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High-Throughput
Volume 8
Issue 2
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High-Throughput, Volume 8, Issue 2 (June 2019) – 10 articles

Cover Story (view full-size image): The mass spectrometric measurement of DNA adducts, the covalent modifications of DNA by environmental and dietary genotoxicants,and endogenously produced electrophiles, provides physico-chemical evidence for DNA damage. As the mass spectrometry-based analytical technology matures and bioinformatics tools become available for large data analysis, it will be feasible to screen for the totality of DNA damage in the human genome through DNA adductomics approaches. As an emerging technology in human exposure assessment, DNA adductomics can advance our understanding about the role of chemical exposures in DNA damage and disease risk. View this paper.
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17 pages, 582 KiB  
Article
Global Properties of Latent Virus Dynamics Models with Immune Impairment and Two Routes of Infection
by Aeshah A. Raezah, Ahmed M. Elaiw and Badria S. Alofi
High-Throughput 2019, 8(2), 16; https://doi.org/10.3390/ht8020016 - 3 Jun 2019
Cited by 3 | Viewed by 3208
Abstract
This paper studies the global stability of viral infection models with CTL immune impairment. We incorporate both productively and latently infected cells. The models integrate two routes of transmission, cell-to-cell and virus-to-cell. In the second model, saturated virus–cell and cell–cell incidence rates are [...] Read more.
This paper studies the global stability of viral infection models with CTL immune impairment. We incorporate both productively and latently infected cells. The models integrate two routes of transmission, cell-to-cell and virus-to-cell. In the second model, saturated virus–cell and cell–cell incidence rates are considered. The basic reproduction number is derived and two steady states are calculated. We first establish the nonnegativity and boundedness of the solutions of the system, then we investigate the global stability of the steady states. We utilize the Lyapunov method to prove the global stability of the two steady states. We support our theorems by numerical simulations. Full article
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11 pages, 1763 KiB  
Article
Development and Optimization of a Miniaturized Western Blot-Based Screening Platform to Identify Regulators of Post-Translational Modifications
by Florencia Villafañez, Vanesa Gottifredi and Gastón Soria
High-Throughput 2019, 8(2), 15; https://doi.org/10.3390/ht8020015 - 3 Jun 2019
Cited by 4 | Viewed by 5348
Abstract
Post-translational modifications (PTMs) are fundamental traits of protein functionality and their study has been addressed using several approaches over the past years. However, screening methods developed to detect regulators of PTMs imply many challenges and are usually based on expensive techniques. Herein, we [...] Read more.
Post-translational modifications (PTMs) are fundamental traits of protein functionality and their study has been addressed using several approaches over the past years. However, screening methods developed to detect regulators of PTMs imply many challenges and are usually based on expensive techniques. Herein, we described the development and optimization of a western blot-based platform for identification of regulators of a specific PTM—mono-ubiquitylation of proliferating cell nuclear antigen (PCNA). This cell-based method does not require specific equipment, apart from the basic western blot (WB) devices and minor accessories, which are accessible for most research labs. The modifications introduced to the classical WB protocol allow the performance of PTM analysis from a single well of a 96-well plate with minimal sample manipulation and low intra- and inter-plate variability, making this method ideal to screen arrayed compound libraries in a 96-well format. As such, our experimental pipeline provides the proof of concept to design small screenings of PTM regulators by improving the quantitative accuracy and throughput capacity of classical western blots. Full article
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21 pages, 1119 KiB  
Article
A Multispecies Biofilm In Vitro Screening Model of Dental Caries for High-Throughput Susceptibility Testing
by Lara A. Heersema and Hugh D. C. Smyth
High-Throughput 2019, 8(2), 14; https://doi.org/10.3390/ht8020014 - 30 May 2019
Cited by 9 | Viewed by 5079
Abstract
There is a current need to develop and optimize new therapeutics for the treatment of dental caries, but these efforts are limited by the relatively low throughput of relevant in vitro models. The aim of this work was to bridge the 96-well microtiter [...] Read more.
