Mage transposon: a novel gene delivery system for mammalian cells.

Transposons, as non-viral integration vectors, provide a secure and efficient method for stable gene delivery. In this study, we have discovered Mage (MG), a novel member of the piggyBac(PB) family, which exhibits strong transposability in a variety of mammalian cells and primary T cells. The wild-type MG showed a weaker insertion preference for near genes, transcription start sites (TSS), CpG islands, and DNaseI hypersensitive sites in comparison to PB, approaching the random insertion pattern. Utilizing in silico virtual screening and feasible combinatorial mutagenesis in vitro, we effectively produced the hyperactive MG transposase (hyMagease). This variant boasts a transposition rate 60% greater than its native counterpart without significantly altering its insertion pattern. Furthermore, we applied the hyMagease to efficiently deliver chimeric antigen receptor (CAR) into T cells, leading to stable high-level expression and inducing significant anti-tumor effects both in vitro and in xenograft mice models. These findings provide a compelling tool for gene transfer research, emphasizing its potential and prospects in the domains of genetic engineering and gene therapy.

Atp23p and Atp10p coordinate to regulate the assembly of yeast mitochondrial ATP synthase.

Two chaperones, Atp23p and Atp10p, were previously shown to regulate the assembly of yeast mitochondrial ATP synthase, and extra expression of ATP23 was found to partially rescue an atp10 deletion mutant, by an unknown mechanism. Here, we identified that the residues 112-115 (LRDK) of Atp23p were required for its function in assisting assembly of the synthase, and demonstrated both functions of Atp23p, processing subunit 6 precursor and assisting assembly of the synthase, were required for the partial rescue of atp10 deletion mutant. By chasing labeling with isotope 35 S-methionine, we found the stability of subunit 6 of the synthase increased in atp10 null strain upon overexpression of ATP23. Further co-immunoprecipitation (Co-IP) and blue native PAGE experiments showed that Atp23p and Atp10p were physically associated with each other in wild type. Moreover, we revealed the expression level of Atp23p increased in atp10 null mutant compared with the wild type. Furthermore, we found that, after 72 hours growth, atp10 null mutant showed leaky growth on respiratory substrates, presence of low level of subunit 6 and partial recovery of oligomycin sensitivity of mitochondrial ATPase activity. Further characterization revealed the expression of Atp23p increased after 24 hours growth in the mutant. These results indicated, in atp10 null mutant, ATP10 deficiency could be partially complemented with increased expression of Atp23p by stabilizing some subunit 6 of the synthase. Taken together, this study revealed the two chaperones Atp23p and Atp10p coordinated to regulate the assembly of mitochondrial ATP synthase, which advanced our understanding of mechanism of assembly of yeast mitochondrial ATP synthase.

A core-shell-structured molecularly imprinted polymer on upconverting nanoparticles for selective and sensitive fluorescence sensing of sulfamethazine.

A core-shell structured molecularly imprinted polymer on upconverting nanoparticles (UCNPs@MIP) was synthesized for the fluorescence (FL) sensing of sulfamethazine (SMZ). Hexagonal UCNPs were synthesized by the solvothermal method, then coated with a thin silica shell and modified with vinyl groups. Finally, surface polymerization was initiated among the vinyl groups, the functional monomers and cross-linking agents by the initiator. The MIP synthesized by this procedure was anchored on the surface of UCNPs, possessed better site accessibility and lower transfer resistance for the target molecule compared to bulk imprinted materials. The obtained UCNPs@MIP showed good binding capacity, fast response, high selectivity and specificity to the SMZ. The FL intensity of the UCNPs@MIP decreased sensitively with the increasing concentration of SMZ in the range of 50-700 ng mL(-1), the detection limit was 34 ng mL(-1) (S/N = 3). The UCNPs@MIP was successfully applied to the detection of SMZ in chicken samples. Thanks to the unique near-infrared (NIR) excitation nature of UCNPs, the chicken meat only needed some simple extraction procedures before FL detection, no complex purifications were required. The average recoveries ranged from 96.01% to 98.90%, with relative standard deviations (RSDs) below 4.5%. This work offers a novel sensing system that combined the advantages of upconverting nanotechnology and molecularly imprinted technology.

[A modified TAIL-PCR and its application in isolating gene promoter of wheat].

Using a modified TAIL-PCR technique, the 5' -flanking region of the X gene in wheat was successfully isolated. Two novel modifications of the TAIL-PCR were introduced here: using a battery of random 10-mers as the short arbitrary primers instead of three degenerate 16-mers; using 29 degrees C instead of 44 degrees C as the annealing temperature for the low-stringency cycle; increasing five high-stringency cycles and reducing five low-stringency cycles; and using single primers for the third round of product identification. Isolated 5' -flanking region was fused to the GUS gene, and tested for expression in Arabidopsis plants. Histochemical analysis of the transgenic plants showed the report gene was driven by isolated 5'-flanking region. Modified TAIL-PCR technique could isolate rapidly the promoter of any gene from organisms with large genomes.