Mechanisms of temperature acclimatisation in the psychrotolerant green alga Coccomyxa subellipsoidea C-169 (Trebouxiophyceae).

Despite the interest in different temperature acclimatisations of higher plants, few studies have considered the mechanisms that allow psychrotolerant microalgae to live in a cold environment. Although the analysis of the genomes of some algae revealed the presence of specific genes that encode enzymes that can be involved in the response to stress, this area has not been explored deeply. This work aims to clarify the acclimatisation mechanisms that enable the psychrotolerant green alga Coccomyxa subellipsoidea C-169 to grow in a broad temperature spectrum. The contents of various biochemical compounds in cells, the lipid composition of the biological membranes of entire cells, and the thylakoid fraction as well as the electron transport rate and PSII efficiency were investigated. The results demonstrate an acclimatisation mechanism that is specific for C. subellipsoidea and that allows the maintenance of appropriate membrane fluidity, for example, in thylakoid membranes. It is achieved almost exclusively by changes within the unsaturated fatty acid pool, like changes from C18:2 into C18:3 and C16:2 into C16:3 or vice versa. This ensures, for example, an effective transport rate through PSII and in consequence a maximum quantum yield of it in cells growing at different temperatures. Furthermore, reactions characteristic for both psychrotolerant and mesophilic microalgae, involving the accumulation of lipids and soluble sugars in cells at temperatures other than optimal, were observed. These findings add substantially to our understanding of the acclimatisation of psychrotolerant organisms to a wide range of temperatures and prove that this process could be accomplished in a species-specific manner.

Capillary zone electrophoresis of bacterial extracellular vesicles: A proof of concept.

The extracellular vesicles (EVs) released by plant pathogens of the Pectobacterium genus were investigated. The isolates were obtained using differential centrifugation followed by filtration and were characterized in terms of total protein content and particle size distribution. The transmission electron microscopy (TEM) analysis revealed the presence of two morphologically differentiated subpopulations of vesicles in the obtained isolates. The proteomic analysis using matrix-assisted laser desorption ionization mass spectrometry with time of flight detector (MALDI-TOF/TOF-MS) enabled to identify 62 proteomic markers commonly found in EVs of Gram-negative rods from the Enterobacteriaceae family. Capillary electrophoresis (CE) was proposed as a novel tool for the characterization of EVs. The method allowed for automated and fast (<15 min per sample) separation of vesicles from macromolecular aggregates with low sample consumption (about 10 nL per analysis). The approach required simple background electrolyte (BGE) composed of 50 mM BTP and 75 mM glycine (pH 9.5) and standard UV detection. The report presents a new opportunity for quality control of samples containing EVs.

Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages.

Established cell lines are widely used in research, however an appealing question is the comparability of the cells between various laboratories, their characteristics and stability in time. Problematic is also the cell line misidentification, genetic and phenotypic shift or Mycoplasma contamination which are often forgotten in research papers. The monocyte/macrophage-like cell line RAW 264.7 has been one of the most commonly used myeloid cell line for more than 40 years. Despite its phenotypic and functional stability is often discussed in literature or at various scientific discussion panels, their stability during the consecutive passages has not been confirmed in any solid study. So far, only a few functional features of these cells have been studied, for example their ability to differentiate into osteoclasts. Therefore, in the present paper we have investigated the phenotype and functional stability of the RAW 264.7 cell line from passage no. 5 till passage no. 50. We found out that the phenotype (expression of particular macrophage-characteristic genes and surface markers) and functional characteristics (phagocytosis and NO production) of RAW 264.7 cell line remains stable through passages: from passage no. 10 up to passage no. 30. Overall, our results indicated that the RAW 264.7 cell line should not be used after the passage no. 30 otherwise it may influence the data reliability.

Dual-Laboratory Validation of a Method for the Determination of Fructans in Infant Formula and Adult Nutritionals: First Action 2016.14.

Until recently, only two AOAC Official MethodsSM have been available for the analysis of fructans: Method 997.08 and Method 999.03. Both are based on the analysis of the fructan component monosaccharides (glucose and fructose) after hydrolysis. The two methods have some limitations due to the strategies used for removing background interferences (such as from sucrose, α-glucooligosaccharides, and free sugars). The method described in this paper has been developed to overcome those limitations. The method is largely based on Method 999.03 and uses combined enzymatic and SPE steps to remove the interfering components without impacting the final analytical result. The method has been validated in two laboratories on infant formula and adult nutritionals. Recoveries were in the range of 86-119%, with most being in the range of 91-104%. RSDr values were in the range of 0.7-2.6%, with one exception when the fructan concentration was close to the LOQ, resulting in an RSDr of 8.9%. The performance is generally within the requirements outlined in the AOAC Standard Method Performance Requirements (SMPR® 2014.002), which specifies recoveries in the range of 90-110% and RSDr values below 6%.

Will the use of double barrier result in sustained release of vancomycin? Optimization of parameters for preparation of a new antibacterial alginate-based modern dressing.

The aim of this research was to prepare and characterize an alginate-based wound dressing containing vancomycin immobilized at the silica surface. The silica samples functionalized with amine, diol and carboxylic acid groups were loaded with 7.8, 5.7 and 7.1wt.% of the antibiotic respectively. The immobilized drug was encapsulated in alginate or gelatin/alginate gels and the average concentration of vancomycin was about 10mg per g of the dried gel. The effect of functional organic groups at the silica surface on the release rate of the drug was investigated. Only the drug immobilized at Si-amine in alginate matrix was found to demonstrate slower release from the proposed wound dressing. The in vitro release profiles for other silica carriers did not show significant differences in relation to the free loaded drug. The presence of gelatin had a favourable impact on the slowing down of the drug release from the dressing with a double barrier. All the gels studied with vancomycin immobilized at the silica surface demonstrated antimicrobial activity against various bacteria. A reduction of the drug dose to a half had no effect on changing microbiological activity of gels.

Vancomycin-modified silica: Synthesis, controlled release and biological activity of the drug.

Vancomycin was immobilized on three different organically functionalized silicas. The materials obtained were used for a controlled release of the antibiotic. The influence of the type of chemical bond on the in vitro drug release was investigated. A weak ionic bonding caused burst release of the drug within one day. A covalent bonding resulted in a slowdown in the release process and uniformity of dosage release. For these two carriers, biological activity of the drug was retained because the minimal inhibitory concentration values against the strains tested were similar to that of a free form of the drug (about 2 µg/mL). A strong ionic bonding of vancomycin adversely affected both the drug release, as well as its biological activity. A strong base on the surface of the silica prevented disconnection of the antibiotic which then became ineffective.