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The aim of this study was to investigate the changes in circulating concentrations of citrulline, neopterin, kynurenine, and tryptophan during the course of chemoradiation in patients with cervical cancer. Sixteen patients with histologically confirmed carcinoma of the uterine cervix, aged 53 ± 15 years (range 29–76 years), were included in this study. Plasma neopterin, kynurenine, and tryptophan were determined with an enzyme-linked immunosorbent assay. Plasma citrulline was measured with high-performance liquid chromatography. Compared to baseline, citrulline concentration was markedly and statistically significantly decreased at visits 2, 3, and 4, while returning to pretreatment concentrations at visit 5. A significant increase in serum neopterin concentrations was observed at visits 4 and 5. With the exception of decreased kynurenine/tryptophan ratio at visit 3, no significant changes were observed in the concentrations of kynurenine, tryptophan, and kynurenine/tryptophan ratio throughout the course of the treatment. In conclusion, present data demonstrate that citrulline concentrations decrease early and neopterin concentrations increase late during the course of chemoradiation in patients with cervical carcinoma. Citrulline represents a biomarker of intestinal toxicity in this population.

Determination of concentration of biomarkers of the activation of immune system, uric acid, and creatinine in the saliva can be useful tool for the diagnosis and monitoring of early manifestations of diseases such as malignant, inflammatory, and periodontal disorders. We have developed and validated a high-performance liquid chromatographic method coupled with fluorescence and diode array detection for the separation and quantification of neopterin, tryptophan, creatinine, uric acid, and kynurenine in the human saliva. A separation of these analytes was achieved within 9 min by using second-generation monolithic stationary phase and elution with phosphate buffer. The present method involves very simple sample preparation requiring small amount of sample matrix. The internal standard 3-nitro- l -tyrosine was used for a more precise quantification. The sensitivity of the present method was demonstrated with lower limits of quantification of 0.6 × 10−3 μmol/L for neopterin, 0.725 μmol/L for tryptophan, 0.12 μmol/L for creatinine, 0.18 μmol/L for uric acid, and 0.135 μmol/L for kynurenine. The method was validated with 67 real-life saliva samples collected from patients suffering from breast, ovarian, colorectal, and renal cancer, and 19 saliva samples from patients with periodontal diseases and allowed monitoring of inflammatory response.

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 μm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in

Cyclic electron transport (CET) around photosystem I (PSI) plays an important role in balancing the ATP/NADPH ratio and the photoprotection of plants. The NAD(P)H dehydrogenase complex (NDH) has a key function in one of the CET pathways. Current knowledge indicates that, in order to fulfill its role in CET, the NDH complex needs to be associated with PSI; however, until now there has been no direct structural information about such a supercomplex. Here we present structural data obtained for a plant PSI-NDH supercomplex. Electron microscopy analysis revealed that in this supercomplex two copies of PSI are attached to one NDH complex. A constructed pseudo-atomic model indicates asymmetric binding of two PSI complexes to NDH and suggests that the low-abundant Lhca5 and Lhca6 subunits mediate the binding of one of the PSI complexes to NDH. On the basis of our structural data, we propose a model of electron transport in the PSI-NDH supercomplex in which the association of PSI to NDH seems to be important for efficient trapping of reduced ferredoxin by NDH.

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.