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.

Nitroreductase Increases Menadione-Mediated Oxidative Stress in Aspergillus nidulans.

Nitroreductases (NTRs) catalyze the reduction of a wide range of nitro-compounds and quinones using NAD(P)H. Although the physiological functions of these enzymes remain obscure, a tentative function of resistance to reactive oxygen species (ROS) via the detoxification of menadione has been proposed. This suggestion is based primarily on the transcriptional or translational induction of an NTR response to menadione rather than on convincing experimental evidence. We investigated the performance of a fungal NTR from Aspergillus nidulans (AnNTR) exposed to menadione to address the question of whether NTR is really an ROS defense enzyme. We confirmed that AnNTR was transcriptionally induced by external menadione. We observed that menadione treatment generated cytotoxic levels of O2•-, which requires well-known antioxidant enzymes such as superoxide dismutase, catalase, and peroxiredoxin to protect A. nidulans against menadione-derived ROS stress. However, AnNTR was counterproductive for ROS defense, since knocking out AnNTR decreased the intracellular O2•- levels, resulting in fungal viability higher than that of the wild type. This observation implies that AnNTR may accelerate the generation of O2•- from menadione. Our in vitro experiments indicated that AnNTR uses NADPH to reduce menadione in a single-electron reaction, and the subsequent semiquinone-quinone redox cycling resulted in O2•- generation. We demonstrated that A. nidulans nitroreductase should be an ROS generator, but not an ROS scavenger, in the presence of menadione. Our results clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous. IMPORTANCE Menadione is commonly used as an O2•- generator in studies of oxidative stress responses. However, the precise mechanism through which menadione mediates cellular O2•- generation, as well as the way in which cells respond, remains unclear. Elucidating these events will have important implications for the use of menadione in biological and medical studies. Our results show that the production of Aspergillus nidulans nitroreductase (AnNTR) was induced by menadione. However, the accumulated AnNTR did not protect cells but instead increased the cytotoxic effect of menadione through a single-electron reduction reaction. Our finding that nitroreductase is involved in the menadione-mediated O2•- generation pathway has clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous.

A self-targeting and controllable drug delivery system constituting mesoporous silica nanoparticles fabricated with a multi-stimuli responsive chitosan-based thin film layer.

Surface modification and functionalization of nanomaterials have been adopted widely in devising smart drug delivery systems. This work examines the fabrication of multi-stimuli responsive surfaces on mesoporous silica nanoparticles (MSN) for environmentally sensitive site specific drug delivery with reduced risk of premature drug leakage. Chitosan cross-linked via disulfide bonds was applied to form a thin film on drug-loaded MSN, realizing a capsulation and stimuli-sensitive regulating gate membrane; that was further conjugated with folate for site specific targeting toward cancer cells. The chitosan thin film was very stable under neutral conditions and could effectively prevent drug leakage, but was sensitive to both pH and GSH stimulations to reach rapid drug release. Thus, drug release could be triggered by changes in such factors that are common to cancer cells. However, complete and accelerated release could only be realized when triggered simultaneously by both acidic pH and GSH. Moreover, tests with HepG-2 cells confirmed that folate-receptor mediated endocytosis successfully enhanced the cellular uptake of the nanoparticle and antitumor activity toward cancer cells. It is expected that this surface chemical modification strategy promises a powerful approach constructing smart drug delivery systems for efficient and safe chemotherapy.