Universal Detection and Imaging for Multiple Targets by Coupling Target-Primed Nicking-Enhanced Rolling Circle Amplification with Self-Powered DNAzyme Walker.

Although promising in monitoring low-abundance analytes, most of the DNAzyme walker is only responsive to a specific target. Herein, a universal, ready-to-use platform is developed by coupling nicking-enhanced rolling circle amplification and a self-powered DNAzyme walker (NERSD). It addressed the issues that DNAzyme strands need to be specifically designed for different biosensing system, allowing highly sensitive analysis of various targets with the same DNAzyme walker components. It is also specific owing to target-dependent ligation of the padlock probe and precise cleavage of a substrate by a DNAzyme strand. As typically demonstrated, the strategy has an equivalent capacity with the qRT-PCR kit in distinguishing plasma miR-21 levels of breast cancer patients from normal subjects and is able to differentiate intracellular miR-21 and ATP levels by confocal imaging. The approach characteristic of programmability, flexibility, and generality indicated the potential in all kinds of biosensing and imaging platform.

[SigE inhibits DNA damage and participates in the regulation of DNA damage repair in Mycobacterium smegmatis].

Objective To investigate the anti-DNA damage role of Sigma factor E (SigE) and its regulation mechanism of DNA damage repair in Mycobacterium smegmatis(MS). Methods The SigE gene of Mycobacterium smegmatis was cloned into plasmid pMV261 to construct recombinant plasmid pMV261(+)-SigE, and the inserted gene was verified by sequencing. The recombinant plasmid was electrically transformed into Mycobacterium smegmatis to construct SigE over-expression strain, and the expression of SigE was detected by Western blot analysis. The Mycobacterium smegmatis containing pMV261 plasmid was used as the control strain. Growth differences between the two stains were monitored by measuring the 600 nm absorbance (A600) value of the bacterial culture suspension. The survival rate differences between two kinds of strains which were treated with three kinds of DNA damaging agents including ultraviolet ray (UV), cisplatin (DDP), and mitomycin C (MMC) were detected by colony forming unit (CFU) counting assay. DNA damage repair pathways of Mycobacteria were analyzed through bioinformatics and SigE-related genes were screened. The relative expression levels of these genes possibly related to the SigE against DNA damage were detected by real-time fluorescence quantitative PCR. Results SigE over-expression strain pMV261(+)-SigE/MS was constructed and the expression of SigE in Mycobacterium smegmatis was detected. Compared with the control strain, the SigE over-expression strain grew more slowly and entered the growth plateau later; survival rate analysis found that SigE over-expression strain was more resistant to three DNA damaging agents including UV, DDP, and MMC. Bioinformatic analysis indicated that SigE gene was closely related to DNA damage repair genes recA, single-strand DNA binding protein, (ssb), and dnaE2; the expression levels of recA, dnaE2, and ssb in SigE over-expression strain all increased with varying degrees compared with those in the control strain. Conclusion SigE plays an important role in inhibiting the DNA damage of Mycobacterium smegmatis, whose mechanism is closely related to the regulation of DNA damage repair.

Inhibition of mycobacteria proliferation in macrophages by low cisplatin concentration through phosphorylated p53-related apoptosis pathway.

BACKGROUND: Drug resistance is a prominent problem in the treatment of tuberculosis, so it is urgent to develop new anti- tuberculosis drugs. Here, we investigated the effects and mechanisms of cisplatin (DDP) on intracellular Mycobacterium smegmatis to tap the therapeutic potential of DDP in mycobacterial infection. RESULTS: Macrophages infected with Mycobacterium smegmatis were treated with DDP alone or combined with isoniazid or rifampicin. The results showed that the bacterial count in macrophages decreased significantly after DDP (≤ 6 µg/mL) treatment. When isoniazid or rifampicin was combined with DDP, the number of intracellular mycobacteria was also significantly lower than that of isoniazid or rifampicin alone. Apoptosis of infected cells increased after 24 h of DDP treatment, as shown by flow cytometry and transmission electron microscopy detection. Transcriptome sequencing showed that there were 1161 upregulated and 645 downregulated differentially expressed genes (DEGs) between the control group and DDP treatment group. A Trp53-centered protein interaction network was found based on the top 100 significant DEGs through STRING and Cytoscape software. The expression of phosphorylated p53, Bax, JAK, p38 MAPK and PI3K increased after DDP treatment, as shown by Western blot analysis. Inhibitors of JAK, PI3K or p38 MAPK inhibited the increase in cell apoptosis and the reduction in the intracellular bacterial count induced by DDP. The p53 promoter Kevetrin hydrochloride scavenges intracellular mycobacteria. If combined with DDP, Kevetrin hydrochloride could increase the effect of DDP on the elimination of intracellular mycobacteria. In conclusion, DDP at low concentrations could activate the JAK, p38 MAPK and PI3K pathways in infected macrophages, promote the phosphorylation of p53 protein, and increase the ratio of Bax to Bcl-2, leading to cell apoptosis, thus eliminating intracellular bacteria and reducing the spread of mycobacteria. CONCLUSION: DDP may be a new host-directed therapy for tuberculosis treatment, as well as the p53 promoter Kevetrin hydrochloride.

