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.     

A Method for Evaluating Lignin Mobility Distributions Obtained by Capillary Zone Electrophoresis

Abstract

The profile of the mobility distribution of lignin-containing samples depends on type of sample. To facilitate comparison, a procedure for determining the average mobility (μ_av), i.e. the average charge density, of lignin is presented. The procedure is applied to black liquor (Sb), isolated dissolved lignin (Sd) and isolated residual (Sr) lignin samples, obtained from flow-through kraft cooks of softwood. The μ_av of the isolated lignin samples is compared with the concentration of phenol and carboxyl groups and relative molecular size. As the cook proceeds the μ_av for a particular type of lignin sample increases, reflecting an increase in average charge density. The μ_av, measured at pH 12, decreases in the order Sd>Sb>Sr, except at the end of the cook, when the average charge densities of the Sb and Sr samples are similar. Associations between lignin and carbohydrate fragments are proposed to cause the lower mobility of black liquor compared to isolated dissolved lignin. Characterisations performed at pH 10 indicate that the isolated dissolved lignin samples may have a higher pK_a in the middle of the cook than the other samples.     

Fourier‐Transform Rheology of Unvulcanized,Carbon Black Filled Styrene Butadiene Rubber

Rubber materials filled with reinforcing fillers display nonlinear rheological behavior at small strain amplitudes below γ₀ < 0.1. Nevertheless, rheological data are analyzed mostly in terms of linear parameters, such as shear moduli (G′, G″), which loose their physical meaning in the nonlinear regime. In this work styrene butadiene rubber filled with carbon black (CB) under large amplitude oscillatory shear (LAOS) is analyzed in terms of the nonlinear parameter I_3/1. Three different CB grades are used and the filler load is varied between 0 and 70 phr. It is found that I_3/1(φ) is most sensitive to changes of the total accessible filler surface area at low strain amplitudes (γ₀ = 0.32). The addition of up to 70 phr CB leads to an increase of I_3/1(φ) by a factor of more than ten. The influence of the measurement temperature on I_3/1 is pronounced for CB levels above the percolation threshold.

     

Bivalent EGFR-Targeting DARPin-MMAE Conjugates

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).     

Commercially available alternatives to palm oil

Since several years there has been a demand for food products free of palm oil, noticeable in the Western European market. Alternatives based on liquid oils, fully hydrogenated fats, and exotic fats like shea and sal etc., have been developed by the research groups of several specialty oils and fats suppliers. This article describes the advantages and disadvantages of those products and compares them to similar products based on palm oil. It is also discussed how reasonable the replacement of palm products would be, since sustainable and 3‐MCPD/glycidolester‐reduced palm based specialty oils are also available on the market.     

Initial scalar lithospheric magnetic anomaly map of China and surrounding regions derived from CSES satellite data

Science China Technological Sciences - The China Seismo-Electromagnetic Satellite (CSES), China’s first satellite to measure geophysical fields with scientific goals in both space and solid...     

Elucidation of Phosphatidylcholine Composition in Krill Oil Extracted from <Emphasis Type=Italic>Euphausia superba</Emphasis>

High performance liquid chromatography-electrospray tandem mass spectrometry was used to elucidate the phospholipids in krill oil extracted from Euphausia superba, an emerging source for human nutritional supplements. The study was carried out in order to map the species of the choline-containing phospholipid classes: phosphatidylcholine and lyso-phosphatidylcholine. In addition, the prevalent phosphatidylcholine class was quantified and the results compared with prior analysis. The qualification was performed with separation on a reverse phase chromatography column, while the quantification was obtained with class separation on a normal phase chromatography column. An Orbitrap system was used for the detection, and pulsed-Q dissociation fragmentation was utilized for the identification of the species. An asymmetrical exclusion list was applied for detection of phospholipid species of lower concentration, significantly improving the number of species observed. A total of 69 choline-containing phospholipids were detected, whereof 60 phosphatidylcholine substances, among others seven with probable omega-3 fatty acids in both sn-1 and sn-2. The phosphatidylcholine concentration was estimated to be 34 ± 5 g/100 g oil (n = 5). These results confirm the complexity of the phospholipid composition of krill oil, and the presence of long chained, heavily unsaturated fatty acids.     

Effect of high temperature and the role of sulfate on adsorption behavior and effectiveness of AMPS®‐based cement fluid loss polymers

The fluid loss control performance of 2‐acrylamido‐2‐methylpropane sulfonic acid (AMPS®)‐based copolymers added to cement slurries was studied at 27 and 100°C, respectively. It was found that effectiveness of these fluid loss additives solely relies on achievement of a high adsorbed amount on the surface of cement. At elevated temperature (100°C), CaAMPS®‐N,N‐dimethyl acrylamide copolymer (CaAMPS®‐co‐NNDMA) exhibits reduced adsorption and hence decreased fluid loss control of the cement slurry. The reason behind this behavior is poor calcium binding capability of the sulfonate anchor groups, which coordinate with calcium atoms present on the mineral surface. Whereas, an increase in the sulfate concentration present in cement pore solution instigates partial coiling of CaAMPS®‐co‐NNDMA and causes only a slight influence on the performance of this copolymer. The elevated sulfate content results from thermal degradation of ettringite, a cement hydrate mineral produced during the early stages of cement hydration. Incorporation of minor amounts (～ 1.3 mol %) of maleic anhydride into this copolymer produces a terpolymer, which exhibits higher and more stable adsorption, even at high temperature. This effect is owed to the presence of homopolymer blocks of polycarboxylates distributed along the polymer trunk. On mineral surfaces, they present much stronger anchor groups than sulfonate functionalities, as evidenced by their higher calcium binding capability. Consequently, fluid loss performance of CaAMPS®‐co‐NNDMA‐co‐MA is little affected by temperature. Understanding the influence of temperature on the physicochemical interactions occurring between additives and the mineral surface can help to design more effective admixtures suitable for high temperature applications. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Friction of linerboard based on recycled fiber

The purpose of this investigation was to study the origin of the differences in paper‐to‐paper friction of linerboards based on old corrugated containers (OCC). The sheets were subjected to two extraction stages and analyzed with respect to, surface roughness, and their content of low‐molecular‐mass lipophilic compounds (LLC). Friction was measured using a friction tester based on the horizontal plane principle. The surface roughness was measured using a Perthometer profiler and the low molecular mass lipophilic constituent of the paper sheets was determined by gas chromatography‐mass spectroscopy. The sheets were imaged using environmental scanning electron microscopy (ESEM), and the relative compositions of inorganic ions on the paper surfaces were determined by energy dispersive X‐ray spectroscopy (EDS). The results showed that a high amount of LLC in the sheets lead to low friction, due to lubrication. It was also observed that large CaCO₃ particles on the surface had a friction‐increasing effect, and that there was no relationship between the surface roughness and the friction. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 85: 1511–1520, 2002