Glutathione metabolism contributes to the induction of trained immunity

The innate immune system displays heterologous memory characteristics, which are characterized by stronger responses to a secondary challenge. This phenomenon termed trained immunity relies on epigenetic and metabolic rewiring of innate immune cells. As reactive oxygen species (ROS) production has been associated with the trained immunity phenotype, we hypothesized that the increased ROS levels and the main intracellular redox molecule glutathione play a role in the induction of trained immunity. Here we show that pharmacological inhibition of ROS in an in vitro model of trained immunity did not influence cell responsiveness; the modulation of glutathione levels reduced pro-inflammatory cytokine production in human monocytes. Single nucleotide polymorphisms (SNPs) in genes involved in glutathione metabolism were found to be associated with changes in pro-inflammatory cytokine production capacity upon trained immunity. Also, plasma glutathione concentrations were positively associated with ex vivo IL-1 beta production, a biomarker of trained immunity, produced by monocytes of BCG-vaccinated individuals. In conclusion, glutathione metabolism is involved in the induction of trained immunity, and future studies are warranted to explore its functional consequences in human diseases.Proteomic

Electron transfer from A(0)(-) to A(1) in Photosystem I from Chlamydomonas reinhardtii occurs in both the A and B branch with 25-30-ps lifetime

We have recorded transient absorption kinetics at 390 nm with picosecond resolution in order to observe electron transfer from the reduced primary acceptor, A-0, to the secondary acceptor,

Protein digestion optimization for characterization of drug-protein adducts using response surface modeling

The formation of drug-protein adducts in vivo may have important clinical and toxicological implications. Consequently, there is a great interest in the detection of these adducts and the elucidation of their role in the processes leading to adverse and idiosyncratic drug reactions. Enzymatic digestion is a crucial step in bottom-up proteomics strategies for the analysis of drug-protein adducts. The chosen proteolytic enzyme and digestion conditions have a large influence on the protein coverage of the modified protein and identification of its modification site. In this work, the enzymatic digestion conditions (pH, temperature and time) of trypsin and thermolysin were optimized specifically for the characterization of Human Serum Albumin (HSA) adducts. Using a Design of Experiments (DOE), it was found that of the three optimized parameters mainly pH and temperature showed strong effects on both responses. The optimized digestion conditions were different from those obtained from the suppliers or literature. Their application to HSA adducts resulted in improved protein coverage and signal intensity regarding the peptide containing the modification site, thereby highlighting the importance of a detailed optimization of digestion conditions. © 2010 Elsevier B.V

Amino acid analysis using chromatography–mass spectrometry: An inter platform comparison study

The analysis of amino acids has become a central task in many aspects. While amino acid analysis has traditionally mainly been carried out using either gas chromatography (GC) in combination with flame ionization detection or liquid chromatography (LC) with either post-column derivatization using ninhydrin or pre-column derivatization using o-phthalaldehyde, many of today's analysis platforms are based on chromatography in combination with mass spectrometry (MS). While derivatization is mandatory for the GC-based analysis of amino acids, several LC platforms have emerged, particularly in the dawn of targeted metabolite profiling using hydrophilic interaction liquid chromatography (HILIC) coupled to MS, allowing the analysis of underivatized amino acids. Among the numerous analytical platforms available for amino acid analysis today, we here compare three prominent approaches, being GC-MS and LC-MS after amino acid derivatization using chloroformate and HILIC-MS of underivatized amino acids. We compare and discuss practical issues as well as performance characteristics, e.g., the use of 13C-labeled internal standards, of the different platforms and present data on their practical implementation in our laboratory. Finally, we compare the real-life applicability of all three platforms for a complex biological sample. While all three platforms are very-well suited for the analysis of complex biological samples they all show advantages and disadvantages for some analytes as discussed in detail in this manuscript

Derivatization of carboxylic acids with 4-APEBA for detection by positive-ion LC-ESI-MS(/MS) applied for the analysis of prostanoids and NSAID in urine

In order to develop a generic positive ionization ESI LC-MS method for a variety of interesting substance classes, a new derivatization strategy for carboxylic acids was developed. The carboxylic acid group is labeled with the bromine containing 4-APEBA reagent based on carbodiimide chemistry. The derivatization reaction can be carried out under aqueous conditions, thereby greatly simplifying sample preparation. In this paper, the derivatization of carboxylic acids is exemplified for the determination of prostanoids and non-steroidal anti-inflammatory drugs (NSAID). Optimization of the derivatization conditions was studied. In order to prove the applicability of the presented approach, we applied the described protocol to urine samples from complex regional pain syndrome (CRPS) patients and were able to detect several prostanoids not visible in the urine of healthy volunteers. Further, the determination of the non-steroidal anti-inflammatory drug ibuprofen in a urine sample was possible. © 2010 Elsevier B.V

Increased PUFA Content and 5-Lipoxygenase Pathway Expression Are Associated with Subcutaneous Adipose Tissue Inflammation in Obese Women with Type 2 Diabetes

Diabetes mellitus: pathophysiological changes and therap

Fc receptors on neutrophils: two sides of the same coin?

Proteomic

Fc receptors on neutrophils: two sides of the same coin?

Proteomic

Stability-indicating study of the anti-Alzheimer's drug galantamine hydrobromide

Galantamine hydrobromide was subjected to different stress conditions (acidic, alkaline, thermal, photolytic and oxidative). Degradation was found to occur under acidic, photolytic and oxidative conditions, while the drug was stable under alkaline and elevated temperature conditions. A stability-indicating reversed-phase liquid chromatographic method was developed for the determination of the drug in the presence of its degradation products. The method was validated for linearity, precision, accuracy, specificity, selectivity and intermediate precision. Additionally, the degradation kinetics of the drug was assessed in relevant cases. The kinetics followed a first order behavior in the case of acidic and photolytic degradation, while a two-phase kinetics behavior was found for the oxidative degradation. The degradation products were characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Dehydration, epimerization and N-oxidation were the main processes observed during the degradation of galantamine. Moreover, if sufficient material could be isolated the inhibitory activity against the target enzyme acetylcholinesterase was also assessed. © 2011 Elsevier B.V