Dean Billheimer

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Scholarly Contributions

Chapters

• Billheimer, D. D., Sinari, S., Nedelkov, D., & Reaven, P. (2016). The Analysis of Human Serum Albumin 1 Proteoforms Using Compositional Framework. In Statistical Analysis of Proteomics, Metabolomics, and Lipidomics Data Using Mass Spectrometry. Springer International Publishing Switzerland.

Journals/Publications

• Hallmark, B., Wegienka, G., Havstad, S., Billheimer, D., Ownby, D., Mendonca, E. A., Gress, L., Stern, D. A., Myers, J. B., Khurana Hershey, G. K., Hoepner, L., Miller, R. L., Lemanske, R. F., Jackson, D. J., Gold, D. R., O'Connor, G. T., Nicolae, D. L., Gern, J. E., Ober, C., , Wright, A. L., et al. (2021). Chromosome 17q12-21 Variants are Associated with Multiple Wheezing Phenotypes in Childhood. American journal of respiratory and critical care medicine.
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  Birth cohort studies have identified several temporal patterns of wheezing, only some of which are associated with asthma. Whether 17q12-21 genetic variants, which are closely associated with asthma, are also associated with childhood wheezing phenotypes remains poorly explored.
• Lieber, C. A., Majumder, S. K., Billheimer, D., Ellis, D. L., & Mahadevan-Jansen, A. (2021). Raman microspectroscopy for skin cancer detection in vitro. Journal of biomedical optics, 13(2), 024013.
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  We investigate the potential of near-infrared Raman microspectroscopy to differentiate between normal and malignant skin lesions. Thirty-nine skin tissue samples consisting of normal, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma from 39 patients were investigated. Raman spectra were recorded at the surface and at 20-microm intervals below the surface for each sample, down to a depth of at least 100 microm. Data reduction algorithms based on the nonlinear maximum representation and discrimination feature (MRDF) and discriminant algorithms using sparse multinomial logistic regression (SMLR) were developed for classification of the Raman spectra relative to histopathology. The tissue Raman spectra were classified into pathological states with a maximal overall sensitivity and specificity for disease of 100%. These results indicate the potential of using Raman microspectroscopy for skin cancer detection and provide a clear rationale for future clinical studies.
• Alkhatib, N. S., McBride, A., Bhattacharjee, S., Ramos, K., Erstad, B., Slack, M., Billheimer, D., & Abraham, I. (2020). Pricing methods in outcome-based contracting: δ5: risk of efficacy failure-based pricing. Journal of medical economics, 23(11), 1246-1255.
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  Six Delta is a six-dimensional independent platform for outcome-based pricing/contracting. The fifth dimension (δ5) estimates prices on the basis of the risk of efficacy failure of a drug. We describe this dimension's methodology and present a proof-of-concept application to the treatment of non-small cell lung cancer (NSCLC) with EGFR mutation with osimertinib.
• Gerald, J. K., Hallmark, B., Billheimer, D., Martinez, F. D., & Gerald, L. B. (2020). Are Latino children of Mexican origin with asthma less responsive to inhaled corticosteroids than white children?. The journal of allergy and clinical immunology. In practice, 7(7), 2419-2421.
• Ivanova, A., Israel, E., LaVange, L. M., Peters, M. C., Denlinger, L. C., Moore, W. C., Bacharier, L. B., Marquis, M. A., Gotman, N. M., Kosorok, M. R., Tomlinson, C., Mauger, D. T., Georas, S. N., Wright, R. J., Noel, P., Rosner, G. L., Akuthota, P., Billheimer, D., Bleecker, E. R., , Cardet, J. C., et al. (2020). The precision interventions for severe and/or exacerbation-prone asthma (PrecISE) adaptive platform trial: statistical considerations. Journal of biopharmaceutical statistics, 1-12.
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  The Precision Interventions for Severe and/or Exacerbation-prone Asthma (PrecISE) study is an adaptive platform trial designed to investigate novel interventions to severe asthma. The study is conducted under a master protocol and utilizes a crossover design with each participant receiving up to five interventions and at least one placebo. Treatment assignments are based on the patients' biomarker profiles and precision health methods are incorporated into the interim and final analyses. We describe key elements of the PrecISE study including the multistage adaptive enrichment strategy, early stopping of an intervention for futility, power calculations, and the primary analysis strategy.
• Khalil, N., Ducatman, A. M., Sinari, S., Billheimer, D., Hu, C., Littau, S., & Burgess, J. L. (2020). Per- and Polyfluoroalkyl Substance and Cardio Metabolic Markers in Firefighters. Journal of occupational and environmental medicine, 62(12), 1076-1081.
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  To evaluate if serum polyfluoroalkyl substances (PFAS) were associated with cardiometabolic markers.
• Kimura, H., Francisco, D., Conway, M., Martinez, F. D., Vercelli, D., Polverino, F., Billheimer, D., & Kraft, M. (2020). Type 2 inflammation modulates ACE2 and TMPRSS2 in airway epithelial cells. The Journal of allergy and clinical immunology, 146(1), 80-88.e8.
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  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has dramatically changed our world, country, communities, and families. There is controversy regarding risk factors for severe COVID-19 disease. It has been suggested that asthma and allergy are not highly represented as comorbid conditions associated with COVID-19.
• Miller, D. C., Beamer, P., Billheimer, D., Subbian, V., Sorooshian, A., Campbell, B. S., & Mosier, J. M. (2020). Aerosol Risk with Noninvasive Respiratory Support in Patients with COVID-19. Journal of the American College of Emergency Physicians open.
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  This study evaluates aerosol production with high flow nasal cannula () and noninvasive positive pressure ventilation () compared to six liters per minute by low-flow nasal cannula.
• Ober, C., McKennan, C. G., Magnaye, K. M., Altman, M. C., Washington, C., Stanhope, C., Naughton, K. A., Rosasco, M. G., Bacharier, L. B., Billheimer, D., Gold, D. R., Gress, L., Hartert, T., Havstad, S., Khurana Hershey, G. K., Hallmark, B., Hogarth, D. K., Jackson, D. J., Johnson, C. C., , Kattan, M., et al. (2020). Expression quantitative trait locus fine mapping of the 17q12-21 asthma locus in African American children: a genetic association and gene expression study. The Lancet. Respiratory medicine, 8(5), 482-492.
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  African ancestry is associated with a higher prevalence and greater severity of asthma than European ancestries, yet genetic studies of the most common locus associated with childhood-onset asthma, 17q12-21, in African Americans have been inconclusive. The aim of this study was to leverage both the phenotyping of the Children's Respiratory and Environmental Workgroup (CREW) birth cohort consortium, and the reduced linkage disequilibrium in African Americans, to fine map the 17q12-21 locus.
• Skiba, M. B., Hopkins, L. L., Hopkins, A. L., Billheimer, D., & Funk, J. L. (2020). Nonvitamin, Nonmineral Dietary Supplement Use in Individuals with Rheumatoid Arthritis. The Journal of nutrition, 150(9), 2451-2459.
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  Over-the-counter, natural product-based (nonvitamin, nonmineral) dietary supplement (NVNM DS) use is common in adults with rheumatoid arthritis (RA), a group at risk for drug-DS interactions, due to polypharmacy, but this use is underreported to health care providers. Recent dramatic changes in US sales of specific NVNM DS suggest that the prevalence and types of NVNM DS used in RA populations may also have shifted.
• Willis, A. L., Calton, J. B., Calton, J., Kim, A. S., Lee, R., Torabzadeh, E., Billheimer, D. D., Le, C. H., Martinez, F. D., & Chang, E. H. (2020). RV-C infections result in greater clinical symptoms and epithelial responses compared to RV-A infections in patients with CRS. Allergy.
• Beamer, P. I., Furlong, M., Lothrop, N., Guerra, S., Billheimer, D., Stern, D. A., Zhai, J., Halonen, M., Wright, A. L., & Martinez, F. D. (2019). CC16 Levels into Adult Life Are Associated with Nitrogen Dioxide Exposure at Birth. American journal of respiratory and critical care medicine, 200(5), 600-607.
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  Lung function and growth are adversely associated with nitrogen dioxide (NO) exposure. Lower levels of circulating club cell secretory protein (CC16) in childhood are also associated with subsequent decreased lung function. NO exposure may induce epithelial damage in lungs and alter club cell proliferation and morphology. To determine if increased ambient NO levels at participants' home addresses in early life were associated with decreased levels of CC16 from age 6 to 32 years. Participants were enrolled at birth in the Tucson Children's Respiratory Study and had circulating CC16 measured at least once between age 6 and 32. Linear mixed models were used to determine the association between estimated ambient NO exposure at participants' home address at birth or age 6 with CC16 levels from age 6 to 32. NO exposures at birth or age 6 were available for 777 children with one or more CC16 measurement. We found a negative association between NO exposure and CC16 levels, with a 4.7% (95% confidence interval, -8.6 to -0.7) decrease in CC16 levels from age 6 to 32 per interquartile range increase in NO exposure (6.0 ppb) at the participants' birth address. We observed modification by race (p interaction = 0.04), with stronger associations among participants with at least one black parent (-29.6% [95% confidence interval, -42.9% to -13.2%] per interquartile range). NO at participant's age 6 address was not significantly associated with CC16 levels (-1.9%; 95% confidence interval, -6.3 to 2.6). Higher exposure to NO at birth is associated with persistently low levels of CC16 from 6 to 32 years.
• Mosier, J. M., Stolz, U., Milligan, R., Roy-Chaudhury, A., Lutrick, K., Hypes, C. D., Billheimer, D., & Cairns, C. B. (2019). Impact of Point-of-Care Ultrasound in the Emergency Department on Care Processes and Outcomes in Critically Ill Nontraumatic Patients. Critical care explorations, 1(6), e0019.
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  Outcomes data on point-of-care ultrasound (POCUS) in critically ill patients are lacking. This study examines the association between POCUS in the emergency department and outcomes in critically ill patients.
• Gerald, J. K., Fisher, J. M., Brown, M. A., Clemens, C. J., Moore, M. A., Carvajal, S. C., Bryson, D., Stefan, N., Billheimer, D., & Gerald, L. B. (2018). School-supervised use of a once-daily inhaled corticosteroid regimen: A cluster randomized trial. The Journal of allergy and clinical immunology.
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  School-supervised use of a once-daily inhaled corticosteroid regimen (supervised therapy) can improve medication adherence and asthma control.
• Huang, S., Bedrick, E. J., Vasquez, M., Hu, C., Bell, M. L., Roe, D., Billheimer, D. D., Guerra, S., Guerra, S., Billheimer, D. D., Roe, D., Bell, M. L., Vasquez, M., Hu, C., Huang, S., & Bedrick, E. J. (2018). Regularized continuous-time Markov model via elastic net. Biometrics.
• Huang, S., Hu, C., Bell, M. L., Billheimer, D., Guerra, S., Roe, D., Vasquez, M. M., & Bedrick, E. J. (2018). Regularized continuous-time Markov Model via elastic net. Biometrics, 74(3), 1045-1054.
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  Continuous-time Markov models are commonly used to analyze longitudinal transitions between multiple disease states in panel data, where participants' disease states are only observed at multiple time points, and the exact state paths between observations are unknown. However, when covariate effects are incorporated and allowed to vary for different transitions, the number of potential parameters to estimate can become large even when the number of covariates is moderate, and traditional maximum likelihood estimation and subset model selection procedures can easily become unstable due to overfitting. We propose a novel regularized continuous-time Markov model with the elastic net penalty, which is capable of simultaneous variable selection and estimation for large number of parameters. We derive an efficient coordinate descent algorithm to solve the penalized optimization problem, which is fully automatic and data driven. We further consider an extension where one of the states is death, and time of death is exactly known but the state path leading to death is unknown. The proposed method is extensively evaluated in a simulation study, and demonstrated in an application to real-world data on airflow limitation state transitions.
• Li, H., Fan, J., Vitali, F., Berghout, J., Aberasturi, D., Li, J., Wilson, L., Chiu, W., Pumarejo, M., Han, J., Kenost, C., Koripella, P. C., Pouladi, N., Billheimer, D., Bedrick, E. J., & Lussier, Y. A. (2018). Novel disease syndromes unveiled by integrative multiscale network analysis of diseases sharing molecular effectors and comorbidities. BMC medical genomics, 11(Suppl 6), 112.
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  Forty-two percent of patients experience disease comorbidity, contributing substantially to mortality rates and increased healthcare costs. Yet, the possibility of underlying shared mechanisms for diseases remains not well established, and few studies have confirmed their molecular predictions with clinical datasets.
• Skiba, M. B., Luis, P. B., Alfafara, C., Billheimer, D., Schneider, C., & Funk, J. L. (2018). Curcuminoid Content and Safety-Related Markers of Quality of Turmeric Dietary Supplements Sold in an Urban Retail Marketplace in the United States. Molecular nutrition & food research, e1800143.
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  Turmeric is a top selling dietary supplement (DS) in the United States with rapidly expanding usage. Therefore, turmeric DS formulations available for sale in an urban US retail marketplace are analyzed, and point of sale information is related to measures of quality relevant to safety.
• Han, J., Dzierlenga, A. L., Lu, Z., Billheimer, D. D., Torabzadeh, E., Lake, A. D., Li, H., Novak, P., Shipkova, P., Aranibar, N., Robertson, D., Reily, M. D., Lehman-McKeeman, L. D., & Cherrington, N. J. (2017). Metabolomic profiling distinction of human nonalcoholic fatty liver disease progression from a common rat model. Obesity (Silver Spring, Md.), 25(6), 1069-1076.
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  Characteristic pathological changes define the progression of steatosis to nonalcoholic steatohepatitis (NASH) and are correlated to metabolic pathways. A common rodent model of NASH is the methionine and choline deficient (MCD) diet. The objective of this study was to perform full metabolomic analyses on liver samples to determine which pathways are altered most pronouncedly in this condition in humans, and to compare these changes to rodent models of nonalcoholic fatty liver disease (NAFLD).
• Li, H., Canet, M. J., Clarke, J. D., Billheimer, D., Xanthakos, S. A., Lavine, J. E., Erickson, R. P., & Cherrington, N. J. (2017). Pediatric Cytochrome P450 Activity Alterations in Nonalcoholic Steatohepatitis. Drug metabolism and disposition: the biological fate of chemicals, 45(12), 1317-1325.
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  Variable drug responses depend on individual variation in the activity of drug-metabolizing enzymes, including cytochrome P450 enzymes (CYP). As the most common chronic liver disease in children and adults, nonalcoholic steatohepatitis (NASH) has been identified as a source of significant interindividual variation in hepatic drug metabolism. Compared with adults, children present age-related differences in pharmacokinetics and pharmacodynamics. The purpose of this study was to determine the impact of fatty liver disease severity on the activity of a variety of CYP enzymes in children and adolescents. Healthy and nonalcoholic fatty liver disease pediatric subjects aged 12-21 years inclusive received an oral cocktail of four probe drugs: caffeine (CYP1A2, 100 mg), omeprazole (CYP2C19, 20 mg), losartan (CYP2C9, 25 mg), and midazolam (CYP3A4, 2 mg). Venous blood and urine were collected before administration and 1, 2, 4, and 6 hours after administration. Concentrations of the parent drugs and CYP-specific metabolites were quantified in plasma and urine using liquid chromatography with tandem mass spectrometry. In plasma, the decreased metabolic area under the curve (AUC) ratio, defined as the metabolite AUC to parent AUC, of omeprazole indicated significant decreases of CYP2C19 (P = 0.002) enzymatic activities in NASH adolescents, while the urine analyses did not show significant differences and were highly variable. A comparison between the present in vivo pediatric studies and a previous ex vivo study in adults indicates distinct differences in the activities of CYP1A2 and CYP2C9. These data demonstrate that pediatric NASH presents an altered pattern of CYP activity and NASH should be considered as a confounder of drug metabolism for certain CYP enzymes. These differences could lead to future investigations that may reveal unexpected variable drug responses that should be considered in pediatric dosage recommendations.
• Lothrop, N., Hussaini, K., Billheimer, D., & Beamer, P. (2017). Community-level characteristics and environmental factors of child respiratory illnesses in Southern Arizona. BMC public health, 17(1), 516.
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  Lower respiratory illnesses (LRIs) and asthma are common diseases in children
• Beamer, P. I., Klimecki, W. T., Loh, M., Van Horne, Y. O., Sugeng, A. J., Lothrop, N., Billheimer, D., Guerra, S., Lantz, R. C., Canales, R. A., & Martinez, F. D. (2016). Association of Children's Urinary CC16 Levels with Arsenic Concentrations in Multiple Environmental Media. International journal of environmental research and public health, 13(5).
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  Arsenic exposure has been associated with decreased club cell secretory protein (CC16) levels in adults. Further, both arsenic exposure and decreased levels of CC16 in childhood have been associated with decreased adult lung function. Our objective was to determine if urinary CC16 levels in children are associated with arsenic concentrations in environmental media collected from their homes. Yard soil, house dust, and tap water were taken from 34 homes. Urine and toenail samples were collected from 68 children. All concentrations were natural log-transformed prior to data analysis. There were associations between urinary CC16 and arsenic concentration in soil (b = -0.43, p = 0.001, R² = 0.08), water (b = -0.22, p = 0.07, R² = 0.03), house dust (b = -0.37, p = 0.07, R² = 0.04), and dust loading (b = -0.21, p = 0.04, R² = 0.04). In multiple analyses, only the concentration of arsenic in soil was associated with urinary CC16 levels (b = -0.42, p = 0.02, R² = 0.14 (full model)) after accounting for other factors. The association between urinary CC16 and soil arsenic may suggest that localized arsenic exposure in the lungs could damage the airway epithelium and predispose children for diminished lung function. Future work to assess this possible mechanism should examine potential associations between airborne arsenic exposures, CC16 levels, lung function, and other possible confounders in children in arsenic-impacted communities.
