Fluorescence-Based Nanoparticle Tracking Analysis and Flow Cytometry for Characterization of Endothelial Extracellular Vesicle Release

As extracellular vesicles (EVs) have become a prominent topic in life sciences, a growing number of studies are published on a regular basis addressing their biological relevance and possible applications. Nevertheless, the fundamental question of the true vesicular nature as well as possible influences on the EV secretion behavior have often been not adequately addressed. Furthermore, research regarding endothelial cell-derived EVs (EndoEVs) often focused on the large vesicular fractions comprising of microvesicles (MV) and apoptotic bodies. In this study we aimed to further extend the current knowledge of the influence of pre-isolation conditions, such as cell density and conditioning time, on EndoEV release from human umbilical vein endothelial cells (HUVECs). We combined fluorescence nanoparticle tracking analysis (NTA) and the established fluorescence-triggered flow cytometry (FT-FC) protocol to allow vesicle-specific detection and characterization of size and surface markers. We found significant effects of cell density and conditioning time on both abundance and size distribution of EndoEVs. Additionally, we present detailed information regarding the surface marker display on EVs from different fractions and size ranges. Our data provide crucial relevance for future projects aiming to elucidate EV secretion behavior of endothelial cells. Moreover, we show that the influence of different conditioning parameters on the nature of EndoEVs has to be considered.     

Engineering a Highly Active Sucrose Isomerase for Enhanced Product Specificity by Using a “Battleship” Strategy

The sucrose isomerase SmuA from Serratia plymuthica efficiently catalyses the isomerisation of sucrose into isomaltulose, an artificial sweetener used in the food industry. However, the formation of a hygroscopic by-product, trehalulose, necessitates additional separation to obtain a crystalline product. Therefore, we have improved the product specificity of SmuA by first introducing a few exploratory amino acid exchanges around the active site and investigating their influence. Then, we devised a second set of mutations, either at promising positions from the preceding cycle, but with a different side chain, or at alternative positions in the vicinity. After seven iterative cycles involving just 55 point mutations, we obtained the triple mutant Y219L/D398G/V465E which showed 2.3 times less trehalulose production but still had high catalytic efficiency (k_cat/K_M=11.8 mM⁻¹ s⁻¹). Not only does this mutant SmuA appear attractive as an industrial biocatalyst, but our semirational protein-engineering strategy, which resembles the battleship board game, should be of interest for other challenging enzyme optimization endeavours.     

Combining Inorganic Chemistry and Biology: The Underestimated Potential of Metal Complexes in Medicine

The vast majority of investigated compounds in modern medicine are based on organic molecules. Within the last decades, the field of medicinial chemistry is shifting towards the application of metal complexes. These compounds offer different mechanisms of action in comparison to organic molecules due to their unique properties, making them novel drug candidates. Herein, the successful combination of metal containing compounds and medicine is highlighted by their application for photodynamic therapy.     

Intraregional trade shares for goods-producing industries: RPC estimates using EU data

The lack of subnational trade data has dampened the development of reliable regional and multiregional models for regional policy development. So, most researchers and vendors of regional and interregional economic models continue to rely on location quotients, supply–demand pool techniques, or minor modifications of them, despite knowing that they under-estimate interregional trade. In this piece, we analyse the relative viability of estimates of intraregional trade—so called “regional purchase coefficients” (RPCs). We do so for manufacturing sectors in 28 EU countries using the World Input–Output Database. We introduce an RPC-estimating technique using a quasi-binomial regression approach for goods-producing industries; we apply standard supply/demand ratios as RPCs for service-based industries. We then apply the estimates to an aggregate EU input–output (I-O) table and measure how closely the results approximate the I-O tables (direct requirements matrices) for each of the 28 EU nations. We compare these findings to those obtained by other conventional approaches. We also evaluate their ability to replicate the country Leontief inverses and output multipliers. We find quasi-binomial regression approaches superior across the board.     

Quadrature for parabolic Galerkin BEM with moving surfaces

The successful implementation of the Galerkin Boundary Element Method hinges on the accurate and effective quadrature of the influence coefficients. For parabolic boundary integral operators quadrature must be performed in space and time where integrals have singularities when source- and evaluation points coincide. For problems where the surface is fixed, the time integration can be performed analytically, but for moving geometries numerical quadrature in space and time must be used. For this case a set of transformations is derived that render the singular space–time integrals into smooth integrals that can be treated with standard tensor product Gauss quadrature rules. This methodology can be applied to the heat equation and to transient Stokes flow.     

