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A thorough characterization of starting materials is the precondition for further research, especially for cement, which contains various phases and presents quite a complex material for fundamental scientific investigation. In the paper at hand, the characterization data of the reference cement CEM I 42.5 R used within the priority program 2005 of the German Research Foundation (DFG SPP 2005) are presented from the aspects of chemical and mineralogical compositions as well as physical and chemical properties. The data were collected based on tests conducted by nine research groups involved in this cooperative program. For all data received, the mean values and the corresponding errors were calculated. The results shall be used for the ongoing research within the priority program.
Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagentsmare expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via Nhydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve.
Cancer cell lipid class homeostasis is altered under nutrient-deprivation but stable under hypoxia
(2019)
Background: Cancer cells modify the balance between fatty acid (FA) synthesis and uptake under metabolic stress, induced by oxygen/nutrient deprivation. These modifications were shown to alter the levels of individual triglyceride (TG) or phospholipid sub-species. To attain a holistic overview of the lipidomic profiles of cancer cells under stress we performed a broad lipidomic assay, comprising 244 lipids from six major classes. This assay allowed us to perform robust analyses and assess the changes in averages of broader lipid-classes, stratified on the basis of saturation index of their fatty-acyl side chains.
Methods: Global lipidomic profiling using Liquid Chromatography-Mass Spectrometry was performed to assess lipidomic profiles of biologically diverse cancer cell lines cultivated under metabolically stressed conditions.
Results: Neutral lipid compositions were markedly modified under serum-deprived conditions and, strikingly, the cellular level of triglyceride subspecies decreased with increasing number of double bonds in their fatty acyl chains.
In contrast and unexpectedly, no robust changes were observed in lipidomic profiles of hypoxic (2% O2) cancer cells despite concurrent changes in proliferation rates and metabolic gene expression.
Conclusions: Serum-deprivation significantly affects lipidomic profiles of cancer cells. Although, the levels of individual lipid moieties alter under hypoxia (2% O2), the robust averages of broader lipid classes remain unchanged.
Molecular MRI is a promising in-vivo modality to detect and quantify morphological and molecular vessel-wall changes in atherosclerosis. The combination of different molecular biomarkers may improve the risk stratification of patients. This study aimed to investigate the feasibility of simultaneous visualization and quantification of plaque-burden and inflammatory activity by dual-probe molecular
MRI in a mouse-model of progressive atherosclerosis and in response-to-therapy. Homozygous apolipoprotein E knockout mice (ApoE−/−) were fed a high-fat-diet (HFD) for up to four-months prior to MRI of the brachiocephalic-artery. To assess response-to-therapy, a statin was administered for the same duration. MR imaging was performed before and after administration of an elastin-specific gadolinium-based and a macrophage-specific iron-oxide-based probe. Following in-vivo MRI, samples were analyzed using histology, immunohistochemistry, inductively-coupled-mass-spectrometry and laser-inductively-coupled-mass-spectrometry. In atherosclerotic-plaques, intraplaque expression
of elastic-fibers and inflammatory activity were not directly linked. While the elastin-specific probe demonstrated the highest accumulation in advanced atherosclerotic-plaques after four-months of HFD, the iron-oxide-based probe showed highest accumulation in early atherosclerotic-plaques after two months of HFD. In-vivo measurements for the elastin and iron-oxide-probe were in good agreement
with ex-vivo histopathology (Elastica-van-Giesson stain: y = 298.2 + 5.8, R2 = 0.83, p < 0.05; Perls‘ Prussian-blue-stain: y = 834.1 + 0.67, R2 = 0.88, p < 0.05). Contrast-to-noise-ratio (CNR) measurements of the elastin probe were in good agreement with ICP-MS (y = 0.11x-11.3, R² = 0.73, p < 0.05). Late stage atherosclerotic-plaques displayed the strongest increase in both CNR and gadolinium concentration (p < 0.05). The gadolinium probe did not affect the visualization of the iron-oxide-probe and vice versa. This study demonstrates the feasibility of simultaneous assessment of plaque-burden.
Xenobiotics and their reactive metabolites are conjugated with native biomolecules such as glutathione and glucoside during phase II metabolism. Toxic metabolites are usually detoxified during this step. On the other hand, these reactive species have a potential health impact by disrupting many enzymatic functions. Thus, it is crucial to understand phase II conjugation reactions of xenobiotics in order to address their fate and possible toxicity mechanisms.
Additionally, conventional methods (in vivo and in vitro) have limitation due to matrix complexity and time-consuming. Hence, developing fast and matrix-free alternative method is highly demandable. In this work, oxidative phase I metabolites and reactive species of chlorpyrifos (insecticide) and fluopyram (fungicide) were electrochemically produced by using a boron-doped diamond electrode coupled online to electrospray mass spectrometry (ESI-MS). Reactive species of the substrates were trapped by biomolecules (glutathione and glucoside) and phase II conjugative metabolites were identified using liquid chromatography (LC)-MS/MS, and/or Triple time of flight (TripleTOF)-MS. Glutathione conjugates and glucosylation of chlorpyrifos, trichloropyridinol, oxon, and monohydroxyl fluopyram were identified successfully. Glutathione and glucoside were conjugated with chlorpyrifos, trichloropyridinol, and oxon by losing a neutral HCl. In the case of fluopyram, its monohydroxyl metabolite was actively conjugated with both glutathione and glucoside. In summary, seven bioconjugates of CPF and its metabolites and two bioconjugates of fluopyram metabolites were identified using electrochemistry (EC)/MS for the first time in this work. The work could be used as an alternative approach to identify glutathione and glucosylation conjugation reactions of other organic compounds too. It is important, especially to predict phase II conjugation within a short time and matrix-free environment.
