Multiple RNA Rapid In Situ Imaging Based on Cas9 Code Key System.

Existing RNA in situ imaging strategies mostly utilize parallel repetitive nucleic acid self-assembly to achieve multiple analysis, with limitations of complicated systems and cumbersome steps. Here, a Cas9 code key system with key probe (KP) encoder and CRISPR/Cas9 signal exporter is developed. This system triggers T-protospacer adjacent motif (T-PAM structural transitions of multiple KP encoders to form coding products with uniform single-guide RNA (sgRNA) target sequences as tandem nodes. Only single sgRNA/Cas9 complex is required to cleave multiple coding products, enabling efficient "many-to-one" tandem signaling, and non-collateral cleavage activity-dependent automatic signaling output through active introduction of mismatched bases. Compared with conventional parallel multiple signaling analysis model, the proposed system greatly simplifies reaction process and enhances detection efficiency. Further, a rapid multiple RNA in situ imaging system is developed by combining the Cas9 code key system with a T-strand displacement amplification (T-SDA) signal amplifier. The constructed system is applied to tumor cells and clinicopathology slices, generating clear multi-mRNA imaging profiles in less than an hour with just one step. Therefore, this work provides reliable technical support for clinical tumor typing and molecular mechanism investigation.

Rapid in situ RNA imaging based on Cas12a thrusting strand displacement reaction.

RNA In situ imaging through DNA self-assembly is advantaged in illustrating its structures and functions with high-resolution, while the limited reaction efficiency and time-consuming operation hinder its clinical application. Here, we first proposed a new strand displacement reaction (SDR) model (Cas12a thrusting SDR, CtSDR), in which Cas12a could overcome the inherent reaction limitation and dramatically enhance efficiency through energy replenishment and by-product consumption. The target-initiated CtSDR amplification was established for RNA analysis, with order of magnitude lower limit of detection (LOD) than the Cas13a system. The CtSDR-based RNA in situ imaging strategy was developed to monitor intra-cellular microRNA expression change and delineate the landscape of oncogenic RNA in 66 clinic tissue samples, possessing a clear advantage over classic in situ hybridization (ISH) in terms of operation time (1 h versus 14 h) while showing comparable sensitivity and specificity. This work presents a promising approach to developing advanced molecular diagnostic tools.

Targeting NOX4 disrupts the resistance of papillary thyroid carcinoma to chemotherapeutic drugs and lenvatinib.

Advanced differentiated thyroid cancer cells are subjected to extreme nutritional starvation which contributes to develop resistance to treatments; however, the underlying mechanism remains unclear. Cells were subjected to serum deprivation by culture in medium containing 0.5% fetal bovine serum. A CCK8 assay, cell death Detection ELISAPLUS kit, and PI staining were conducted to determine cell viability, cell apoptosis, and cell cycle, respectively. NADPH oxidase 4 (NOX4) knockdown-stable cell lines were generated by lentivirus-mediated shRNA knockdown in BCPAP cells and TPC-1 cells. Etoposide and doxorubicin, two chemotherapeutic drugs, as well as lenvatinib were utilized to determine the effect of NOX4 on drug resistance. Lenvatinib-resistant BCPAP cells (LRBCs) were established to confirm this effect. The underlining mechanisms of NOX4 under starvation were explored using western blot. Finally, GLX351322, an inhibitor targeting NOX4, was used to inhibit NOX4-derived ROS in vitro and detect its effect on drug resistance of tumor cells in vivo. NOX4 is overexpressed under serum deprivation in BCPAP or TPC-1 cells. NOX4 knockdown impairs cell viability, increases cell apoptosis, extends G1 phase during cell cycle and modulates the level of energy-associated metabolites in starved cells. When the starved cells or LRBCs are treated with chemotherapeutic drugs or Lenvatinib, NOX4 knockdown inhibits cell viability and aggravates cell apoptosis depending on NOX4-derived ROS production. Mechanistically, starvation activates TGFß1/SMAD3 signal, which mediates NOX4 upregulation. The upregulated NOX4 then triggers ERKs and PI3K/AKT pathway to influence cell apoptosis. GLX351322, a NOX4-derived ROS inhibitor, has an inhibitory effect on cell growth in vitro and the growth of BCPAP-derived even LRBCs-derived xenografts in vivo. These findings highlight NOX4 and NOX4-derived ROS as a potential therapeutic target in resistance to PTC.

