Human tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL) might be developed as a novel anti-tumor drug due to its selective cytotoxicity in tumor cells. The predicted Macaca mulatta TRAIL (mmTRAIL) is highly homologous to hTRAIL in nucleotide acid as well as amino acid sequence, suggesting that mmTRAIL might induce apoptosis of human cancer cells. However, the cytotoxicity of mmTRAIL in human cancer cells has not been investigated. In this paper, it is reported that the gene encoding mmTRAIL has been cloned by using reverse-transcriptase polymerase chain reaction (RT-PCR) from monkey peripheral blood mononuclear cells (PBMCs) in our laboratory. Subsequently, an expression plasmid was constructed by inserting mmTRAIL gene into pQE30 plasmid. After induction by addition of Isopropyl β-D-1-Thiogalactopyranoside (IPTG), mmTRAIL was expressed. MmTRAIL was recovered from supernatant of sonicated bacteria by Ni-NTA agarose affinity chromatography. SDS-PAGE and gel filtration chromatography demonstrated that mmTRAIL forms trimer in solution. In vitro assays indicated that mmTRAIL was cytotoxic to human COLO205 tumor cells but not to normal cells at low concentration of nanomole. In addition, antitumor effect of mmTRAIL was evaluated in mice bearing human COLO205 tumor xenografts. Intratumorally injected mmTRAIL significantly inhibited growth of tumor grafts. These results suggested that mmTRAIL was valuable as candidate drug for cancer-targeted therapy.
Animals ; Antineoplastic Agents ; Apoptosis ; Cell Line, Tumor ; Cloning, Molecular ; Humans ; Leukocytes, Mononuclear ; Macaca mulatta ; Mice ; Plasmids ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Xenograft Model Antitumor Assays

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification in molecular diagnostics. The PCR includes multiple reaction stages (denaturation, annealing, and extension), and a complicated thermalcycler is required to repetitively provide different temperatures for different stages for 30-40 cycles within at least 1-2 hours. Due to the complicated devices and the long amplification time, it is difficult to adopt conventional PCR in point-of-care testing (POCT). Comparing to conventional PCR, isothermal amplification is able to provide a much faster and more convenient nucleic acid detection because of highly efficient amplification at a constant reaction temperature provided by a simple heating device. When isothermal amplification is combined with microfluidics, a more competent platform for POCT can be established. For example, various diagnosis devices based on isothermal amplification have been used to rapidly and conveniently detect SARS-CoV-2 viruses. This review summarized the recent development and applications of the microfluidics-based isothermal amplification. First, different typical isothermal amplification methods and related detection methods have been introduced. Subsequently, different types of microfluidic systems with isothermal amplification were discussed based on their characteristics, for example, functionality, system structure, flow control, and operation principles. Furthermore, detection of pathogens (e.g. SARS-CoV-2 viruses) based on isothermal amplification was introduced. Finally, the combination of isothermal amplification with other new technologies, e.g. CRISPR, has been introduced as well.
COVID-19/diagnosis* ; Humans ; Microfluidics ; Nucleic Acid Amplification Techniques ; Polymerase Chain Reaction ; SARS-CoV-2/genetics*