Terminal deoxynucleotidyl transferase-mediated CRISPR sensing platform for simple and point-of-care detection of cobalt pollution.

The excessive use of cobalt in various chemical industries and arbitrary discharge of industrial wastewater have led to increased cobalt pollution in soil and water resources, increasing the risk of human exposure to high concentrations of cobalt and necessitating an urgent need for on-site monitoring platform for cobalt pollution. In this study, the terminal deoxynucleotidyl transferase (TdT)-CRISPR platform has been developed. In this platform, cobalt as a cofactor of TdT, can significantly improve the tailing efficiency of TdT-mediated extension. Therefore, when cobalt is present, the detection probe can be extended with poly(T) tails through the TdT-mediated extension, which can be subsequently served as the DNA activator for Cas12a, leading to the cleavage of fluorescence reporter molecules and triggering turn-on fluorescence signals. Consequently, this dual amplification sensing strategy of TdT-CRISPR platform demonstrated exceptional sensitivity (0.83 nM) and high specificity for cobalt over other ions. Furthermore, the method was successfully employed for the detection of cobalt in tap water and river samples. CRISPR-lateral flow assays (CRISPR-LFAs) were evaluated in this study for the simple and point-of-care detection of cobalt pollution. The assays are capable of detecting cobalt concentrations as low as 50 nM, which is significantly lower than the environmental standards of 16.9 µM, through strip analysis with the naked eye. These results commonly suggest that the TdT-CRISPR platform holds significant promise for monitoring cobalt pollution, providing a robust and sensitive solution for on-site detection and contributing to the mitigation of cobalt contamination risks in environmental matrices.

CMTM6 promotes hepatocellular carcinoma progression through stabilizing ß-catenin.

CMTM6, a regulator of PD-L1 stability, has been implicated in the development of various cancers. However, the expression and role of CMTM6 in hepatocellular carcinoma (HCC) remains controversial. Our study revealed a negative correlation between CMTM6 expression and HCC prognosis through bioinformatics analysis and immunofluorescence staining. CMTM6 expression was also positively associated with alpha-fetoprotein (AFP) levels, supporting its potential as a prognostic marker for HCC. Using Cmtm6 knockout mice, we found that Cmtm6 deficiency inhibited HCC formation and cell proliferation in primary liver cancer models induced by DEN and DEN/CCl4. In HCC cell lines, CMTM6 promoted cell proliferation and interacted with ß-catenin, stabilizing it by preventing ubiquitination. In conclusion, our study suggested that CMTM6 upregulation promotes HCC cell proliferation through the ß-catenin pathway, making it a potential therapeutic target for HCC treatment.

Fluorescence turn-off strategy for sensitive detection of DNA methyltransferase activity based on DNA-templated gold nanoclusters.

DNA methylation results in a variety of human diseases and the DNA methylation process is mediated by DNA methyltransferases, which have therefore become potential targets for disease treatment. In this study, a turn-off nanogold biological probe system was successfully created for determining the activity of DNA methyltransferases (M.SssI MTase). A dumbbell-shaped DNA probe with a site-recognizable region of M. SssI MTase and a fluorescent signal probe based on a DNA-templated gold nanocluster (DNA-AuNC) probe combined for the quantitative detection of M. SssI MTase. This dumbbell-shaped DNA probe was methylated by M. SssI MTase, and the dumbbell-shaped DNA probe with a methyl group was recognized by an endonuclease (GlaI) and cleaved into hairpin DNA. The dGTP was added to the 3'-OH terminus of hairpin DNA fragments in the presence of terminal deoxynucleotidyl transferase (TdT), and the hairpin DNA was extended with a G-rich sequence that can be used as an inactivation probe. When the inactivation probe was combined with the signal probe, the fluorescent signal disappeared due to the photoinduced electron transfer effect. Methyltransferase activity was then detected based on the turn-off principle of the fluorescence signal from the DNA-AuNCs. The bioprobe enabled sensitive detection of M. SssI MTase with a detection limit of 0.178 U mL-1 and good specificity. The bioprobe demonstrated good detection efficiency in both human serum and cell lysates, and its unique fluorescence turn-off mechanism provided good resistance to interference, thus increasing its potential application in complex biological samples. Moreover, it is suitable for screening and assessing the inhibitory activity of M. SssI MTase inhibitors, and therefore has significant potential for disease diagnosis and drug discovery.

Co-Expression with Membrane CMTM6/4 on Tumor Epithelium Enhances the Prediction Value of PD-L1 on Anti-PD-1/L1 Therapeutic Efficacy in Gastric Adenocarcinoma.

Anti-PD-1/L1 immunotherapy has been intensively used in heavily treated population with advanced gastric adenocarcinoma. However, the immunotherapeutic efficacy is low even in PD-L1 positive patients. We aimed to establish a new strategy based on the co-expression of CMTM6/4 and PD-L1 for patient stratification before immunotherapy. By analyzing the data obtained from TCGA and single-cell RNA sequencing at the mRNA level, and 6-color multiplex immunofluorescence staining of tumor tissues in tissue array and 48-case pre-immunotherapy patients at the protein level, we found that CMTM6/4 and PD-L1 co-expressed in both epithelial and mesenchymal regions of gastric adenocarcinoma. The tumor tissues had higher levels of CMTM6/4 expression than their adjacent ones. A positive correlation was found between the expression of CMTM6/4 and the expression of PD-L1 in tumor epithelium. Epithelial co-expression of CMTM6/4 and PD-L1 in gastric tumor region was associated with shorter overall survival but better short-term response to anti-PD-1/L1 immunotherapy. Thus, we developed a predictive model and three pathological patterns based on the membrane co-expression of CMTM6/4 and PD-L1 in tumor epithelial cells for pre-immunotherapy patient screening in gastric adenocarcinoma.