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Abbas, Ioana M. ; Vranic, M. ; Hoffmann, Holger ; El-Khatib, Ahmed H. ; Montes-Bayón, M. ; Möller, H. M. ; Weller, Michael G.
Hepcidin-25 was identified as the main iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry (MS/MS), high-resolution mass spectrometry HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D)model of hepcidin-25with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.
Alnajjar, M. A. ; Bartelmeß, Jürgen ; Hein, R. ; Ashokkumar, Pichandi ; Nilam, M. ; Nau, W. M. ; Rurack, Knut ; Hennig, A.
We introduce herein boron-dipyrromethene (BODIPY) dyes as a new class of fluorophores for the design of reporter dyes for supramolecular host–guest complex formation with cucurbit[7]uril (CB7). The BODIPYs contain a protonatable aniline nitrogen in the meso-position of the BODIPY chromophore, which was functionalized with known binding motifs for CB7. The unprotonated dyes show low fluorescence due to photoinduced electron transfer (PET), whereas the protonated dyes are highly fluorescent. Encapsulation of the binding motif inside CB7 positions the aniline nitrogen at the carbonyl rim of CB7, which affects the pKa value, and leads to a host-induced protonation and thus to a fluorescence increase. The possibility to tune binding affinities and pKa values
is demonstrated and it is shown that, in combination with the beneficial photophysical properties of BODIPYs, several new applications of host–dye reporter pairs can be implemented. This includes indicator displacement assays with favourable absorption and
emission wavelengths in the visible spectral region, fluorescence correlation spectroscopy, and noncovalent surface functionalization
with fluorophores.
To keep pace with the rising number of detected mycotoxins, there is a growing need for fast and reliable toxicity tests to assess the potential threat to food safety. Toxicity tests with the bacterial-feeding nematode Caenorhabditis elegans as model organism are well established. In this study the C. elegans wildtype strain N2 (var. Bristol) was used to investigate the toxic effects of the food-relevant mycotoxins citrinin (CIT) and zearalenone-14-sulfate (ZEA-14-S) and zearalenone (ZEA) on different life cycle parameters including reproduction, thermal and oxidative stress resistance and lifespan. The metabolization of the mycotoxins by the nematodes in vivo was investigated using HPLC-MS/MS. ZEA was metabolized in vivo to the reduced isomers α-zearalenol (α-ZEL) and β-ZEL. ZEA 14-S was reduced to α-/β-ZEL 14-sulfate and CIT was metabolized to mono-hydroxylated CIT. All mycotoxins tested led to a significant decrease in the number of nematode offspring produced. ZEA and CIT displayed negative effects on stress tolerance levels and for CIT an additional shortening of the mean lifespan was observed. In the case of ZEA-14-S, however, the mean lifespan was prolonged. The presented study shows the applicability of C. elegans for toxicity testing of emerging food mycotoxins for the purpose of assigning potential health threats.
Neamtu, Mariana ; Nadejde, C. ; Hodoroaba, Vasile-Dan ; Schneider, Rudolf ; Verestiuc, L. ; Panne, Ulrich
Cost-effective water cleaning approaches using improved Treatment technologies, for instance based on catalytic processes with high activity catalysts, are urgently needed. The aim of our study was to synthesize efficient Fenton-like photo-catalysts for rapid degradation of persistent organic micropollutants in aqueous medium. Iron-based nanomaterials were chemically synthesized through simple procedures by immobilization of either iron(II) oxalate (FeO) or iron(III) citrate (FeC) on magnetite (M) nanoparticles stabilized with polyethylene glycol (PEG). Various investigation techniques were performed in order to characterize the freshly prepared catalysts. By applying advanced oxidation processes, the effect of catalyst dosage, hydrogen peroxide concentration and UV-A light exposure were examined for Bisphenol A (BPA) conversion, at laboratory scale, in mild conditions. The obtained results revealed that BPA degradation was rapidly enhanced in the presence of low-concentration H2O2, as well as under UV-A light, and is highly dependent on the surface characteristics of the catalyst. Complete photo-degradation of BPA was achieved over the M/PEG/FeO catalyst in less than 15 minutes. Based on the catalytic performance, a hierarchy of the tested catalysts was established: M/PEG/FeO > M/PEG/FeC > M/PEG. The results of cytotoxicity assay using MCF-7 cells indicated that the aqueous samples after treatment are less cytotoxic.
Beyer, Sebastian ; Schürmann, Robin ; Feldmann, Ines ; Blocki, A. ; Bald, Ilko ; Schneider, Rudolf ; Emmerling, Franziska
Equipping ZIF particles with a polyelectrolyte membrane provides functional groups at their interface, enabling further conjugations necessary for applications such as targeted drug delivery. Previous approaches to coat ZIF particles with polyelectrolytes led to surface corrosion of the template material. This work overcomes previous limitations by performing a Layer-by-Layer (LbL) polyelectrolyte coating onto ZIF-8 and ZIF-67 particles in nonaqueous environment. Using the 2-methylimidazolium salt of polystyrensulfonic acid instead of the acid itself and polyethyleneimine in methanol led to intact ZIF particles after polyelectrolyte coating. This was verified by electron microscopy. Further, zetapotential and atomic force microscopy measurements confirmed a continuous polyelectrolyte multilayer built up. The here reported adaption to the well-studied (LbL) polyelectrolyte selfassembly process provides a facile method to equip ZIF particles with a nanometer thin polyelectrolyte multilayer membrane.
