Proton-Detected Solid-State NMR of the Cell-Free Synthesized α-Helical Transmembrane Protein NS4B from Hepatitis C Virus

Proton-detected 100 kHz magic-angle-spinning (MAS) solid-state NMR is an emerging analysis method for proteins with only hundreds of microgram quantities, and thus allows structural investigation of eukaryotic membrane proteins. This is the case for the cell-free synthesized hepatitis C virus (HCV) nonstructural membrane protein 4B (NS4B). We demonstrate NS4B sample optimization using fast reconstitution schemes that enable lipid-environment screening directly by NMR. 2D spectra and relaxation properties guide the choice of the best sample preparation to record 2D ¹H-detected ¹H,¹⁵N and 3D ¹H,¹³C,¹⁵N correlation experiments with linewidths and sensitivity suitable to initiate sequential assignments. Amino-acid-selectively labeled NS4B can be readily obtained using cell-free synthesis, opening the door to combinatorial labeling approaches which should enable structural studies.     

Is the particle deposition in a cell exposure facility comparable to the lungs? A computer model approach

Abstract

Cell exposure experiments at the air-liquid interface (ALI) are used increasingly as indicators for health effects and for the impact of aerosols on the lung. Thereby the aerosol particles are kept airborne and can deposit on a cell surface area similar to the human respiratory tract (RT). However, geometry and air flow rates of an ALI system deviate considerably from the RT. As the tissue-delivered particle dose to the lungs (TD) can hardly be measured, computer models of particle deposition are used here to mimic both the particle deposition at ALI and in the RT. An ALI exposure setup (VitroCell GmbH) for an airflow rate of 100 cm³ min^?1 is selected, where the particle deposition model has been verified experimentally. For the RT we use the hygroscopic lung deposition model of Ferron et al. (2013 Ferron, G. A., S. Upadhyay, R. Zimmermann, and E. Karg. 2013. Model of the deposition of aerosol particles in the respiratory tract of the rat. II. Hygroscopic particle deposition. J. Aerosol Med. Pulm. Drug Deliv. 26 (2):101–19. doi:10.1089/jamp.2011.0965.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]). Model runs are performed for the particle deposition and for the deposited particles per surface area in both the ALI and the RT. The results show that the ALI-deposited mass is 1-2 orders of magnitude higher than in the alveolar region, because the surface area of the lung region is substantially larger. A particle size range from 40 to 450 nm is identified, where the ratio of both the deposition in a lung region and the deposition at the ALI varies by a factor less than two. Mean values for this ratio are 31 and 101 for the tracheo-bronchial and the alveolar region, respectively. The same size range is found for the ratio of the deposited particles per surface area in a lung region and at the ALI. For this range the mean surface deposition at the ALI is 23- and 1575-times larger than in the tracheo-bronchial and the alveolar lung region, respectively. The effect is partly compensated by different flow rate and cell size.

Copyright © 2020 American Association for Aerosol Research     

Oxide ceramic fibers via dry spinning process—From lab to fab

Oxide fibers preparation and manufacturing capabilities at Fraunhofer-Center HTL are introduced, showing the development and preparation of oxide ceramic fibers from lab scale to pilot scale up to near production scale. As a specific example, the development of an aluminosilicate fiber with mullite composition is discussed in more detail. Fiber development started from nonaqueous sol-gel precursors in the early lab scale. With increasing fiber spinning volume, precursors were switched to water-soluble systems. Transformation from green fiber to ceramic fiber was monitored by thermogravimetric and differential thermal analysis, X-ray diffraction, and scanning electron microscopy. The evolution of ceramic phases, microstructure formation, and the effects on tensile strength and Young's modulus were investigated. Weibull statistics and fracture analysis helped to understand the results. Next step will be the transition from large lab scale to pilot scale, demonstrating manufacturing capability.     

SiCN ceramics with controllable carbon nanomaterials for electromagnetic absorption performance

Different kinds of carbon nanomaterials, free carbon (C_free), graphene, and N-containing graphene (NG), in single-source-precursors-derived SiCN ceramics, were in situ generated by modifying polysilazane with divinylbenzene, dopamine hydrochloride and melamine, respectively. Adjusting the carbon source brings phase structure and electromagnetic wave absorption (EMA) properties differences of SiCN/C ceramics. In situ C_free enhances the EMA capacity of SiCN ceramics by improving their electrical conductivity of 9.2 × 10⁻⁴ S/cm. The electrical conductivity of SiCN ceramics with 2D graphene sheets balloons to 2.5 × 10⁻³ S/cm, causing poor impedance match thus leading to a worse EMA performance. In situ NG in SiCN ceramics has a low electrical conductivity of 5.6 × 10⁻⁸ S/cm, making for excellent impedance match. The corrugated NG boosts dielectric loss, interfacial, and dipole polarization. NG-SiCN nanocomposites possess an outstanding EMA performance with RL_min of −61.08 dB and effective absorption bandwidth of 4.05 GHz, which are ∼2.4 times lower and ∼4 times higher than those of SiCN, respectively.     

A Novel Coenzyme A Analogue in the Anaerobic,Sulfate-Reducing,Marine Bacterium Desulfobacula toluolica Tol2T

Coenzyme A (CoA) thioesters are formed during anabolic and catabolic reactions in every organism. Degradation pathways of growth-supporting substrates in bacteria can be predicted by differential proteogenomic studies. Direct detection of proposed metabolites such as CoA thioesters by high-performance liquid chromatography coupled with high-resolution mass spectrometry can confirm the reaction sequence and demonstrate the activity of these degradation pathways. In the metabolomes of the anaerobic sulfate-reducing bacterium Desulfobacula toluolica Tol2^T grown with different substrates various CoA thioesters, derived from amino acid, fatty acid or alcohol metabolism, have been detected. Additionally, the cell extracts of this bacterium revealed a number of CoA analogues with molecular masses increased by 1 dalton. By comparing the chromatographic and mass spectrometric properties of synthetic reference standards with those of compounds detected in cell extracts of D. toluolica Tol2^T and by performing co-injection experiments, these analogues were identified as inosino-CoAs. These CoA thioesters contain inosine instead of adenosine as the nucleoside. To the best of our knowledge, this finding represents the first detection of naturally occurring inosino-CoA analogues.     