• Beamer, P. I., Klimecki, W. T., Loh, M., Van Horne, Y. O., Sugeng, A. J., Lothrop, N., Billheimer, D., Guerra, S., Lantz, R. C., Canales, R. A., & Martinez, F. D. (2016). Response to García-Nieto et al. Comments on Beamer et al. Association of Children's Urinary CC16 Levels with Arsenic Concentrations in Multiple Environmental Media. Int. J. Environ. Res. Public Health 2016, 13, 521. International journal of environmental research and public health, 13(10).
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  We would like to thank the editors for providing us with the opportunity to respond to the points raised by Dr. García Nieto.[...].
• Beamer, P. I., Lothrop, N., Lu, Z., Ascher, R., Ernst, K., Stern, D. A., Billheimer, D., Wright, A. L., & Martinez, F. D. (2016). Spatial clusters of child lower respiratory illnesses associated with community-level risk factors. Pediatric pulmonology, 51(6), 633-42.
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  Identifying geographic areas with increased incidence of disease may elucidate community-level risk factors for intervention development. Lower respiratory illnesses (LRIs) are the leading cause of death in children and are associated with other morbidities. We assessed geographic clustering of LRIs and evaluated if these spatial patterns and associated risk factors differed by phenotype. Participants enrolled at birth in the Tucson Children's Respiratory Study were followed through age three for physician diagnosed LRIs. Spatial clustering analysis, based upon each participant's birth address, was performed for four LRI phenotypes. We conducted principal component analysis at the census tract level to generate indices for lower socioeconomic status (SES), poorer housing conditions, and increased air pollution. Enrollment addresses were mapped for 812 subjects, of whom 58.4%, 33.5%, 34.2%, and 23.4% had any LRI, a wheezing LRI, a viral LRI, and a respiratory syncytial virus (RSV) LRI, respectively. Patterns of spatial clustering and associated risk factors differed by LRI phenotype. Multivariable regression analyses showed that wheezing LRI clusters were associated with increased air pollution (OR = 1.18, P = 0.01). Being in a viral cluster was associated with poorer housing conditions (OR = 1.28, P = 0.01), while being in a RSV cluster was associated with increased air pollution (OR = 1.14, P = 0.006), poorer housing conditions (OR = 1.54, P = 0.003), and higher SES (OR = 0.77, P = 0.001). Our use of social and environmental indices allowed us to identify broad contextual factors that may contribute to increased incidence of LRIs in specific geographic regions. To reduce LRI incidence, multifaceted interventions should be developed at the community level. Pediatr Pulmonol. 2016;51:633-642. © 2015 Wiley Periodicals, Inc.
• Beamer, P., Loh, M. M., Klimecki, W., Ornelas Van Horne, Y., Sugeng, A. J., Lothrop, N. Z., Billheimer, D. D., Guerra, S., Lantz, R. C., Canales, R. A., & Martinez, F. (2016). Association of children's urinary CC16 levels with arsenic concentrations in multiple environmental media. International Journal of Environmental Research and Public Health.
• Berry, C. E., Billheimer, D., Jenkins, I. C., Lu, Z. J., Stern, D. A., Gerald, L. B., Carr, T. F., Guerra, S., Morgan, W. J., Wright, A. L., & Martinez, F. D. (2016). A Distinct Low Lung Function Trajectory from Childhood to the Fourth Decade of Life. American journal of respiratory and critical care medicine, 194(5), 607-12.
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  Low maximally attained lung function increases the risk of chronic obstructive pulmonary disease irrespective of the subsequent rate of lung function decline.
• Koska, J., Yassine, H., Trenchevska, O., Sinari, S., Schwenke, D. C., Yen, F. T., Billheimer, D., Nelson, R. W., Nedelkov, D., & Reaven, P. D. (2016). Disialylated apolipoprotein C-III proteoform is associated with improved lipids in prediabetes and type 2 diabetes. Journal of lipid research, 57(5), 894-905.
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  The apoC-III proteoform containing two sialic acid residues (apoC-III2) has different in vitro effects on lipid metabolism compared with asialylated (apoC-III0) or the most abundant monosialylated (apoC-III1) proteoforms. Cross-sectional and longitudinal associations between plasma apoC-III proteoforms (by mass spectrometric immunoassay) and plasma lipids were tested in two randomized clinical trials: ACT NOW, a study of pioglitazone in subjects with impaired glucose tolerance (n = 531), and RACED (n = 296), a study of intensive glycemic control and atherosclerosis in type 2 diabetes patients. At baseline, higher relative apoC-III2 and apoC-III2/apoC-III1 ratios were associated with lower triglycerides and total cholesterol in both cohorts, and with lower small dense LDL in the RACED. Longitudinally, changes in apoC-III2/apoC-III1 were inversely associated with changes in triglycerides in both cohorts, and with total and small dense LDL in the RACED. apoC-III2/apoC-III1 was also higher in patients treated with PPAR-γ agonists and was associated with reduced cardiovascular events in the RACED control group. Ex vivo studies of apoC-III complexes with higher apoC-III2/apoC-III1 showed attenuated inhibition of VLDL uptake by HepG2 cells and LPL-mediated lipolysis, providing possible functional explanations for the inverse association between a higher apoC-III2/apoC-III1 and hypertriglyceridemia, proatherogenic plasma lipid profiles, and cardiovascular risk.
• Pulko, V., Davies, J. S., Martinez, C., Lanteri, M. C., Busch, M. P., Diamond, M. S., Knox, K., Bush, E. C., Sims, P. A., Sinari, S., Billheimer, D., Haddad, E. K., Murray, K. O., Wertheimer, A. M., & Nikolich-Žugich, J. (2016). Human memory T cells with a naive phenotype accumulate with aging and respond to persistent viruses. Nature immunology, 17(8), 966-75.
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  The number of naive T cells decreases and susceptibility to new microbial infections increases with age. Here we describe a previously unknown subset of phenotypically naive human CD8(+) T cells that rapidly secreted multiple cytokines in response to persistent viral antigens but differed transcriptionally from memory and effector T cells. The frequency of these CD8(+) T cells, called 'memory T cells with a naive phenotype' (TMNP cells), increased with age and after severe acute infection and inversely correlated with the residual capacity of the immune system to respond to new infections with age. CD8(+) TMNP cells represent a potential new target for the immunotherapy of persistent infections and should be accounted for and subtracted from the naive pool if truly naive T cells are needed to respond to antigens.
• Schissler, A. G., Li, Q., Chen, J. L., Kenost, C., Achour, I., Billheimer, D. D., Li, H., Piegorsch, W. W., & Lussier, Y. A. (2016). Analysis of aggregated cell-cell statistical distances within pathways unveils therapeutic-resistance mechanisms in circulating tumor cells. Bioinformatics (Oxford, England), 32(12), i80-i89.
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  As 'omics' biotechnologies accelerate the capability to contrast a myriad of molecular measurements from a single cell, they also exacerbate current analytical limitations for detecting meaningful single-cell dysregulations. Moreover, mRNA expression alone lacks functional interpretation, limiting opportunities for translation of single-cell transcriptomic insights to precision medicine. Lastly, most single-cell RNA-sequencing analytic approaches are not designed to investigate small populations of cells such as circulating tumor cells shed from solid tumors and isolated from patient blood samples.
• Xu, H., Radabaugh, T., Lu, Z., Galligan, M., Billheimer, D., Vercelli, D., Wright, A. L., Monks, T. J., Halonen, M., & Lau, S. S. (2016). Exploration of early-life candidate biomarkers for childhood asthma using antibody arrays. Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology, 27(7), 696-701.
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  Proteomic approaches identifying biomarkers have been applied to asthma to only a very limited extent.
• Beamer, P., Lothrop, N. Z., Lu, Z., Ascher, R., Ernst, K. C., Stern, D. A., Billheimer, D. D., Wright, A. L., & Martinez, F. D. (2015). Spatial Clusters of Child Lower Respiratory Illnesses associated with Community-Level Risk Factors. Pediatric Pulmonology, DOI: 10.1002/ppul.23332.
• Beamer, P., Lothrop, N. Z., Lu, Z., Ascher, R., Ernst, K. C., Stern, D. A., Billheimer, D. D., Wright, A. L., & Martinez, F. D. (2015). Spatial Clusters of Child Lower Respiratory Illnesses associated with Community-Level Risk Factors. Pediatric Pulmonology. doi:10.1002/ppul.23332
• Beamer, P., Lothrop, N. Z., Stern, D., Billheimer, D. D., Wright, A., & Martinez, F. (2014). Increased wheezing risk associated with diesel exposure among children of younger mothers. European Respiratory Journal, 46(3), 853-855. doi:10.1183/09031936.00227214
• Beamer, P., Lothrop, N. Z., Stern, D., Billheimer, D. D., Wright, A., & Martinez, F. (2015). Increased wheezing risk associated with diesel exposure among children of younger mothers. European Respiratory Journal, 46(3), 853-855. doi:10.1183/09031936.00227214
• Yassine, H. N., Trenchevska, O., Ramrakhiani, A., Parekh, A., Koska, J., Walker, R. W., Billheimer, D., Reaven, P. D., Yen, F. T., Nelson, R. W., Goran, M. I., & Nedelkov, D. (2015). The Association of Human Apolipoprotein C-III Sialylation Proteoforms with Plasma Triglycerides. PloS one, 10(12), e0144138.
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  Apolipoprotein C-III (apoC-III) regulates triglyceride (TG) metabolism. In plasma, apoC-III exists in non-sialylated (apoC-III0a without glycosylation and apoC-III0b with glycosylation), monosialylated (apoC-III1) or disialylated (apoC-III2) proteoforms. Our aim was to clarify the relationship between apoC-III sialylation proteoforms with fasting plasma TG concentrations.
• Billheimer, D., Gerner, E. W., McLaren, C. E., & LaFleur, B. (2014). Combined benefit of prediction and treatment: a criterion for evaluating clinical prediction models. Cancer informatics, 13(Suppl 2), 93-103.
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  Clinical treatment decisions rely on prognostic evaluation of a patient's future health outcomes. Thus, predictive models under different treatment options are key factors for making good decisions. While many criteria exist for judging the statistical quality of a prediction model, few are available to measure its clinical utility. As a consequence, we may find that the addition of a clinical covariate or biomarker improves the statistical quality of the model, but has little effect on its clinical usefulness. We focus on the setting where a treatment decision may reduce a patient's risk of a poor outcome, but also comes at a cost; this may be monetary, inconvenience, or the potential side effects. This setting is exemplified by cancer chemoprevention, or the use of statins to reduce the risk of cardiovascular disease. We propose a novel approach to assessing a prediction model using a formal decision analytic framework. We combine the predictive model's ability to discriminate good from poor outcome with the net benefit afforded by treatment. In this framework, reduced risk is balanced against the cost of treatment. The relative cost-benefit of treatment provides a useful index to assist patient decisions. This index also identifies the relevant clinical risk regions where predictive improvement is needed. Our approach is illustrated using data from a colorectal adenoma chemoprevention trial.
• Spivey, C. A., Chisholm-Burns, M. A., Damadzadeh, B., & Billheimer, D. (2014). Determining the effect of immunosuppressant adherence on graft failure risk among renal transplant recipients. Clinical Transplantation, 28(1), 96-104.
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  Abstract: The objective was to use the United States Renal Data System (USRDS) to quantify the relationship between immunosuppressant therapy (IST) adherence and risk of graft failure among adult renal transplant recipients (RTRs). A secondary objective was to examine the relationship among select patient characteristics and IST adherence. The study sample included adult RTRs who: received primary transplant between January 1, 1999 and December 31, 2005; experienced graft survival for at least 12 months post-transplant and had at least 12 months of data in the USRDS; utilized Medicare coverage for IST; and were prescribed cyclosporine or tacrolimus. IST adherence was measured by medication possession ratio (MPR). Pearson chi-square tests were used to examine associations between patient characteristics and MPR quartiles. Cox proportional hazards regression was used to assess relationships among time to graft failure, MPR, and patient characteristics. Thirty-one thousand nine hundred and thirteen RTRs met inclusion criteria. Older age, female gender, white race, deceased donors, and tacrolimus were associated with greater adherence (p < 0.001). Cox proportional hazard modeling indicated greater adherence, white race, and having a living donor were significantly associated with longer graft survival (p < 0.05). Future prospective studies should further examine the clinical significance of IST nonadherence as it relates to graft failure. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
• Yassine, H. N., Jackson, A. M., Borges, C. R., Billheimer, D., Koh, H., Smith, D., Reaven, P., Lau, S. S., & Borchers, C. H. (2014). The application of multiple reaction monitoring and multi-analyte profiling to HDL proteins. Lipids in Health and Disease, 13(1).
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  Abstract: Background: HDL carries a rich protein cargo and examining HDL protein composition promises to improve our understanding of its functions. Conventional mass spectrometry methods can be lengthy and difficult to extend to large populations. In addition, without prior enrichment of the sample, the ability of these methods to detect low abundance proteins is limited. Our objective was to develop a high-throughput approach to examine HDL protein composition applicable to diabetes and cardiovascular disease (CVD). Methods. We optimized two multiplexed assays to examine HDL proteins using a quantitative immunoassay (Multi-Analyte Profiling- MAP) and mass spectrometric-based quantitative proteomics (Multiple Reaction Monitoring-MRM). We screened HDL proteins using human xMAP (90 protein panel) and MRM (56 protein panel). We extended the application of these two methods to HDL isolated from a group of participants with diabetes and prior cardiovascular events and a group of non-diabetic controls. Results: We were able to quantitate 69 HDL proteins using MAP and 32 proteins using MRM. For several common proteins, the use of MRM and MAP was highly correlated (p < 0.01). Using MAP, several low abundance proteins implicated in atherosclerosis and inflammation were found on HDL. On the other hand, MRM allowed the examination of several HDL proteins not available by MAP. Conclusions: MAP and MRM offer a sensitive and high-throughput approach to examine changes in HDL proteins in diabetes and CVD. This approach can be used to measure the presented HDL proteins in large clinical studies. © 2014 Yassine et al.; licensee BioMed Central Ltd.
• Hanusch, K., Janssen, C. H., Billheimer, D., Jenkins, I., Spurgeon, E., Lowry, C. A., & Raison, C. L. (2013). Whole-body hyperthermia for the treatment of major depression: Associations with thermoregulatory cooling. American Journal of Psychiatry, 170(7), 802-804.
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  PMID: 23820835;
• Hanusch, K., Janssen, C. H., Billheimer, D., Jenkins, I., Spurgeon, E., Lowry, C. A., & Raison, C. L. (2013). Whole-body hyperthermia for the treatment of major depression: associations with thermoregulatory cooling.. American Journal of Psychiatry, 170(7), 802-4.
• Jacquemin, J., Ammiraju, J. S., Haberer, G., Billheimer, D. D., Yu, Y., Liu, L. C., Rivera, L. F., Mayer, K., Chen, M., & Wing, R. A. (2013). Fifteen Million Years of Evolution in the Oryza Genus Shows Extensive Gene Family Expansion. Molecular plant.
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  In analyzing gene families in the whole-genome sequences available for O. sativa (AA), O. glaberrima (AA), and O. brachyantha (FF), we observed large size expansions in the AA genomes compared to FF genomes for the super-families F-box and NB-ARC, and five additional families: the Aspartic proteases, BTB/POZ proteins (BTB), Glutaredoxins, Trypsin α-amylase inhibitor proteins, and Zf-Dof proteins. Their evolutionary dynamic was investigated to understand how and why such important size variations are observed between these closely related species. We show that expansions resulted from both amplification, largely by tandem duplications, and contraction by gene losses. For the F-box and NB-ARC gene families, the genes conserved in all species were under strong purifying selection while expanded orthologous genes were under more relaxed purifying selection. In F-box, NB-ARC, and BTB, the expanded groups were enriched in genes with little evidence of expression, in comparison with conserved groups. We also detected 87 loci under positive selection in the expanded groups. These results show that most of the duplicated copies in the expanded groups evolve neutrally after duplication because of functional redundancy but a fraction of these genes were preserved following neofunctionalization. Hence, the lineage-specific expansions observed between Oryza species were partly driven by directional selection.
• Marri, P. R., Stern, D. A., Wright, A. L., Billheimer, D., & Martinez, F. D. (2013). Asthma-associated differences in microbial composition of induced sputum. Journal of Allergy and Clinical Immunology, 131(2), 346-352.e3.
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  PMID: 23265859;Abstract: Background: It is increasingly evident that microbial colonization of the respiratory tract might have a role in the pathogenesis of asthma. Objective: We sought to characterize and compare the microbiome of induced sputum in asthmatic and nonasthmatic adults. Methods: Induced sputum samples were obtained from 10 nonasthmatic subjects and 10 patients with mild active asthma (8/10 were not using inhaled corticosteroids). Total DNA was extracted from sputum supernatants and amplified by using primers specific for the V6 hypervariable region of bacterial 16s rRNA. Samples were barcoded, and equimolar concentrations of 20 samples were pooled and sequenced with the 454 GS FLX sequencer. Sequences were assigned to bacterial taxa by comparing them with 16s rRNA sequences in the Ribosomal Database Project. Results: All sputum samples contained 5 major bacterial phyla: Firmicutes, Proteobacteria, Actinobacteria, Fusobacterium, and Bacteroidetes, with the first 3 phyla accounting for more than 90% of the total sequences. Proteobacteria were present in higher proportions in asthmatic patients (37% vs 15%, P < .001). In contrast, Firmicutes (47% vs 63%, P = .17) and Actinobacteria (10% vs 14%, P = .36) were found more frequently in samples from nonasthmatic subjects, although this was not statistically significant. Hierarchical clustering produced 2 significant clusters: one contained primarily asthmatic samples and the second contained primarily nonasthmatic samples. In addition, samples from asthmatic patients had greater bacterial diversity compared with samples from nonasthmatic subjects. Conclusion: Patients with mild asthma have an altered microbial composition in the respiratory tract that is similar to that observed in patients with more severe asthma. © 2012 American Academy of Allergy, Asthma & Immunology..
