GC–MS analysis of oxysterols and their formation in cultivated liver cells (HepG2)

Oxysterols play a key role in many (patho)physiological processes and they are potential biomarkers for oxidative stress in several diseases. Here we developed a rapid gas chromatographic-mass spectrometry-based method for the separation and quantification of 11 biologically relevant oxysterols bearing hydroxy, epoxy, and dihydroxy groups. Efficient chromatographic separation (resolution ≥ 1.9) was achieved using a medium polarity 35%-diphenyl/65%-dimethyl polysiloxane stationary phase material (30 m × 0.25 mm inner diameter and 0.25 μm film thickness). Based on thorough analysis of the fragmentation during electron ionization we developed a strategy to deduce structural information of the oxysterols. Optimized sample preparation includes (i) extraction with a mixture of n-hexane/iso-propanol, (ii) removal of cholesterol by solid phase extraction with unmodified silica, and (iii) trimethylsilylation. The method was successfully applied on the analysis of brain samples, showing consistent results with previous studies and a good intra- and interday precision of ≤20%. Finally, we used the method for the investigation of oxysterol formation during oxidative stress in HepG2 cells. Incubation with tert-butyl hydroperoxide led to a massive increase in free radical formed oxysterols (7-keto-chol > 7β-OH-chol >> 7α-OH-chol), while 24 h incubation with the glutathione peroxidase 4 inhibitor RSL3 showed no increase in oxidative stress based on the oxysterol pattern. Overall, the new method described here enables the robust analysis of a biologically meaningful pattern of oxysterols with high sensitivity and precision allowing us to gain new insights in the biological formation and role of oxysterols.     

Leakage of Metallic Ball Seat Valves with Anisotropic Surfaces

Metallic ball seat valves are an essential component of many hydraulic systems. They are used for many different purposes such as pressure relief valves or check valves. Despite their universal usage, their sealing mechanism is not yet fully understood. In previous works, a successful method for the simulation of the fluid leakage of metallic ball seat valves has been developed and confirmed experimentally. The method is based on Persson's contact mechanics theory, which is based on surface roughness power spectrum C(q). This theory takes a wide range of roughness values at different length scales into account. The previous method has been restricted to isotropic surfaces, but most surfaces of practical interest are highly anisotropic. A method for the calculation of pressure flow factors using Persson's method is presented. Based on these, a model for the leakage calculation can be developed. The simulation results are validated using an experiment.     

Phase separation of a hafnium alkoxide-modified polysilazane upon polymer-to-ceramic transformation—A case study

The polymer-to-ceramic transformation of a hafnium alkoxide-modified polysilazane was investigated via thermogravimetric analysis coupled with in situ mass spectrometry (TG/MS), nuclear magnetic resonance (MAS NMR) and transmission electron microscopy (TEM). The results indicate that the structural evolution of the polysilazane upon ceramization is strongly affected by the modification with hafnium alkoxide. Thus, the content of carbon in the ceramic backbone was relatively low, whereas a large amount of SiN₄ sites and a segregated carbon phase was present in the sample. Furthermore, this study revealed the formation of a SiHfCNO amorphous single phase ceramic via pyrolysis of the polymer at 700 °C, whereas at higher pyrolysis temperatures precipitation of hafnia was observed, leading to an amorphous hafnia/silicon carbonitride ceramic nanocomposite. The precipitation of hafnia was shown to not rely on decomposition processes, but to be a result of rearrangement reactions occurring within the ceramic material.     

Enabling resilient P2P video streaming: survey and analysis

Peer-to-Peer (P2P) techniques for multimedia streaming have been shown to be a good enhancement to the traditional client/server methods when trying to reduce costs and increase robustness. Due to the fact that P2P systems are highly dynamic, the main challenge that has to be addressed remains supporting the general resilience of the system. Various challenges arise when building a resilient P2P streaming system, such as network failures and system dynamics. In this paper, we first classify the different challenges that face P2P streaming and then present and analyze the possible countermeasures. We classify resilience mechanisms as either core mechanisms, which are part of the system, or as cross-layer mechanisms that use information from different communication layers, which might inflict additional costs. We analyze and present resilience mechanisms from an engineering point of view, such that a system engineer can use our analysis as a guide to build a resilient P2P streaming system with different mechanisms and for various application scenarios.     

Kochen-Specker theorem studied with neutron interferometer

The Kochen-Specker theorem shows the incompatibility of noncontextual hidden variable theories with quantum mechanics. Quantum contextuality is a more general concept than quantum non-locality which is quite well tested in experiments using Bell inequalities. Within neutron interferometry we performed an experimental test of the Kochen-Specker theorem with an inequality, which identifies quantum contextuality, by using spin-path entanglement of single neutrons. Here entanglement is achieved not between different particles, but between degrees of freedom of a single neutron, i.e., between spin and path degree of freedom. Appropriate combinations of the spin analysis and the position of the phase shifter allow an experimental verification of the violation of an inequality derived from the Kochen-Specker theorem. The observed violation 2.291±0.008?1 clearly shows that quantum mechanical predictions cannot be reproduced by noncontextual hidden variable theories.     

Determination of flux directions by thermodynamic network analysis: Computing informative metabolite pools

Network thermodynamics focuses on the energetic analysis of complex metabolic networks. The method connects free Gibbs energies, metabolite concentrations and flux directions by fundamental thermodynamic laws. Here, a new application of network thermodynamics is presented that identifies those metabolite pools that have to be measured in order to determine as many flux directions as possible. For a medium-scaled reaction network such informative metabolite pools are computed with an approach based on Monte Carlo sampling. It turns out that some reactions can be directed with only a few measurements whereas other reactions cannot be directed even with a complete data set. High connectivity in metabolic reaction networks in alliance with concentration ranges make it impossible to intuitively foresee such results. In particular, the impact of measurements of a special type of metabolites being involved in many reactions, so called energetic currency metabolites is investigated.     

New Anacardic Acid‐Inspired Benzamides: Histone Lysine Acetyltransferase Activators

A series of N‐(4‐cyano‐3‐trifluoromethyl‐phenyl)‐2‐ethoxy‐6‐alkyl (and alkenyl) benzamides related to the anacardic acid derivative CTPB have been prepared from 2,6‐dihydroxybenzoic acid with a Suzuki coupling and addition of the anion of 4‐cyano‐3‐trifluoromethylphenylamine to a benzodioxinone as the key steps. In U937 cells, these analogues, in particular 7 c , 7 d , 7 f and 7 j , induced cell‐cycle arrest in the G1 phase, caused apoptosis in about 20 % of the cells, and increased the acetylation levels of H3. These activities correlate with the enzymatic activation of histone lysine acetyltransferases (KATs): CBP and PCAF.