Polysilazane derived micro/nano Si3N4/SiC composites

The pyrolised polysilazanes poly(hydridomethyl)silazane NCP 200 and poly(urea)silazane CERASET derived Si–C–N amorphous powders were used for preparation of micro/nano Si₃N₄/SiC composites by hot pressing. Y₂O₃–Al₂O₃ and Y₂O₃–Yb₂O₃ were used, as sintering aids. The resulting ceramic composites of all compositions were dense and polycrystalline with fine microstructure of average grain size <1 μm of both Si₃N₄ and SiC phases. The fine SiC nano-inclusions were identified within the Si₃N₄ micrograins. Phase composition of both composites consist of , β modifications of Si₃N₄ and SiC. High weight loss was observed during the hot pressing cycle, 12 and 19 wt.% for NCP 200 and CERASET precursors, respectively. The fracture toughness of both nanocomposites (NCP 2000 and CERASET derived) was not different. Indentation method measured values are from 5 to 6 MPa m^1/2, with respect to the sintering additive system. Fracture toughness is slightly sensitive to the SiC content of the nanocomposite. Hardness increases with the content of SiC in the nanocomposite. The highest hardness was achieved for pyrolysed CERASET precursor with 2 wt.% Y₂O₃ and 6 wt.% Yb₂O₃, HV 23 GPa. This is a consequence of the highest SiC content as well as the chemical composition of additives.     

Crystallization of Polymer-Derived Silicon Carbonitride at 1873 K under Nitrogen Overpressure

The chemical stability of an amorphous silicon carbonitride ceramic, having the composition 0.57SiC·0.43Si₃N₄·0.49C is studied as a function of nitrogen overpressure at 1873 K. The ceramic suffers a weight loss at p _N2 < 3.5 bar (1 bar = 100 kPa), does not show a weight change from 3.5 to 11 bar, and gains weight above 11 bar. The structure of the ceramic changes with pressure: it is crystalline from 1 to 6 bar, amorphous at ∼10 bar, and is crystalline above ∼10 bar. The weight-loss transition, at 3.5 bar, is in excellent agreement with the prediction from thermodynamic analysis when the activities of carbon, SiC, and Si₃N₄ are set equal to those of the crystalline forms; this implies that the material crystallizes before decomposition. The amorphous to crystalline transition that occurs at ∼10 bar, and which is accompanied by weight gain, is likely to have taken place by a different mechanism. A nucleation and growth reaction with the atmospheric nitrogen is proposed as the likely mechanism. The supersaturation required to nucleate α-Si₃N₄ crystals is calculated to be 30 kJ/mol.     

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.     

DNA Primer Extension with Cyclopropenylated 7‐Deaza‐2′‐deoxyadenosine and Efficient Bioorthogonal Labeling in Vitro and in Living Cells

A deoxyadenosine triphosphate (dATP) analogue for DNA labeling was synthesized with the 1‐methylcyclopropene (1MCP) group at the 7‐position of 7‐deaza‐2′‐deoxyadenosine and applied for primer extension experiments. The real‐time kinetic data reveals that this 1MCP‐modified dATP analogue is incorporated into DNA much faster than that of the similarly 1MCP‐modified deoxyuridine triphosphate (dUTP) analogue. The postsynthetic fluorescent labeling of these oligonucleotides works efficiently according to PAGE analysis, and can be applied for immobilization of a functional antibody on a surface. Site‐specific labeling at two different positions in DNA was achieved and the bioorthogonality of the postsynthetic fluorescent labeling was demonstrated in living HeLa cells.     

Oxidation Kinetics of an Amorphous Silicon Carbonitride Ceramic

The oxidation kinetics of amorphous silicon carbonitride (SiCN) was measured at 1350°C in ambient air. Two types of specimens were studied: one in the form of thin disks, the other as a powder. Both specimens contained open nanoscale porosity. The disk specimens exhibited weight gain that saturated exponentially with time, analogous to the oxidation behavior of reaction-bonded Si₃N₄. The saturation value of the weight gain increased linearly with specimen volume, suggesting the nanoscale pore surfaces oxidized uniformly throughout the specimen. This interpretation was confirmed by high-resolution electron microscopy and secondary ion mass spectroscopy. Experiments with the powders (having a particle size much larger than the scale of the nanopores) were also consistent with measurements of the disks. However, the powder specimens, having a high surface-to-volume ratio, continued to show measurable weight gain due to oxidation of the exterior surface. The wide range of values for the surface-to-volume ratio, which included all specimens, permitted a separation of the rate of oxidation of the free surface and the oxidation of the internal surfaces of the nanopores. Surface oxidation data were used to obtain the rate constant for parabolic growth of the oxidation scale. The values for the rate constant obtained for SiCN lay at the lower end of the spectrum of oxidation rates reported in the literature for several Si₃N₄ and SiC materials. Convergence in the behavior of SiCN and CVD-SiC is ascribed to the purity of both materials. Conversely, it is proposed that the high rates of oxidation of sintered polycrystalline silicon carbides and nitrides, as well as the high degree of variability of these rates, might be related to the impurities introduced by the sintering aids.