• Marri, P. R., Stern, D. A., Wright, A. L., Billheimer, D., & Martinez, F. D. (2013). Asthma-associated differences in microbial composition of induced sputum. The Journal of Allergy and Clinical Immunology, 131(2).
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  It is increasingly evident that microbial colonization of the respiratory tract might have a role in the pathogenesis of asthma. We observe differences in microbial community composition between adults diagnosed with asthma and normal healthy adults.
• Spivey, C. A., Chisholm-Burns, M. A., Damadzadeh, B., & Billheimer, D. (2014). Determining the effect of immunosuppressant adherence on graft failure risk among renal transplant recipients. Clinical transplantation, 28(1).
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  The objective was to use the United States Renal Data System (USRDS) to quantify the relationship between immunosuppressant therapy (IST) adherence and risk of graft failure among adult renal transplant recipients (RTRs). A secondary objective was to examine the relationship among select patient characteristics and IST adherence. The study sample included adult RTRs who: received primary transplant between January 1, 1999 and December 31, 2005; experienced graft survival for at least 12&amp;amp;#160;months post-transplant and had at least 12&amp;amp;#160;months of data in the USRDS; utilized Medicare coverage for IST; and were prescribed cyclosporine or tacrolimus. IST adherence was measured by medication possession ratio (MPR). Pearson chi-square tests were used to examine associations between patient characteristics and MPR quartiles. Cox proportional hazards regression was used to assess relationships among time to graft failure, MPR, and patient characteristics. Thirty-one thousand nine hundred and thirteen RTRs met inclusion criteria. Older age, female gender, white race, deceased donors, and tacrolimus were associated with greater adherence (p&amp;amp;#160;&amp;lt;&amp;amp;#160;0.001). Cox proportional hazard modeling indicated greater adherence, white race, and having a living donor were significantly associated with longer graft survival (p&amp;amp;#160;&amp;lt;&amp;amp;#160;0.05). Future prospective studies should further examine the clinical significance of IST nonadherence as it relates to graft failure.
• Yassine, H. N., Jackson, A. M., Borges, C. R., Billheimer, D., Koh, H., Smith, D., Reaven, P., Lau, S. S., & Borchers, C. H. (2013). The application of multiple reaction monitoring and multi-analyte profiling to HDL proteins. Lipids in health and disease, 13.
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  HDL carries a rich protein cargo and examining HDL protein composition promises to improve our understanding of its functions. Conventional mass spectrometry methods can be lengthy and difficult to extend to large populations. In addition, without prior enrichment of the sample, the ability of these methods to detect low abundance proteins is limited. Our objective was to develop a high-throughput approach to examine HDL protein composition applicable to diabetes and cardiovascular disease (CVD).
• Yassine, H., Borges, C. R., Schaab, M. R., Billheimer, D., Stump, C., Reaven, P., Lau, S. S., & Nelson, R. (2013). Mass spectrometric immunoassay and MRM as targeted MS-based quantitative approaches in biomarker development: Potential applications to cardiovascular disease and diabetes. Proteomics - Clinical Applications, 7(7-8), 528-540.
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  PMID: 23696124;Abstract: Type 2 diabetes mellitus (T2DM) is an important risk factor for cardiovascular disease (CVD)-the leading cause of death in the United States. Yet not all subjects with T2DM are at equal risk for CVD complications; the challenge lies in identifying those at greatest risk. Therapies directed toward treating conventional risk factors have failed to significantly reduce this residual risk in T2DM patients. Thus newer targets and markers are needed for the development and testing of novel therapies. Herein we review two complementary MS-based approaches-mass spectrometric immunoassay (MSIA) and MS/MS as MRM-for the analysis of plasma proteins and PTMs of relevance to T2DM and CVD. Together, these complementary approaches allow for high-throughput monitoring of many PTMs and the absolute quantification of proteins near the low picomolar range. In this review article, we discuss the clinical relevance of the high density lipoprotein (HDL) proteome and Apolipoprotein A-I PTMs to T2DM and CVD as well as provide illustrative MSIA and MRM data on HDL proteins from T2DM patients to provide examples of how these MS approaches can be applied to gain new insight regarding cardiovascular risk factors. Also discussed are the reproducibility, interpretation, and limitations of each technique with an emphasis on their capacities to facilitate the translation of new biomarkers into clinical practice. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Yassine, H., Borges, C. R., Schaab, M. R., Billheimer, D., Stump, C., Reaven, P., Lau, S. S., & Nelson, R. (2013). Mass spectrometric immunoassay and MRM as targeted MS-based quantitative approaches in biomarker development: potential applications to cardiovascular disease and diabetes. Proteomics. Clinical applications, 7(7-8).
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  Type 2 diabetes mellitus (T2DM) is an important risk factor for cardiovascular disease (CVD)--the leading cause of death in the United States. Yet not all subjects with T2DM are at equal risk for CVD complications; the challenge lies in identifying those at greatest risk. Therapies directed toward treating conventional risk factors have failed to significantly reduce this residual risk in T2DM patients. Thus newer targets and markers are needed for the development and testing of novel therapies. Herein we review two complementary MS-based approaches--mass spectrometric immunoassay (MSIA) and MS/MS as MRM--for the analysis of plasma proteins and PTMs of relevance to T2DM and CVD. Together, these complementary approaches allow for high-throughput monitoring of many PTMs and the absolute quantification of proteins near the low picomolar range. In this review article, we discuss the clinical relevance of the high density lipoprotein (HDL) proteome and Apolipoprotein A-I PTMs to T2DM and CVD as well as provide illustrative MSIA and MRM data on HDL proteins from T2DM patients to provide examples of how these MS approaches can be applied to gain new insight regarding cardiovascular risk factors. Also discussed are the reproducibility, interpretation, and limitations of each technique with an emphasis on their capacities to facilitate the translation of new biomarkers into clinical practice.
• Chisholm-Burns, M. A., Spivey, C. A., Billheimer, D., Schlesselman, L. S., Flowers, S. K., Hammer, D., Engle, J. P., Nappi, J. M., Pasko, M. T., Ross, L. A., Sorofman, B., Rodrigues, H. A., & Vaillancourt, A. M. (2012). Multi-institutional study of women and underrepresented minority faculty members in academic pharmacy. American Journal of Pharmaceutical Education, 76(1).
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  Abstract: Objectives. To examine trends in the numbers of women and underrepresented minority (URM) pharmacy faculty members over the last 20 years, and determine factors influencing women faculty members' pursuit and retention of an academic pharmacy career. Methods. Twenty-year trends in women and URM pharmacy faculty representation were examined. Women faculty members from 9 public colleges and schools of pharmacy were surveyed regarding demographics, job satisfaction, and their academic pharmacy career, and relationships between demographics and satisfaction were analyzed. Results. The number of women faculty members more than doubled between 1989 and 2009 (from 20.7% to 45.5%), while the number of URM pharmacy faculty members increased only slightly over the same time period. One hundred fifteen women faculty members completed the survey instrument and indicated they were generally satisfied with their jobs. The academic rank of professor, being a nonpharmacy practice faculty member, being tenured/tenure track, and having children were associated with significantly lower satisfaction with fringe benefits. Women faculty members who were tempted to leave academia for other pharmacy sectors had significantly lower salary satisfaction and overall job satisfaction, and were more likely to indicate their expectations of academia did not match their experiences (p
• Chisholm-Burns, M. A., Spivey, C. A., Billheimer, D., Schlesselman, L. S., Flowers, S. K., Hammer, D., Engle, J. P., Nappi, J. M., Pasko, M. T., Ross, L. A., Sorofman, B., Rodrigues, H. A., & Vaillancourt, A. M. (2012). Multi-institutional study of women and underrepresented minority faculty members in academic pharmacy.. American journal of pharmaceutical education, 76(1), 7-.
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  PMID: 22412206;PMCID: PMC3298405;Abstract: To examine trends in the numbers of women and underrepresented minority (URM) pharmacy faculty members over the last 20 years, and determine factors influencing women faculty members' pursuit and retention of an academic pharmacy career. Twenty-year trends in women and URM pharmacy faculty representation were examined. Women faculty members from 9 public colleges and schools of pharmacy were surveyed regarding demographics, job satisfaction, and their academic pharmacy career, and relationships between demographics and satisfaction were analyzed. The number of women faculty members more than doubled between 1989 and 2009 (from 20.7% to 45.5%), while the number of URM pharmacy faculty members increased only slightly over the same time period. One hundred fifteen women faculty members completed the survey instrument and indicated they were generally satisfied with their jobs. The academic rank of professor, being a nonpharmacy practice faculty member, being tenured/tenure track, and having children were associated with significantly lower satisfaction with fringe benefits. Women faculty members who were tempted to leave academia for other pharmacy sectors had significantly lower salary satisfaction and overall job satisfaction, and were more likely to indicate their expectations of academia did not match their experiences (p
• Chisholm-Burns, M., Spivey, C., Billheimer, D., Schlesselman, L., Hammer, D., Engle, J., Nappi, J., Pasko, M., Ann, R. L., Sorofman, B., Rodrigues, H., & Vaillancourt, A. (2012). Multi-Institutional Study of Women and Underrepresented Minority Faculty Members in Academic Pharmacy. Am J Pharm Educ, 76(1), 7.
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  PMID: 22412206
• Gomez-Rubio, P., Gomez-Rubio, P., Klimentidis, Y., Klimentidis, Y., Cantu-Soto, E., Cantu-Soto, E., Meza-Montenegro, M., Meza-Montenegro, M., Billheimer, D., Billheimer, D., Lu, Z., Lu, Z., Chen, Z., Chen, Z., Klimecki, W., & Klimecki, W. (2012). Indigenous American Ancestry is Associated with Arsenic Methylation Efficiency in an Admixed Population of Northwest Mexico. J Toxicol Environ Health A, 75(1), 36-49.
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  PMID 22047162
• Gomez-Rubio, P., Klimentidis, Y. C., Cantu-Soto, E., Meza-Montenegro, M. M., Billheimer, D., Zhenqiang, L. u., Chen, Z., & Klimecki, W. T. (2012). Indigenous American Ancestry is Associated with arsenic methylation efficiency in an admixed population of northwest Mexico. Journal of Toxicology and Environmental Health - Part A: Current Issues, 75(1), 36-49.
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  PMID: 22047162;PMCID: PMC3572940;Abstract: Many studies provide evidence relating lower human arsenic (As) methylation efficiency, represented by high percent urinary monomethylarsonic acid (MMA(V)), with several As-induced diseases, possibly due to the fact that MMA(V) serves as a proxy for MMA(III), the most toxic As metabolite. Some epidemiological studies suggested that indigenous Americans (AME) methylate As more efficiently; however, data supporting this have been equivocal. The aim of this study was to characterize the association between AME ancestry and As methylation efficiency using a panel of ancestry informative genetic markers to determine individual ancestry proportions in an admixed population (composed of two or more isolated ancestral populations) of 746 individuals environmentally exposed to As in northwest Mexico. Total urinary As (TAs) mean and range were 170.4 and 2.3-1053.5 μg/L, while percent AME (%AME) mean and range were 72.4 and 23-100. Adjusted (gender, age, AS3MT 7388/M287T haplotypes, body mass index [BMI], and TAs) multiple regression model showed that higher AME ancestry is significantly associated with lower percentage of urinary As excreted as MMA(V) (%uMMA) in this population (p < .01). Data also demonstrated a significant interaction between BMI and gender, indicating negative association between BMI and %uMMA, stronger in women than men (p < .01). Moreover, age and the AS3MT variants 7388 (intronic) and M287T (nonsynonymous) were also significantly associated with As methylation efficiency (p < .01). This study highlights the importance of BMI and indigenous American ancestry in some of the observed variability in As methylation efficiency, underscoring the need to be considered in epidemiology studies, particularly those carried out in admixed populations. Copyright © Taylor & Francis Group, LLC.
• Marri, P., Stern, D., Wright, A., Billheimer, D., & Martinez, F. (2012). Asthma-associated Differences in Microbial Composition of Induced Sputum. J Allergy Clin Immunol.
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  [Epub ahead of print] PMID: 23265859
• Gomez-Rubio, P., Roberge, J., Arendell, L., Harris, R. B., O'Rourke, M. K., Chen, Z., Cantu-Soto, E., Meza-Montenegro, M. M., Billheimer, D., Zhenqiang, L. u., & Klimecki, W. T. (2011). Association between body mass index and arsenic methylation efficiency in adult women from southwest U.S. and northwest Mexico. Toxicology and Applied Pharmacology, 252(2), 176-182.
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  PMID: 21320519;PMCID: PMC3075343;Abstract: Human arsenic methylation efficiency has been consistently associated with arsenic-induced disease risk. Interindividual variation in arsenic methylation profiles is commonly observed in exposed populations, and great effort has been put into the study of potential determinants of this variability. Among the factors that have been evaluated, body mass index (BMI) has not been consistently associated with arsenic methylation efficiency; however, an underrepresentation of the upper BMI distribution was commonly observed in these studies. This study investigated potential factors contributing to variations in the metabolism of arsenic, with specific interest in the effect of BMI where more than half of the population was overweight or obese. We studied 624 adult women exposed to arsenic in drinking water from three independent populations. Multivariate regression models showed that higher BMI, arsenic (+. 3 oxidation state) methyltransferase (AS3MT) genetic variant 7388, and higher total urinary arsenic were significantly associated with low percentage of urinary arsenic excreted as monomethylarsonic acid (%uMMA) or high ratio between urinary dimethylarsinic acid and uMMA (uDMA/uMMA), while AS3MT genetic variant M287T was associated with high %uMMA and low uDMA/uMMA. The association between BMI and arsenic methylation efficiency was also evident in each of the three populations when studied separately. This strong association observed between high BMI and low %uMMA and high uDMA/uMMA underscores the importance of BMI as a potential arsenic-associated disease risk factor, and should be carefully considered in future studies associating human arsenic metabolism and toxicity. © 2011 Elsevier Inc.
• Gomez-Rubio, P., Roberge, J., Arendell, L., Harris, R., O'Rourke, M., Chen, Z., Cantu-Soto, E., Meza-Montenegro, M., Billheimer, D., Lu, Z., & Klimecki, W. (2011). Association between body mass index and arsenic methylation efficiency in adult women from southwest U.S. and northwest Mexico. Toxicology Applied Pharmacology, 252(2), 176-182.
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  PMID 21320519
• Jiang, J., Yang, E. S., Jiang, G., Nowsheen, S., Wang, H., Wang, T., Wang, Y., Billheimer, D., Chakravarthy, A. B., Brown, M., Haffty, B., & Xia, F. (2011). p53-dependent BRCA1 nuclear export controls cellular susceptibility to DNA damage. Cancer Research, 71(16), 5546-5557.
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  PMID: 21742769;Abstract: Subcellular localization regulates BRCA1 function, and BRCA1 is exported to the cytoplasm following DNA damage in a p53-dependent manner. Because more than 50% of solid tumors harbor p53 mutations, it is possible that genetically wild-type (wt) BRCA1 is functionally abnormal through compromised nuclear-cytoplasmic shuttling in sporadic breast cancer patients with dysfunctional p53. In this study, we have investigated the mechanisms of p53-dependent BRCA1 subcellular distribution and DNA damage-induced nuclear export, as well as the impact on the resulting cytotoxic response to therapy in human breast cancer. We first show that p53 mediates BRCA1 nuclear export via protein - protein binding, rather than by modulation of its transcription. Furthermore, it is the C-terminal (BRCT) region of BRCA1 that is critical for its interaction with p53, and p53 may promote BRCA1 nuclear export by interrupting the association of BRCA1 with BARD1. In sporadic breast cancer specimens, dysfunctional p53 strongly correlates with nuclear retention of sequence-verified wt BRCA1. This p53-dependent BRCA1 shuttling determines cellular susceptibility to DNA damage as augmentation of cytosolic BRCA1 significantly enhances cancer cell susceptibility to ionizing radiation. Taken together, our data suggest that p53 dysfunction compromises nuclear export of wt BRCA1 as a mechanism to increase cellular resistance to DNA damage in sporadic breast cancer. We propose that targeting nuclear BRCA1 to the cytoplasm may offer a unique strategy to sensitize p53-deficient sporadic breast cancers to DNA damage - based therapy. ©2011 AACR.
• Jiang, J., Yang, E., Jiang, G., Nowsheen, S., Wang, H., Wang, T., Wang, Y., Billheimer, D., Chakravarthy, A., Brown, M., Haffty, B., & Xia, F. (2011). p53-dependent BRCA1 nuclear export controls cellular susceptibility to DNA damage. Cancer Res, 71(16), 5546-5557.
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  PMID: 21742769
• Lafleur, B., Lee, W., Billheimer, D., Lockhart, C., Liu, J., & Merchant, N. (2011). Statistical methods for assays with limits of detection: Serum bile acid as a differentiator between patients with normal colons, adenomas, and colore. J Carcinog, 10, 12.
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  PMID 21712958
• Lafleur, B., Lee, W., Billhiemer, D., Lockhart, C., Liu, J., & Merchant, N. (2011). Statistical methods for assays with limits of detection: Serum bile acid as a differentiator between patients with normal colons, adenomas, and colorectal cancer. Journal of Carcinogenesis, 10.