There is a current need to develop and optimize new therapeutics for the treatment of dental caries, but these efforts are limited by the relatively low throughput of relevant in vitro models. The aim of this work was to bridge the 96-well microtiter plate system with a relevant multispecies dental caries model that could be reproducibly grown to allow for the high-throughput screening of anti-biofilm therapies. Various media and inoculum concentrations were assessed using metabolic activity, biomass, viability, and acidity assays to determine the optimal laboratory-controlled conditions for a multispecies biofilm composed of Streptococcus gordonii, Streptococcus mutans, and Candida albicans. The selected model encompasses several of the known fundamental characteristics of dental caries-associated biofilms. The 1:1 RPMI:TSBYE 0.6% media supported the viability and biomass production of mono- and multispecies biofilms best. Kinetic studies over 48 h in 1:1 RPMI:TSBYE 0.6% demonstrated a stable biofilm phase between 10 and 48 h for all mono- and multispecies biofilms. The 1:1:0.1 S. gordonii: S. mutans: C. albicans multispecies biofilm in 1:1 RPMI:TSBYE 0.6% is an excellent choice for a high-throughput multispecies model of dental caries. This high-throughput multispecies model can be used for screening novel therapies and for better understanding the treatment effects on biofilm interactions and stability. Full article
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25 pages, 4250 KiB  
Review
Emerging Technologies in Mass Spectrometry-Based DNA Adductomics
by Jingshu Guo and Robert J. Turesky
High-Throughput 2019, 8(2), 13; https://doi.org/10.3390/ht8020013 - 14 May 2019
Cited by 35 | Viewed by 7989
Abstract
The measurement of DNA adducts, the covalent modifications of DNA upon the exposure to the environmental and dietary genotoxicants and endogenously produced electrophiles, provides molecular evidence for DNA damage. With the recent improvements in the sensitivity and scanning speed of mass spectrometry (MS) [...] Read more.
The measurement of DNA adducts, the covalent modifications of DNA upon the exposure to the environmental and dietary genotoxicants and endogenously produced electrophiles, provides molecular evidence for DNA damage. With the recent improvements in the sensitivity and scanning speed of mass spectrometry (MS) instrumentation, particularly high-resolution MS, it is now feasible to screen for the totality of DNA damage in the human genome through DNA adductomics approaches. Several MS platforms have been used in DNA adductomic analysis, each of which has its strengths and limitations. The loss of 2′-deoxyribose from the modified nucleoside upon collision-induced dissociation is the main transition feature utilized in the screening of DNA adducts. Several advanced data-dependent and data-independent scanning techniques originated from proteomics and metabolomics have been tailored for DNA adductomics. The field of DNA adductomics is an emerging technology in human exposure assessment. As the analytical technology matures and bioinformatics tools become available for analysis of the MS data, DNA adductomics can advance our understanding about the role of chemical exposures in DNA damage and disease risk. Full article
(This article belongs to the Special Issue Adductomics: Elucidating the Environmental Causes of Disease)
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15 pages, 4843 KiB  
Article
The Adductomics of Isolevuglandins: Oxidation of IsoLG Pyrrole Intermediates Generates Pyrrole–Pyrrole Crosslinks and Lactams
by Wenzhao Bi, Geeng-Fu Jang, Lei Zhang, John W. Crabb, James Laird, Mikhail Linetsky and Robert G. Salomon
High-Throughput 2019, 8(2), 12; https://doi.org/10.3390/ht8020012 - 10 May 2019
Cited by 3 | Viewed by 4998
Abstract
Isoprostane endoperoxides generated by free radical-induced oxidation of arachidonates, and prostaglandin endoperoxides generated through enzymatic cyclooxygenation of arachidonate, rearrange nonenzymatically to isoprostanes and a family of stereo and structurally isomeric γ-ketoaldehyde seco-isoprostanes, collectively known as isolevuglandins (isoLGs). IsoLGs are stealthy toxins, and free [...] Read more.
