We examined the responsiveness of unicellular green alga Dunalliela tertiolecta to selected stressors employing confocal- and time-resolved imaging of endogenous fluorescence. Our aim was to monitor cell endogenous fluorescence changes under exposure to heavy metal Cd, acidification, as well as light by laser-induced photobleaching. The accumulation of Cd in algae cells was confirmed by the secondary ion mass spectroscopy technique. For the first time, custom-made computational techniques were employed to evaluate separately the fluorescence in the flagella vs. the body region. In the presence of Cd, we recorded increase in the green fluorescence in the flagella region in the form of opacities, without change in the fluorescence lifetimes, suggesting higher availability of the fluorescent molecules. Under acidification, we noted significant rise in the green fluorescence in the flagella region, but associated with longer fluorescence lifetimes, pointing to changes in the algae environment. Photobleaching experiments corroborated gathered observations. Obtained data support a differential responsiveness of the flagella vs. the body region to stressors and enable us to better understand the pathophysiological changes of algal cells in culture under stress conditions.
Hydrophobic interaction chromatography is a versatile method to polish antibodies. Here, we present a polishing procedure in order to obtain an ultra-pure preparation of antitumor necrosis factor (TNF) alpha IgG1. Hydrophobic interaction chromatography (HIC) was used with Toyopearl® Phenyl 650M adsorbent in the presence of ammonium sulfate. Adsorption isotherms, breakthrough curves and chromatographic runs were carried out. The eluted antibody was recovered with 99.9 % purity and 96.2 % yield. In the main peak, aggregates, host cell proteins (HCP) and DNA content were below the limit of detection of the analytical methods used. Thus, the method proposed here shows potential to be employed in a downstream process when an ultra-pure antibody preparation is required.
Core fucosylation of an Fc N-linked glycan affects antibody effector functions, as the absence of fucose increases the antibody dependent cell cytotoxicity (ADCC) response with increased binding to the Fcγ receptors. The work presented here compares two different approaches to incrementally reduce core fucosylation of a camelid heavy chain antibody, EG2-hFc expressed in CHO cells which targets the EGFR receptor. The first method uses a fucosyltransferase (FUT) inhibitor, 2- fluoro peracetylated fucose (2FF), which was added to cell cultures expressing the EG2-hFc antibody in increasing concentrations up to 50μM. At this concentration there was no observed effect on cell growth. Glycan analysis was performed on antibodies collected from culture samples using HILIC-HPLC. The inhibitor reduced total fucosylation from 80% to 17.5% at 20μM 2FF. The second method involved transfecting the EG2-hFc producing cells with a prokaryotic GDP- 6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene in order to deflect the fucose de novo pathway into producing rhamnose which is not incorporated into a glycan. Stable clones from transfected pools were isolated following flow cytometry using the green fluorescent protein (GFP) marker which was co-expressed with the RMD gene. High expressing RMD clones reduced the fucosylation of the antibody glycan to as low as 16%. The addition of 2FF to cultures of these RMD clones reduced the fucosylation level even further to 3% of the antibody glycan. An incremental increase in fucosylation was obtained by step-wise addition of fucose (up to 1 mM) to the RMD cells, in which the fucosylation level increased to a maximum of 87%. We also used ESI-MS to analyze the fucosylation pattern of EG2-hFc with addition of increasing concentrations of 2FF. This showed that 2FF inhibits the addition of fucose in a concentration- dependent and specific manner with the inhibition of fucose occurring one fucose at a time. Control cultures showed the presence of a predominant peak indicating two fucose moieties per antibody. As the 2FF inhibitor concentration was increased peaks corresponding to one fucose per antibody and non-fucosylated antibody predominated with a gradual decrease of the 2 fucose peak to insignificance at 15 μM 2FF.
Sugar nucleotide-dependent (Leloir) glycosyltransferases are powerful catalysts for glycoside synthesis. Their applicability can be limited due to elaborate production of enzyme preparations deployable in biocatalytic processes. Here, we show that efficient enzyme formulation promotes glycosyltransferases for the synthesis of the natural C-glycoside nothofagin. Adding Brij-35 detergent (1 %, w/v) during sonication of the E. coli BL21-Gold (DE3) expression strain, recovery of Oryza sativa C-glycosyltransferase was enhanced by ∼3-fold, partly due to the release of enzyme activity trapped in insoluble pellet. Freeze drying of the resulting cell-free extract (∼17 U ml-1) reduced the volume ∼20-fold and gave ∼55 mg solids ml-1 liquid processed, with 83 % retention of the original activity and a specific activity of 0.20 U mg-1 solids. The Glycine max sucrose synthase was processed analogously, giving a solid enzyme preparation of 0.28 U mg-1 in 63 % yield. Both enzyme formulations were stable for several weeks. The glycosyltransferase cascade reaction for 3'-β-C-glucosylation of phloretin (60 mM; as inclusion complex with hydroxypropyl-β-cyclodextrin) from UDP-glucose (generated in situ by sucrose synthase from 500 mM sucrose and 0.5 mM UDP) showed excellent performance metrics (≥ 98 % yield; 3.2 g l-1 h-1 space-time yield; ∼90 regeneration cycles for UDP). Collectively, our study demonstrates a facile procedure for solid glycosyltransferase formulations practically usable in glycoside synthesis.
Dengue virus (DENV), an arthropod-borne virus, has rapidly spread in recent years. DENV diagnosis is performed through virus serology, isolation or molecular detection, while genotyping is usually done through Sanger sequencing of the envelope gene. This study aimed to optimize the use of shotgun metagenomics and subsequent bioinformatics analysis to detect and type DENV directly from clinical samples without targeted amplification. Additionally, presence of DENV quasispecies (intra-host variation) was revealed by detecting single nucleotide variants. Viral RNA was isolated with or without DNase-I treatment from 17 DENV (1-4) positive blood samples. cDNA libraries were generated using either a combination of the NEBNext® RNA to synthesize cDNA followed by Nextera XT DNA library preparation, or the TruSeq RNA V2 (TS) library preparation kit. Libraries were sequenced using both the MiSeq and NextSeq. Bioinformatic analysis showed complete ORFs for all samples by all approaches, but longer contigs and higher sequencing depths were obtained with the TS kit. No differences were observed between MiSeq and NextSeq sequencing. Detection of multiple DENV serotypes in a single sample was feasible. Finally, results were obtained within three days with associated reagents costs between €130-170/sample. Therefore, shotgun metagenomics is suitable for identification and typing of DENV in a clinical setting.
