Laboratory Fellow, Chemist, Pathogen Biology Team Leader
Laboratory Fellow, Chemist, Pathogen Biology Team Leader

Biography

David Wunschel’s primary research focus has been on forensics and attribution of biological and biochemical threat agents. Specific focus has been on the development of forensic laboratory methods using proteomic, lipid, carbohydrate, and toxic metabolite profiling of microbial and toxic agents. Other topic areas include biomarker discovery, bacterial identification/taxonomy and bacterial interactions with their environment. More recent project work focused on untargeted metabolite identification for new toxin analogs and the development of novel activity assays. He has been an invited participant on multiple national biodefense panels and edited multiple special journal issues and book chapters on microbial and toxinforensics.

Research Interest

  • forensics and attribution
  • microbial response to environment
  • toxin activity assays
  • host response to infection
  • aquatic toxins

Disciplines and Skills

  • Liquid Chromatography-Mass Spectrometry (LC-MS)
  • Mass Spectrometry
  • Metabolite Identification
  • Microbial Structure
  • Protein Characterization
  • Bacterial Taxonomy
  • Microbial and Plant Toxins

Education

PhD in biomedical sciences, University of South Carolina School of Medicine

BS in biological sciences, Clemson University

Affiliations and Professional Service

  • Member and Committee Organizer, American Chemical Society
  • Member, American Society for Microbiology
  • Member, American Society for Mass Spectrometry
  • Member and Committee Organizer, International Symposium on the Interface between Analytical Chemistry and Microbiology
  • Editor, Journal of Microbiological Methods
  • Editor, Frontiers in Bioengineering and Biotechnology

Awards and Recognitions

PNNL business developer (2015) and project manager (2018)

Patents

Beck K.M. and D.S. Wunschel. January 20, 2004. High Spatial Resolution Matrix Assisted Laser Desorption/Ionization (MALDI). US Patent No. 6,680,477.

Publications

2022

  • Kaiser B.L.D., D. Birdsell, J.R. Hutchison, M. Forsman, J. Thelaus, S.C. Jenson, V. Andrianaivoarimanana, M. Bystrom, K. Myrtennas, R.F. McDonough, R.D. Nottingham, J.W. Sahl, H.P. Schweizer, M. Rajerison, M. Forsman, D.S. Wunschel, and D.M. Wagner. 2022. “Proteomic signatures of antimicrobial resistance in Yersinia pestis and Francisella tularensis.” Frontiers in Medicine 9:821071. https://doi.org/10.3389/fmed.2022.821071  

2019

  • Merkley E.D., D.S. Wunschel, K.L. Wahl, and K.H. Jarman. 2019. “Applications and Challenges of Forensic Proteomics.” (Review). Forensic Science International 297:350-363. PNNL-SA-139574. https://doi.org/10.1016/j.forsciint.2019.01.022 

2018

  • Dunbar J., S. Pillai, D. Wunschel, M. Dickens, S. Morse, D. Franz, A. Bartko, J. Challacombe, T. Persons, M.S. Hughes, S.R. Blanke, R. Holland, J. Hutchison, E. Merkley, K. Campbell, C. Branda, S. Sharma, L. Lindler, K. Anderson, and D. Hodge. 2018. “Perspective on improving environmental monitoring of biothreats.” Frontiers in Bioengineering and Biotechnology, (Biosafety Biosec.) 6:147. https://doi.org/10.3389/fbioe.2018.00147 Accepted: 25 Sep 2018.
  • Fredriksson S.A., D.S. Wunschel, S.W. Lindström, C. Nilsson, K.L. Wahl, and C. KL Åstot. 2018. “A Ricin Forensic Profiling Approach Based on a Complex Set of Biomarkers.” Talanta. 186:628-635. PNNL-SA-130526. https://doi.org/10.1016/j.talanta.2018.03.070. PNNL-SA-130526.
  • Jarman K.H., N.C. Heller, S.C. Jenson, J.R. Hutchison, B.L. Deatherage Kaiser, S.H. Payne, D.S. Wunschel, and E.D. Merkley. 2018. “Proteomics Goes to Court: A Statistical Foundation for Forensic Toxin/Organism Identification Using Bottom-Up Proteomics.” Journal of Proteome Research 17(9):3075-3085. https://doi.org/10.1021/acs.jproteome.8b00212
  • Leiser O.P., J.K. Blackburn, T.L. Hadfield, H.W. Kreuzer, D.S. Wunschel, and C.J. Bruckner-Lea. 2018. “Laboratory strains of Bacillus anthracis exhibit pervasive alteration in expression of proteins related to sporulation under laboratory conditions relative to genetically related wild strains.” PLoS ONE 13(12): e0209120. https://doi.org/10.1371/journal.pone.0209120
  • Wunschel D.S., J.R. Hutchison, B.L. Deatherage Kaiser, B.M. Hess, M.A. Sydor, A. Lin, and M.G. Warner. 2018. “Proteomic Signatures Differentiating Bacillus anthracis Sterne Sporulation on Soil Relative to Laboratory Media.” Analyst 143(1):123-132. Dec 18. PNNL-SA-113414. https://doi.org/10.1039/C7AN01412K
  • Wunschel D.S., B.R. Valenzuela, B.L. Kaiser, and K. Victry. 2018. “Method Development for Comprehensive Extraction and Analysis of Marine Toxins: Liquid-Liquid Extraction and Tandem Liquid Chromatography Separations Coupled to Electrospray Tandem Mass Spectrometry.” Talanta 187:302-307. PNNL-SA-131966https://doi.org/10.1016/j.talanta.2018.05.019

