Abstract. Adoptive transfer of NK cells has been widely applied clinically for cancer immunotherapy. However, the difficulties to obtain a large number of activated NK cells impede the successful application of such therapy. In the present study, we implemented a novel method involving the use of immobilized human 4-1BBL and interleukin-21 to amplify NK cells from the peripheral blood mononuclear cells (PBMCs) of healthy donors. Following stimulation for 21 days, we achieved considerable expansion of NK cells with high purity and strong cytotoxicity. This is the first time solid phase cytokines were used to augment NK cells, and this method has the advantage of no need to introduce feeder cells, without prior purification of NK cells and it effectively stimulated and expanded NK cells. The strategy of cell proliferation and activation could lead to a safer and more effective application of NK cells clinically.
Hepatic ischemia-reperfusion (I/R) injury, a common clinical complication of liver transplantation, gravely affects patient prognosis. Krüppel-like factors (KLFs) constitute a family of C2/H2 zinc finger DNA-binding proteins. KLF6, a member of the KLF protein family, plays crucial roles in proliferation, metabolism, inflammation, and injury responses; however, its role in HIR is largely remains unknown. After I/R injury, we found that KLF6 expression in mice and hepatocytes was significantly upregulated. Mice were then subjected to I/R following injection of shKLF6- and KLF6-overexpressing adenovirus through the tail vein. KLF6 deficiency markedly exacerbated liver damage, cell apoptosis, and activation of hepatic inflammatory responses, whereas hepatic overexpression of KLF6 in mice produced the opposite results. In addition, we knocked out or overexpressed KLF6 in AML12 cells before exposing them to a hypoxia-reoxygenation challenge. KLF6 knockout decreased cell viability and increased hepatocyte inflammation, apoptosis, and ROS, whereas KLF6 overexpression had the opposite effects. Mechanistically, KLF6 inhibited the overactivation of autophagy at the initial stage, and the regulatory effect of KLF6 on I/R injury was autophagy-dependent. CHIP-qPCR and luciferase reporter gene assays confirmed that KLF6 bound to the promoter region of Beclin1 and inhibited its transcription. Additionally, KLF6 activated the mTOR/ULK1 pathway. Finally, we performed a retrospective analysis of the clinical data of liver transplantation patients and identified significant associations between KLF6 expression and liver function following liver transplantation. In conclusion, KLF6 inhibited the overactivation of autophagy via transcriptional regulation of Beclin1 and activation of the mTOR/ULK1 pathway, thereby protecting the liver from I/R injury. KLF6 is expected to serve as a biomarker for estimating the severity of I/R injury following liver transplantation.
The cell envelope of gram-negative bacteria consists of the outer membrane (OM), inner membrane (IM), and periplasm. The β-barrel outer membrane proteins (OMPs) embedded in the OM perform diverse and significant functions such as signaling, transporting, and proteolysis. The OMPs of gram-negative bacteria share similar folding pathways with that of mitochondria and chloroplasts. Therefore, the study of the OMP folding mechanism not only provides insights into antimicrobial drug design but also helps elucidate mitochondrial and chloroplast biogenesis. Most knowledge about OMP folding was obtained from ensemble experiments where OMPs were usually at micromolar concentrations and prone to aggregate, which is different from the physiological environment in the cells. Unlike ensemble techniques, single-molecule detection (SMD) can measure OMPs from nano-to picomolar concentrations and prevent aggregation. In this work, we investigated the folding of OmpT, one of the OMPs, in Tween-20 and n-dodecyl β-d-maltopyranoside (DDM) micelles by SMD. We prepared monodisperse OmpT and observed both unfolded and folded OmpT in Tween-20 and DDM micelles under different urea concentrations by single-molecule fluorescence resonance energy transfer (FRET). The folded OmpT in Tween-20 is structurally similar to the native OmpT folded in DDM but exhibits weaker resistance to urea. In contrast, OmpA barely folds and OmpC hardly folds in Tween-20 micelles. We confirmed that folded OmpT forms complexes with detergent micelles and estimated the number of bound Tween-20 and DDM molecules per OmpT by fluorescence correlation spectroscopy. We compared the enzymatic activity of OmpT folded in two detergents with a fluorescent peptide as substrate, and found that the folded form of OmpT in Tween-20 possesses weaker enzymatic activity than that in DDM. We also investigated the folding properties of OmpT, OmpA, and OmpC in the presence of the β-barrel assembly machine (BAM) complex. OmpT folds efficiently in liposome even without the BAM complex; OmpA only folds with the help of the BAM complex; and OmpC does not fold with or without the BAM complex. Based on the comparison of the folding of OmpT, OmpA, and OmpC in detergent micelles and in the presence of the BAM complex, we propose that OmpT has stronger folding tendency than OmpA and OmpC, which supports the idea that the exact role of the BAM complex is dependent on the distinct folding properties of individual OMPs. Since Tween-20 is a widely used reagent to block nonspecific adsorption in SMD experiments, our results also remind people to exercise caution to prevent possible wrong interpretations caused by the interaction between proteins and Tween-20.
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