Isoprostane endoperoxides generated by free radical-induced oxidation of arachidonates, and prostaglandin endoperoxides generated through enzymatic cyclooxygenation of arachidonate, rearrange nonenzymatically to isoprostanes and a family of stereo and structurally isomeric γ-ketoaldehyde seco-isoprostanes, collectively known as isolevuglandins (isoLGs). IsoLGs are stealthy toxins, and free isoLGs are not detected in vivo. Rather, covalent adducts are found to incorporate lysyl ε-amino residues of proteins or ethanolamino residues of phospholipids. In vitro studies have revealed that adduction occurs within seconds and is uniquely prone to cause protein–protein crosslinks. IsoLGs accelerate the formation of the type of amyloid beta oligomers that have been associated with neurotoxicity. Under air, isoLG-derived pyrroles generated initially are readily oxidized to lactams and undergo rapid oxidative coupling to pyrrole–pyrrole crosslinked dimers, and to more highly oxygenated derivatives of those dimers. We have now found that pure isoLG-derived pyrroles, which can be generated under anoxic conditions, do not readily undergo oxidative coupling. Rather, dimer formation only occurs after an induction period by an autocatalytic oxidative coupling. The stable free-radical TEMPO abolishes the induction period, catalyzing rapid oxidative coupling. The amine N-oxide TMAO is similarly effective in catalyzing the oxidative coupling of isoLG pyrroles. N-acetylcysteine abolishes the generation of pyrrole–pyrrole crosslinks. Instead pyrrole-cysteine adducts are produced. Two unified single-electron transfer mechanisms are proposed for crosslink and pyrrole-cysteine adduct formation from isoLG-pyrroles, as well as for their oxidation to lactams and hydroxylactams. Full article
(This article belongs to the Special Issue Adductomics: Elucidating the Environmental Causes of Disease)
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15 pages, 3289 KiB  
Article
Efficient Screening of Combinatorial Peptide Libraries by Spatially Ordered Beads Immobilized on Conventional Glass Slides
by Timm Schwaar, Maike Lettow, Dario Remmler, Hans G. Börner and Michael G. Weller
High-Throughput 2019, 8(2), 11; https://doi.org/10.3390/ht8020011 - 30 Apr 2019
Cited by 8 | Viewed by 6942
Abstract
Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves [...] Read more.
Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput “all-on-one chip” system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide. Full article
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13 pages, 872 KiB  
Review
Mass Spectrometry-Based Methodologies for Targeted and Untargeted Identification of Protein Covalent Adducts (Adductomics): Current Status and Challenges
by João Nunes, Catarina Charneira, Judit Morello, João Rodrigues, Sofia A. Pereira and Alexandra M. M. Antunes
High-Throughput 2019, 8(2), 9; https://doi.org/10.3390/ht8020009 - 23 Apr 2019
Cited by 17 | Viewed by 5868
Abstract
Protein covalent adducts formed upon exposure to reactive (mainly electrophilic) chemicals may lead to the development of a wide range of deleterious health outcomes. Therefore, the identification of protein covalent adducts constitutes a huge opportunity for a better understanding of events underlying diseases [...] Read more.
Protein covalent adducts formed upon exposure to reactive (mainly electrophilic) chemicals may lead to the development of a wide range of deleterious health outcomes. Therefore, the identification of protein covalent adducts constitutes a huge opportunity for a better understanding of events underlying diseases and for the development of biomarkers which may constitute effective tools for disease diagnosis/prognosis, for the application of personalized medicine approaches and for accurately assessing human exposure to chemical toxicants. The currently available mass spectrometry (MS)-based methodologies, are clearly the most suitable for the analysis of protein covalent modifications, providing accuracy, sensitivity, unbiased identification of the modified residue and conjugates along with quantitative information. However, despite the huge technological advances in MS instrumentation and bioinformatics tools, the identification of low abundant protein covalent adducts is still challenging. This review is aimed at summarizing the MS-based methodologies currently used for the identification of protein covalent adducts and the strategies developed to overcome the analytical challenges, involving not only sample pre-treatment procedures but also distinct MS and data analysis approaches. Full article
(This article belongs to the Special Issue Adductomics: Elucidating the Environmental Causes of Disease)
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24 pages, 986 KiB  
Review
The Mercapturomic Profile of Health and Non-Communicable Diseases
by Clara Gonçalves-Dias, Judit Morello, Valdir Semedo, M. João Correia, Nuno R. Coelho, Emilia C. Monteiro, Alexandra M. M. Antunes and Sofia A. Pereira
High-Throughput 2019, 8(2), 10; https://doi.org/10.3390/ht8020010 - 23 Apr 2019
Cited by 8 | Viewed by 4573
Abstract
The mercapturate pathway is a unique metabolic circuitry that detoxifies electrophiles upon adducts formation with glutathione. Since its discovery over a century ago, most of the knowledge on the mercapturate pathway has been provided from biomonitoring studies on environmental exposure to toxicants. However, [...] Read more.