2017

  • Merkley E., S. Jenson, J. Arce, A. Melville, O. Leiser, D. Wunschel, and K. Wahl. 2017. “Ricin-like proteins from the castor plant do not influence liquid chromatography-mass spectrometry detection of ricin in forensically relevant samples.” Toxicon 140:18-31. PNNL-SA-124754. https://doi.org/10.1016/j.toxicon.2017.10.004

2016

  • Mo K.F., Z. Dai, and D.S. Wunschel. 2016. “Production and Characterization of Desmalonichrome Relative Binding Affinity for Uranyl Ions in Relation to Other Siderophores.” Journal of National Products 79(6):1492–1499. PNNL-SA-113689. https://doi.org/10.1021/acs.jnatprod.5b00933

2015

  • Kaiser Deatherage, B.L., D.S. Wunschel, M.A. Sydor, M.G. Warner, K.L. Wahl KL, and J.R. Hutchison. 2015. “Improved Proteomic Analysis Following Trichloroacetic Acid Extraction of Bacillus anthracis spore proteins.” Journal of Microbiological Methods 118:18-24. https://doi.org/10.1016/j.mimet.2015.08.008
  • Hess B.M., B.L. Deatherage Kaiser, M.A. Sydor, D.S. Wunschel, C.J. Bruckner-Lea, and J.R. Hutchison. 2015. “Discerning Viable from Nonviable Yersinia pestis pgm- and Bacillus anthracis. Journal of Bioterrorism Biodefense 6:3. https://doi.org/10.4172/2157-2526.1000138

2014

  • Kooken J., K. Fox, A. Fox, D. Altomare, K. Creek, D. Wunschel, S. Pajares-MerinoS, I. Martínez-Ballesteros, J. Garaizar, O. Oyarzabal, and M. Samadpour. 2014. “Identification of Staphylococcal Species Based on Variations in Protein Sequence (Tandem Mass Spectrometry) and DNA Sequence (Microarray).” Molecular and Cellular Probes 28(1):41-50. doi: 10.1016/j.mcp.2013.10.003 
  • Kooken J., K. Fox, A. Fox, and D. Wunschel. 2014. “Assessment of Marker Proteins Identified in Whole Cell Extracts for Bacterial Speciation Using Liquid Chromatography Electrospray Tandem Mass Spectrometry.” Molecular and Cellular Probes 28(1):34-40. doi:10.1016/j.mcp.2013.08.002.
  • Webb-Robertson B.J.M., C.D. Corley, L.A. McCue, B.H. Clowers, C.P. Dowling, K.L. Wahl, D.S. Wunschel, and H.W. Kreuzer. 2014. “Forensic Signature Detection of Yersinia pestis Culturing Practices across Institutions Using a Bayesian Network.” Journal of Forensic Investigation 2(1):7.
  • Wunschel D., H. Engelmann, K. Victry, B. Clowers, C. Sorensen, N.B. Valentine, C. Mahoney, T.W. Wietsma, and K.L. Wahl. 2014. “Protein markers for identification of Y. pestis and their variation related to culture.” Molecular and Cellular Probes 28:65-72. doi:10.1016/j.mcp.2013.12.001

