Identification strategy using combined mass spectrometric techniques for elucidation of phase I and phase II in vitro metabolites of lipophilic marine biotoxins

Combining mass spectrometric tools, a total of 47 in vitro metabolites of okadaic acid (OA), dinophysistoxins 1 and 2 (DTX1 and DTX2), yessotoxin (YTX), azaspiracid1 (AZA1), and pectenotoxin 2 (PTX2) could be detected and confirmed after an incubation with rat liver S9-mix. In a first step, liquid chromatography (LC) combined with tandem mass spectrometry (MS/MS) was used as a screening tool for the identification of in vitro metabolites of lipophilic marine biotoxins. Metabolic phase I and phase II reactions were screened for metabolites by calculating and subsequently monitoring theoretical MS transitions. In a second step, metabolites were confirmed by determination of accurate masses using high resolution MS provided by Orbitrap technology. Subsequently, product ion spectra, precursor ion spectra, and MS3 spectra were recorded for structure elucidation of metabolites. While all investigated toxins were found to form various oxygenated metabolites during the oxidative phase I metabolism, those metabolites varied in the number of added oxygen atoms and in the number of individual isomers. No hints were obtained concerning the formation of glutathione adducts, and a conjugation with glucuronic acid was detected for AZA1 only.     

Fabrication and structural characterization of interdigitated thin film La1 − x Sr x CoO 3 (LSCO) electrodes

For the prospective use as micro-Solid Oxide Fuel Cell (μ-SOFC) cathodes and for the investigation of reaction kinetics, La_{1 − x}Sr_xCoO₃ (LSCO) mixed ionic electronic conducting thin films were deposited by DC and RF sputtering onto a number of different substrate materials and characterized. Standard photolithographic and wet chemical etching methods were utilized to microstructure the LSCO films and XRD, SEM, AFM, WDS, and RBS were used to characterize their structure, topography, and chemistry. Sputtering resulted in very homogeneous and smooth thin crystalline films with Sr deficiency and submicron sized grains. Hydrochloric acid was found to readily etch LSCO with the etching quality strongly dependent on substrate material. LSCO films were most easily etched when deposited directly on silicon substrates, etched at intermediate rates when deposited on Gd:CeO₂ films, and most resistant to etching after deposition onto single crystal yttria stabilized zirconia (YSZ) substrates. Imperfect etching was attributed to interface formation and the presence of impurities.     

A Xanthohumol-Rich Hop Extract Diminishes Endotoxin-Induced Activation of TLR4 Signaling in Human Peripheral Blood Mononuclear Cells: A Study in Healthy Women

Infections with Gram-negative bacteria are still among the leading causes of infection-related deaths. Several studies suggest that the chalcone xanthohumol (XN) found in hop (Humulus lupulus) possesses anti-inflammatory effects. In a single-blinded, placebo controlled randomized cross-over design study we assessed if the oral intake of a single low dose of 0.125 mg of a XN derived through a XN-rich hop extract (75% XN) affects lipopolysaccharide (LPS)-induced immune responses in peripheral blood mononuclear cells (PBMCs) ex vivo in normal weight healthy women (n = 9) (clinicaltrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT04847193","term_id":"NCT04847193"}}NCT04847193) and determined associated molecular mechanisms. LPS-stimulation of PBMCs isolated from participants 1 h after the intake of the placebo for 2 h resulted in a significant induction of pro-inflammatory cytokine release which was significantly attenuated when participants had consumed XN. The XN-dependent attenuation of proinflammatory cytokine release was less pronounced 6 h after the LPS stimulation while the release of sCD14 was significantly reduced at this timepoint. The LPS-dependent activation of hTLR4 transfected HEK293 cells was significantly and dose-dependently suppressed by the XN-rich hop extract which was attenuated when cells were co-challenged with sCD14. Taken together, our results suggest even a one-time intake of low doses of XN consumed in a XN-rich hop extract can suppress LPS-dependent stimulation of PBMCs and that this is related to the interaction of the hop compound with the CD14/TLR4 signaling cascade.     