Long non-coding RNA SNHG9 regulates viral replication in rhabdomyosarcoma cells infected with enterovirus D68 via miR-150-5p/c-Fos axis.

Background: The Enterovirus D68 (EV-D68) epidemic has increased knowledge of the virus as a pathogen capable of causing serious respiratory and neurological illnesses. It has been shown that long noncoding RNAs (lncRNAs) regulate viral replication and infection via multiple mechanisms or signaling pathways. However, the precise function of lncRNAs in EV-D68 infection remains unknown. Methods: The differential expression profiles of lncRNA in EV-D68-infected and uninfected rhabdomyosarcoma (RD) cells were studied using high-throughput sequencing technology. The knockdown through small interfering RNA (siRNA) and overexpression of lncRNA SNHG9 (small ribonucleic acid host gene 9) were applied to investigate how lncRNA SNHG9 regulates EV-D68 propagation. The targeted interactions of lncRNA SNHG9 with miR-150-5p and miR-150-5p with c-Fos were validated using dual luciferase reporter system. LncRNA SNHG9 knockdown and miR-150-5p inhibitor were co-transfected with RD cells. QRT-PCR and western blot were used to detect RNA and protein levels, of c-Fos and VP1, respectively. Median tissue culture infectious dose (TCID50) was applied to detect viral titers. Results: The results demonstrated that a total of 375 lncRNAs were highly dysregulated in the EV-D68 infection model. In the EV-D68 infection model, lncRNA SNHG9 and c-Fos were increased in EV-D68-infected RD cells. However, the expression level of miR-150-5p was downregulated. In addition, overexpression of SNHG9 in RD cells resulted in decreased viral replication levels and viral titers following infection with EV-D68, and further experiments revealed that overexpression of SNHG9 inhibited the viral replication by targeting increased miR-150-5p binding and significantly increased c-Fos expression in RD cells. Conclusion: Our findings indicate that the SNHG9/miR-150-5p/c-Fos axis influences EV-D68 replication in host cells and that SNHG9 may be a possible target for anti-EV-D68 infection therapies.

High throughput sequencing of whole transcriptome and construct of ceRNA regulatory network in RD cells infected with enterovirus D68.

BACKGROUND: With the advancement of sequencing technologies, a plethora of noncoding RNA (ncRNA) species have been widely discovered, including microRNAs (miRNAs), circular RNAs (circRNAs), and long ncRNAs (lncRNAs). However, the mechanism of these non-coding RNAs in diseases caused by enterovirus d68 (EV-D68) remains unclear. The goal of this research was to identify significantly altered circRNAs, lncRNAs, miRNAs, and mRNAs pathways in RD cells infected with EV-D68, analyze their target relationships, demonstrate the competing endogenous RNA (ceRNA) regulatory network, and evaluate their biological functions. METHODS: The total RNAs were sequenced by high-throughput sequencing technology, and differentially expressed genes between control and infection groups were screened using bioinformatics method. We discovered the targeting relationship between three ncRNAs and mRNA using bioinformatics methods, and then built a ceRNA regulatory network centered on miRNA. The biological functions of differentially expressed mRNAs (DEmRNAs) were discovered through GO and KEGG enrichment analysis. Create a protein interaction network (PPI) to seek for hub mRNAs and learn more about protein-protein interactions. The relative expression was verified using RT-qPCR. The effects of Fos and ARRDC3 on virus replication were confirmed using RT-qPCR, virus titer (TCID50/ml), Western blotting. RESULTS: 375 lncRNAs (154 upregulated and 221 downregulated), 33 circRNAs (32 upregulated and 1 downregulated), 96 miRNAs (49 upregulated and 47 downregulated), and 239 mRNAs (135 upregulated and 104 downregulated) were identified as differently in infected group compare to no-infected group. A single lncRNA or circRNA can be connected with numerous miRNAs, which subsequently coregulate additional mRNAs, according to the ceRNA regulatory network. The majority of DEmRNAs were shown to be connected to DNA binding, transcription regulation by RNA polymerase II, transcription factor, MAPK signaling pathways, Hippo signal pathway, and apoptosis pathway, according to GO and KEGG pathway enrichment analysis. The hub mRNAs with EGR1, Fos and Jun as the core were screened through PPI interaction network. We preliminarily demonstrated that the Fos and ARRDC3 genes can suppress EV-D68 viral replication in order to further verify the results of full transcriptome sequencing. CONCLUSION: The results of whole transcriptome analysis after EV-D68 infection of RD cells were first reported in this study, and for the first time, a ceRNA regulation network containing miRNA at its center was established for the first time. The Fos and ARRDC3 genes were found to hinder viral in RD cells. This study establishes a novel insight host response during EV-D68 infection and further investigated potential drug targets.