2013

  • Clowers B.H., D.S. Wunschel, H.W. Kreuzer, H.E. Engelmann, N. Valentine, and K.L. Wahl. 2013. “Characterization of Residual Medium Peptides from Yersinia pestis Cultures.” Analytical Chemistry 85(8):3933-9. doi.org/10.1021/ac3034272.
  • Schultz I.R., J. Nagler, P. Swanson, D. Wunschel, A.D. Skillman, V. Burnett, D. Smith, and R. Barry. 2013. “Toxicokinetic, Toxicodynamic and Toxicoproteomic Aspects of Short-term Exposure to Trenbolone in Female Fish.” Toxicological Sciences 136 (2):413-429. doi: 10.1093/toxsci/kft220. http://toxsci.oxfordjournals.org/content/early/2013/09/26/toxsci.kft220.long
  • Wunschel D.S., E. Tulman, H. Engelmann, B.H. Clowers, S. Geary, A. Robinson, and X. Liao. 2013. “Forensic Proteomics of Poxvirus Production.” Analyst 138:6385. DOI: 10.1039/C3AN00711A

2012

  • Varnum S.M., B.J.M. Webb-Robertson, J.G. Pounds, R.J. Moore, R.D. Smith, C.W. Frevert, S.J. Skerrett, and D.S. Wunschel. 2012. “Proteomic Analysis of bronchoalveolar lavage fluid proteins from mice infected with Francisella tularensis. Journal of Proteome Research 11(7):3690-703. DOI10.1021/pr3001767.
  • Wunschel D.S., A.M. Melville, C.J. Ehrhardt, H.A. Colburn, K.D. Victry, K.C. Antolick, J.H. Wahl, and K.L. Wahl. 2012. “Integration of Gas Chromatography Mass Spectrometry Methods for Differentiating Ricin Preparation Methods.” Analyst 137(9):2077-2085. DOI: 10.1039/C2AN16186A

2011

  • Colburn H.A., D.S. Wunschel, K.C. Antolick, A.M. Melville, and N.B. Valentine. 2011. “The Effect of Growth Medium on B. anthracis Sterne Spore Carbohydrate Content.” Journal of Microbial Methods 85(3):183-9. doi: 10.1016/j.mimet.2011.02.015
  • Wunschel D.S., K.L. Wahl, H.A. Colburn, A.M. Melville, C.M. Sorensen, N.B. Valentine, and C.L. Stamper. 2011. “Determination of Post-Culture Processing with Carbohydrates by MALDI-MS and TMS derivatization GC-MS.” Talanta 85:2352-2360. https://doi.org/10.1016/j.talanta.2011.07.073

2010

  • Colburn H.A., D.S Wunschel, H.W. Kreuzer, J.J. Moran, K.C. Antolick, and A.M. Melville. 2010. “Analysis of carbohydrate and fatty acid marker abundance in ricin toxin preparations for forensic information.” Analytical Chemistry 82(14):6040-6047. https://doi.org/10.1021/ac1006206
  • Wahl K.L., H.A. Colburn, D.S. Wunschel, C.E. Petersen, K.H. Jarman, and N.B. Valentine. 2010. “Residual Agar Determination in Bacterial Spores by Electrospray Ionization Mass Spectrometry.” Analytical Chemistry 82(4):1200-1206. https://doi.org/10.1021/ac901491c