Oligomer Dynamics of LL-37 Truncated Fragments Probed by α-Hemolysin Pore and Molecular Simulations

Oligomerization of antimicrobial peptides (AMPs) is critical in their effects on pathogens. LL-37 and its truncated fragments are widely investigated regarding their structures, antimicrobial activities, and application, such as developing new antibiotics. Due to the small size and weak intermolecular interactions of LL-37 fragments, it is still elusive to establish the relationship between oligomeric states and antimicrobial activities. Here, an α-hemolysin nanopore, mass spectrometry (MS), and molecular dynamic (MD) simulations are used to characterize the oligomeric states of two LL-37 fragments. Nanopore studies provide evidence of trapping events related to the oligomer formation and provide further details on their stabilities, which are confirmed by MS and MD simulations. Furthermore, simulation results reveal the molecular basis of oligomer dynamics and states of LL-37 fragments. This work provides unique insights into the relationship between the oligomer dynamics of AMPs and their antimicrobial activities at the single-molecule level. The study demonstrates how integrating methods allows deciphering single molecule level understanding from nanopore sensing approaches.     

Sulfation Pattern Dependent Iron(III) Mediated Interleukin-8 Glycan Binding

Cytokines such as interleukin-8 activate the immune system during infection and interact with sulfated glycosaminoglycans with specific sulfation patterns. In some cases, these interactions are mediated by metal ion binding which can be used to tune surface-based glycan-protein interactions. We evaluated the effect of both hyaluronan sulfation degree and Fe³⁺ on interleukin-8 binding by electrochemical impedance spectroscopy and surface characterizations. Our results show that sulfation degree and metal ion interactions have a synergistic effect in tuning the electrochemical response of the glycated surfaces to the cytokine.     

Aspects of reproducibility and stability for partial cure of epoxy matrix resin

Partial cure of thermosets is a promising approach to enhance manufacturing possibilities of reinforced and unreinforced polymers. If partial cure is taken into consideration as a genuine process parameter, novel manufacturing technologies can be developed by exploiting the specific properties of incomplete polymer networks. A main concern in this context is to control the kinetic reaction avoiding inhomogeneous or instable degrees of cure. Based on a combination of numerical simulations and experiments, a methodology is presented that enables a systematic assessment of the reproducibility and stability of partial cure. Special attention is paid to the interaction of thermal boundary conditions and the cure kinetic of thick samples as well as the storability of partially cured resin under different conditions. Guidelines for cure cycle selection, mold design, and storage are derived. The possibility to use complex multistep cure schedules and extended storage periods is demonstrated for an unmodified noninhibited epoxy resin. © 2019 The Authors. Journal of Applied Polymer Science published by Wiley Periodicals Inc. J. Appl. Polym. Sci. 2020 , 137, 48342.     

The Central Region of Testican-2 Forms a Compact Core and Promotes Cell Migration

Testicans are modular proteoglycans of the extracellular matrix of various tissues where they contribute to matrix integrity and exert cellular effects like neurite outgrowth and cell migration. Using testican-2 as a representative member of the family, we tackle the complete lack of general structural information and structure–function relationship. First, we show using isothermal titration calorimetry and modeling that extracellular calcium-binding domain (EC) has only one active calcium-binding site, while the other potential site is inactive, and that testican-2 is within extracellular matrix always in the calcium-loaded form. Next, we demonstrate using various prediction methods that N- and C-terminal regions plus interdomain connections are flexible. We support this by small-angle X-ray-scattering analysis of C-terminally truncated testican-2, which indicates that the triplet follistatin-EC-thyroglobulin domain forms a moderately compact core while the unique N-terminal is disordered. Finally, using cell exclusion zone assay, we show that it is this domain triplet that is responsible for promoting cell migration and not the N- and C-terminal regions.     

First Ring-Expanded Maytansin Lactone Accessed by a New Mutasynthetic Variant

A multiblocked mutant strain (ΔAHBA and Δasm12, asm21) of Actinosynnema pretiosum, the producer of the highly toxic maytansinoid ansamitocin, has been used for the mutasynthetic production of new proansamitocin derivatives. The use of mutant strains that are blocked in the biosynthesis of an early building block as well as in the expression of two tailoring enzymes broadens the scope of chemo-biosynthetic access to new maytansinoids. Remarkably, a ring-expanded macrolactone derived from ansamitocin was created for the first time.