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  PMID: 21712958;PMCID: PMC3122101;Abstract: In analytic chemistry a detection limit (DL) is the lowest measurable amount of an analyte that can be distinguished from a blank; many biomedical measurement technologies exhibit this property. From a statistical perspective, these data present inferential challenges because instead of precise measures, one only has information that the value is somewhere between 0 and the DL (below detection limit, BDL). Substitution of BDL values, with 0 or the DL can lead to biased parameter estimates and a loss of statistical power. Statistical methods that make adjustments when dealing with these types of data, often called left-censored data, are available in many commercial statistical packages. Despite this availability, the use of these methods is still not widespread in biomedical literature. We have reviewed the statistical approaches of dealing with BDL values, and used simulations to examine the performance of the commonly used substitution methods and the most widely available statistical methods. We have illustrated these methods using a study undertaken at the Vanderbilt-Ingram Cancer Center, to examine the serum bile acid levels in patients with colorectal cancer and adenoma. We have found that the modern methods for BDL values identify disease-related differences that are often missed, with statistically naive approaches.
• Lake, A. D., Novak, P., Fisher, C. D., Jackson, J. P., Hardwick, R. N., Billheimer, D. D., Klimecki, W. T., & Cherrington, N. J. (2011). Analysis of global and absorption, distribution, metabolism, and elimination gene expression in the progressive stages of human nonalcoholic fatty liver disease. Drug Metabolism and Disposition, 39(10), 1954-1960.
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  PMID: 21737566;PMCID: PMC3186211;Abstract: Nonalcoholic fatty liver disease (NAFLD) is characterized by a series of pathological changes that range from simple fatty liver to nonalcoholic steatohepatitis (NASH). The objective of this study is to describe changes in global gene expression associated with the progression of human NAFLD. This study is focused on the expression levels of genes responsible for the absorption, distribution, metabolism, and elimination (ADME) of drugs. Differential gene expression between three clinically defined pathological groups - normal, steatosis, and NASH - was analyzed. Genome-wide mRNA levels in samples of human liver tissue were assayed with Affymetrix GeneChip Human 1.0ST arrays. A total of 11,633 genes exhibited altered expression out of 33,252 genes at a 5% false discovery rate. Most gene expression changes occurred in the progression from steatosis to NASH. Principal component analysis revealed that hepatic disease status was the major determinant of differential ADME gene expression rather than age or sex of sample donors. Among the 515 drug transporters and 258 drug-metabolizing enzymes (DMEs) examined, uptake transporters but not efflux transporters or DMEs were significantly over-represented in the number of genes down-regulated. These results suggest that uptake transporter genes are coordinately targeted for down-regulation at the global level during the pathological development of NASH and that these patients may have decreased drug uptake capacity. This coordinated regulation of uptake transporter genes is indicative of a hepatoprotective mechanism acting to prevent accumulation of toxic intermediates in disease- compromised hepatocytes. Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics.
• Lake, A. D., Novak, P., Fisher, C. D., Jackson, J. P., Hardwick, R. N., Billheimer, D. D., Klimecki, W. T., & Cherrington, N. J. (2011). Analysis of global and absorption, distribution, metabolism, and elimination gene expression in the progressive stages of human nonalcoholic fatty liver disease. Drug metabolism and disposition: the biological fate of chemicals, 39(10), 1954-60.
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  Nonalcoholic fatty liver disease (NAFLD) is characterized by a series of pathological changes that range from simple fatty liver to nonalcoholic steatohepatitis (NASH). The objective of this study is to describe changes in global gene expression associated with the progression of human NAFLD. This study is focused on the expression levels of genes responsible for the absorption, distribution, metabolism, and elimination (ADME) of drugs. Differential gene expression between three clinically defined pathological groups-normal, steatosis, and NASH-was analyzed. Genome-wide mRNA levels in samples of human liver tissue were assayed with Affymetrix GeneChip Human 1.0ST arrays. A total of 11,633 genes exhibited altered expression out of 33,252 genes at a 5% false discovery rate. Most gene expression changes occurred in the progression from steatosis to NASH. Principal component analysis revealed that hepatic disease status was the major determinant of differential ADME gene expression rather than age or sex of sample donors. Among the 515 drug transporters and 258 drug-metabolizing enzymes (DMEs) examined, uptake transporters but not efflux transporters or DMEs were significantly over-represented in the number of genes down-regulated. These results suggest that uptake transporter genes are coordinately targeted for down-regulation at the global level during the pathological development of NASH and that these patients may have decreased drug uptake capacity. This coordinated regulation of uptake transporter genes is indicative of a hepatoprotective mechanism acting to prevent accumulation of toxic intermediates in disease-compromised hepatocytes.
• Lake, A., Novak, P., Fisher, C., Jackson, J., Hardwick, R., Billheimer, D., Klimecki, W., & Cherrington, N. (2011). Analysis of Global and ADME Gene Expression In the Progressive Stages of Human Non-Alcoholic Fatty Liver Disease. Drug Metab Dispos, 39(10), 1954-1960.
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  PMID: 21737566
• Lake, A., Novak, P., Fisher, C., Jackson, J., Hardwick, R., Billheimer, D., Klimecki, W., & Cherrington, N. (2011). Analysis of Global and ADME Gene Expression In the Progressive Stages of Human Non-Alcoholic Fatty Liver Disease. Drug Metab Dispos.
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  PMID: 21737566
• Ware, L., Koyama, T., Billheimer, D., Landeck, M., Johnson, E., Brady, S., Bernard, G., & Matthay, M. (2011). Advancing donor management research: design and implementation of a large, randomized, placebo-controlled trial. Ann Intensive Care, 1(1), 20.
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  PMID: 21906362
• Zhang, H., Liu, Q., Zimmerman, L. J., Ham, A. J., Slebos, R. J., Rahman, J., Kikuchi, T., Massion, P. P., Carbone, D. P., Billheimer, D., & Liebler, D. C. (2011). Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry. Molecular & cellular proteomics : MCP, 10(6), M110.006593.
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  Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633-641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20-30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications.
• Zhang, H., Liu, Q., Zimmerman, L. J., Ham, A. L., J., R., Rahman, J., Kikuchi, T., Massion, P. P., Carbone, D. P., Billheimer, D., & Liebler, D. C. (2011). Methods for peptide and protein quantitation by liquid chromatography- multiple reaction monitoring mass spectrometry. Molecular and Cellular Proteomics, 10(6).
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  PMID: 21357624;PMCID: PMC3108838;Abstract: Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633-641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20-30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
• Zhang, H., Liu, Q., Zimmerman, L., Ham, A., Slebos, R., Rahman, J., Kikuchi, T., Massion, P., Carbone, D., Billheimer, D., & Liebler, D. (2011). Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry. Mol Cell Proteomics, 10(6).
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  M110.006593 ; PMID: 21357624
• Carbone, D. P., Salmon, J. S., Billheimer, D., Chen, H., Sandler, A., Roder, H., Roder, J., Tsypin, M., Herbst, R. S., Tsao, A. S., Tran, H. T., & Dang, T. P. (2010). VeriStrat^® classifier for survival and time to progression in non-small cell lung cancer (NSCLC) patients treated with erlotinib and bevacizumab. Lung Cancer, 69(3), 337-340.
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  PMID: 20036440;PMCID: PMC2891357;Abstract: We applied an established and commercially available serum proteomic classifier for survival after treatment with erlotinib (VeriStrat®) in a blinded manner to pretreatment sera obtained from recurrent advanced NSCLC patients before treatment with the combination of erlotinib plus bevacizumab. We found that VeriStrat® could classify these patients into two groups with significantly better or worse outcomes and may enable rational selection of patients more likely to benefit from this costly and potentially toxic regimen. © 2009 Elsevier Ireland Ltd.
• Ming, L. i., Gray, W., Zhang, H., Chung, C. H., Billheimer, D., Yarbrough, W. G., Liebler, D. C., Shyr, Y., & J., R. (2010). Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling. Journal of Proteome Research, 9(8), 4295-4305.
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  PMID: 20586475;PMCID: PMC2920032;Abstract: Shotgun proteomics provides the most powerful analytical platform for global inventory of complex proteomes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and allows a global analysis of protein changes. Nevertheless, sampling of complex proteomes by current shotgun proteomics platforms is incomplete, and this contributes to variability in assessment of peptide and protein inventories by spectral counting approaches. Thus, shotgun proteomics data pose challenges in comparing proteomes from different biological states. We developed an analysis strategy using quasi-likelihood Generalized Linear Modeling (GLM), included in a graphical interface software package (QuasiTel) that reads standard output from protein assemblies created by IDPicker, an HTML-based user interface to query shotgun proteomic data sets. This approach was compared to four other statistical analysis strategies: Student t test, Wilcoxon rank test, Fisher's Exact test, and Poisson-based GLM. We analyzed the performance of these tests to identify differences in protein levels based on spectral counts in a shotgun data set in which equimolar amounts of 48 human proteins were spiked at different levels into whole yeast lysates. Both GLM approaches and the Fisher Exact test performed adequately, each with their unique limitations. We subsequently compared the proteomes of normal tonsil epithelium and HNSCC using this approach and identified 86 proteins with differential spectral counts between normal tonsil epithelium and HNSCC. We selected 18 proteins from this comparison for verification of protein levels between the individual normal and tumor tissues using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS). This analysis confirmed the magnitude and direction of the protein expression differences in all 6 proteins for which reliable data could be obtained. Our analysis demonstrates that shotgun proteomic data sets from different tissue phenotypes are sufficiently rich in quantitative information and that statistically significant differences in proteins spectral counts reflect the underlying biology of the samples. © 2010 American Chemical Society.
• Ming, L. i., Gray, W., Zhang, H., Chung, C. H., Billheimer, D., Yarbrough, W. G., Liebler, D. C., Shyr, Y., & J., R. (2010). Erratum: Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling (Journal of Proteome Research (2010) 9 (4295-4305)). Journal of Proteome Research, 9(11), 6090-.
• Paulovich, A. G., Billheimer, D., Ham, A. L., Vega-Montoto, L., Rudnick, P. A., Tabb, D. L., Wang, P., Blackman, R. K., Bunk, D. M., Cardasis, H. L., Clauser, K. R., Kinsinger, C. R., Schilling, B., Tegeler, T. J., Variyath, A. M., Wang, M., Whiteaker, J. R., Zimmerman, L. J., Fenyo, D., , Carr, S. A., et al. (2010). Interlaboratory study characterizing a yeast performance standard for benchmarking LC-MS platform performance. Molecular and Cellular Proteomics, 9(2), 242-254.
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  PMID: 19858499;PMCID: PMC2830837;Abstract: Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize preanalytical and analytical variation in comparative proteomics experiments.
• Rudnick, P. A., Clauser, K. R., Kilpatrick, L. E., Tchekhovskoi, D. V., Neta, P., Blonder, N., Billheimer, D. D., Blackman, R. K., Bunk, D. M., Cardasis, H. L., Ham, A. L., Jaffe, J. D., Kinsinger, C. R., Mesri, M., Neubert, T. A., Schilling, B., Tabb, D. L., Tegeler, T. J., Vega-Montoto, L., , Variyath, A. M., et al. (2010). Performance metrics for liquid chromatography-tandem mass spectrometry systems in proteomics analyses. Molecular & cellular proteomics : MCP, 9(2), 225-41.
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  A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.
• Rudnick, P. A., Clauser, K. R., Kilpatrick, L. E., Tchekhovskoi, D. V., Neta, P., Blonder, N., Billheimer, D. D., Blackman, R. K., Bunk, D. M., Cardasis, H. L., Ham, A. L., Jaffe, J. D., Kinsinger, C. R., Mesri, M., Neubert, T. A., Schilling, B., Tabb, D. L., Tegeler, T. J., Vega-Montoto, L., , Variyath, A. M., et al. (2010). Performance metrics for liquid chromatography-tandem mass spectrometry systems in proteomics analyses. Molecular and Cellular Proteomics, 9(2), 225-241.
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  PMID: 19837981;PMCID: PMC2830836;Abstract: A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.
• Tabb, D. L., Vega-Montoto, L., Rudnick, P. A., Variyath, A. M., Ham, A. L., Bunk, D. M., Kilpatrick, L. E., Billheimer, D. D., Blackman, R. K., Cardasis, H. L., Carr, S. A., Clauser, K. R., Jaffe, J. D., Kowalski, K. A., Neubert, T. A., Regnier, F. E., Schilling, B., Tegeler, T. J., Wang, M., , Wang, P., et al. (2010). Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry. Journal of Proteome Research, 9(2), 761-776.
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  PMID: 19921851;PMCID: PMC2818771;Abstract: The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies. © 2010 American Chemical Society.
• Tabb, D. L., Vega-Montoto, L., Rudnick, P. A., Variyath, A. M., Ham, A. L., Bunk, D. M., Kilpatrick, L. E., Billheimer, D. D., Blackman, R. K., Cardasis, H. L., Carr, S. A., Clauser, K. R., Jaffe, J. D., Kowalski, K. A., Neubert, T. A., Regnier, F. E., Schilling, B., Tegeler, T. J., Wang, M., , Wang, P., et al. (2010). Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry. Journal of proteome research, 9(2), 761-76.
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  The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.
• Ware, L. B., Koyama, T., Billheimer, D. D., William, W. u., Bernard, G. R., Thompson, B. T., Brower, R. G., Standiford, T. J., Martin, T. R., Matthay, M. A., & Bernard, G. R. (2010). Prognostic and pathogenetic value of combining clinical and biochemical indices in patients with acute lung injury. Chest, 137(2), 288-296.
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  PMID: 19858233;PMCID: PMC2816641;Abstract: Background: No single clinical or biologic marker reliably predicts clinical outcomes in acute lung injury (ALI)/ARDS. We hypothesized that a combination of biologic and clinical markers would be superior to either biomarkers or clinical factors alone in predicting ALI/ARDS mortality and would provide insight into the pathogenesis of clinical ALI/ARDS. Methods: Eight biologic markers that reflect endothelial and epithelial injury, inflammation, and coagulation (von Willebrand factor antigen, surfactant protein D [SP-D]), tumor necrosis factor receptor-1, interleukin [IL]-6, IL-8, intercellular adhesion molecule-1, protein C, plasminogen activator inhibitor-1) were measured in baseline plasma from 549 patients in the ARDSNet trial of low vs high positive end-expiratory pressure. Mortality was modeled with multivariable logistic regression. Predictors were selected using backward elimination. Comparisons between candidate models were based on the receiver operating characteristics (ROC) and tests of integrated discrimination improvement. Results: Clinical predictors (Acute Physiology And Chronic Health Evaluation III [APACHE III], organ failures, age, underlying cause, alveolar-arterial oxygen gradient, plateau pressure) predicted mortality with an area under the ROC curve (AUC) of 0.82; a combination of eight biomarkers and the clinical predictors had an AUC of 0.85. The best performing biomarkers were the neutrophil chemotactic factor, IL-8, and SP-D, a product of alveolar type 2 cells, supporting the concept that acute inflammation and alveolar epithelial injury are important pathogenetic pathways in human ALI/ARDS. Conclusions: A combination of biomarkers and clinical predictors is superior to clinical predictors or biomarkers alone for predicting mortality in ALI/ARDS and may be useful for stratifying patients in clinical trials. From a pathogenesis perspective, the degree of acute inflammation and alveolar epithelial injury are highly associated with the outcome of human ALI/ARDS. © 2010 American College of Chest Physicians.
• Ware, L. B., Koyama, T., Billheimer, D. D., Wu, W., Bernard, G. R., Thompson, B. T., Brower, R. G., Standiford, T. J., Martin, T. R., Matthay, M. A., & , N. A. (2010). Prognostic and pathogenetic value of combining clinical and biochemical indices in patients with acute lung injury. Chest, 137(2), 288-96.
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  No single clinical or biologic marker reliably predicts clinical outcomes in acute lung injury (ALI)/ARDS. We hypothesized that a combination of biologic and clinical markers would be superior to either biomarkers or clinical factors alone in predicting ALI/ARDS mortality and would provide insight into the pathogenesis of clinical ALI/ARDS.
• Woodhams, D. C., Kenyon, N., Bell, S. C., Alford, R. A., Chen, S., Billheimer, D., Shyr, Y., & Rollins-Smith, L. A. (2010). Adaptations of skin peptide defences and possible response to the amphibian chytrid fungus in populations of Australian green-eyed treefrogs, Litoria genimaculata. Diversity and Distributions, 16(4), 703-712.
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  Abstract: Aim: Rapidly evolving pathogens may exert diversifying selection on genes involved in host immune defence including those encoding antimicrobial peptides (AMPs). Amphibian skin peptides are one important defence against chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). We examined the population-level variation in this innate immune defence to understand its relationship with disease dynamics. Location: Queensland, Australia. Methods: We examined skin peptide defences in five geographically distinct populations of Australian green-eyed treefrogs, Litoria genimaculata. Skin peptide samples were collected from 52 frogs from three upland populations that previously declined as chytridiomycosis emerged, but subsequently recovered, and from 34 frogs in two lowland populations that did not decline. Historical samples of skin peptides preceding Bd emergence were not available from any population. Results: In general, lowland populations had more effective peptide defences than upland populations. Peptide profiles were similar among populations, although relative amounts of peptides expressed differed significantly among populations and were more variable in the uplands. Infected frogs in upland populations carried a significantly higher infection burden compared to lowland populations. The presence of effective AMPs in the skin of L. genimaculata does not eliminate infection; however, more effective peptide defences may limit infection intensity and the progression of disease. Main conclusions: The population bottleneck in upland populations caused by chytridiomycosis emergence did not appear to produce responses to selection for more effective peptide defences against chytridiomycosis compared to lowland populations of L. genimaculata. This does not exclude the possibility that current peptide defences have adapted in response to disease emergence. A suggestive (P < 0.10) interaction between infection status and population indicates that in lowland populations, infected individuals tend to be those with lower relative intensities of AMPs, whereas in the upland populations, infected and uninfected individuals are similar. Thus, both the AMPs and the environment may act to mediate resistance to Bd infection. © 2010 Blackwell Publishing Ltd.