2009

  • Johnson T.J., Y.F. Su, N.B. Valentine, H.W. Kreuzer-Martin, K.L. Wahl, S.D. Williams, B.H. Clowers, and D.S. Wunschel. 2009. “The Infrared Spectra of Bacillus Bacteria Part I: Vegetative Bacillus versus Sporulated Cells and the Contributions of Phospholipids to Vegetative Infrared Spectra.” Applied Spectroscopy 63(8):899-907. doi: 10.1366/000370209788964430
  • Webb-Robertson B.J.M., L.A. McCue, N. Beagley, J.E. Mcdermott, D.S. Wunschel, S.M. Varnum, J.Z. Hu, N.G. Isern, G.W. Buchko, K. Mcateer, J.G. Pounds, S.J. Skerrett, D. Liggett, and C.W. Frevert. 2009. “A Bayesian Integration Model of High Throughput Proteomics and Metabolomics Data for Improved Early Detection of Microbial Infections.” Biocomputing 2009:451-463. https://doi.org/10.1142/9789812836939_0042
  • Wunschel D.S., B.J.M. Webb-Robertson, C.W. Frevert, S.J. Skerrett, N. Beagley, A. Willse, H.A. Colburn, and K. Antolick. 2009. “Differentiation of Gram-Negative Bacterial Aerosol Exposure Using Detected Markers in Bronchial-Alveolar Lavage Fluid.” Public Library of Science ONE 4(9): e7047.

2008

  • Wunschel D.S., H.C. Edberg, A. Fox, K.F. Fox, W.M. Harley, K.L. Wahl, and J.H. Wahl. 2008. “Detection of Agar, by Analysis of Sugar Markers, Associated with Bacillus anthracis spores after culture.” Journal of Microbiological Methods 74(2-3):57-63. https://doi.org/10.1016/j.mimet.2008.04.003
  • Jarman K.H., N.B. Valentine, J.B. Cliff, H. Kreuzer, C.E. Petersen, H.C. Edberg, D.S. Wunschel, and K.L. Wahl. 2008. “Bayesian-Integrated Microbial Forensics.” Applied and Environmental Microbiology. 74 (11):3573-3582. doi: 10.1128/AEM.02526-07

2007

  • Campbell J.A., D.S. Wunschel, and C.E. Petersen. 2007. Analysis of Melamine, Cyanuric Acid, Ammelide, and Ammeline Using Matrix-Assisted Laser Desorption Ionization/Time-of-Flight Mass Spectrometry (MALDI/TOFMS).Analytical Letters 40(16-18):3107-3118. https://doi.org/10.1080/00032710701646131

2006

  • Liu G., J. Wang, D. Wunschel, and Y. Lin. 2006. Electrochemical Proteolytic Beacon for Detection of Matrix Metalloproteinase Activities. Journal of the American Chemical Society 128(38):12382-12383. https://doi.org/10.1021/ja0626638
  • Shi L., U. Mayer-Cumblidge M. Marshall, S. Ni, A. Beliaev, E. Hill, B. Chen, Y.A. Gorby, B.H. Lower, D.W. Kennedy, H.M. Mottaz, Z. Wang, D.S. Wunschel, T.C. Squier, J.K. Fredrickson, and J.M. Zachara. 2006. Isolation of High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-type Cytochromes of Shewanella oneidensis MR-1.” Journal of Bacteriology 188(13):4705-4713. doi: 10.1128/JB.01966-05
  • Wunschel D.S., J. Wahl, A. Willse, N. Valentine, and K. Wahl. 2006. “Small protein biomarkers of culture in Bacillus spores detected using capillary liquid chromatography coupled with matrix assisted laser desorption/ionization mass spectrometry.” Journal of Chromatography B 843:25-33. doi: 10.1016/j.jchromb.2006.05.021 

2005

  • Ahram M., J.N. Adkins, D.L. Auberry, D.S. Wunschel, and D.L. Springer. 2005. “A proteomic approach for discovery to characterize protein shedding.” Proteomics 5:123-131. DOI: 10.1002/pmic.200400912
  • Cliff J., D.S. Wunschel, D.J. Gaspar, K.H. Jarman, S. Golledge, N.B. Valentine, and K.L. Wahl. 2005. “Growth media differentiation of Bacillus subtilis spores by elemental characterization using time of flight secondary ion mass spectrometry.” Applied and Environmental Microbiology 71(11):6524-30. DOI: 10.1128/AEM.71.11.6524-6530.2005
  • Fox, Alvin, Larsson, Lennart, Allmaier, Guenter, Wunschel, David S., and Wahl, Karen L. 2005. "Preface: 5th International Symposium on the Interface between Analytical Chemistry and Microbiology - April 19th to 21st, 2004: Hosted at Pacific Northwest National Laboratory". United States. https://doi.org/10.1016/j.mimet.2005.04.010.
  • Valentine N.B., S. Wunschel, D. Wunschel, C. Petersen, and K. Wahl. 2005. “The Effect of Culture Conditions on Microorganism Identification by MALDI Mass Spectrometry.” Applied and Environmental Microbiology 71(1):58-64https://doi.org/10.1128/AEM.71.1.58-64.2005
  • Wunschel D.S., E.A. Hill, J.S. McLean, K.H. Jarman, Y.A. Gorby, N.B. Valentine, and K.L. Wahl. 2005. “Effects of Varied pH, Growth Rate and Temperature using Controlled fermentation and Batch culture on Matrix Assisted Laser Desorption/Ionization Whole Cell Protein Fingerprints.” Journal of Microbiological Methods 62(3):259-271. https://doi.org/10.1016/j.mimet.2005.04.033
  • Wunschel D.S., I.R. Schultz, A.S. Skillman, and K.L. Wahl. 2005 “Method for Detection and Quantitation of Fathead Minnow Vitellogenin (Vtg) by Liquid Chromatography and Matrix Assisted Laser Desorption/ Ionization Mass Spectrometry.” Aquatic Toxicology 73:256-267. DOI: 10.1016/j.aquatox.2005.03.015

