GO-META-TiO2 composite monolithic columns for in-tube solid-phase microextraction of phosphopeptides.

A novel composite monolithic column based on graphene oxide-trimethyl-2-methacroyloxyethylammonium chloride-titania (GO-META-TiO2) was developed for the enrichment of phosphopeptides. META was proposed as a "bridge" to connect GO and TiO2 species to prepare GO-META-TiO2 composite. This high surface area composite (surface area = 196.93 m2 g-1) was fixed in the monolithic column via an in situ UV polymerization process. In-tube solid phase microextraction (IT-SPME) using this composite was coupled with matrix-assisted laser desorption ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) for the enrichment and detection of phosphorylated peptides from a digestion mixture of α-casein, ß-casein, and bovine serum albumin (BSA) in molar ratios of 1:1:1, 1:1:10, and 1:1:100. The key factors affecting the IT-SPME of the phosphopeptides, such as the elution solution concentrations, the extraction flow rate, and the elution flow rate were comprehensively investigated. For further demonstration, this method was employed for the enrichment and detection of phosphorylated peptides from digested chicken egg white. The obtained results indicated that the GO-META-TiO2 composite monolithic column rapidly and efficiently captured the phosphopeptides present in these complex biological samples, even in the 10 fmol ß-casein tryptic digest. We therefore propose that the reported GO-META-TiO2 composite monolithic column possesses a suitable affinity for the selective extraction of phosphopeptides from biological samples. This method paves a way in extending the application of nanomaterials.

Dynamic metabolic changes in seeds and seedlings of Brassica napus (oilseed rape) suppressing UGT84A9 reveal plasticity and molecular regulation of the phenylpropanoid pathway.

In Brassica napus, suppression of the key biosynthetic enzyme UDP-glucose:sinapic acid glucosyltransferase (UGT84A9) inhibits the biosynthesis of sinapine (sinapoylcholine), the major phenolic component of seeds. Based on the accumulation kinetics of a total of 158 compounds (110 secondary and 48 primary metabolites), we investigated how suppression of the major sink pathway of sinapic acid impacts the metabolome of developing seeds and seedlings. In UGT84A9-suppressing (UGT84A9i) lines massive alterations became evident in late stages of seed development affecting the accumulation levels of 58 secondary and 7 primary metabolites. UGT84A9i seeds were characterized by decreased amounts of various hydroxycinnamic acid (HCA) esters, and increased formation of sinapic and syringic acid glycosides. This indicates glycosylation and ß-oxidation as metabolic detoxification strategies to bypass intracellular accumulation of sinapic acid. In addition, a net loss of sinapic acid upon UGT84A9 suppression may point to a feedback regulation of HCA biosynthesis. Surprisingly, suppression of UGT84A9 under control of the seed-specific NAPINC promoter was maintained in cotyledons during the first two weeks of seedling development and associated with a reduced and delayed transformation of sinapine into sinapoylmalate. The lack of sinapoylmalate did not interfere with plant fitness under UV-B stress. Increased UV-B radiation triggered the accumulation of quercetin conjugates whereas the sinapoylmalate level was not affected.

Interaction between total body gamma-irradiation and choline deficiency triggers immediate modulation of choline and choline-containing moieties.

PURPOSE: The objective of this study was to examine the effect of 60Co-gamma (γ) radiation on acute phase modulation, if any, of choline and choline-containing moieties in choline-deficient subjects. Corresponding results could provide information that might be useful in the management of adverse effects of γ-radiation. MATERIALS AND METHODS: Male Swiss mice maintained on a choline-sufficient diet (CSD) and choline-free diet (CFD) based on AIN-93M formula, were subjected to whole body γ-irradiation (2-6 Gy). Liver, serum and brain samples from each group were then tested for: (i) Alterations in choline and choline-containing moieties such as phosphatidylcholine (PC) and sphingomyeline (SM); and (ii) modulation of choline profile modulating enzymes such as phospholipase D (PLD) and total sphingomyelinase (t-SMase). Liver and brain samples were also subjected to histo-pathological examinations. RESULTS: No significant changes were observed in folate, choline, choline-containing moieties and choline-modulating enzymes in choline-sufficient mice. In contrast, interaction between cytotoxic effects of γ-radiation and choline deficiency modulated choline and choline-containing moieties. Feeding CFD reduced hepatic concentrations of choline, PC and SM whereas PLD and t-SMase activities were significantly raised. The decrease in liver choline and choline-containing moieties was accompanied by an increase in blood choline concentration. Despite choline deficiency, the level of choline and acetylcholine synthesizing enzyme choline acetyltransfease (ChAT) significantly increased in the brain. CONCLUSIONS: We propose that choline deprivation and γ-radiation interact to modulate choline reserves of hepatic tissue, which might release choline to blood. Our studies also clearly showed that interaction between choline deficiency and γ-radiation might substantially enhance liver adipogenesis.

1H MR spectroscopy of mesial temporal lobe epilepsies treated with Gamma knife.

Proton MR spectroscopy was used to observe long-term post-irradiation metabolic changes in epileptogenic tissue and in the contralateral parts of the brain which are not available with conventional imaging methods. We studied these changes in the temporal lobe in six patients, following radiosurgery on the amygdala and hippocampus. (1)H MR spectroscopy at 1.5 T with short and long echo times (TE=10 and 135 ms) were used together with standard MR imaging sequences (T1-, T2-weighted). The treatment was performed by Leksell Gamma Knife with a dose of 50 Gy to the center and a 50% isodose to the margin of the target, represented by the mean volume of approximately 7.5 ml. Magnetic resonance imaging and MR spectroscopy examinations were performed at least once per year for 3 years. The most significant changes in spectra were observed approximately 1 year after the irradiation when edema in irradiated area was observed and strong signal of lipids was identified. Later, edema and lipid signals disappeared and follow-up was characterized by a decrease of NAA, Cr, and Cho concentrations in the ipsilateral region of the brain to the irradiation (LCModel calculation from voxel of interest 3.8-4.5 ml positioned into the centrum of target volume). The concentration of NAA, Cr, and Cho after radiosurgery was significantly different from control values ( p<0.05) and also from concentrations in the contralateral part of the brain ( p<0.05). In the contralateral part, the concentration of NAA was significantly increased ( p<0.05) (NAA: before treatment 8.81, after treatment 11.33 mM). No radiotoxic changes were observed in the contralateral part of the brain or behind the area of target volume. The MR spectroscopy findings precluded MRI observation and MRS results completed data about the development of radiotoxic changes in the target volume.