Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease Ⅳ-assisted exponential signal amplification

Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 30-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 30-PO_(4) end of the substrate and generates 30-OH,TdT can effectively elongate the 30-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 30-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10^(-3) U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.

A cost-effective detection of low-abundance mutation with DNA three-way junction structure and lambda exonuclease

We presented a low-abundance mutation detection method with lambda exonuclease and DNA threeway junction structure.The assistant strand in the DNA three-way junction structure could regulate the reaction system from the kinetics and thermodyna mics aspects.The optimization of the assista nt strand helps to improve the selectivity of the mutant-type DNA to the wild-type DNA about 35 times.Moreover,the cost of the optimization process could be saved by about 90%.The method was applied to the detection of a human ovarian cancer-related gene mutation BRCA1(rs1799949,c.2082 C>T).The limit of detection to the mutation abundance in the DNA three-way junction structure system(0.2%) was one order lower compared with that in the double-stranded DNA structure system(2%).The mutation abundance in different standard samples was quantitively measured,and the results were consistent with the initial abundance in the standard samples.

The OX40-TRAF6 axis promotes CTLA-4 degradation to augment antitumor CD8^(+)T-cell immunity

Immune checkpoint blockade(ICB),including anti-cytotoxic T-lymphocyte associated protein 4(CTLA-4),benefits only a limited number of patients with cancer.Understanding the in-depth regulatory mechanism of CTLA-4 protein stability and its functional significance may help identify ICB resistance mechanisms and assist in the development of novel immunotherapeutic modalities to improve ICB efficacy.Here,we identified that TNF receptor-associated factor 6(TRAF6)mediates Lys63-linked ubiquitination and subsequent lysosomal degradation of CTLA-4.Moreover,by using TRAF6-deficient mice and retroviral overexpression experiments,we demonstrated that TRAF6 promotes CTLA-4 degradation in a T-cell-intrinsic manner,which is dependent on the RING domain of TRAF6.This intrinsic regulatory mechanism contributes to CD8+T-cell-mediated antitumor immunity in vivo.Additionally,by using an OX40 agonist,we demonstrated that the OX40-TRAF6 axis is responsible for CTLA-4 degradation,thereby controlling antitumor immunity in both tumor-bearing mice and patients with cancer.Overall,our findings demonstrate that the OX40-TRAF6 axis promotes CTLA-4 degradation and is a potential therapeutic target for the improvement of T-cell-based immunotherapies.