High-temperature phase and microstructure evolution of polymer-derived SiZrCN and SiZrBCN ceramic nanocomposites

A zirconium and a zirconium/boron containing single-source precursor were synthesized via chemical modification of a commercially available polysilazane (Durazane 1800) with tetrakis (dimethylamido) zirconium (IV) (TDMAZ) as well as with both TDMAZ and borane dimethyl sulfide complex, respectively. The polymer-to-ceramic transformation of the precursors into SiZrCN and SiZrBCN ceramics as well as the thermal evolution of their phase composition and microstructure was studied. The pyrolysis of the precursors led to the formation of amorphous SiZrCN and SiZrBCN ceramics. Interestingly, the as prepared SiZrBCN ceramic was single-phasic and fully featureless; whereas SiZrCN exhibited the presence of nano-sized ZrO₂ particles; however, only very localized in close proximity to internal surfaces. Heat treatment at higher temperatures induced crystallization processes in both prepared ceramics. Thus, at temperatures beyond 1500°C, cubic ZrC_xN_y, β-Si₃N₄ as well as β-SiC were generated. It was shown that the incorporation of B into SiZrCN suppressed the crystallization of ZrC_xN_y and, in addition, impeded the reaction of SiN_x with C, resulting in an improved thermal stability of SiZrBCN compared to SiZrCN ceramic. Moreover boron was shown to be mainly located in the sp²-hybridized “free” carbon present in SiZrBCN, forming a turbostratic BCN phase which has been unequivocally detected by means of high-resolution transmission electron microscopy (HRTEM) and energy-dispersive X-ray spectroscopy (EDS).     

Design and Synthesis of Dihydroxamic Acids as HDAC6/8/10 Inhibitors

We report the synthesis and evaluation of a class of selective multitarget agents for the inhibition of HDAC6, HDAC8, and HDAC10. The concept for this study grew out of a structural analysis of the two selective inhibitors Tubastatin A (HDAC6/10) and PCI-34051 (HDAC8), which we recognized share the same N-benzylindole core. Hybridization of the two inhibitor structures resulted in dihydroxamic acids with benzyl-indole and -indazole core motifs. These substances exhibit potent activity against HDAC6, HDAC8, and HDAC10, while retaining selectivity over HDAC1, HDAC2, and HDAC3. The best substance inhibited the viability of the SK-N-BE(2)C neuroblastoma cell line with an IC₅₀ value similar to a combination treatment with Tubastatin A and PCI-34051. This compound class establishes a proof of concept for such hybrid molecules and could serve as a starting point for the further development of enhanced HDAC6/8/10 inhibitors.     

First-Generation Bispidine Chelators for 213BiIII Radiopharmaceutical Applications

Hepta- and octadentate bispidines (3,7-diazabicyclo[3.3.1]nonane, diaza-adamantane) with acetate, methyl-pyridine, and methyl-picolinate pendant groups at the amine donors of the bispidine platform have been prepared and used to investigate Bi^III coordination chemistry. Crystal structure and solution spectroscopic data (NMR spectroscopy and mass spectrometry) confirm that the rigid and relatively large bispidine cavity with an axially distorted geometry is well suited for Bi^III and in all cases forms nine-coordinate complexes; this is supported by an established hole size and shape analysis. It follows that nonadentate bispidines probably will be more suited as bifunctional chelators for ²¹³Bi^III-based radiopharmaceuticals. However, two isomeric picolinate-/acetate-based heptadentate ligands already show very efficient complexation kinetics with ²¹³Bi^III at ambient temperature and kinetic stability that is comparable with the standard ligands used in this field. The experimentally determined hydrophilicities (log D_7.4 values) show that the Bi^III complexes reported are relatively hydrophilic and well suited for medicinal applications. We also present a very efficient and relatively accurate method to compute charge distributions and hydrophilicities, and this will help to further optimize the systems reported here.     

Effect of simulated brazing on the microstructure and corrosion behavior of twin roll cast AA3003

Aluminum alloy (AA) 3003 is widely used as fin and tube material in heat exchangers. In these heat exchangers, the tube and fin material are brazed together by means of an AA4xxx alloy. In this study, the effect of a simulated brazing step on the microstructure and corrosion behavior of a twin roll casted (TRC) AA3003 was studied. In particular, the effect of simulated brazing on the corrosion properties of the centre line segregation (CLS) of alloying elements and the interaction of the intermetallic particles with the surrounding matrix is explored. It is shown that the cross sections are significantly more active than the surface of the TRC AA3003, revealing the importance of the CLS on the corrosion behavior. Furthermore, it is shown that the number of pits and their depths decreased considerably on the exposed cross sections after simulated brazing, especially near the CLS. Scanning Kelvin probe force microscopy measurements show that after the simulated brazing step the Volta potential difference between the intermetallic particles and the matrix reduced significantly. This change in the Volta potential could be caused by the slight increase of Si content in the matrix.