Combining X-ray Absorption Fine Spectroscopy (XAFS) with Anomalous Small-Angle X-ray Scattering (ASAXS) determines the location of Cu2+ ions in silicoaluminophosphate (SAPO-5) frameworks prepared by hydrothermal crystallization or impregnation. As expected, for the hydrothermally prepared sample, incorporation in the SAPO-5 framework was observed. For the first time preferential location of Cu2+ ions at the inner and outer surfaces of the framework is determined. Temperature-Programmed Reduction (TPR) and X-ray Photoelectron Spectroscopy (XPS) investigations demonstrated that such Cu2+ is stable in an argon (Ar) atmosphere up to 550 °C and can only be reduced under a hydrogen atmosphere. In contrast, Cu2+ deposited by impregnation on the pure SAPO-5 framework can be easily reduced to Cu+ in an Ar atmosphere. At lower Cu amounts, mononuclear tetrahedrally coordinated Cu species were formed which are relatively stable in the monovalent form. In contrast, at higher Cu amounts, CuO particles were found which change easily between the mono- and bivalent species.
Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput “all-on-one chip” system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide.
Commercial bead-based assays are commonly built upon polystyrene particles. The polymeric carrier can be encoded with organic dyes and has ideal material properties for cytometric applications such as low density and high refractive index. However, functional groups are conventionally integrated during polymerization and subsequent modification is limited to the reactivity of those groups. Additionally, polystyrene as the core material leads to many hydrophobic areas still being present on the beads’ surfaces even after functionalization, Rendering the particles prone to nonspecific adsorption during an application. The latter calls for several washing steps and the use of additives in (bio)analytical assays. In this contribution, we show how these limitations can be overcome by using monodisperse polystyrene (PS) core/silica (SiO2) shell particles (SiO2@PS). Two different hydrophobic BODIPY (boron−dipyrromethene) dyes were encapsulated inside a poly(vinylpyrrolidone) (PVP) -stabilized polystyrene core in different concentrations to create 5-plex arrays in two separate detection channels of a cytometer. A subsequent modification of the silica shell with an equimolar APTES/PEGS (aminopropyltriethoxysilane/polyethylene glycol silane) blend added multifunctional properties to the hybrid core/Shell microparticles in a single step: APTES provides amino groups for the attachment of a caffeine derivative (as a hapten) to create antigen-coupled microspheres; the PEG moiety effectively suppresses nonspecific binding of antibodies, endowing the surface with antifouling properties. The particles were applied in a competitive fluorescence immunoassay in suspension, and a highly selective wash-free assay for the detection of caffeine in beverages was developed as a proof of concept.
The movement of the macroscopic magnetic moment in ferromagnetic systems can be described by the Landau-Lifshitz (LL) or Landau-Lifshitz-Gilbert (LLG) equation. These equations are strictly valid only at absolute zero temperature. To include temperature effects a stochastic version of the LL or LLG equation for a spin density of one per unit cell can be used instead. To apply the stochastic LL to micromagnetic simulations, where the spin density per unit cell is generally higher, a conversion regarding simulation cell size and temperature has to be established. Based on energetic considerations, a conversion for ferromagnetic bulk and thin film systems is proposed. The conversion is tested in micromagnetic simulations which are performed with the Object Oriented Micromagnetic Framework (OOMMF). The Curie temperatures of bulk Nickel, Cobalt and Iron systems as well as Nickel thin-film systems with thicknesses between 6.3 mono layer (ML) and 31ML are determined from micromagnetic simulations. The results show a good agreement with experimentally determined Curie temperatures of bulk and thin film systems when temperature scaling is performed according to the presented model.
As humanity contemplates manned missions to Mars, strategies need to be developed for the design and operation of hospitable environments to safely work in space for years. The supply of spare parts for repair and replacement of lost equipment will be one key need, but in-space manufacturing remains the only option for a timely supply. With high flexibility in design and the ability to manufacture ready-to-use components directly from a computeraided model, additive manufacturing (AM) technologies appear extremely attractive. For the manufacturing of metal parts, laser-beam melting is the most widely used AM process. However, the handling of metal powders in the absence of gravity is one prerequisite for its successful application in space. A gas flow throughout the powder bed is successfully applied to compensate for missing gravitational forces in microgravity experiments. This so-called gas-flow-assisted powder deposition is based on a porous Building platform acting as a filter for the fixation of metal particles in a gas flow driven by a pressure difference maintained by a vacuum pump.