• Chen, S., Ming, L. i., Hong, D., Billheimer, D., Huiming, L. i., Xu, B. J., & Shyr, Y. (2009). A novel comprehensive wave-form MS data processing method. Bioinformatics, 25(6), 808-814.
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  PMID: 19176559;PMCID: PMC2732299;Abstract: Motivation: Mass spectrometry (MS) can generate high-throughput protein profiles for biomedical research to discover biologically related protein patterns-biomarkers. The noisy functional MS data collected by current technologies, however, require consistent, sensitive and robust data-processing techniques for successful biomedical application. Therefore, it is important to detect features precisely for each spectrum, quantify them well and assign a unique label to features from the same protein-peptide across spectra. Results: In this article, we propose a new comprehensive MS data preprocessing package, Wave-spec, which includes several novel algorithms. It can overcome several conventional difficulties. Wave-spec can be applied to multiple types of MS data generated with different MS technologies. Results from this new package were evaluated and compared to several existing approaches based on a MALDI-TOF MS dataset. © The Author 2009. Published by Oxford University Press. All rights reserved.
• Codreanu, S. G., Zhang, B., Sobecki, S. M., Billheimer, D. D., & Liebler, D. C. (2009). Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal. Molecular & cellular proteomics : MCP, 8(4), 670-80.
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  Lipid peroxidation yields a variety of electrophiles, which are thought to contribute to the molecular pathogenesis of diseases involving oxidative stress, yet little is known of the scope of protein damage caused by lipid electrophiles. We identified protein targets of the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE) in RKO cells treated with 50 or 100 mum HNE. HNE Michael adducts were biotinylated by reaction with biotinamidohexanoic acid hydrazide, captured with streptavidin, and the captured proteins were resolved by one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and identified by liquid chromatography-tandem mass spectrometry. Of the 1500+ proteins identified, 417 displayed a statistically significant increase in adduction with increasing HNE exposure concentration. We further identified 18 biotin hydrazide-modified, HNE-adducted peptides by specific capture using anti-biotin antibody and analysis by high resolution liquid chromatography-tandem mass spectrometry. A subset of the identified HNE targets were validated with a streptavidin capture and immunoblotting approach, which enabled detection of adducts at HNE exposures as low as 1 mum. Protein interaction network analysis indicated several subsystems impacted by endogenous electrophiles in oxidative stress, including the 26 S proteasomal and chaperonin containing TCP-1 (CCT) systems involved in protein-folding and degradation, as well as the COP9 signalosome, translation initiation complex, and a large network of ribonucleoproteins. Global analyses of protein lipid electrophile adducts provide a systems-level perspective on the mechanisms of diseases involving oxidative stress.
• Codreanu, S. G., Zhang, B., Sobecki, S. M., Billheimer, D. D., & Liebler, D. C. (2009). Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal. Molecular and Cellular Proteomics, 8(4), 670-680.
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  PMID: 19054759;PMCID: PMC2667350;Abstract: Lipid peroxidation yields a variety of electrophiles, which are thought to contribute to the molecular pathogenesis of diseases involving oxidative stress, yet little is known of the scope of protein damage caused by lipid electrophiles. We identified protein targets of the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE) in RKO cells treated with 50 or 100 μM HNE. HNE Michael adducts were biotinylated by reaction with biotinamidohexanoic acid hydrazide, captured with streptavidin, and the captured proteins were resolved by one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and identified by liquid chromatography-tandem mass spectrometry. Of the 1500+ proteins identified, 417 displayed a statistically significant increase in adduction with increasing HNE exposure concentration. We further identified 18 biotin hydrazide-modified, HNE-adducted peptides by specific capture using anti-biotin antibody and analysis by high resolution liquid chromatography-tandem mass spectrometry. A subset of the identified HNE targets were validated with a streptavidin capture and immunoblotting approach, which enabled detection of adducts at HNE exposures as low as 1 μM. Protein interaction network analysis indicated several subsystems impacted by endogenous electrophiles in oxidative stress, including the 26 S proteasomal and chaperonin containing TCP-1 (CCT) systems involved in protein-folding and degradation, as well as the COP9 signalosome, translation initiation complex, and a large network of ribonucleoproteins. Global analyses of protein lipid electrophile adducts provide a systems-level perspective on the mechanisms of diseases involving oxidative stress. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
• Micalizzi, D. S., Christensen, K. L., Jedlicka, P., Coletta, R. D., Barón, A. E., Harrell, J. C., Horwitz, K. B., Billheimer, D., Heichman, K. A., Welm, A. L., Schiemann, W. P., & Ford, H. L. (2009). The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-β signaling. Journal of Clinical Investigation, 119(9), 2678-2690.
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  PMID: 19726885;PMCID: PMC2735914;Abstract: Inappropriate activation of developmental pathways is a well-recognized tumor-promoting mechanism. Here we show that overexpression of the homeoprotein Six1, normally a developmentally restricted transcriptional regulator, increases TGF-β signaling in human breast cancer cells and induces an epithelial-mesenchymal transition (EMT) that is in part dependent on its ability to increase TGF-β signaling. TGF-β signaling and EMT have been implicated in metastatic dissemination of carcinoma. Accordingly, we used spontaneous and experimental metastasis mouse models to demonstrate that Six1 overexpression promotes breast cancer metastasis. In addition, we show that, like its induction of EMT, Six1-induced experimental metastasis is dependent on its ability to activate TGF-β signaling. Importantly, in human breast cancers Six1 correlated with nuclear Smad3 and thus increased TGF-β signaling. Further, breast cancer patients whose tumors overexpressed Six1 had a shortened time to relapse and metastasis and an overall decrease in survival. Finally, we show that the effects of Six1 on tumor progression likely extend beyond breast cancer, since its overexpression correlated with adverse outcomes in numerous other cancers including brain, cervical, prostate, colon, kidney, and liver. Our findings indicate that Six1, acting through TGF-β signaling and EMT, is a powerful and global promoter of cancer metastasis.
• Salmon, S., Chen, H., Chen, S., Herbst, R., Tsao, A., Tran, H., Sandler, A., Billheimer, D., Shyr, Y., Lee, J., Massion, P., Brahmer, J., Schiller, J., Carbone, D., & Dang, T. P. (2009). Classification by mass spectrometry can accurately and reliably predict outcome in patients with non-small cell lung cancer treated with erlotinib-containing regimen. Journal of Thoracic Oncology, 4(6), 689-696.
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  PMID: 19404214;PMCID: PMC3563261;Abstract: Purpose: Although many lung cancers express the epidermal growth factor receptor and the vascular endothelial growth factor, only a small fraction of patients will respond to inhibitors of these pathways. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) has shown promise in biomarker discovery, potentially allowing the selection of patients who may benefit from such therapies. Here, we use a matrix-assisted laser desorption/ ionization MS proteomic algorithm developed from a small dataset of erlotinib-bevacizumab treated patients to predict the clinical outcome of patients treated with erlotinib alone. Methods: Pretreatment serum collected from patients in a phase I/II study of erlotinib in combination with bevacizumab for recurrent or refractory non-small cell lung cancer was used to develop a proteomic classifier. This classifier was validated using an independent treatment cohort and a control population. Result: A proteomic profile based on 11 distinct m/z features was developed. This predictive algorithm was associated with outcome using the univariate Cox proportional hazard model in the training set (p = 0.0006 for overall survival; p = 0.0012 for progression-free survival). The signature also predicted overall survival and progression-free survival outcome when applied to a blinded test set of patients treated with erlotinib alone on Eastern Cooperative Oncology Group 3503 (n = 82, p < 0.0001 and p = 0.0018, respectively) but not when applied to a cohort of patients treated with chemotherapy alone (n = 61, p = 0.128). Conclusion: The independently derived classifier supports the hypothesis that MS can reliably predict the outcome of patients treated with epidermal growth factor receptor kinase inhibitors. © 2009 by the International Association for the Study of Lung Cancer.
• Tennessen, J. A., Woodhams, D. C., Chaurand, P., Reinert, L. K., Billheimer, D., Shyr, Y., Caprioli, R. M., Blouin, M. S., & Rollins-Smith, L. A. (2009). Variations in the expressed antimicrobial peptide repertoire of northern leopard frog (Rana pipiens) populations suggest intraspecies differences in resistance to pathogens. Developmental and Comparative Immunology, 33(12), 1247-1257.
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  PMID: 19622371;PMCID: PMC2927990;Abstract: The northern leopard frog (Rana pipiens or Lithobates pipiens) is historically found in most of the provinces of Canada and the northern and southwest states of the United States. In the last 50 years, populations have suffered significant losses, especially in the western regions of the species range. Using a peptidomics approach, we show that the pattern of expressed antimicrobial skin peptides of frogs from three geographically separated populations are distinct, and we report the presence of four peptides (brevinin-1Pg, brevinin-1Pl, ranatuerin-2Pb, and ranatuerin-2Pc) that have not previously been found in skin secretions. The differences in expressed peptides reflect differences in the distribution of alleles for the newly described Brevinin1.1 locus in the three populations. When enriched peptide mixtures were tested for their ability to inhibit growth of the pathogenic amphibian chytrid (Batrachochytrium dendrobatidis), peptides from Minnesota or Vermont frogs were more effective that peptides from Michigan frogs. Four of the purified peptides were tested for their ability to inhibit growth of two bacterial pathogens (Aeromonas hydrophila and Staphylococcus epidermidis) and B. dendrobatidis. Three of the four were effective inhibitors of B. dendrobatidis and S. epidermidis, but none inhibited A. hydrophila. We interpret these differences in expression and activity of antimicrobial peptides as evidence to suggest that each population may have been selected to express a suite of peptides that reflects current and past encounters with skin microbes. © 2009 Elsevier Ltd. All rights reserved.
• Biswas, S., Trobridge, P., Romero-Gallo, J., Billheimer, D., Myeroff, L. L., K., J., Markowitz, S. D., & Grady, W. M. (2008). Mutational inactivation of TGFBR2 in microsatellite unstable colon cancer arises from the cooperation of genomic instability and the clonal outgrowth of transforming growth factor β resistant cells. Genes Chromosomes and Cancer, 47(2), 95-106.
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  PMID: 17985359;Abstract: The mutational inactivation of transforming growth factor β receptor type II (TGFBR2) occurs in ∼30% of colon cancers and promotes the formation of colon cancer by inhibiting the tumor suppressor activity of the TGFB signaling pathway. TGFBR2 mutations occur in >90% of microsatellite unstable (MSI) colon cancers and affect a polyadenine tract in exon 3 of TGFBR2, called BAT-RII, which is vulnerable to mutation in the setting of DNA mismatch repair (MMR) system deficiency. In light of the vulnerable nature of the BAT-RII tract in the setting of MMR inactivation and the favorable effects of TGFBR2 inactivation in colon cancer, analysis of TGFBR2 inactivation provides an opportunity to assess the roles of genomic instability vs. clonal selection in cells acquiring TGFBR2 BAT-RII tract mutations in MSI colon cancer formation. The contribution of genomic instability and/or clonal evolution to the mutational inactivation of TGBFR2 in MSI colon cancers has not been studied in a systematic way that would allow a determination of the relative contribution of these two mechanisms in the formation of MSI colon cancer. It has not been demonstrated whether the BAT-RII tract mutations are strictly a consequence of the BAT-RII region being hypermutable in the setting of MMR deficiency or if the mutations are rather a consequence of clonal selection pressure against the TGFB receptor. Through the use of defined cell line systems, we show that both genomic instability and clonal selection of TGFB resistant cells contribute to the high frequency of TGFBR2 mutations in MSI colon cancer. © 2007 Wiley-Liss, Inc.
• Burgess, E. F., Ham, A. L., Tabb, D. L., Billheimer, D., Roth, B. J., Chang, S. S., Cookson, M. S., Hinton, T. J., Cheek, K. L., Hill, S., & Pietenpol, J. A. (2008). Prostate cancer serum biomarker discovery through proteomic analysis of alpha-2 macroglobulin protein complexes. Proteomics - Clinical Applications, 2(9), 1223-1233.
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  Abstract: Alpha-2 macroglobulin (A2M) functions as a universal protease inhibitor in serum and is capable of binding various cytokines and growth factors. In this study, we investigated if immunoaffinity enrichment and proteomic analysis of A2M protein complexes from human serum could improve detection of biologically relevant and novel candidate protein biomarkers in prostate cancer. Serum samples from six patients with androgen-independent, metastatic prostate cancer and six control patients without malignancy were analyzed by immunoaffinity enrichment of A2M protein complexes and MS identification of associated proteins. Known A2M substrates were reproducibly identified from patient serum in both cohorts, as well as proteins previously undetected in human serum. One example is heat shock protein 90 alpha (HSP90α), which was identified only in the serum of cancer patients in this study. Using an ELISA, the presence of HSP90α in human serum was validated on expanded test cohorts and found to exist in higher median serum concentrations in prostate cancer (n = 18) relative to control (n = 13) patients (median concentrations 50.7 versus 27.6 ng/mL, respectively, p = 0.001). Our results demonstrate the technical feasibility of this approach and support the analysis of A2M protein complexes for proteomic-based serum biomarker discovery. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Lieber, C. A., Majumder, S. K., Billheimer, D., Ellis, D. L., & Mahadevan-Jansen, A. (2008). Raman microspectroscopy for skin cancer detection in vitro. Journal of Biomedical Optics, 13(2).
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  PMID: 18465976;Abstract: We investigate the potential of near-infrared Raman microspectroscopy to differentiate between normal and malignant skin lesions. Thirty-nine skin tissue samples consisting of normal, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma from 39 patients were investigated. Raman spectra were recorded at the surface and at 20-μm intervals below the surface for each sample, down to a depth of at least 100μm. Data reduction algorithms based on the nonlinear maximum representation and discrimination feature (MRDF) and discriminant algorithms using sparse multinomial logistic regression (SMLR) were developed for classification of the Raman spectra relative to histopathology. The tissue Raman spectra were classified into pathological states with a maximal overall sensitivity and specificity for disease of 100%. These results indicate the potential of using Raman microspectroscopy for skin cancer detection and provide a clear rationale for future clinical studies. © 2008 Society of Photo-Optical Instrumentation Engineers.
• Lieber, C. A., Majumder, S. K., Ellis, D. L., Billheimer, D. D., & Mahadevan-Jansen, A. (2008). In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy. Lasers in Surgery and Medicine, 40(7), 461-467.
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  PMID: 18727020;PMCID: PMC2782422;Abstract: Background and Objectives: Nonmelanoma skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are the most common skin cancers, presenting nearly as many cases as all other cancers combined. The current gold-standard for clinical diagnosis of these lesions is histopathologic examination, an invasive, time-consuming procedure. There is thus considerable interest in developing a real-time, automated, noninvasive tool for nonmelanoma skin cancer diagnosis. In this study, we explored the capability of Raman microspectroscopy to provide differential diagnosis of BCC, SCC, inflamed scar tissue, and normal tissue in vivo. Study Design: Based on the results of previous in vitro studies, we developed a portable confocal Raman system with a handheld probe for clinical study. Using this portable system, we measured Raman spectra of 21 suspected nonmelanoma skin cancers in 19 patients with matched normal skin spectra. These spectra were input into nonlinear diagnostic algorithms to predict pathological designation. Results: All of the BCC (9/9), SCC (4/4), and inflamed scar tissues (8/8) were correctly predicted by the diagnostic algorithm, and 19 out of 21 normal tissues were correctly classified. This translates into a 100% (21/21) sensitivity and 91% (19/21) specificity for abnormality, with a 95% (40/ 42) overall classification accuracy. Conclusions: These findings reveal Raman microspectroscopy to be a viable tool for real-time diagnosis and guidance of nonmelanoma skin cancer resection. © 2008 Wiley-Liss, Inc.
• Lieber, C. A., Majumder, S. K., Ellis, D. L., Billheimer, D. D., & Mahadevan-Jansen, A. (2008). In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy. Lasers in surgery and medicine, 40(7), 461-7.
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  Nonmelanoma skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are the most common skin cancers, presenting nearly as many cases as all other cancers combined. The current gold-standard for clinical diagnosis of these lesions is histopathologic examination, an invasive, time-consuming procedure. There is thus considerable interest in developing a real-time, automated, noninvasive tool for nonmelanoma skin cancer diagnosis. In this study, we explored the capability of Raman microspectroscopy to provide differential diagnosis of BCC, SCC, inflamed scar tissue, and normal tissue in vivo.
• Meyrick, B. O., Friedman, D. B., Billheimer, D. D., Cogan, J. D., Prince, M. A., A., J., & Loyd, J. E. (2008). Proteomics of transformed lymphocytes from a family with familial pulmonary arterial hypertension. American Journal of Respiratory and Critical Care Medicine, 177(1), 99-107.
