Of about one dozen human P450 s that catalyze biotransformations of xenobiotics, CYP2D6 is one of the more important ones based on the number of its drug substrates. It shows a very high degree of interindividual variability, which is primarily due to the extensive genetic polymorphism that influences expression and function. This so-called debrisoquine/sparteine oxidation polymorphism has been extensively studied in many different populations and over 80 alleles and allele variants have been described. CYP2D6 protein and enzymatic activity is completely absent in less than 1% of Asian people and in up to 10% of Caucasians with two null alleles, which do not encode a functional P450 protein product. The resulting "poor metabolizer" (PM) phenotype is characterized by the inability to use CYP2D6-dependent metabolic pathways for drug elimination, which affect up to 20% of all clinically used drugs. The consequences are increased risk of adverse drug reactions or lack of therapeutic response. Today, genetic testing predicts the PM phenotype with over 99% certainty. At the other extreme, the "Ultrarapid Metabolizer" (UM) phenotype can be caused by alleles carrying multiple gene copies. "Intermediate Metabolizers" (IM) are severely deficient in their metabolism capacity compared to normal "Extensive Metabolizers" (EM), but in contrast to PMs they express a low amount of residual activity due to the presence of at least one partially deficient allele. Whereas the intricate genetics of the CYP2D6 polymorphism is becoming apparent at ever greater detail, applications in clinical practice are still rare. More clinical studies are needed to show where patients benefit from drug dose adjustment based on their genotype. Computational approaches are used to predict and rationalize substrate specificity and enzymatic properties of CYP2D6. Pharmacophore modeling of ligands and protein homology modeling are two complementary approaches that have been applied with some success. CYP2D6 is not only expressed in liver but also in the gut and in brain neurons, where endogenous substrates with high-turnover have been found. Whether and how brain functions may be influenced by polymorphic expression are interesting questions for the future.

Cytochrome P450 2D6: overview and update on pharmacology, genetics, biochemistry

Molecularly imprinted polymers are synthetic receptors for a targeted molecule. As such, they are analogues of the natural antibody–antigen systems. In this review, after a recounting of the early history of the general field, we specifically focus on the application of these polymers as sensors. In these applications, the polymers are paired with a reporting system, which may be electrical, electrochemical, optical, or gravimetric. The presence of the targeted molecule effects a change in the reporting agent, and a calibrated quantity of the target is recorded. In this review, we describe the imprinted polymer production processes, the techniques used for reporting, and the applications of the reported sensors. A brief survey of recent applications to gas-phase sensing is included, but the focus is primarily on the development of sensors for targets in solution. Included among the applications are those designed to detect toxic chemicals, toxins in foods, drugs, explosives, and pathogens. The application...

Molecularly Imprinted Polymers

The generation of polymer brushes by surface-initiated controlled radical polymerization (SI-CRP) techniques has become a powerful approach to tailor the chemical and physical properties of interfaces and has given rise to great advances in surface and interface engineering. Polymer brushes are defined as thin polymer films in which the individual polymer chains are tethered by one chain end to a solid interface. Significant advances have been made over the past years in the field of polymer brushes. This includes novel developments in SI-CRP, as well as the emergence of novel applications such as catalysis, electronics, nanomaterial synthesis and biosensing. Additionally, polymer brushes prepared via SI-CRP have been utilized to modify the surface of novel substrates such as natural fibers, polymer nanofibers, mesoporous materials, graphene, viruses and protein nanoparticles. The last years have also seen exciting advances in the chemical and physical characterization of polymer brushes, as well as an ev...

Surface-Initiated Controlled Radical Polymerization: State-of-the-Art, Opportunities, and Challenges in Surface and Interface Engineering with Polymer Brushes

Electrochemical reduction of an exemplary ATRP catalyst, C 11 Br 2 /Me 6 TREN, is shown to be an efficient process to mediate and execute an ATRP. The onset of polymerization occurs only through passage of a cathodic current achieved under a reductive potential to form Cu 1 Br 2 /Me 6 TREN, within the reaction medium. Unprecedented control over the polymerization kinetics can be attained through electrochemical methods by modulating the magnitude of the applied potential allowing polymerization rate enhancement or retardation. Additional polymerization control is gained through electrochemical “dials” allowing polymerization rate enhancements achieved by larger applied potentials and the ability to successfully switch a polymerization “on” and “off between dormant and active states by application of multistep intermittent potentials.

https://science.sciencemag.org/content/sci/332/6025/81.full.pdf

Electrochemically mediated atom transfer radical polymerization

The interstitium describes the fluid, proteins, solutes, and the extracellular matrix (ECM) that comprise the cellular microenvironment in tissues. Its alterations are fundamental to changes in cell function in inflammation, pathogenesis, and cancer. Interstitial fluid (IF) is created by transcapillary filtration and cleared by lymphatic vessels. Herein we discuss the biophysical, biomechanical, and functional implications of IF in normal and pathological tissue states from both fluid balance and cell function perspectives. We also discuss analysis methods to access IF, which enables quantification of the cellular microenvironment; such methods have demonstrated, for example, that there can be dramatic gradients from tissue to plasma during inflammation and that tumor IF is hypoxic and acidic compared with subcutaneous IF and plasma. Accumulated recent data show that IF and its convection through the interstitium and delivery to the lymph nodes have many and diverse biological effects, including in ECM reorganization, cell migration, and capillary morphogenesis as well as in immunity and peripheral tolerance. This review integrates the biophysical, biomechanical, and biological aspects of interstitial and lymph fluid and its transport in tissue physiology, pathophysiology, and immune regulation.

