High-throughput evolutionary optimization of the induction medium towards recombinant protein production in BY-2 tobacco.

Bright yellow (BY-2) tobacco cells combined with the XVE chemically inducible system are one of the most promising plant-based platforms for recombinant protein production. This offers a range of benefits, including the separation of the cell growth and heterologous gene expression, lack of risk of infecting the end product with prions and human viruses or appropriate protein glycosylation and folding. However, low protein productivity remains a major obstacle that limits the extensive commercialization of bioproduction in plants. A number of molecular, cell culture and down processing approaches have been made to overcome this problem. Media development for the specific nutritional and hormonal requirements of transgenic plant cells is one of the most efficient cell-culture approaches. We optimized the induction medium towards recombinant protein production in BY-2 and demonstrated the usefulness of evolutionary medium optimization for high-yield protein production in liquid plant cultures. A reliable XVE/GFP model, parallel conducting experiments in a microscale on 96-well plates, and dedicated Gene Game evolutionary optimization software allowed for an effective search of 7611 possible solutions of 11-component media. Within the 4608 formulations tested, the Induct X medium was found with a significant 107.14% increase in protein expression in relation to the standard BY-2 medium.

Effects of the activation of heme oxygenase-1 on hormonal and metabolic changes in rats fed a high-fat diet.

Heme oxygenase-1 (HO-1) is the inducible isoform of the heme oxygenase system, which catalyzes heme degradation. Up-regulation of this enzyme under pathological conditions is associated with beneficial effects in the whole organism. However, the potential of HO-1 in the alleviation of disturbances induced by a high-fat diet (HFD) is poorly elucidated. The present study was undertaken to determine the effects of pharmacological activation of HO-1 by hemin on some hormones and metabolic parameters in rats fed an HFD for 8 weeks. It was demonstrated that, in rats fed an HFD, blood glucose levels were increased compared with control animals. However, this hyperglycemic effect was alleviated by induction of HO-1. The observed decrease in glycemia was not associated with an increase in blood insulin concentrations, but was accompanied by improved glucose tolerance, which points to the potentiation of insulin action. Concentrations of free fatty acids were elevated in response to HFD; however, this effect appeared to be mitigated by hemin. Rats fed an HFD displayed clear-cut hyperleptinemia, which is a hallmark of leptin resistance. This derangement was effectively prevented by hemin therapy. Feeding with an HFD also increased blood ghrelin levels, whereas hemin slightly reduced blood ghrelin concentration. Carbohydrate and lipid metabolism in the liver of rats on an HFD was found to be disturbed, leading to increased lipid accumulation and reduced glycogen stores. However, negative changes in liver metabolism were partially attenuated as a result of induction of HO-1. Our results show that activation of HO-1 by hemin ameliorates some changes induced by HFD feeding. Normalization of blood leptin levels in these animals seems to be the most relevant finding, since hyperleptinemia is associated with dysregulation of energy homeostasis and with numerous other disorders. These results indicate that the HO system holds great potential to alleviate alterations induced by HFD.

Variability of lysozyme and lactoferrin bioactive protein concentrations in equine milk in relation to LYZ and LTF gene polymorphisms and expression.

BACKGROUND: Equine milk is considered to be an interesting product for human nutrition, mainly owing to its low allergenicity and significant amounts of bioactive proteins, including lysozyme (LYZ) and lactoferrin (LTF). The present study assessed the effect of genetic factors on LYZ and LTF concentration variability in mare's milk. RESULTS: Significant effects of horse breed and lactation stage on milk LYZ and LTF contents were observed. The highest level of LTF and the lowest concentration of LYZ were recorded for the Polish Warmblood Horse breed. The highest amounts of both proteins were found for the earliest investigated time point of lactation (5th week). Altogether 13 (nine novel) polymorphisms were found in the 5'-flanking regions of both genes, but they showed no significant relationship with milk LYZ and LTF contents. Several associations were found between selected SNPs and the LYZ gene relative transcript level. CONCLUSION: While the present study indicated the existence of intra- and interbreed variability of LYZ and LTF contents in mare's milk, this variation is rather unrelated to the 5'-flanking variants of genes encoding both proteins. This study is a good introduction for broader investigations focused on the genetic background for variability of bioactive protein contents in mare's milk. © 2016 Society of Chemical Industry.