2004

  • Springer D.L., D.L. Auberry, M. Ahram, J.N. Adkins, J.M. Feldhaus, J.H. Wah, D.S. Wunschel, and K.D. Rodland. 2004. “Characterization of plasma membrane proteins from ovarian cancer cells using mass spectrometry.” Disease Markers 19:219-228. https://doi.org/10.1155/2004/725617
  • Wunschel D.S., C. Masselon, B.B. Feng, and R.D. Smith. 2004. “Proofreading activity of Pfu thermostable DNA polymerase on a 6-O-Methyl Guanine Containing Template Monitored by ESI-FTICR Mass Spectrometry.” Chembiochem 5(7):1012-1015. https://doi.org/10.1002/cbic.200400059

2003

  • Adkins J.N., S.P. Varnum, K.J. Auberry, R.J. Moore, N.H. Angell, D.S. Wunschel, R.D. Smith, D.L. Springer, and J.G. Pounds. 2003. “Global proteomics for human blood serum I: Multidimensional separation of peptides coupled with mass spectrometry.” Tox. Sci.  72 (1S):240
  • Springer D.L., D. Auberry, J. Adkins, D. Wunschel, J. Feldhaus, J.H. Wahl, and K.D. Rodland. 2003. “Characterization of membrane proteins from ovarian cancer cells using mass spectrometry.” FASEB JOURNAL 17(5):A986-A986.

2002

  • Zangar R.C., A.L. Kimzey, J.R. Okita, D.S. Wunschel, R.J. Edwards, H.S. Kim, and R.T. Okita. 2002. “Formation of High Molecular Weight Cytochrome P450 3A Protein Conjugates in a Process Distinct from the Classical Ubiquitination Pathway.” Molecular Pharmacology 61(4):892-904.

2000

  • Krahmer M., J. Walters, K.F. Fox, A. Fox, K. Creek, L. Pirisi-Creek, D. Wunschel, R. Smith, D. Tabb, and J. Yates III. 2000. “MS for Identification of Single Nucleotide Polymorphisms and MS/MS for Discrimination of Isomeric PCR Product.” Analytical Chemistry 72(17):4033-4040. DOI: 10.1021/ac000142b
  • Wunschel D.S., L. Pasa Tolic, B.B. Feng, and R.D. Smith. 2000. “ESI-FTICR Analysis of Large PCR Products.” Journal of American Society for Mass Spectrometry 11:333-337.

1999

  • Feng B.B., D.S. Wunschel, C.D. Masselon, L. Pasa Tolic, and R.D. Smith. 1999. “Retrieval of DNA Using Soft-Landing after Mass Analysis by ESI-FTICR for Enzymatic Manipulation.” Journal of the American Chemical Society121: 8961-8962. https://doi.org/10.1021/ja9923888

