Characterization of endogenous promoters of GapC1 and GS for recombinant protein expression in Phaeodactylum tricornutum.

Although diatoms have been utilized as a cellular factory to produce biopharmaceuticals, recombinant proteins, and biofuels, only a few numbers of gene promoters are available. Therefore, the development of novel endogenous promoters is essential for the production of a range of bioactive substances. Here, we characterized the activities of endogenous promoters glyceraldehyde-3-phosphate dehydrogenase (GapC1) and glutamine synthetase (GS) of Phaeodactylum tricornutum using green fluorescent protein (GFP) under different culture conditions. Compared with the widely used fucoxanthin chlorophyll-binding protein A (fcpA) promoter, the GS promoter constitutively drove the expression of GFP throughout all growth phases of P. tricornutum, regardless of culture conditions. Additionally, the GFP level driven by the GapC1 promoter was the highest at the log phase, similar to the fcpA promoter, and increased light and nitrogen-starvation conditions reduced GFP levels by inhibiting promoter activity. These results suggested that the GS promoter could be utilized as a strong endogenous promoter for the genetic engineering of P. tricornutum.

Improvement in host metabolic homeostasis and alteration in gut microbiota in mice on the high-fat diet: A comparison of calcium supplements.

Despite the previously reported health benefits of calcium intake for the attenuation of metabolic disease, few studies have investigated the relationships among calcium intake, gut microbiota, and host metabolism. In this study, we assessed the effects of calcium supplementation on host microbial community composition and metabolic homeostasis. Mice were fed a high-fat diet with different calcium concentrations (4 and 12 g/kg) of 2 calcium supplements, calcium carbonate and calcium citrate. Supplementation with the higher concentration of calcium citrate significantly prevented body weight gain and decreased plasma biomarkers for metabolic disorder compared to calcium carbonate supplementation. Both calcium supplementation led to changes in microbial composition, increased propionate production and increased anorexigenic GLP-1 gene expression. The calcium citrate groups also experienced less metabolic endotoxemia. Our findings suggested that calcium supplementation could ameliorate host metabolic disorder caused by a high-fat diet, due to gut microbiota changes as well as decreased intestinal inflammation.

Identification and characterisation of the novel endogenous promoter HASP1 and its signal peptide from Phaeodactylum tricornutum.

Although diatoms have been extensively studied as bioreactors, only a limited number of efficient gene promoters are available. Therefore, the development of new endogenous promoters is important for the heterologous production of a variety of recombinant proteins. Herein, we identified the most abundant secreted protein in Phaeodactylum tricornutum, designated 'highly abundant secreted protein 1' (HASP1), and characterised the activities of its promoter and signal peptide using green fluorescent protein (GFP) as a reporter. The HASP1 promoter strongly drove GFP expression during all growth phases of P. tricornutum in culture, in contrast to the commonly used fcpA promoter, which is less active during the stationary phase. The HASP1 signal peptide was also sufficient for facilitating efficient secretion of GFP by P. tricornutum. Our findings suggest that both the promoter and the signal peptide of HASP1 can be utilized as novel tools for the overexpression and secretion of recombinant proteins in P. tricornutum.

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells.

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer's disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.