The mercapturate pathway is a unique metabolic circuitry that detoxifies electrophiles upon adducts formation with glutathione. Since its discovery over a century ago, most of the knowledge on the mercapturate pathway has been provided from biomonitoring studies on environmental exposure to toxicants. However, the mercapturate pathway-related metabolites that is formed in humans—the mercapturomic profile—in health and disease is yet to be established. In this paper, we put forward the hypothesis that these metabolites are key pathophysiologic factors behind the onset and development of non-communicable chronic inflammatory diseases. This review goes from the evidence in the formation of endogenous metabolites undergoing the mercapturate pathway to the methodologies for their assessment and their association with cancer and respiratory, neurologic and cardiometabolic diseases. Full article
(This article belongs to the Special Issue Adductomics: Elucidating the Environmental Causes of Disease)
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30 pages, 5464 KiB  
Article
Dark Proteome Database: Studies on Dark Proteins
by Nelson Perdigão and Agostinho Rosa
High-Throughput 2019, 8(2), 8; https://doi.org/10.3390/ht8020008 - 27 Mar 2019
Cited by 13 | Viewed by 6280
Abstract
The dark proteome, as we define it, is the part of the proteome where 3D structure has not been observed either by homology modeling or by experimental characterization in the protein universe. From the 550.116 proteins available in Swiss-Prot (as of July 2016), [...] Read more.
The dark proteome, as we define it, is the part of the proteome where 3D structure has not been observed either by homology modeling or by experimental characterization in the protein universe. From the 550.116 proteins available in Swiss-Prot (as of July 2016), 43.2% of the eukarya universe and 49.2% of the virus universe are part of the dark proteome. In bacteria and archaea, the percentage of the dark proteome presence is significantly less, at 12.6% and 13.3% respectively. In this work, we present a necessary step to complete the dark proteome picture by introducing the map of the dark proteome in the human and in other model organisms of special importance to mankind. The most significant result is that around 40% to 50% of the proteome of these organisms are still in the dark, where the higher percentages belong to higher eukaryotes (mouse and human organisms). Due to the amount of darkness present in the human organism being more than 50%, deeper studies were made, including the identification of ‘dark’ genes that are responsible for the production of so-called dark proteins, as well as the identification of the ‘dark’ tissues where dark proteins are over represented, namely, the heart, cervical mucosa, and natural killer cells. This is a step forward in the direction of gaining a deeper knowledge of the human dark proteome. Full article
(This article belongs to the Special Issue Algorithms and Tools in Computational Proteomics)
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11 pages, 2711 KiB  
Article
Vertical Scanning Interferometry for Label-Free Detection of Peptide-Antibody Interactions
by Andrea Palermo, Richard Thelen, Laura K. Weber, Tobias Foertsch, Simone Rentschler, Verena Hackert, Julia Syurik and Alexander Nesterov-Mueller
High-Throughput 2019, 8(2), 7; https://doi.org/10.3390/ht8020007 - 27 Mar 2019
Cited by 2 | Viewed by 4441
Abstract
Peptide microarrays are a fast-developing field enabling the mapping of linear epitopes in the immune response to vaccinations or diseases and high throughput studying of protein-protein interactions. In this respect, a rapid label-free measurement of protein layer topographies in the array format is [...] Read more.
Peptide microarrays are a fast-developing field enabling the mapping of linear epitopes in the immune response to vaccinations or diseases and high throughput studying of protein-protein interactions. In this respect, a rapid label-free measurement of protein layer topographies in the array format is of great interest but is also a great challenge due to the extremely low aspect ratios of the peptide spots. We have demonstrated the potential of vertical scanning interferometry (VSI) for a detailed morphological analysis of peptide arrays and binding antibodies. The VSI technique is shown to scan an array area of 5.1 square millimeters within 3–4 min at a resolution of 1.4 μm lateral and 0.1 nm vertical in the full automation mode. Topographies obtained by VSI do match the one obtained by AFM measurements, demonstrating the accuracy of the technique. A detailed topology of peptide-antibody layers on single spots was measured. Two different measurement regions are distinguished according to the antibody concentration. In the case of weakly diluted serum, the thickness of the antibody layer is independent of the serum dilution and corresponds to the physical thickness of the accumulated antibody layer. In strongly diluted serum, the thickness measured via VSI is linearly proportional to the serum dilution. Full article
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