Mast1 mediates radiation-induced gastric injury via the P38 MAPK pathway.

Radiation-induced gastric injury is a serious adverse effect and reduces the efficacy of radiotherapy treatment. However, the mechanisms underlying radiation-induced stomach injury remain unclear. Here, mouse stomach and gastric epithelial cells were irradiated with different doses of X-ray radiation. The results showed that radiation induced gastric injury in vivo and in vitro. Differentially expressed functional mRNAs in irradiation-induced gastric tissues were screened from the Gene Expression Omnibus (GEO) database. We found that the expression of microtubule-associated serine/threonine kinase 1 (Mast1) was downregulated in mouse gastric tissues and gastric epithelial cells after irradiation. Furthermore, functional assays showed that knockdown of Mast1 inhibited growth and promoted apoptosis in gastric epithelial cells, while overexpression of Mast1 protected gastric epithelial cells from radiation damage. Mechanistically, Mast1 negatively regulated radiation-induced injury in gastric epithelial cells by inhibiting the activation of P38. The apoptosis caused by knockdown of Mast1 in gastric epithelial cells could be partially reversed by the P38 inhibitor SB203580. Moreover, data from several gastric cancer cell lines and online databases revealed that Mast1 was not involved in the development of gastric cancer. Collectively, our findings demonstrated that Mast1 is essential for radiation-induced gastric injury, providing a promising prognostic and therapeutic target.

Quikgene: a gene synthesis method integrated with ligation-free cloning.

Gene synthesis is a convenient tool that is widely used to make genes for a variety of purposes. All current protocols essentially take inside-out approaches to assemble complete genes using DNA oligonucleotides or intermediate fragments. Here we present an efficient method that integrates gene synthesis and cloning into one step. Our method, which is evolved from QuikChange mutagenesis, can modify, extend, or even de novo synthesize relatively large genes. The genes are inserted directly into vectors without ligations or subcloning. We de novo synthesized a 600-bp gene through multiple steps of polymerase chain reaction (PCR) directly into a bacterial expression vector. This outside-in gene synthesis method is called Quikgene. Furthermore, we have defined an overlap region of a minimum of nine nucleotides in insertion primers that is sufficient enough to circularize PCR products for efficient transformation, allowing one to significantly reduce the lengths of primers. Taken together, our protocol greatly extends the current length limit for QuikChange insertion. More importantly, it combines gene synthesis and cloning into one step. It has potential applications for high-throughput structural genomics.

Recovery of steviol glycosides from industrial stevia by-product via crystallization and reversed-phase chromatography.

Mother liquor sugar (MLS), as the by-product of stevia production, contained ~65% steviol glycosides (SGs). Recovery of the SGs from MLS was achieved by crystallization coupled with reversed-phase chromatography. Crystallization was done by dissolving MLS in methanol solution and SGs were crystallized due to the polarity difference from the medium. Composition of SGs crystals differed with various temperature, time, solid-to-liquid ratio and water content. SGs were 42.25% recovered with high purity of 96.89% under optimal conditions (solid-to-liquid ratio = 1:5 (w/v), T = 25 °C, t = 24 h) in absolute methanol. The liquid phase after crystallization was subsequently subjected to reversed-phase chromatography, whereby the impurities were firstly eluted with 35% (v/v) ethanol solution and the purified SGs were then desorbed by absolute ethanol, finally recovering 95.20% of SGs in the purity of 98.08%. The total SGs recovery of the whole procedure was 97.23%. The two-step purification was easy-to-operate and feasible to scale-up for industrial application.