Tuning the Dynamic Reaction Balance of CRISPR/Cas12a and RPA in One Pot: A Key to Switch Nucleic Acid Quantification.

Excavating nucleic acid quantitative capabilities by combining clustered regularly interspaced short palindromic repeats (CRISPR) and isothermal amplification in one pot is of common interest. However, the mutual interference between CRISPR cleavage and isothermal amplification is the primary obstacle to quantitative detection. Though several works have demonstrated enhanced detection sensitivity by reducing the inhibition of CRISPR on amplification in one pot, few paid attention to the amplification process and even dynamic reaction processes between the two. Herein, we find that DNA quantification can be realized by regulating either recombinase polymerase amplification (RPA) efficiency or CRISPR/Cas12a cleaving efficiency (namely, tuning the dynamic reaction balance) in one pot. The sensitive quantification is realized by utilizing dual PAM-free crRNAs for CRISPR/Cas12a recognition. The varied RPA primer concentration with stabilized CRISPR systems significantly affects the amplification efficiency and quantitative performances. Alternatively, quantitative detection can also be achieved by stabilizing the amplification process while regulating the CRISPR/Cas12a concentration. The quantitative capability is proved by detecting DNA targets from Lactobacillus acetotolerans and SARS-CoV-2. The quantitative performance toward real samples is comparable to quantitative real-time PCR for detecting L. acetotolerans spiked in fermented food samples and SARS-CoV-2 clinical samples. We expect that the presented method will be a powerful tool for quantifying other nucleic acid targets.

Effect of gut microbiome regulated Taohong Siwu Decoction metabolism on glioma cell phenotype.

Introduction: To establish a new model for exploring the mechanism of the gut microbiome and drug metabolism, we explored whether Taohong Siwu Decoction acts after metabolism by intestinal flora under the premise of clarifying the interaction between intestinal flora and drug metabolism. Methods: Taohong Siwu Decoction (TSD) was fed to germ-free mice and conventional mice, respectively. The serum from both groups of mice was removed and co-cultured with glioma cells in vitro. The co-cultured glioma cells were compared separately for changes at the RNA level using RNA-seq technology. The genes of interest in the comparison results were selected for validation. Results: The differences in the phenotypic alterations of glioma cells between serum from TSD-fed germ-free mice and normal mice were statistically significant. In vitro experiments showed that Taohong Siwu Decoction-fed normal mouse serum-stimulated glioma cells, which inhibited proliferation and increased autophagy. RNA-seq analysis showed that TSD-fed normal mouse serum could regulate CDC6 pathway activity in glioma cells. The therapeutic effect of TSD is significantly influenced by intestinal flora. Conclusion: The treatment of tumors by TSD may be modulated by intestinal flora. We established a new method to quantify the relationship between intestinal flora and the regulation of TSD efficacy through this study.

The deubiquitinating enzyme USP1 modulates ERα and modulates breast cancer progression.

Breast cancer is one of the most common malignancies worldwide, while the luminal types (ERα positive) accounts for two third of all breast cancer cases. Although ERα positive breast cancer could be effective controlled by endocrine therapy, most of the patients will develop endocrine resistance, which becomes a headache clinical issue for breast cancer field. Endocrine resistance could be caused by multiple pathway disorders, the dys-regulation of ERα signaling might be a critical factor, which makes it urgent and important to reveal the potential molecular mechanism of ERα signaling. In our current study, we identified a new deubiquitination enzyme USP1 through screening the whole DUB (Deubiquitinases) siRNA library. The expression of USP1 is elevated in human breast cancer compared with normal mammary tissues. Importantly, USP1 expression levels are specially correlated with poor survival in ERα positive patients. USP1 depletion inhibited breast cancer cell progression and ERα signaling activity. Immuno-precipitation assays indicate that USP1 associates with ERα and promotes its stability possibly via inhibiting ERα K48-linked poly-ubiquitination. In conclusion, our data implicate a non-genomic mechanism by USP1 via stabilizing ERα protein controls ERα target gene expression linked to breast cancer progression.

DLC2 inhibits development of glioma through regulating the expression ratio of TAp73α/TAp73ß.

To date, the anti-tumor mechanism of the deleted in liver cancer 2 (DLC2) in gliomas is still unclear. The study shows that TAp73α expression and TAp73α/TAp73ß ratio are frequently high in gliomas and that TAp73α and TAp73ß have opposite roles in regulating proliferation and apoptosis of glioma cells. Moreover, DLC2 is low-expressed in gliomas, which negatively correlates with TAp73α expression and TAp73α/TAp73ß ratio. More importantly, DLC2 inhibits development of glioma by decreasing expression of TAp73α, which changes the expression ratio of TAp73α/TAp73ß in glioma cells. Mechanically, DLC2 interacts directly with TAp73α and induces TAp73α ubiquitination and degradation, which is mediated through SAM domain of DLC2 and TAp73α. In detail, DLC2 with SAM domain deletion fails to interact with TAp73α and induce TAp73α ubiquitination and degradation, and SAM deletion decreased tumorigenesis-inhibition effect of DLC2. In conclusion, DLC2 inhibits glioma development by inducing TAp73α degradation and subsequent change of TAp73α/TAp73ß expression ratio.