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.

Screening for Active Compounds Targeting Human Natural Killer Cell Activation Identifying Daphnetin as an Enhancer for IFN-γ Production and Direct Cytotoxicity.

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ+ NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments.

A combination of pirfenidone and TGF-ß inhibition mitigates cystic echinococcosis-associated hepatic injury.

Cystic echinococcosis (CE) occurs in the intermediate host's liver, assuming a bladder-like structure surrounded by the host-derived collagen capsule mainly derived from activated hepatic stellate cells (HSCs). However, the effect of CE on liver natural killer (NK) cells and the potential of transforming growth factor-ß (TGF-ß) signalling inhibition on alleviating CE-related liver damage remain to be explored. Here, by using the CE-mouse model, we revealed that the inhibitory receptors on the surface of liver NK cells were up-regulated, whereas the activating receptors were down-regulated over time. TGF-ß1 secretion was elevated in liver tissues and mainly derived from macrophages. A combination of TGF-ß signalling inhibitors SB525334 and pirfenidone could reduce the expression of TGF-ß1 signalling pathway-related proteins and collagen production. Based on the secretion of TGF-ß1, only the pirfenidone group showed a depressing effect. Also, the combination of SB525334 and pirfenidone exhibited a higher potential in effectively alleviating the senescence of the hepatocytes and restoring liver function. Together, TGF-ß1 may be a potential target for the treatment of CE-associated liver fibrosis.

[Study on effect of echinococcus granulosus protoscolices on fibrosis of bone marrow mesenchymal stem cells].

OBJECTIVE: To investigate the effect of echinococcus granulosus protoscolices on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into fibroblasts. METHODS: Femur bone marrow of 4-week-old C57BL/6 mice was taken and BMSCs were isolated and cultured by adherent culture. Echinococcus granulosus protoscolices was extracted from the liver of sheep infected with echinococcus granulosus. The experiment was divided into two groups. The experimental group was co-cultured with the 3rd generation BMSCs and the echinococcus granulosus protoscolices, and the control group was the 3rd generation BMSCs. Before and after co-culture, the morphology of BMSCs and the activity of echinococcus granulosus protoscolices were observed by inverted microscope. After cultured for 1, 3, 5, and 7 days, the mRNA expressions of transforming growth factor ß 1 (TGF-ß 1), collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescent quantitative PCR, the protein expressions of TGF-ß 1, collagen type Ⅰ, collagen type Ⅲ, Smad7, and phosphorylated Smad2/3 were detected by Western blot, and the contents of collagen type Ⅰ and collagen type Ⅲ in the supernatant of the two groups were detected by ELISA. RESULTS: After 7 days of co-culture, the morphology of BMSCs changed into fusiform and irregular triangle, which was closer to the mouse fibroblasts. The relative mRNA expressions of TGF-ß 1, collagen type Ⅰ, and collagen type Ⅲ in the experimental group were significantly higher than those in the control group; the relative protein expressions of TGF-ß 1, collagen type Ⅰ, collagen type Ⅲ, and phosphorylated Smad2/3 in the experimental group were significantly higher than those in the control group, and the relative protein expression of Smad7 in the experimental group was significantly lower than that in the control group; the contents of collagen type Ⅰ and collagen type Ⅲ in the supernatant of the experimental group were significantly higher than those in the control group. The differences between the two groups were significant ( P<0.05). CONCLUSION: Echinococcus granulosus protoscolices may promote the secretion of collagen type Ⅰ, collagen type Ⅲ, and TGF-ß 1 by TGF-ß 1/Smad signal pathway, which can promote the fibrosis of BMSCs that related to the formation of fibrocystic wall by echinococcosis.

Estrogen and propofol combination therapy inhibits endoplasmic reticulum stress and remarkably attenuates cerebral ischemia-reperfusion injury and OGD injury in hippocampus.

AIM: Endoplasmic reticulum stress (ERS) is vital in inducing apoptosis via caspase-12 and C/EBP homologous protein (CHOP) apoptotic pathway in the hippocampus after ischemia-reperfusion injury. The study aimed to estimate the efficacy of estrogen and propofol combination therapy against ERS-induced apoptosis after cerebral ischemia-reperfusion injury and oxygen-glucose deprivation (OGD) injury in the hippocampus in vivo and in vitro. METHODS: Rat model of cerebral ischemia-reperfusion injury was generated by middle cerebral artery occlusion (MCAO) strategy with ischemic intervention for 90 min and reperfusion for 24 h. Propofol processing ischemia-reperfusion group (Propofol group) infused 50 mg/kg/h of propofol via the femoral vein at the onset of reperfusion for 30 min. Estrogen processing ischemia-reperfusion group (estrogen group) received 0.0125 mg/kg of estrogen via tail vein at 30 min prior to MCAO. Combination therapy for ischemia-reperfusion group (combination group) received simultaneous processing with propofol and estrogen. In vitro, brain slices were randomly exposed to dimethylsulfoxide (DSMO), 10 µm of propofol, 10 nm of estrogen, or propofol and estrogen. Changes in the orthodromic population spike (OPS) at the end of reoxygenation were recorded. Neurological deficit examination, Nissl staining, and 2,3,5-triphenyltetrazolium chloride (TTC) staining were employed to evaluate the level of cerebral ischemia-reperfusion injury. The expression of caspase-3, caspase-12, glucose-regulated protein 78 (GRP78), and CHOP were investigated by Western blot and immunofluorescence staining assays. Neural apoptotic rate in hippocampus was detected by the flow cytometry trial. RESULTS: Neurological deficit score, infarct volume, the expression of caspase-3 (P < 0.05), caspase-12, GRP78, CHOP, and neural apoptotic rate of I/R group increased markedly (P < 0.01). When obtaining drug treatment, neurological deficit score (P < 0.05), infarct volume, the expression levels of caspase-12 and GRP78, and neural apoptotic rate of the propofol group decreased significantly (P < 0.01). Furthermore, neurological deficit score, infarct volume, expression levels of caspase-3, caspase-12, GRP78, and CHOP (P < 0.05), and neural apoptotic rate decreased in the estrogen group (P < 0.01) and especially in the combination group (P < 0.01). Compared with the propofol group, the neurological deficit score (P < 0.05), infarct volume, caspase-3, caspase-12, GRP78, CHOP, and neural apoptotic rate of the combination group decreased (P < 0.01). Compared with the estrogen group, the infarct volume, caspase-3 (P < 0.05), GRP78, CHOP, and neural apoptotic rate (P < 0.05) of the combination group decreased (P < 0.01). Compared with the propofol group, the infarct volume, caspase-3, caspase-12 (P < 0.05), and GRP78 (P < 0.05) of the estrogen group decreased (P < 0.01). Propofol and estrogen treatment can delay the abolishing time of OPS and increase the recovery rate and amplitude of OPS, compared with OGD group (P < 0.01), especially in the combination therapy (P < 0.01). CONCLUSION: The neuroprotection of propofol and estrogen combination therapy inhibited excessive ERS-induced apoptosis against cerebral ischemia-reperfusion injury and OGD injury in the hippocampus of rats. Furthermore, the outcomes demonstrated that combination therapy yielded synergistic effects.