Online immunocapture ICP‑MS for the determination of the metalloprotein ceruloplasmin in human serum
(2018)
The human copper-protein ceruloplasmin (Cp) is the major copper-containing protein in the human body. The accurate determination of Cp is mandatory for the reliable diagnosis of several diseases. However, the analysis of Cp has proven to be difficult. The aim of our work was a proof of concept for the determination of a metalloprotein-based on online immunocapture ICP-MS. The immuno-affinity step is responsible for the enrichment and isolation of the analyte from serum, whereas the compound-independent quantitation with ICP-MS delivers the sensitivity,
precision, and large dynamic range. Off-line ELISA (enzyme-linked immunosorbent assay) was used in parallel to confirm the elution profile of the analyte with a structure-selective method. The total protein elution was observed with the 32S mass trace. The ICP-MS signals were normalized on a 59Co signal. The human copper-protein Cp could be selectively determined. This was shown with pure Cp and with a sample of human serum. The good correlation with off-line ELISA shows that Cp could be captured and eluted selectively from the anti-Cp affinity column and subsequently determined by the copper signal of ICP-MS.
The quality of research antibodies is an issue for decades. Although several papers have been published to improve the situation, their impact seems to be limited. This publication makes the effort to simplify the description of validation criteria in a way that the occasional antibody user is able to assess the validation level of an immunochemical reagent. A simple, 1-page checklist is supplied for the practical application of these criteria.
Borzekowski, Antje ; Drewitz, T. ; Keller, Julia ; Pfeifer, Dietmar ; Kunte, Hans-Jörg ; Koch, Matthias ; Rohn, S. ; Maul, R.
Zearalenone (ZEN) and its phase II sulfate and glucoside metabolites have been detected in food and feed commodities. After consumption, the conjugates can be hydrolyzed by the human intestinal microbiota leading to liberation of ZEN that implies an underestimation of the true ZEN exposure. To include ZEN conjugates in routine analysis, reliable standards are needed, which are currently not available. Thus, the aim of the present study was to develop a facilitated biosynthesis of ZEN-14-sulfate, ZEN-14-glucoside and ZEN-16-glucoside. A metabolite screening was conducted by adding ZEN to liquid fungi cultures of known ZEN conjugating Aspergillus and Rhizopus strains. Cultivation conditions and ZEN incubation time were varied. All media samples were analyzed for metabolite formation by HPLC-MS/MS. In addition, a consecutive biosynthesis was developed by using Fusarium graminearum for ZEN biosynthesis with subsequent conjugation of the toxin by utilizing Aspergillus and Rhizopus species. ZEN-14-sulfate (yield: 49%) is exclusively formed by Aspergillus oryzae. ZEN-14-glucoside (yield: 67%) and ZEN-16-glucoside (yield: 39%) are formed by Rhizopus oryzae and Rhizopus oligosporus, respectively. Purities of ≥73% ZEN-14-sulfate, ≥82% ZEN-14-glucoside and ≥50% ZEN-16-glucoside were obtained by 1H-NMR. In total, under optimized cultivation conditions, fungi can be easily utilized for a targeted and regioselective synthesis of ZEN conjugates.
Single-cell analysis by ICP-MS/MS as a fast tool for cellular bioavailability studies of arsenite
(2018)
Single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) has become a powerful and fast tool to evaluate the elemental composition at a single-cell level. In this study, the cellular bioavailability of arsenite (incubation of 25 and 50 μM for 0-48 h) has been successfully assessed by SC-ICP-MS/MS for the first time directly after re-suspending the cells in water. This procedure avoids the normally arising cell membrane permeabilization caused by cell fixation methods (e.g. methanol fixation). The reliability and feasibility of this SC-ICP-MS/MS approach with a limit of detection of 0.35 fg per cell was validated by conventional bulk ICP-MS/MS analysis after cell digestion and parallel measurement of sulfur and phosphorus.
Laux, P. ; Tentschert, J. ; Riebeling, Ch. ; Braeuning, A. ; Creutzenberg, O. ; Epp, A. ; Fessard, V. ; Haas, K.-H. ; Haase, A. ; Hund-Rinke, K. ; Jakubowski, Norbert ; Kearns, P. ; Lampen, A. ; Rauscher, H. ; Schoonjans, R. ; Störmer, A. ; Thielmann, A. ; Mühle, U. ; Luch, A.
Development and market introduction of new nanomaterials trigger the need for an adequate risk assessment of such products alongside suitable risk communication measures. Current application of classical and new nanomaterials is analyzed in context of regulatory requirements and standardization for chemicals, food and consumer products. The challenges of nanomaterial characterization as the main bottleneck of risk assessment and regulation are presented. In some areas, e.g., quantification of nanomaterials within complex matrices, the establishment and adaptation of analytical techniques such as laser ablation inductively coupled plasma mass spectrometry and others are potentially suited to meet the requirements. As an example, we here provide an approach for the reliable characterization of human exposure to nanomaterials resulting from food packaging. Furthermore, results of nanomaterial toxicity and ecotoxicity testing are discussed, with concluding key criteria such as solubility and fiber rigidity as important parameters to be considered in material development and regulation. Although an analysis of the public opinion has revealed a distinguished rating depending on the particular field of application, a rather positive perception of nanotechnology could be ascertained for the German public in general. An improvement of material characterization in both toxicological testing as well as end-product control was concluded as being the main obstacle to ensure not only safe use of materials, but also wide acceptance of this and any novel technology in the general public.