerge.

DESCRIPTION (provided by applicant): Although oxygenated fatty acids and oxidized phospholipids are critical signaling molecules (and/or biomarkers) in acute lung injury (ALI), their extraordinary diversity along with limitations in biochemical methodologies have prohibited their systematic study. We recently described new methodology of oxidative lipidomics that combines the use of liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI- MS). We now propose to develop a new enabling technology of imaging mass spectrometry (IMS) for oxidative lipidomics that will resolve issues of spatial confinements of peroxidation reactions in lipids in regions of the lung and ultimately near limits of individual pulmonary cells. Accordingly, the overall aim of this proposal is to: develop IMS of oxidative lipidomics and define spatial distribution of oxidized, oxidizable and non-oxidizable phospholipids - particularly of anionic cardiolipin (CL) and phosphatidylserine (PS) - in lungs of intact mice after oxidant (H2O2-generating glucose oxidase)-mediated ALI. To this end, we will utilize multiple approaches of increasing power in resolution: a) Matrix assisted laser desorption ionization (MALDI)-IMS-TOF-TOF(~50mm); b) the high mass resolving power and measurement accuracy of Fourier Transform Ion Cyclotron Resonance MS (~20mm resolution, Bruker Daltonics SolariX, Billerica, MA); and c) high spatial resolving power of nano-scale matrices combined with MALDI-post-ionization mobility-orthogonal Time of Flight MS (~10mm Ionwerks, Inc, NIH NIDA) and oversampling-stepping (<10mm). Serial sections will be used for: a) quantitative measures of oxidative lipidomics via LC-ESI-MS; and b) fluorescence immunohistochemistry that will be co-registered with IMS spectra to reveal structure and function of lung in ALI. The use of mitochondrial targeted antioxidants will serve as a validation of this technology and expand application of such imaging to metabolomics and molecular pharmacology. This proposal will establish a new enabling technology (IMS) for oxidative lipidomics that will: a) resolve issues of spatial confinements of peroxidation in lung that cannot be readily examined otherwise; b) provide a panoramic snap-shot of hundreds of signals simultaneously; c) be informative about oxidation of critical phospholipids (CL and PS) that are early signaling molecules in cell death (apoptosis) and inflammation (clearance of apoptotic cells); and d) be used in a metabolomic mode to reveal molecular pharmacological information on disposition of small molecule protectants in ALI.

DESCRIPTION (provided by applicant): The International Society of Zinc Biology (ISZB) was founded in 2007, as a nonprofit organization. Our fundamental goal is to bring together scientists from diverse fields with common interest in the structural, biochemical, genetic, physiological and medical aspects of zinc biology. Membership is open to individuals from any nation. Our Society is a consensus-based organization that strongly encourages exchange of information and ideas among zinc researchers. To that aim, the ISZB has organized and supported one of a kind biennial international conferences (ISZBC) dating back to 2008. Work at these meetings has shown that fundamental mechanisms of zinc biology are of critical relevance to understanding health and disease across a broad spectrum that includes but is not limited to cancer, immunology, neurology, as well as metabolic and endocrine disorders. Bringing these investigators together has contributed to substantial advances in these fields, and promoted new collaborations between fields. The number of attendees has steadily grown and now exceeds 140 with an intentional balance of graduate students, postdoctoral fellows, junior faculty, and established investigators. The 4th ISZBC is being held in Pacific Grove, California at Asilomar from September 14th-19th, 2014, and will be organized by a diverse and experienced team of investigators. Asilomar is ideally suited for a meeting of this size and has been chosen because it provides an exceptional environment for participant interaction and scientific exchange. Our conference is the premier and only scientific venue focused exclusively on the field of zinc biology. The ISZBC strongly supports the development of the next generation of researchers including those from underrepresented populations and highly values trainee development and diversity. The ISZBC; 1) encourages the exchange of unpublished data; 2) is highly interactive between young and established investigators to foster meaningful collaborations; and 3) promotes networking between investigators, trainees and their respective institutions from around the world. As a society we are driven by innovation and discovery. As a testament to this, we have a separate scientific program committee that has recruited the top senior and junior talent in established and emerging areas of zinc biology regardless of society affiliation. The 2014 conference will integrate junior faculty/trainee podium presentations into every scientific session. Five graduate student or postdoctoral fellows chosen from submitted abstracts are also invited to present orally, providing these trainees with an invaluable opportunity to showcase their research. We propose to select from abstract submissions ten U.S. trainees for registration waivers, focusing on underrepresented minorities. Importantly, this year's ISZBC will have an interactive forum focused on career development for trainee attendees. Special efforts have been made in general to encourage attendance by underrepresented minorities in science, including women. Thus, the ISZBC supports a variety of activities that are directly relevant to the scientific missions of the National Institutes of Healh and public health.