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  PMID: 17932379;PMCID: PMC2176118;Abstract: Rationale: Not all family members with BMPR2 mutations develop pulmonary arterial hypertension (PAH), implying that additional modifier genes or proteins are necessary for full expression of the disease. Objectives: To determine whether protein expression is altered in patients with familial PAH (FPAH) compared with obligate carriers and nondiseased control subjects. Methods: Protein extractsfrom transformed blood lymphocytes from four patients with FPAH, three obligate carriers, and three marriedin control subjects from one family with a known BMPR2 mutation (exon 3 T354G) were labeled with either Cy3 or Cy5. Cy3/5 pairs were separated by standard two-dimensional differential gel electrophoresis using a Cy2-labeled internal standard of all patient samples. Log volume ratios were analyzed using a linear mixed effects model. Proteins were identified by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI-TOF MS) and tandem TOF/TOF MS/MS. Measurements and Main Results: Hierarchical clustering, heat-map, and principal components analysis revealed marked changes in protein expression in patients with FPAH when compared with obligate carriers. Significant changes were apparent in expression of 16 proteins (P
• Sanders, M. E., Dias, E. C., Xu, B. J., Mobley, J. A., Billheimer, D., Roder, H., Grigorieva, J., Dowsett, M., Arteaga, C. L., & Caprioli, R. M. (2008). Differentiating Proteomic Biomarkers in Breast Cancer by Laser Capture Microdissection and MALDI MS. Journal of Proteome Research, 7(4), 1500-1507.
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  PMID: 18386930;PMCID: PMC2738605;Abstract: We assessed proteomic patterns in breast cancer using MALDI MS and laser capture microdissected cells. Protein and peptide expression in invasive mammary carcinoma versus normal mammary epithelium and estrogen-receptor positive versus estrogen-receptor negative tumors were compared. Biomarker candidates were identified by statistical analysis and classifiers were developed and validated in blinded test sets. Several of the mlz features used in the classifiers were identified by LC-MS/MS and two were confirmed by immunohistochemistry. © 2008 American Chemical Society.
• Frangoul, H., Nemecek, E. R., Billheimer, D., Pulsipher, M. A., Khan, S., Woolfrey, A., Manes, B., Cole, C., Walters, M. C., Ayas, M., Ravindranath, Y., Levine, J. E., & Grupp, S. A. (2007). A prospective study of G-CSF-primed bone marrow as a stem-cell source for allogeneic bone marrow transplantation in children: A Pediatric Blood and Marrow Transplant Consortium (PBMTC) study. Blood, 110(13), 4584-4587.
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  PMID: 17827386;Abstract: A prospective multicenter trial was conducted to evaluate the safety and feasibility of granulocyte colony-stimulating factor (G-CSF)-primed bone marrow (G-BM) in children receiving allogeneic bone marrow transplantation (BMT). A total of 42 children with a median age of 9.8 years (range, 0.8-17 years) were enrolled. Donors with median age of 9.2 years (range, 1.1-22 years) received 5 μg/kg per day of subcutaneous G-CSF for 5 consecutive days. BM was harvested on the fifth day. No donor experienced complications related to G-CSF administration or marrow harvest. Median nucleated (NC) and CD34 cells infused was 6.7 × 108/kg (range, 2.4-18.5 × 108/kg) and 7.4 × 106/kg (range, 2-27.6 × 106/kg), respectively. Neutrophil and platelet engraftment was at a median of 19 days (range, 13-28 days) and 20 days (range, 9-44 days), respectively. A total of 13 (32%) patients developed grade 2 graft-versus-host disease (GVHD), and 5 (13%) of 40 evaluable patients developed chronic GVHD (3 limited and 2 extensive). Higher cell dose was not associated with increased risk of acute or chronic GVHD. Overall survival and event-free survival at 2 years were 81% and 69%, respectively. Collection of G-BM from pediatric donors is safe, and can result in high NC and CD34 cell doses that facilitate engraftment after myeloablative BMT without a discernable increase in the risk of GVHD. © 2007 by The American Society of Hematology.
• Li, J., Xu, B. J., Shakhtour, B., Deane, N., Merchant, N., Heslin, M. J., Washington, K., Coffey, R. J., Beauchamp, R. D., Shyr, Y., & Billheimer, D. (2007). Variability of in situ proteomic profiling and implications for study design in colorectal tumors. International Journal of Oncology, 31(1), 103-111.
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  PMID: 17549410;Abstract: Knowledge of intrinsic tumor heterogeneity is vital for understanding of tumor progression mechanisms as well as for providing efficient treatments. In situ proteomic profiling of tumors is a powerful technology with potential to enhance our understanding of tumor biology, but sources of variability due to patient and tumor heterogeneity are poorly understood and are the topic of this investigation. Clarification of variability within case and between cases is also important for designing future studies. Direct protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a sensitive and powerful technology for obtaining hundreds of protein expression peaks from a thin tissue section. By combining robotic microspotting and laser capture microdissection with MALDI MS, we acquired multiple spectra per case to evaluate inter- and intra-case variability in human colorectal tumor and murine cecal carcinoma. We detected 256 peaks from 164 samples of 111 patients, which consisted of 55 normal colorectal mucosal samples, 24 adenomas, 71 primary carcinomas, and 14 hepatic metastases. In addition, we detected 291 peptide/protein peaks from 34 orthotopically transplanted murine cecal carcinomas and 14 hepatic metastases. In human colorectal samples, we observed that proteomic profiling in adenomas was more homogeneous across patients than in normal mucosa specimens (p=0.0008), but primary carcinoma exhibited greater heterogeneity than normal mucosa and adenomas (both p
• M'Koma, A. E., Blum, D. L., Norris, J. L., Koyama, T., Billheimer, D., Motley, S., Ghiassi, M., Ferdowsi, N., Bhowmick, I., Chang, S. S., Fowke, J. H., Caprioli, R. M., & Bhowmick, N. A. (2007). Detection of pre-neoplastic and neoplastic prostate disease by MADI profiling of urine. Biochemical and Biophysical Research Communications, 353(3), 829-834.
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  PMID: 17194448;PMCID: PMC2562600;Abstract: The heterogeneous progression to the development of prostate cancer (PCa) has precluded effective early detection screens. Existing prostate cancer screening paradigms have relatively poor specificity for cancer relative to other prostate diseases, commonly benign prostatic hyperplasia (BPH). A method for discrimination of BPH, HGPIN, and PCa urine proteome was developed through testing 407 patient samples using matrix assisted laser desorption-mass spectrometry time of flight (MALDI-TOF). Urine samples were adsorbed to reverse phase resin, washed, and the eluant spotted directly for MALDI-TOF analysis of peptides. The processing resolved over 130 verifiable signals of a mass range of 1000-5000 m/z to suggest 71.2% specificity and 67.4% sensitivity in discriminating PCa vs. BPH. Comparing BPH and HGPIN resulted in 73.6% specificity and 69.2% sensitivity. Comparing PCa and HGPIN resulted in 80.8% specificity and 81.0% sensitivity. The high throughput, low-cost assay method developed is amenable for large patient numbers required for supporting biomarker identification. © 2006.
• Robichaux-Viehoever, A., Kanter, E., Shappell, H., Billheimer, D., III, H. J., & Mahadevan-Jansen, A. (2007). Characterization of Raman spectra measured in vivo for the detection of cervical dysplasia. Applied Spectroscopy, 61(9), 986-993.
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  PMID: 17910796;Abstract: Raman spectroscopy has been shown to have the potential for providing differential diagnosis in the cervix with high sensitivity and specificity in previous studies. The research presented here further evaluates the potential of near-infrared Raman spectroscopy to detect cervical dysplasia in a clinical setting. Using a portable system, Raman spectra were collected from the cervix of 79 patients using clinically feasible integration times (5 seconds on most patients). Multiple Raman measurements were taken from colposcopically normal and abnormal areas prior to the excision of tissue. Data were processed to extract Raman spectra from measured signal, which includes fluorescence and noise. The resulting spectra were correlated with the corresponding histopathologic diagnosis to determine empirical differences between different diagnostic categories. Using histology as the gold standard, logistic regression discrimination algorithms were developed to distinguish between normal ectocervix, squamous metaplasia, and high-grade dysplasia using independent training and validation sets of data. An unbiased estimate of the accuracy of the model indicates that Raman spectroscopy can distinguish between high-grade dysplasia and benign tissue with sensitivity of 89% and specificity of 81%, while colposcopy in expert hands was able to discriminate with a sensitivity and specificity of 87% and 72%. © 2007 Society for Applied Spectroscopy.
• Woodhams, D. C., Vredenburg, V. T., Simon, M., Billheimer, D., Shakhtour, B., Shyr, Y., Briggs, C. J., Rollins-Smith, L. A., & Harris, R. N. (2007). Symbiotic bacteria contribute to innate immune defenses of the threatened mountain yellow-legged frog, Rana muscosa. Biological Conservation, 138(3-4), 390-398.
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  Abstract: Symbiotic microorganisms influence health and disease and may contribute to the innate immune defenses of amphibians. The mountain yellow-legged frog, Rana muscosa, is currently undergoing unprecedented population declines. One cause of recent declines is the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd). Skin swabs for detection of Bd, skin peptide secretions, and symbiotic skin bacteria were collected from 70 adult R. muscosa from two populations designated "Sixty Lake" and "Conness" in 2004-2005. The Conness population has persisted with the presence of Bd for at least 6 years whereas the Sixty Lake population is newly infected and declining. Of the frogs sampled at Conness, 67.5% were infected; whereas 96.7% of the Sixty Lake frogs were infected. Sixty Lake frogs were also more intensely infected than frogs at Conness. We isolated symbiotic bacteria that may contribute to immune defense. A significantly greater proportion of individuals with at least one anti-Bd bacterial species present were found at Conness (85%) than at Sixty Lake (62%). We observed no apparent differences in total skin peptides recovered; however, peptide mixtures from frogs at Sixty Lake showed better growth inhibitory activity against Bd than peptides from frogs at Conness. By MALDI-TOF MS analysis, there were no differences between the two populations in the previously described antimicrobial peptides (ranatuerin-2Ma, ranatuerin-2Mb, and temporin-1M). Antimicrobial skin peptides are only one factor in the resistance of R. muscosa to Bd infection. We suggest that symbiotic bacteria with the ability to persist in the presence of mucosal peptides may inhibit infection and colonization of the skin by Bd and increase the effectiveness of innate defense mechanisms in the skin. © 2007 Elsevier Ltd. All rights reserved.
• Yang, L., Amann, J. M., Kikuchi, T., Porta, R., Guix, M., Gonzalez, A., Park, K., Billheimer, D., Arteaga, C. L., Tai, H., DuBois, R., Carbone, D. P., & Johnson, D. H. (2007). Inhibition of epidermal growth factor receptor signaling elevates 15-hydroxyprostaglandin dehydrogenase in non-small-cell lung cancer. Cancer Research, 67(12), 5587-5593.
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  PMID: 17575121;Abstract: Evidence indicates that the induction of cyclooxygenase-2 (COX-2) and high prostaglandin E2 (PGE2) levels contribute to the pathogenesis of non-small-cell lung cancer (NSCLC). In addition to overproduction by COX-2, PGE2 concentrations also depend upon the levels of the PGE2 catabolic enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). We find a dramatic down-regulation of PGDH protein in NSCLC cell lines and in resected human tumors when compared with matched normal lung. Affymetrix array analysis of 10 normal lung tissue samples and 49 resected lung tumors revealed a much lower expression of PGDH transcripts in all NSCLC histologic groups. In addition, treatment with the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) erlotinib increased the expression of 15-PGDH in a subset of NSCLC cell lines. This effect may be due in part to an inhibition of the extracellular signal-regulated kinase (ERK) pathway as treatment with mitogen-activated protein kinase kinase (MEK) inhibitor U0126 mimics the erlotinib results. We show by quantitative reverse transcription-PCR that the transcript levels of ZEB1 and Slug transcriptional repressors are dramatically reduced in a responsive cell line upon EGFR and MEK/ERK inhibition. In addition, the Slug protein, but not ZEB1, binds to the PGDH promoter and represses transcription. As these repressors function by recruiting histone deacetylases to promoters, it is likely that PGDH is repressed by an epigenetic mechanism involving histone deacetylation, resulting in increased PGE 2 activity in tumors. This effect is reversible in a subset of NSCLC upon treatment with an EGFR TKI. ©2007 American Association for Cancer Research.
• Chakravarthy, A. B., Kelley, M. C., McLaren, B., Truica, C. I., Billheimer, D., Mayer, I. A., Grau, A. M., Johnson, D. H., Simpson, J. F., Beauchamp, R. D., Jones, C., & Pietenpol, J. A. (2006). Neoadjuvant concurrent paclitaxel and radiation in stage II/III breast cancer. Clinical Cancer Research, 12(5), 1570-1576.
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  PMID: 16533783;Abstract: Purpose: The aim of this study was to determine the safety and pathologic response rates following neoadjuvant paclitaxel and radiation in patients with stage II/III breast cancer and to evaluate the use of sequential biopsies to allow an in vivo assessment of biological markers as potential predictive markers of response to this regimen. Patients and Methods: Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel 175 mg/m2 every 3 weeks, followed by twice-weekly paclitaxel 30 mg/m2 and concurrent radiation. Core biopsies were obtained at baseline and 24 to 72 hours after the first cycle of paclitaxel. After completing neoadjuvant treatment, patients underwent definitive surgery. The primary end point was pathologic complete response, which is defined as the absence of any invasive cancer at surgery. Potential markers of therapeutic response were evaluated including markers of proliferation, apoptosis, p21, HER2, estrogen receptor, and progesterone receptor status. Results: Of the 38 patients enrolled, 13 (34%) had a pathologic complete response. There was no significant difference in baseline Ki-67 between responders (35%) and nonresponders (28%; P = 0.45). There was also no significant change in Ki-67 following paclitaxel administration. Despite this lack of immunohistologic change in proliferative activity, baseline mitotic index was higher for patients with pathologic complete response over nonresponders (27 versus 10, P = 0.003). Moreover, the increase in mitotic index following paclitaxel administration was associated with pathologic complete response. Conclusions: Neoadjuvant paclitaxel/radiation is effective and well tolerated. Tumor proliferation at baseline and response to chemotherapy as measured by mitotic activity may serve as an important indicator of pathologic response to neoadjuvant paclitaxel/radiation. ©2006 American Association for Cancer Research.
• Fowler, K., Poehling, K., Billheimer, D., Hamilton, R., Huiyun, W. u., Mulder, J., & Frangoul, H. (2006). Hospice referral practices for children with cancer: A survey of pediatric oncologists. Journal of Clinical Oncology, 24(7), 1099-1104.
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  PMID: 16505429;Abstract: Purpose: To examine hospice referral patterns among pediatric oncologists and identify barriers to referral. Methods: A self-administered survey was sent to 1,200 pediatric oncologists who are members of Children's Oncology Group. Two electronic mail messages followed by traditional mail surveys were sent to eligible physicians. Pediatricians and pediatric oncologists developed, pretested, and modified the survey for item clarification. Results: Of 944 eligible pediatric oncologists surveyed, 632 replied, yielding a response rate of 67%. Most respondents reported having access to palliative care programs (65%) and hospice services (85%), but few (27%) had access to inpatient hospice services. More respondents reported feeling comfortable managing end-of-life pain than psychological issues (86% v 67%, respectively). Many pediatric oncologists (62%) reported that half or more of their patients died in the hospital. In multivariate analysis, physicians with access to hospice that accepts patients receiving chemotherapy had more patients die at home than in hospital compared with physicians without access to such services (P = .007). The probability of hospice referral was positively associated with the presence of a hospice facility (P < .001) and with a larger size oncology group (P = .024). Only 2.5% of respondents referred patients at the time of relapse. Continued therapy was cited as the most common reason for not making a referral, and was significantly higher when hospice did not admit children receiving chemotherapy (P = .002). Conclusion: Hospice referral for children with cancer is usually made late in the course of their disease and might improve if hospice admits patients who are actively receiving chemotherapy. © 2006 by American Society of Clinical Oncology.
• Johnson, J. C., Schmidt, C. R., Shrubsole, M. J., Billheimer, D. D., Joshi, P. R., Morrow, J. D., Heslin, M. J., Washington, M. K., Ness, R. M., Zheng, W., Schwartz, D. A., Coffey, R. J., Beauchamp, R. D., & Merchant, N. B. (2006). Urine PGE-M: A Metabolite of Prostaglandin E₂ as a Potential Biomarker of Advanced Colorectal Neoplasia. Clinical Gastroenterology and Hepatology, 4(11), 1358-1365.
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  PMID: 16996805;Abstract: Background & Aims The enzyme cyclooxygenase-2 is expressed in a majority of colorectal carcinomas (CRCs) and is important in prostaglandin production. We have developed an accurate method to measure the urinary metabolite of prostaglandin E2 (PGE-M) using recently developed mass spectrometric techniques. The purpose of this pre-validation study was to determine if urinary PGE-M levels can be used as a biomarker to discriminate between healthy patients and those with colorectal disease. Methods Urine PGE-M was assessed in a total of 228 patients with CRC, colonic adenomatous polyps, Crohn's disease, and in subjects with no endoscopically detectable disease. Thirteen rectal carcinoma patients were treated with celecoxib and urinary PGE-M was measured before and after treatment. Results Urine PGE-M levels were increased among healthy men compared with healthy women (median, 8.59 [interquartile range (IQR), 5.67-22.3] vs 4.25 [IQR, 2.35-6.03], P = .0027). Urine PGE-M levels among patients with Crohn's disease (median, 19.85 [IQR, 6.89-90.2]), CRC (median, 14.65 [IQR, 5.94-92.1]), or large adenomas greater than 1 cm in size (median, 18.85 [IQR, 11.9-25.6]) were significantly increased when compared with patients who had either small polyps less than 1 cm in size (median, 9.69 [IQR, 6.41-22.2]), or no polyps (median, 7.05 [IQR, 2.35-24.7]) (P = .0001). PGE-M levels decreased significantly after celecoxib treatment in patients with rectal cancer (median, 21.7 [IQR, 16.2-29.9] vs 9.14 [IQR, 7.14-13.2], P = .009). Conclusions The increase in urinary PGE-M in patients with colorectal cancers and large adenomas suggests that urinary PGE-M is a potentially useful biomarker for the detection of advanced colorectal neoplasia. © 2006 AGA Institute.
