SBC2007-175993 MEASUREMENT OF IN-VIVO PULMONARY VASCULAR IMPEDANCE IN TWO ANIMAL MODELS OF PULMONARY HYPERTENSION

INTRODUCTION Pulmonary vascular input impedance has been increasingly promoted as an important diagnostic for pulmonary arterial hypertension (PAH) The human studies noted above have understandably not examined detailed associations between impedance and vascular behavior and structure, since the latter data are obtainable only through focused drug studies or ex-vivo measurements. Mechanical changes to a vascular network should be reflected in its input impedance; thus, such investigation should be useful in determining how impedance varies with changes in vascular condition, such as chronic stiffening due to vascular remodeling or acute stiffening due to smooth muscle cell response and/or pressure-induced strain-stiffening. Naturally, clinical identification of such stiffness changes on a routine basis could greatly impact diagnosis. Here, we demonstrate simple-toimplement impedance measurements in two animal models as part of a larger effort to establish said links between clinically-viable diagnostics, such as impedance, and physiological changes that occur to the vasculature as part of the progression of PAH. METHODS Animal Preparation: The two animal models examined here develop PAH due to chronic exposure to a hypoxic environment. The first model consisted of 10 male Sprague-Dawley rats (300-400g), half exposed to hypoxia via hypobaric chamber for 3-4 weeks (barometric pressure ≈ 410 mmHg) and half retained at standard conditions in Denver, CO (barometric pressure ≈ 630 mmHg). The second model utilized 4 male Holstein calves (70-110lb), again with half exposed to hypoxia for two weeks (barometric pressure ≈ 460 mmHg) and the other half remaining normoxic. Both models were exposed to a 12:12-h light-dark cycle, and water and appropriate food were made available ad libitum. Animal care and use committees at both the University of Colorado Health Science Center (rat) and Colorado State University (calf) approved all protocols and procedures. Animal Data Collection and Analysis: The measurements obtained from each animal are identical; the main differences between collection methods are equipment size and type. For all measurements, rats are anesthetized with ketamine hydrochloride (40 mg/kg) and xylazine (10 mg/kg) intraperitoneally, while cows remain conscious. Right jugular access is then obtained in each animal, and a fluid filled catheter, consisting of PV1 tubing for the rat or a commercial Swan-Ganz catheter for the calf, is inserted into the main pulmonary artery (MPA) for pressure measurements. During collection of MPA pressure, blood velocity at the midline of the MPA is obtained with pulse-wave Doppler echocardiography using an FPA probe on a commercial ultrasound scanner (Vivid 5, GE Medical Systems Inc). The imaging depth dictates the probe frequency

Progress report no. 4

Statement of responsibility on title-page reads: editors: M.J. Driscoll, D.D. Lanning, I. Kaplan, A.T. Supple ; contributors: A. Alvim, G.J. Brown, J.K. Chan, T.P. Choong, M.J. Driscoll, G. A. Ducat, I.A. Forbes, M.V. Gregory, S.Y. Ho, C.M. Hove, O. K. Kadiroglu, R.J. Kennerley, D.D. Lanning, J.L. Lazewatsky, L. Lederman, A.S. Leveckis, V.A. Miethe, P. A. Scheinert, A.M. Thompson, N.E. Todreas, C.P. Tzanos, and P.J. WoodIncludes bibliographical referencesProgress report; June 30, 1973U.S. Atomic Energy Commission contract: AT(11-1)225

Demonstration of Protein-Based Human Identification Using the Hair Shaft Proteome

YesHuman identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.The Technology Commercialization Innovation Program (Contracts #121668, #132043) of the Utah Governors Office of Commercial Development, the Scholarship Activitie