[Zinc-dependent metalloprotease 1 (Zmp1) inhibits the proliferation and phagosome-lysosome fusion of mouse macrophage RAW264.7].

Objective To observe the effect of zinc-dependent metalloprotease 1 (Zmp1) of Bacillus Calmette Guerin vaccine (BCG) on the proliferation and phagosome-lysosome fusion of RAW264.7 mouse macrophage. Methods Zmp1 was expressed in E.coli BL21 (DE3) and purified by metal chelate magnetic beads. RAW264.7 cells were incubated with the purified Zmp1. Cell proliferation was detected at 0, 24, 48, 72 and 96 hours by cell counting kit-8 (CCK-8) assay and cell cycle distribution was detected by flow cytometry (FCM). The formation of phagosome was induced after RAW264.7 cells were infected with attenuated Salmonella. Attenuated Salmonella in phagosomes and lysosome associated membrane protein 1 (LAMP1) were marked by Alexa FluorR350-anti-Salmonella antibodies (blue fluorescence) and Cy5-anti-LAMP1 antibodies (red fluorescence), respectively. Two kind of fluorescence in RAW264.7 cells were observed under a fluorescent microscope and the fusion of phagosome and lysosome was analyzed by overlaying two kind of fluorescence (purple fluorescence). Results RAW264.7 cell proliferation decreased obviously 48 hours after treated with Zmp1, and the cell count in G1 phase declined, but the number of S-phase cells increased. In RAW264.7 cells infected with attenuated Salmonella and labeled by double immunofluorescent staining, phagolysosomes exhibiting purple fluorescence were reduced after treated by Zmp1. Conclusion Zmp1 can inhibit mouse RAW264.7 cell proliferation and phagosome-lysosome fusion.

Cloning and characterization of Clp protease proteolytic subunit 2 and its implication in clinical diagnosis of tuberculosis.

OBJECTIVE: To clone, express, and characterize Mycobacterium tuberculosis (Mtb) ClpP2, and evaluated the potential usage of ClpP2 in clinical diagnosis of tuberculosis. METHODS: Mtb ClpP2 was cloned into recombinant plasmid pET32a (+) and transformed into E. coli BL21 (DE3). SDS-PAGE and Western blot analysis were performed to detect the expression of the recombinant protein. The immunogenicity of Mtb ClpP2 was assessed with epitope prediction and antibody titer assay. Quantitative real-time PCR was performed to detect the influence of stress conditions on ClpP2 expression. ClpP2 antigen and antibody in patients with pulmonary diseases were detected by indirect ELISA. ROC curve was constructed to assess the diagnostic accuracy of Mtb ClpP2 for tuberculosis. RESULTS: We had cloned and expressed recombinant Mtb ClpP2 in E. coli. Our results showed that Mtb ClpP2 had potent immunogenicity, and our own prepared polyclonal antibody could be used in detection and diagnostic tests. Results from Western blot showed that ClpP2 was mainly located in M. bovis BCG cytoplasm, and real-time PCR indicated that stress conditions could enhance the mRNA expression of ClpP2. Indirect ELISA suggested that, in tuberculosis patients, both the levels of ClpP2 antigen and antibody were increased, and the positive rates of ClpP2 were elevated. ROC curve had demonstrated satisfactory sensitivity and specificity of ClpP2-based diagnosis for tuberculosis. CONCLUSION: Our results suggest that Mtb ClpP2 antigens would be used as a biomarker in tuberculosis pathogenesis. These findings highlight the feasibility of the application of Mtb ClpP2 in the clinical diagnosis of tuberculosis.