1998

  • Fox K.F., D.S. Wunschel, A. Fox, and GC Stewart. 1998. “Complementarity of GC-MS and LC-MS Analyses for Determination of Carbohydrate Profiles of Vegetative Cells and Spores of bacilli.” Journal of Microbiology Methods 33(1):1-11. https://doi.org/10.1016/S0167-7012(98)00030-X
  • Wunschel D.S., D.C. Muddiman, K.F. Fox, A. Fox, and R.D. Smith. 1998. “Heterogeneity in Bacillus cereus PCR Products Detected by ESI-FTICR Mass Spectrometry.” Analytical Chemistry 70(6): 1203-1207. https://doi.org/10.1021/ac971156t
  • Wunschel D.S., D.C. Muddiman, and R.D. Smith. 1998. “Mass Spectrometric Characterization of DNA for Molecular Biological Applications: Advances using MALDI and ESI.” Advanced Mass Spectrometry, E.J. Karjalainen, A.E. Hesso, J.E. Jalonen, U.P. Karjalainen, Eds., Elsevier Science Publishers B.V. Amsterdam, v14: 377-406.

1997

  • Wunschel D.S., K.F. Fox, A. Fox,M.L. Nagpal, K.K. Kim, G.C. Stewart, and M. Shahgholi. 1997. “Quantitative Analysis of Neutral and Acidic Sugars in Whole Cell Bacterial Hydrolysates Using Anion Exchange Liquid Chromatography Electrospray Tandem Mass Spectrometry.” Journal of Chromatography 776:205-219. DOI: 10.1016/s0021-9673(97)00356-7

1996

  • Muddiman D.C., D.S. Wunschel, C. Liu, L. Pasa Tolic, K.F. Fox, A. Fox, and R.D. Smith. 1996. “Characterization of PCR Products From bacilli Using Electrospray Ionization FT-ICR Mass Spectrometry.” Analytical Chemistry 68(21):3805-3812. https://doi.org/10.1021/ac960689j

1995

  • Wunschel D.S., K.F. Fox, A. Fox, J.E. Bruce, D.C. Muddiman, and R.D. Smith. 1995. “Analysis of Double-Stranded Polymerase Chain Reaction Products from the Bacillus cereus Group by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.” Rapid Communications in Mass Spectrometry 10(1):29-35.

1994

  • Wunschel D.S., K.F. Fox, G.E. Black, and A. Fox. 1994. “Discrimination Among the B. cereus Group, in Comparison to B. subtilis, by Structural Carbohydrate Profiles and Ribosomal RNA Spacer Region PCR.” Systematic and Applied Microbiology 17: 625-635. https://doi.org/10.1016/S0723-2020(11)80085-8 

Other Publications

2019

  • Merkley ED, Kaiser BLD, Wahl KL, Wunschel DS. (2019) Proteomics for Bioforensics. In: Microbial Forensics 3rd ed.  Ed. S. Schutzer B. Budowle. Elsevier. Oxford, UK. ISBN 9780128153796. 775 pp.

2012

  • Cover Story for Chemical and Engineering News, “Tracing A Threat” on the research within the DHS S&T chemical forensics program. Volume 90 Issue 6:10-15. Issue Date: February 6, 2012.

2011

  • Wunschel D.S. and A. Fox. 2011. “Carbohydrate markers of organism purity and growth environment.” In. Chemical and physical signatures for microbial forensics. Ed. Cliff J., Kreuzer H. Wunschel D., Ehrhardt C Humana Press, NY, NY. ISBN 978-1-60327-217-9; Library of Congress Control Number 2011943307.
  • Wunschel D.S., A. Fox, and K.L. Wahl. 2011. “Glycoprotein and protein markers for strain differentiation and growth environment or media attribution.” In Chemical and physical signatures for microbial forensics. Ed. J. Cliff, H. Kreuzer, D. Wunschel, C. Ehrhardt. Humana Press, NY, NY. ISBN 978-1-60327-217-9; Library of Congress Conrtol Number 2011943307.

2010

  • Wahl K.L., D.S. Wunschel, and B.H. Clowers. 2010. “Proteomics Development and Application for Bioforensics.” In Microbial Forensics 2nd ed. Ed. S. Schutzer B. Budowle. Elsevier. Oxford, UK. Chapter 26, pp 449.

2005

  • Fox, Alvin, Larsson, Lennart, Allmaier, Guenter, Wunschel, David S., and Wahl, Karen L. 2005. "Preface: 5th International Symposium on the Interface between Analytical Chemistry and Microbiology - April 19th to 21st, 2004: Hosted at Pacific Northwest National Laboratory". United States. https://doi.org/10.1016/j.mimet.2005.04.010.