• Johnson, J. C., Schmidt, C. R., Shrubsole, M. J., Billheimer, D. D., Joshi, P. R., Morrow, J. D., Heslin, M. J., Washington, M. K., Ness, R. M., Zheng, W., Schwartz, D. A., Coffey, R. J., Beauchamp, R. D., & Merchant, N. B. (2006). Urine PGE-M: A metabolite of prostaglandin E2 as a potential biomarker of advanced colorectal neoplasia. Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association, 4(11), 1358-65.
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  The enzyme cyclooxygenase-2 is expressed in a majority of colorectal carcinomas (CRCs) and is important in prostaglandin production. We have developed an accurate method to measure the urinary metabolite of prostaglandin E(2) (PGE-M) using recently developed mass spectrometric techniques. The purpose of this pre-validation study was to determine if urinary PGE-M levels can be used as a biomarker to discriminate between healthy patients and those with colorectal disease.
• Yankeelov, T. E., DeBusk, L. M., Billheimer, D. D., Luci, J. J., Lin, P. C., Price, R. R., & Gore, J. C. (2006). Repeatability of a reference region model for analysis of murine DCE-MRI data at 7T. Journal of Magnetic Resonance Imaging, 24(5), 1140-1147.
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  PMID: 17024660;Abstract: Purpose: To test the repeatability of a reference region (RR) model for the analysis of dynamic contrast-enhanced MRI (DCE-MRI) in a mouse model of cancer at high field. Materials and Methods: Seven mice were injected with 10 6 4T1 mammary carcinoma cells and imaged eight to 10 days later on a Varian 7.0T scanner. Two DCE-MRI studies were performed for each mouse (separated by 2.5 hours). The RR model was used to analyze the data, and returned estimates on the perfusion-permeability index (Ktrans) for the RR and the tissue of interest (TOI), as well as the extravascular extracellular volume fraction (ve) for the TOI. Results: When the first injection was compared with the second injection, all parameters tested were highly correlated (r2 = 0.90, 0.62, 0.82 for the RR K trans, TOI Ktrans, and TOI ve, respectively, with P < 0.001 for all). To observe a statistically significant change (at the 5% level) in a treatment study with seven animals in each group, log 10 changes of 0.084 and 0.077 in the tumor Ktrans and ve. respectively, are required. Conclusion: If a reliable arterial input function (AIF) is unavailable, the RR model is a reasonable alternative to measuring MRI contrast-agent (CA) kinetics in mouse models of cancer at high field. © 2006 Wiley-Liss, Inc.
• Barbieri, C. E., Perez, C. A., Johnson, K. N., Ely, K. A., Billheimer, D., & Pietenpol, J. A. (2005). IGFBP-3 is a direct target of transcriptional regulation by ΔNp63α in squamous epithelium. Cancer Research, 65(6), 2314-2320.
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  PMID: 15781645;Abstract: ΔNp63α is a nuclear transcription factor that maintains epithelial progenitor cell populations, is overexpressed in several epithelial cancers, and can negatively regulate apoptosis. However, the mechanisms by which ΔNp63α promotes cell survival are unclear. ΔNp63α has been reported to act as a transcriptional repressor, but specific target genes directly repressed by ΔNp63α remain unidentified. Here, we present evidence that ΔNp63α functions to negatively regulate the proapoptotic protein IGFBP-3. Disruption of p63 expression in squamous epithelial cells increases IGFBP-3 expression, whereas ectopic expression of ΔNp63α down-regulates IGFBP-3. ΔNp63α binds to sites in the IGFBP-3 gene in vivo and can modulate transcription through these sites. Furthermore, ΔNp63α and IGFBP-3 expression patterns are inversely correlated in normal squamous epithelium and squamous cell carcinomas. These data suggest that IGFBP-3 is a target of transcriptional repression by ΔNp63α and that this repression represents a mechanism by which tumors that overexpress p63 may be protected from apoptosis. ©2005 American Association for Cancer Research.
• Etzioni, R., Hawley, S., Billheimer, D., True, L. D., & Knudsen, B. (2005). Analyzing patterns of staining in immunohistochemical studies: Application to a study of prostate cancer recurrence. Cancer Epidemiology Biomarkers and Prevention, 14(5), 1040-1046.
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  PMID: 15894650;Abstract: Background: Immunohistochemical studies use antibodies to stain tissues with the goal of quantifying protein expression. However, protein expression is often heterogeneous resulting in variable degrees and patterns of staining. This problem is particularly acute in prostate cancer, where tumors are infiltrative and heterogeneous in nature. In this article, we introduce analytic approaches that explicitly consider both the frequency and intensity of tissue staining. Methods: Compositional data analysis is a technique used to analyze vectors of unit-sum proportions, such as those obtained from soil sample studies or species abundance surveys. We summarized specimen staining patterns by the proportion of cells staining at mild, moderate, and intense levels and used compositional data analysis to summarize and compare the resulting staining profiles. Results: In a study of Syndecan-1 staining patterns among 44 localized prostate cancer cases with Gleason score 7 disease, compositional data analysis did not detect a statistically significant difference between the staining patterns in recurrent (n = 22) versus nonrecurrent (n = 22) patients. Results indicated only modest increases in the proportion of cells staining at a moderate intensity in the recurrent group. In contrast, an analysis that compared quantitative scores across groups indicated a (borderline) significant increase in staining in the recurrent group (P = 0.05, t test). Conclusions: Compositional data analysis offers a novel analytic approach for immunohistochemical studies, providing greater insight into differences in staining patterns between groups, but possibly lower statistical power than existing, score-based methods. When appropriate, we recommend conducting a compositional data analysis in addition to a standard score-based analysis. Copyright © 2005 American Association for Cancer Research.
• Chaurand, P., Schwartz, S. A., Billheimer, D., Xu, B. J., Crecelius, A., & Caprioli, R. M. (2004). Integrating Histology and Imaging Mass Spectrometry. Analytical Chemistry, 76(4), 1145-1155.
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  PMID: 14961749;Abstract: MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry (IMS) is a new technology that generates molecular profiles and two-dimensional ion density maps of peptide and protein signals directly from the surface of thin tissue sections. This allows specific information to be obtained on the relative abundance and spatial distribution of proteins. One important aspect of this is the opportunity to correlate these specific ion images with histological features observed by optical microscopy. To facilitate this, we have developed protocols that allow MALDI mass spectrometry imaging and optical microscopy to be performed on the same section. Key components of these protocols involve the use of conductive glass slides as sample support for the tissue sections and MS-friendly tissue staining protocols. We show the effectiveness of these with protein standards and with several types of tissue sections. Although stain-specific intensity variations occur, the overall protein pattern and spectrum quality remain consistent between stained and control tissue samples. Furthermore, imaging mass spectrometry experiments performed on stained sections showed good image quality with minimal delocalization of proteins resulting from the staining protocols.
• Scarfone, C., Lavely, W. C., Cmelak, A. J., Delbeke, D., Martin, W. H., Billheimer, D., & Hallahan, D. E. (2004). Prospective feasibility trial of radiotherapy target definition for head and neck cancer using 3-dimensional PET and CT imaging. Journal of Nuclear Medicine, 45(4), 543-552.
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  PMID: 15073248;Abstract: The aim of this investigation was to evaluate the influence and accuracy of 18F-FDG PET in target volume definition as a complementary modality to CT for patients with head and neck cancer (HNC) using dedicated PET and CT scanners. Methods: Six HNC patients were custom fitted with head and neck and upper body immobilization devices, and conventional radiotherapy CT simulation was performed together with 18F-FDG PET imaging. Gross target volume (GTV) and pathologic nodal volumes were first defined in the conventional manner based on CT. A segmentation and surface-rendering registration technique was then used to coregister the 18F-FDG PET and CT planning image datasets. 18F-FDG PET GTVs were determined and displayed simultaneously with the CT contours. CT GTVs were then modified based on the PET data to form final PET/CT treatment volumes. Five-field intensity-modulated radiation therapy (IMRT) was then used to demonstrate dose targeting to the CT GTV or the PET/CT GTV. Results: One patient was PET-negative after induction chemotherapy. The CT GTV was modified in all remaining patients based on 18F-FDG PET data. The resulting PET/CT GTV was larger than the original CT volume by an average of 15%. In 5 cases, 18F-FDG PET identified active lymph nodes that corresponded to lymph nodes contoured on CT. The pathologically enlarged CT lymph nodes were modified to create final lymph node volumes in 3 of 5 cases. In 1 of 6 patients, 18F-FDG-avid lymph nodes were not identified as pathologic on CT. In 2 of 6 patients, registration of the independently acquired PET and CT data using segmentation and surface rendering resulted in a suboptimal alignment and, therefore, had to be repeated. Radiotherapy planning using IMRT demonstrated the capability of this technique to target anatomic or anatomic/physiologic target volumes. In this manner, metabolically active sites can be intensified to greater daily doses. Conclusion: Inclusion of 18F-FDG PET data resulted in modified target volumes in radiotherapy planning for HNC. PET and CT data acquired on separate, dedicated scanners may be coregistered for therapy planning; however, dual-acquisition PET/CT systems may be considered to reduce the need for reregistrations. It is possible to use IMRT to target dose to metabolically active sites based on coregistered PET/CT data.
• Tsiatis, A. C., Manes, B., Calder, C., Billheimer, D., Wilkerson, K. S., & Frangoul, H. (2004). Incidence and clinical complications of vancomycin-resistant enterococcus in pediatric stem cell transplant patients. Bone Marrow Transplantation, 33(9), 937-941.
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  PMID: 15034540;Abstract: Vancomycin-resistant enterococcus (VRE) are increasingly important pathogens in stem cell transplant (SCT). In all, 61 pediatric SCT patients had surveillance stool cultures for VRE between July 1999 and November 2002. When VRE was identified, the patients were placed on strict contact isolation. VRE was detected in 15 patients (24.6%). The median age was 3.6 years (range 0.6-18.5 years). Of the 15, 13 (87%) received an allogeneic transplant (six unrelated and seven related). Five of the 15 (33%) colonized patients developed VRE bacteremia. The bacteremia resolved in all five patients after therapy with quinupristin/dalfopristin; three patients required central line removal. Four patients died (38-153 days) post-SCT due to relapse or transplant complication not related to VRE. Of the 11 surviving patients, seven cleared the colonization at a median of 144 days (range 61-198 days) postcolonization. Four patients remain colonized at 68-702 days after the first positive culture. Intestinal colonization with VRE occurred commonly in pediatric SCT patients. Although the morbidity from VRE was not substantial, transplant patients were colonized for prolonged periods. Our results indicate that surveillance for VRE is an effective way to identify colonized patients and may lead to a decrease in transmission to other patients. © 2004 Nature Publishing Group. All rights reserved.
• Goldstein, R. E., Billheimer, D., Martin, W. H., Richards, K., Jacobs, J. K., Udelsman, R., & L., G. (2003). Sestamibi Scanning and Minimally Invasive Radioguided Parathyroidectomy Without Intraoperative Parathyroid Hormone Measurement. Annals of Surgery, 237(5), 722-731.
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  PMID: 12724639;PMCID: PMC1514518;Abstract: Objective: To evaluate the results of a large series of patients undergoing minimally invasive radioguided parathyroidectomy (MIRP) in which routine use of the intraoperative parathyroid hormone assay was not used, and to investigate characteristics between patients who had positive preoperative parathyroid scans versus those with negative scans. Summary Background Data: The technique of parathyroidectomy has traditionally involved bilateral exploration of the neck under general endotracheal anesthesia. Parathyroid imaging using technetium-99m sestamibi (MIBI) has evolved and can localize the adenomas in 80% to 90% of patients. The MIRP technique combines parathyroid scintigraphy with a hand-held gamma detector used intraoperatively to guide the surgeon to the adenoma in patients with positive MIBI scans. Central to this technique or other unilateral approaches is a positive MIBI scan. Methods: One hundred seventy-three atients with primary hyperparathyroidism operated on by a single surgeon between January 1998 and July 2002 were included. One hundred twelve patients underwent the MIRP procedure and by definition had a positive preoperative parathyroid scan. The technique involved injecting 20 mCi MIBI 1 hour before the surgical procedure in patients who preoperatively had positive MIBI imaging. Patients had the choice of general or MAC anesthesia. Using an incision of less than 4 cm, the dissection to the adenoma was guided by the Navigator 11-mm probe. These 112 patients and 4 additional patients who for various reasons did not have the MIRP procedure yet had positive MIBI scans were compared to 57 patients who had clearly negative MIBI parathyroid imaging. Results: Follow-up data were available for 108 of 112 patients who underwent MIRP. No patients had persistent hypercalcemia. The long-term success rate for the MIRP group was 98%. Fifty-two percent of the MIRP procedures were performed using MAC anesthesia. Overall, gland weight and serum PTH were related to the probability of a positive MIBI scan. Multiple logistic regression revealed that females were more likely to exhibit positive scans than were males for any fixed serum PTH level. For females, there was a significant relationship between increasing serum parathyroid hormone and a positive MIBI scan. Conversely, in males, the relationship between scan positivity and serum parathyroid hormone was weaker. Conclusions: The MIRP technique without routine intraoperative serum parathyroid hormone measurement resulted in an excellent cure rate for primary hyperparathyroidism. As the MIRP technique as well as other techniques for unilateral cervical exploration are predicated on a positive parathyroid scan, the possible effect of gender on the sensitivity of MIBI scintigraphy for the detection of parathyroid adenomas warrants further investigation.
• Billheimer, D. (2001). Compositional receptor modeling. Environmetrics, 12(5), 451-467.
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  Abstract: Receptor models apportion an ambient mixture of pollutants to the contributing pollution sources. Often, neither the number of sources nor their chemical profiles are known precisely. The dual goals of modeling are to estimate the chemical 'signature' of the sources, and to characterize the mixing process. The author develops a novel modeling approach for receptor data where all model components are compositions (i.e. vectors of proportions). This approach maintains positivity and summation constraints for source contributions and chemical profiles. Further, it incorporates available prior knowledge regarding the source chemical profiles. Including prior knowledge allows parameter estimation while avoiding restrictive assumptions regarding presence or absence of chemical tracers. This approach is illustrated by modeling air pollution data collected from a receptor near Juneau, Alaska. The compositional model produces point estimates of source profiles and mixing proportions similar to those obtained in a previous study. However, interval estimates for mixing proportions are roughly 30 per cent shorter than those found previously. Copyright © 2001 John Wiley & Sons, Ltd.
• Billheimer, D., Guttorp, P., & Fagan, W. F. (2001). Statistical Interpretation of Species Composition. Journal of the American Statistical Association, 96(456), 1205-1213.
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  Abstract: The relative abundance of different species characterizes the structure of a biological community. We analyze an experiment addressing the relationship between omnivorous feeding linkages and community stability. Our goal is to determine whether communities with different predator compositions respond similarly to environmental disturbance. To evaluate these data, we develop a hierarchical statistical model that combines Aitchison's logistic normal distribution with a conditional multinomial observation distribution. In addition, we present an algebra for compositions that includes addition, scalar multiplication, and a metric for differences in compositions. The algebra aids interpretation of treatment effects, treatment interactions, and covariates. Markov chain Monte Carlo (MCMC) is used for inference in a Bayesian framework. Our experimental results indicate that a high degree of omnivory can help to stabilize community dynamics and prevent radical shifts in community composition. This result is at odds with classical food-web predictions, but agrees with recent theoretical formulations.
• Uchino, K., Billheimer, D., & Cramer, S. C. (2001). Entry criteria and baseline characteristics predict outcome in acute stroke trials. Stroke, 32(4), 909-916.
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  PMID: 11283391;Abstract: Background and Purpose - We sought to study the range of entry criteria and baseline characteristics in acute stroke trials and to understand their effects on patient outcomes. Methods - Randomized, placebo-controlled therapeutic trials in patients with acute ischemic stroke were identified. Entry criteria, baseline clinical characteristics, and outcome were extracted for the placebo group of each trial. The relationship between key variables was then determined. Results - Across 90 placebo groups identified, there was great variation in entry criteria and outcome measures. This was associated with divergent outcomes; for example, in some studies most placebo group patients died, while in other studies nearly all had no disability. Entry criteria were significantly correlated with outcome; for example, higher age cutoff for study entry correlated with 3-month mortality. Entry criteria also predicted baseline clinical characteristics; for example, wider time window for study entry correlated directly with time to treatment and inversely with stroke severity (initial National Institutes of Health Stroke Scale score). Baseline characteristics predicted outcome. Greater stroke severity predicted higher 3-month mortality rate; despite this, successful thrombolytic trials have enrolled more severe strokes than most trials. The mean age of enrollees also predicted 3-month mortality and was inversely related to percentage of patients with 3-month Barthel Index score ≥95. The strongest predictors of 3-month mortality were obtained with multivariate models. Conclusions - Acute stroke studies vary widely in entry criteria and outcome measures. Across multiple studies, differences in entry criteria, and the baseline clinical characteristics they predict, influence patient outcomes along a continuum. In some studies, enrolling a specific subset of patients may have improved the chances of identifying a treatment-related effect, while in others, such chances may have been reduced. These findings may be useful in the design of future stroke therapeutic trials.
• Billheimer, D., Cardoso, T., Freeman, E., Guttorp, P., Ko, H., & Silkey, M. (1997). Natural variability of benthic species composition in the Delaware Bay. Environmental and Ecological Statistics, 4(2), 95-115.
