Purification of Recombinant Mouse C-Reactive Protein from Pichia Pastoris GS115 by Nickel Chelating Sepharose Fast-Flow Affinity Chromatography and P-Aminophenyl Phosphoryl Choline Agarose Resin Affinity Chromatography in Tandem

C-reactive protein (CRP) is a circulating marker of inflammation yet with ill-defined biological functions. This is partly due to the uncharacterized activities of endogenous CRP in mice, the major animal model used to define protein function. The hurdles for purification and characterization of mouse CRP are its low circulating levels and the lack of specific antibodies. To clear these hurdles, here we developed an efficient expression system by constructing recombinant Pichia pastoris cells for secretion of native conformation mouse CRP. The recombinant expression of mouse CRP in Escherichia coli failed to yield sufficient amount of native protein, reflecting the importance of post-translational modification of glycosylation in aiding proper folding. By contrast, sufficient amount of native mouse CRP was successfully purified from P. pastoris. Preliminary purification was performed by Nickel Chelating Sepharose Fast-Flow affinity chromatography with 6 × His tags attached to the protein. Subsequently, p-Aminophenyl Phosphoryl Choline Agarose resin affinity chromatography was used for tandem purification. The purified mouse CRP showed native pentamer and capabilities of PC binding. Moreover, the 6 × His tag provides a convenient tool for detecting the interactions of mouse CRP with ligands.

Comparison of Serum Choline and Trimethylamine N-oxide after ingestion of Alpha Glyceryl Phosphoryl Choline and Choline Salts.

Background: Choline supplements may provide potential improvements to health outcomes and exercise performance, yet the bioavailability of choline supplements is poorly understood. Objective: The objective of the present investigation was to examine the levels of serum Choline and Trimethylamine N-oxide (TMAO) after two dose of Alpha Glyceryl Phosphoryl Choline (A-GPC) in comparison to common choline salts. Methods: High-dose and low-dose A-GPC along with choline salt supplements were administered to participants over the course of four weeks in a randomized double-blind fashion. Serum levels of choline and TMAO, the gut byproduct of choline were examined acutely over the course of four hours and again following one week and four weeks of supplementation. Results: High-dose A-GPC and the choline chloride supplement yielded significantly higher choline levels compared to low-dose A-GPC and choline bitartate (F=31.31, p<0.01) though the effect for time was not significant. TMAO levels were not significantly different between supplements (F=1.96. p=0.1361) or across time (F=0.0795, p=0.7795). Conclusion: A-GPC increases serum choline similar to that seen with high-dose choline chloride ingestion without a concomitant increase in TMAO levels and therefore, may be a desirable option as a dietary supplement.

Discrimination between Fresh and Frozen-Thawed Fish Involved in Food Safety and Fraud Protection

This study aims to discriminate fresh fish from frozen/thawed by identification of the key metabolites that are altered during the freezing/thawing processing. Atlantic salmon (Salmo salar) and bullet tuna (Auxis rochei) were selected as they are representative of broad consumption, and susceptible to pathogen contamination. Atlantic salmon samples were subjected to the following regimes: −20 °C (24h) and −35 °C (15 h) freezing, then thawed respectively in the blast chiller and in the cold room and analyzed immediately or after 10 days; (2) bullet tuna samples were frozen at −18 °C and thawed after 15, 30 and 90 days. High resolution mass spectrometry based on untargeted metabolomic analyses and statistical data treatment confirmed significant variations in the quantity of certain metabolites: the amount of l-phenylalanine in salmon increased immediately after thawing while that of anserine decreased. The concentration of l-arginine and its metabolites was altered at the 10th day after thawing rendering them promising markers of salmon freezing/thawing. As regards bullet tuna, compounds resulting from lipid degradation (l-α-Glyceryl-phosphoryl-choline and N-methyl-ethanolamine phosphate) increased notably during the storage period. This approach could be used to reveal common fraudulent incidents such as deliberate replacement of fresh fish with frozen/thawed, with food safety risks as the primary implication.

Recent Advances in Manufacturing Innovative Stents

Cardiovascular diseases are the most distributed cause of death worldwide. Stenting of arteries as a percutaneous transluminal angioplasty procedure became a promising minimally invasive therapy based on re-opening narrowed arteries by stent insertion. In order to improve and optimize this method, many research groups are focusing on designing new or improving existent stents. Since the beginning of the stent development in 1986, starting with bare-metal stents (BMS), these devices have been continuously enhanced by applying new materials, developing stent coatings based on inorganic and organic compounds including drugs, nanoparticles or biological components such as genes and cells, as well as adapting stent designs with different fabrication technologies. Drug eluting stents (DES) have been developed to overcome the main shortcomings of BMS or coated stents. Coatings are mainly applied to control biocompatibility, degradation rate, protein adsorption, and allow adequate endothelialization in order to ensure better clinical outcome of BMS, reducing restenosis and thrombosis. As coating materials (i) organic polymers: polyurethanes, poly(ε-caprolactone), styrene-b-isobutylene-b-styrene, polyhydroxybutyrates, poly(lactide-co-glycolide), and phosphoryl choline; (ii) biological components: vascular endothelial growth factor (VEGF) and anti-CD34 antibody and (iii) inorganic coatings: noble metals, wide class of oxides, nitrides, silicide and carbide, hydroxyapatite, diamond-like carbon, and others are used. DES were developed to reduce the tissue hyperplasia and in-stent restenosis utilizing antiproliferative substances like paclitaxel, limus (siro-, zotaro-, evero-, bio-, amphi-, tacro-limus), ABT-578, tyrphostin AGL-2043, genes, etc. The innovative solutions aim at overcoming the main limitations of the stent technology, such as in-stent restenosis and stent thrombosis, while maintaining the prime requirements on biocompatibility, biodegradability, and mechanical behavior. This paper provides an overview of the existing stent types, their functionality, materials, and manufacturing conditions demonstrating the still huge potential for the development of promising stent solutions.

Uptake and transcytosis of functionalized superparamagnetic iron oxide nanoparticles in an in vitro blood brain barrier model

Phosphoryl choline grafting secured high cellular uptake and increased transcytosis of superparamagnetic iron oxide nanoparticles through a model blood brain barrier.