Interstitial Fluid and Lymph Formation and Transport: Physiological Regulation and Roles in Inflammation and Cancer

Electromembrane extraction of peptides.

High performance liquid chromatography-electrospray tandem mass spectrometry was used to elucidate the phospholipids in krill oil extracted from Euphausia superba, an emerging source for human nutritional supplements. The study was carried out in order to map the species of the choline-containing phospholipid classes: phosphatidylcholine and lyso-phosphatidylcholine. In addition, the prevalent phosphatidylcholine class was quantified and the results compared with prior analysis. The qualification was performed with separation on a reverse phase chromatography column, while the quantification was obtained with class separation on a normal phase chromatography column. An Orbitrap system was used for the detection, and pulsed-Q dissociation fragmentation was utilized for the identification of the species. An asymmetrical exclusion list was applied for detection of phospholipid species of lower concentration, significantly improving the number of species observed. A total of 69 choline-containing phospholipids were detected, whereof 60 phosphatidylcholine substances, among others seven with probable omega-3 fatty acids in both sn-1 and sn-2. The phosphatidylcholine concentration was estimated to be 34 ± 5 g/100 g oil (n = 5). These results confirm the complexity of the phospholipid composition of krill oil, and the presence of long chained, heavily unsaturated fatty acids.

/pdf/elucidation-of-phosphatidylcholine-composition-in-krill-oil-3q94uejxt9.pdf

Elucidation of Phosphatidylcholine Composition in Krill Oil Extracted from Euphausia superba

Rapid isolation of angiotensin peptides from plasma by electromembrane extraction

Proteomics is defined as the large-scale study of proteins in particular for their structures and functions (Anderson and Anderson 1998), and investigations of proteins have become very important since they are the main components of the physiological metabolic pathways in eukaryotic cells. Proteomics increasingly plays an important role in areas like protein interaction studies, biomarker discovery, cancer prevention, drug treatment and disease screening medical diagnostics (Capelo et al. 2009). Proteomics can be performed either in a comprehensive or “shotgun” mode, where proteins are identified in complex mixtures, or as “targeted proteomics” where “selective reaction monitoring” (SRM) is used to choose in advance the proteins to observe, and then measuring them accurately, by optimizing the sample preparation as well as the LC-MS method in accordance to the specific proteins (Mitchell 2010). Whether “MS-based shotgun proteomics” has accomplished anything at all regarding clinically useful results was recently addressed by Peter Mitchell in a feature article (Mitchell 2010), and he states that the field needs to make a further step or even change direction. Referring to discussions with among others John Yates and Matthias Mann, Mitchell addresses the failure in the search for biomarkers as indicators of disease, the difficulties of protein arrays, the uncertainty of quantification in “shotgun proteomics” (due to among others the efficiency of ionization in the mass spectrometers), database shortcomings, the problems of detecting post translational modifications (PTMs), and finally the huge disappointment in the area of drug discovery. The field points in the direction of targeted proteomics, but targeted proteomics will not be the solution to all our questions and comprehensive proteomics will still be needed. In order to get as much information, with as high quality as possible, from a biological sample, both the sample preparation and the final LC-MS analyses need to be optimized. The most important step in the sample preparation for proteomics is the conversion of proteins to peptides and in most cases trypsin is used as enzyme. Trypsin is a protease that specifically cleaves the proteins creating peptides both in the preferred mass range for MS sequencing and with a basic residue at the carboxyl terminus of the peptide, producing information-rich, easily interpretable peptide fragmentation mass spectra. Some other proteases can be used as well, such as Lys-C, which is active in more harsh conditions with 8 M urea, and give larger fragments than trypsin. Asp-N and Glu-C are also highly sequence-

/pdf/a-critical-review-of-trypsin-digestion-for-lc-ms-based-1dl2oly60k.pdf

A Critical Review of Trypsin Digestion for LC-MS Based Proteomics

Early diagnosis is important for effective disease management. Measurement of biomarkers present at the local level of the skin could be advantageous in facilitating the diagnostic process. The analysis of the proteome of suction blister fluid, representative for the interstitial fluid of the skin, is therefore a desirable first step in the search for potential biomarkers involved in biological pathways of particular diseases. Here, we describe a global analysis of the suction blister fluid proteome as potential body fluid for biomarker proteins. The suction blister fluid proteome was compared with a serum proteome analyzed using identical protocols. By using stringent criteria allowing less than 1% false positive identifications, we were able to detect, using identical experimental conditions and amount of starting material, 401 proteins in suction blister fluid and 240 proteins in serum. As a major result of our analysis we construct a prejudiced list of 34 proteins, relatively highly and uniquely detected in suction blister fluid as compared to serum, with established and putative characteristics as biomarkers. We conclude that suction blister fluid might potentially serve as a good alternative biomarker body fluid for diseases that involve the skin.

/pdf/suction-blister-fluid-as-potential-body-fluid-for-biomarker-1w64bw0rpv.pdf

Léon Reubsaet

Papers

Electromembrane extraction of peptides.

Elucidation of Phosphatidylcholine Composition in Krill Oil Extracted from Euphausia superba

Rapid isolation of angiotensin peptides from plasma by electromembrane extraction

A Critical Review of Trypsin Digestion for LC-MS Based Proteomics

Suction blister fluid as potential body fluid for biomarker proteins