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  Abstract: Biological monitoring of aquatic biota is used to assess the impact of changes in the environment. Critical to the development of a sound biological monitoring protocol is the judicious selection of organisms and organism characteristics to be monitored. Accurate interpretations of change necessitate description of the natural variability of the system. We introduce a state-space model for compositional monitoring data, and illustrate how one can incorporate spatial structure and covariates to assess natural variability. The methods are illustrated on benthic survey data from Delaware Bay, and applied to proportional composition at the genus level. The distribution of benthic macroinvertebrates in Delaware Bay depends significantly on salinity. There is residual spatial dependence in the data after accounting for the salinity effect.
• Billheimer, D., & Guttorpz, P. (1995). Zooplankton proportion estimates from non-uniform sample volumes. Environmental and Ecological Statistics, 2(2), 117-124.
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  Abstract: The relative abundance of organisms from different taxa provides information about ecosystem health and diversity. When the numbers of sampled organisms are modelled as Poisson counts, and the sample volumes are not uniform, variance for the proportion attributable to each taxon is difficult to compute. We present a method for computing approximate variances for this situation. The point estimates and their standard errors reduce to the standard multinomial maximum likelihood results when sample volumes are uniform. Further, given initial estimates of population densities for the taxa of interest, optimal sample volumes can be computed. The methods are illustrated for zooplankton counts from Andrus Lake, Michigan. © 1995 Chapman & Hall.
• Gibbens, R. P., Havstad, K. M., Billheimer, D. D., & Herbel, C. H. (1993). Creosotebush vegetation after 50 years of lagomorph exclusion. Oecologia, 94(2), 210-217.
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  Abstract: In 1939, an experiment was established on the Jornada Experimental Range to evaluate the effects of shrub removal, rabbit exclusion, furrowing, and seeding in creosotebush [Larrea tridentata (DC.) Cov] vegetation. Sixteen plots (21.3×21.3 m) were laid out in four rows of four plots per row with a buffer zone of 7.6 m between plots and rows. A barbed wire fence excluded cattle and poultry wire fencing excluded lagomorphs. Treatments were factorially applied at two levels. Plant cover in the plots was sampled in 1938 (before treatment), 1947, 1956, 1960, 1967 and 1989 with randomly located, line-intercept transects. Data from all sampling dates were analyzed as a split plot in time and main effects for 1989 tested by analysis of variance for a 2×4 factorial experiment. There were significant (P

Presentations

• Beamer, P., Yazzie, J., Clausen, R., Begay, M., Charley, P., Begay, M., Teufel-Shone, N., Billheimer, D. D., Ingram, J., & Chief, K. (2019, April). Community-driven University Partnerships to Assess Exposures and Risk Perceptions following the Gold King Mine Spill. Department of Chemistry Seminar Series. Missoula, MT: University of Montana.
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  Chief, K., J. Ingram, D. Billheimer, N. Teufel-Shone, M.G. Begay, P. Charley, M. Begay, R. Clausen, J. Yazzie, and P. Beamer. 2019.Community-driven University Partnerships to Assess Exposures and Risk Perceptions following the Gold King Mine Spill. Department of Chemistry Seminar Series, University of Montana, April 8, 2019, Missoula, MT. (Invited Talk)
• Beamer, P., Yazzie, J., Clausen, R., Begay, M., Charley, P., Begay, M., Teufel-Shone, N., Billheimer, D. D., Ingram, J., & Chief, K. (2019, April). K’é bee da’ahííníítą: Strength through the Diné clan system to respond to the Gold King Mine Spill: Video and Childrens’ Story. Agnese Nelms Haury Foundation Celebrating 5 years of Impact. Tucson, AZ: University of Arizona.
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  Chief, K., J. Ingram, D. Billheimer, N. Teufel-Shone, M.G. Begay, P. Charley, M. Begay, R. Clausen, J. Yazzie, and P. Beamer. 2019. K’é bee da’ahííníítą: Strength through the Diné clan system to respond to the Gold King Mine Spill: Video and Childrens’ Story. Agnese Nelms Haury Foundation Celebrating 5 years of Impact, April 24, 2019, University of Arizona, Tucson, AZ.
• Beamer, P., Yazzie, J., Clausen, R., Begay, M., Charley, P., Begay, M., Teufel-Shone, N., Billheimer, D. D., Ingram, J., & Chief, K. (2019, February). Tó’Łítso, the water is yellow: Investigating short-term exposure and risk perception of Navajo communities to the Gold King Mine Toxic Spill.. Winter Institute and Tribal Forum, Convergence and Divergence in Native Health Research,. Hotel Tucson City Center, Tucson, AZ.: University of Arizona Native American Research and Training Center (NARTC).
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  Chief, K., J. Ingram, D. Billheimer, N. Teufel-Shone, M.G. Begay, P. Charley, M. Begay, R. Clausen, J. Yazzie, and P. Beamer. 2019. Tó’Łítso, the water is yellow: Investigating short-term exposure and risk perception of Navajo communities to the Gold King Mine Toxic Spill. University of Arizona Native American Research and Training Center (NARTC) Winter Institute and Tribal Forum, Convergence and Divergence in Native Health Research, The Confluence of Traditional and Contemporary Medicine, February 26, 2019, Hotel Tucson City Center, Tucson, AZ.
• Chief, K., Hou, X., Ingram, J., Froyum, J., VanHorne, Y. O., Billheimer, D. D., Teufel-Shone, N., & Beamer, P. (2019, January). To' Litso the Water Is Yellow: Arsenic and Lead in Sediment and Agricultural Soil Along the San Juan River on the Navajo Nation One Year after the Gold King Mine Spill. 2018-2019 International Soils Meeting. San Diego, CA.
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  Chief, K., X. Hou, J. Ingram, P. Beamer, J.D. Froyum, Y. Ornelas Van Horne, D. Billheimer and N. Teufel-Shone. 2019. To' Litso the Water Is Yellow: Arsenic and Lead in Sediment and Agricultural Soil Along the San Juan River on the Navajo Nation One Year after the Gold King Mine Spill. 2018-2019 International Soils Meeting, January 6-9, 2019, San Diego, CA.
• Beamer, P., Lothrop, N., Yazzie, J., Begay, M., Charley, P., Begay, M., Teufel-Shone, N., Billheimer, D. D., Ingram, J., Yazzie, C. D., & Chief, K. (2018, June). Tó’Łítso, the water is yellow: Environmental sampling results for soil and sediment. New Mexico Water Resources Research Institute Conference, Environmental Conditions of the Animas and San Juan River Watersheds with Emphasis on Gold King Mine and Other Mine Waste Issues. Shiprock Chapter House, Shiprock, NM: New Mexico Water Resources Research Institute Conference.
• Beamer, P., Lothrop, N., Yazzie, J., Begay, M., Charley, P., Begay, M., Teufel-Shone, N., Billheimer, D. D., Ingram, J., Yazzie, C. D., & Chief, K. (2018, September). Overview of the Gold King Mine Spill Diné Exposure Project. Navajo Emergency Response Executive Session: Lessons learned from the Gold King Mine Spill. Northern Arizona University, Flagstaff, AZ.: University of Arizona.
• Beamer, P., Yazzie, J., Begay, M., Charley, P., Begay, M., Teufel-Shone, N., Billheimer, D. D., Ingram, J., & Chief, K. (2018, April). Desecration of Tó Baka’e and Diné way of life: How the Navajo Sacred Male River of the San Juan became the Yellow River. Restoring K’e Conference. University of Arizona, Tucson, AZ: University of Arizona Superfund Research Program.
• Beamer, P., Yazzie, J., Begay, M., Charley, P., Begay, M., Teufel-Shone, N., Billheimer, D. D., Ingram, J., & Chief, K. (2018, August). Incorporating Diné Perspectives in Assessing Temporal and Spatial Changes of Contaminants after the Gold King Mine Spill in Navajo Agricultural Communities. Water in the Native World: A Symposium on Indigenous Water Knowledge and Hydrologic Science. Salish Kootenai College, Pablo, MT: University of Arizona.
• Beamer, P., Yazzie, J., Begay, M., Charley, P., Begay, M., Teufel-Shone, N., Billheimer, D. D., Ingram, J., & Chief, K. (2018, November). Community-driven university partnerships to assess exposures and risk perceptions of Diné communities following the Gold King Mine Spill. Stanford University Department of Earth System Science Seminar Series. Stanford University, Stanford, CXA: Stanford University.
• Beamer, P., Yazzie, J., Begay, M., Charley, P., Begay, M., Teufel-Shone, N., Billheimer, D. D., Ingram, J., & Chief, K. (2018, October). Cultural values of water. Indigenous Water Ethics: Sacred waters connecting culture, people, & place. University of Arizona, Tucson, AZ: University of Arizona Superfund Research Program.
• Beamer, P., Yazzie, J., Begay, M., Charley, P., Begay, M., Teufel-Shone, N., Billheimer, D. D., Ingram, J., Chief, K., & Yazzie, C. D. (2018, December). Incorporating Dine’ Perspectives in Assessing Environmental Impact of the Gold King Mine Spill in Navajo Agricultural Communities. American Geophysical Union Fall Meeting. Washington D.C: American Geophysical Union.
• Billheimer, D. D. (2018, April). How to BAD: Bayesian Adaptive Design for Clinical Trials. Bayesian Statistics: A Paradigm for 21st Century Science. University of Arizona, Tucson, AZ: UA Statistics Consulting Laboratory (Stat Lab).
• Chief, K., Murphy, M., Ingram, J., Billheimer, D. D., Lothrop, N., Van Horne, Y. O., Pine, B., Begay, M., & Beamer, P. (2018, March). Honoring Traditional Knowledge in Understanding Short-Term Exposures of Navajo Communities after the Gold King Mine Spill. Bridging the Cultural Divide: The Role of Community Health Representatives/Workers in Environmental Public Health. Webinar for NIH/NIEHS Partners in Environmental and Public HealthNIH/NIEHS.
• Chief, K., Yazzie, J., Begay, M., Charley, P., Begay, M., Ornelas Van Horne, Y., Billheimer, D. D., Ingram, J., & Beamer, P. (2018, March). Arsenic and Lead in Sediment in river and canals in 3 Navajo communities 1 year after the Gold King Mine Spill. University of Arizona Cooperative Extension Shiprock Agriculture Days. Shiprock, NM: University of Arizona Cooperative Extension Shiprock Agriculture Days.
• Gerald, L. B., Martinez, F., Billheimer, D. D., Hallmark, B., & Gerald, J. K. (2018, May). Are hispanic children of mexican origin less responsive to ICS treatment than non-hispanic white children: A Meta-Analysis of CARE trials. American Thoracic Society International Conference. San Diego, CA: American Thoracic Society.
• Gerald, L. B., Moore, M. A., Clemens, C. J., Brown, M., Billheimer, D. D., Fisher, J., & Gerald, J. K. (2018, May). Controller medication prescribing and adherence among medicaid insured children enrolled in the supervised asthma medicine in schools study. American Thoracic Society International Conference. San Diego, CA: American Thoracic Society.
• Martinez, F., Wright, A. L., Halonen, M., Billheimer, D. D., Zhai, J., Stern, D. A., O'Rourke, M. K., Lothrop, N., Guerra, S., Furlong, M., & Beamer, P. (2018, August). CC16 levels into adult life are associated with nitrogen dioxide exposure at birth. Joint meeting of the International Society of Exposure Science and the International Society of Environmental Epidemiology. Ottawa, Canada.
• Chief, K., Begay, M., Ornelas Van Horne, Y., Lothrop, N. Z., Billheimer, D. D., Ingram, J., Murphy, M., & Beamer, P. (2017, September/Fall). Honoring traditional knowledge in understanding short term exposures of Navajo communities after the Gold King Mine Spill. National Native Health Research Training Conference. Denver, CO.
• Chief, K., Chief, K., Beamer, P., Beamer, P., Torabzadehkhorasani, E., Torabzadehkhorasani, E., Lothrop, N. Z., Lothrop, N. Z., Billheimer, D. D., Billheimer, D. D., Ornelas Van Horne, Y., Ornelas Van Horne, Y., Ingram, J., & Ingram, J. (2017, June/Summer). Tó’Łítso, the water is yellow: Water quality results of the San Juan River on the Navajo Nation one year after the Gold King Mine Spill. New Mexico Water Resources Research Institute. San Juan College, Farmington, NM.
• Gerald, J. K., Brown, M., Fisher, J., Billheimer, D. D., Clemens, C. J., Moore, M. A., Carvajal, S. C., Bryson, D., Stefan, N., & Gerald, L. B. (2017, May). Supervised Medicine in Schools: The SAMS Study. American Thoracic Society International Conference. Washington, DC: American Thoracic Society.
• Ingram, J., Chief, K., Torbzadehkhorasani, E., Lothrop, N., Billheimer, D. D., Ornelas Van Horne, Y., Settimo, A., & Beamer, P. (2017, Fall). Tó'Łítso, the water is yellow: Water, agricultural, and sediment quality results of the San Juan River on the Navajo Nation one year after the Gold King Mine Spill. Navajo Nation Human Research Review Board Conference. Window Rock, AZ.
• Lothrop, N., Hussaini, K., Billheimer, D. D., & Beamer, P. (2017, Fall). Community-Level Characteristics and Environmental Factors of Child Respiratory Illnesses in Southern Arizona. International Society of Exposure Science Conference. Research Triangle Park, NC.
• Lowe, A., Gerald, J. K., Fisher, J., Billheimer, D. D., Carvajal, S. C., & Gerald, L. B. (2017, May). Among children with asthma, greater acculturation is associated with having a medical home. American Thoracic Society International Conference. Washington, DC: American Thoracic Society.
• Oren, E., Barrett, E., Fisher, J., Billheimer, D. D., Laudenslager, M., & Gerald, L. B. (2017, May). Hair Biomarkers Among Middle-School Children in Relation to School Stress and Asthma. Society for Behavioral Medicine International Conference. San Diego, CA: Society for Behavioral Medicine.
• Chief, K., Beamer, P., Ingram, J., Teufel-Shone, N. I., Billheimer, D. D., & Begay, M. A. (2016, December). What happened at the Gold King Mine Spill and to the Navajo People? -- University Response to assist through Navajo Project Partnerships. National Institute of Environmental Health Sciences “Environmental Health Science FEST” (EHS FEST). Durham, North Carolina.
• Beamer, P., Guerra, S., Lothrop, N. Z., Stern, D. A., Lu, Z., Billheimer, D. D., Halonen, M., Wright, A. L., & Martinez, F. D. (2015, May). Childhood CC16 levels are associated with diesel exposure at birth. American Thoracic Society International Conference 2015. Denver, CO: American Thoracic Society.
• Beamer, P., Lothrop, N. Z., Stern, D. A., Billheimer, D. D., Wright, A. L., & Martinez, F. D. (2014, May). Wheezing Lower Respiratory Illnesses Associated With Diesel Exposure In Children Of Younger Mothers. American Thoracic Society International Conference 2014. San Diego, CA: American Thoracic Society.

Poster Presentations

• Oren, E., Oren, E., Combs, D. A., Combs, D. A., Fisher, J., Fisher, J., Goodwin, J., Goodwin, J., Billheimer, D. D., Billheimer, D. D., Gerald, J. K., Gerald, J. K., Clemens, C. J., Clemens, C. J., Brown, M. A., Brown, M. A., Gerald, L. B., Gerald, L. B., Oren, E., , Combs, D. A., et al. (2016, May). Impact of supervised asthma medication use on sleep outcomes of elementary school children. 2016 International Meeting of the American Thoracic Society. San Francisco, CA.
• Beamer, P., Beamer, P., Guerra, S., Guerra, S., Lothrop, N., Lothrop, N., Stern, D., Stern, D., Lu, Z., Lu, Z., Billheimer, D. D., Billheimer, D. D., Halonen, M., Halonen, M., Wright, A. L., Wright, A. L., Martinez, F., Martinez, F., Beamer, P., , Beamer, P., et al. (2015, May). Childhood CC16 Levels are Associated with Diesel Exposure at Birth. American Thoracic Society International Conference.
• Beamer, P., Guerra, S., Lothrop, N. Z., Stern, D., Lu, Z., Billheimer, D. D., Halonen, M., Wright, A. L., & Martinez, F. (2015, May). Levels are Associated with Diesel Exposure at Birth. American Thoracic Society. Denver, CO.
• Berry, C. E., Jenkins, I., Billheimer, D. D., Stern, D., Guerra, S., Wright, A. L., Morgan, W. J., & Martinez, F. (2015, May). Lung Function Trajectories in the Tucson Children’s Respiratory Study. American Thoracic Society. Denver, CO.
• Lothrop, N. Z., Hussaini, K., Billheimer, D. D., & Beamer, P. I. (2014, October). Respiratory Illness Prevalence and Geographic Risk Factors in Southern Arizona. International Society of Exposure Science Annual Meeting 2014International Society of Exposure Science.

Others

• Beamer, P. I., Lothrop, N., Stern, D. A., Billheimer, D., Wright, A. L., & Martinez, F. D. (2015, September). Increased wheezing risk with diesel exposure among children of younger mothers. The European respiratory journal.
• Yankeelov, T. E., DeBusk, L. M., Billheimer, D. D., Luci, J. J., Lin, P. C., Price, R. R., & Gore, J. C. (2006, Nov). Repeatability of a reference region model for analysis of murine DCE-MRI data at 7T. Journal of magnetic resonance imaging : JMRI.
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  To test the repeatability of a reference region (RR) model for the analysis of dynamic contrast-enhanced MRI (DCE-MRI) in a mouse model of cancer at high field.