Construction of an exogenously and endogenously Co-activated DNA logic amplifier for highly reliable intracellular MicroRNA imaging.

DNA-based molecular amplifiers offer significant promise for molecular-level disease diagnosis and treatment, yet tailoring their activation for precise timing and localization remains a challenge. Herein, we've pioneered a dual activation strategy harnessing external light and internal ATP to create a highly controlled DNA logic amplifier (FDLA) for accurate miRNA monitoring in cancer cells. The FDLA was constructed by tethered the two functionalized catalytic hairpin assembly (CHA) hairpin modules (ATP aptamer sealed hairpin aH1 and photocleavable (PC-linker) sites modified hairpin pH2) to DNA tetrahedron (DTN). The FDLA system incorporates ATP aptamers and PC-linkers as logic control units, allowing them to respond to both exogenous UV light and endogenous ATP present within cancer cells. This response triggers the release of CHA hairpin modules, enabling amplified FRET miRNA imaging through an AND-AND gate. The DTN structure could improve the stability of FDLA and accelerate the kinetics of the strand displacement reaction. It is noteworthy that the UV and ATP co-gated DNA circuit can control the DNA bio-computing at specific time and location, offering spatial and temporal capabilities that can be harnessed for miRNA imaging. Furthermore, the miRNA-sensing FDLA amplifier demonstrates reliable imaging of intracellular miRNA with minimal background noise and false-positive signals. This highlights the feasibility of utilizing both exogenous and endogenous regulatory strategies to achieve spatial and temporal control of DNA molecular circuits within living cancer cells. Such advancements hold immense potential for unraveling the correlation between miRNA and associated diseases.

[Impact of Tetracycline Antibiotic on the Transcriptional Expression of Tetracycline Resistance Genes in Shigella flexneri].

The tetracycline (TC) antibiotic has been widely found in different environmental matrices. The tetracycline resistant bacterium (TRB) of Shigella flexneri was screened and purified from activated sludge, and was then used to study the impact of TC stress on the gene abundances and expression levels of TC resistance genes (TC-ARGs), including tetC, tetO, and tetX, which were respectively quantified by quantitative PCR and reverse transcriptional PCR. Correlations between the TC concentration and gene abundances of TC-ARGs and their expression levels were discussed. The results showed that TC stress had an inhibiting effect on the growth of Shigella flexneri during the entire culture cycle (24 h) and that the growth rate of the bacterial concentration decreased with increasing TC concentration. However, less impact on the gene abundance of TC-ARGs was found. TC stress could promote the expression of TC-ARGs in Shigella flexneri, and the expression levels of tetC, tetO, and tetX genes first increased and then decreased. The correlation results indicated that no significant correlation was observed between the TC concentration and gene abundances of TC-ARGs and their expression levels. Nevertheless, the gene abundances of tetC and tetO were significantly correlated with their expression levels, thus indicating that they can be used to evaluate and assess expression levels to a certain extent.

[Effect of Tetracycline Antibiotic on Abundance and Transcriptional Expression Level of Tetracycline Resistance Genes in Activated Sludge].

Tetracycline resistant bacteria (TRB) screened from activated sludge were used to study the effect of tetracycline (TC) antibiotic on the transcriptional expression of tetracycline resistance genes (TC-ARGs). The gene abundances of seven TC-ARGs including tetA, tetC, tetG, tetM, tetO, tetW, and tetX, as well as their expression levels, were quantified by quantitative PCR (qPCR) and reverse transcriptional PCR (RT-PCR). The correlations between TC concentrations and gene abundance of TC-ARGs and their expression levels were discussed. The results showed that the gene abundances of tetA, tetG, and tetW generally increased with increasing TC exposure concentrations during the entire culture cycle, whereas other TC-ARGs fluctuated greatly. The impact of TC stress on the transcriptional expression level of different TC-ARGs varied to a great extent. The gene expression of tetA was relatively stable and exhibited an upregulated trend with increasing TC concentrations. When the TC concentration was 100 mg·L-1, the upregulation of tetA expression was as high as 5.3-fold compared with the control. Under short-term TC stress (one day), the transcriptional expression level was upregulated with increasing TC concentration. The correlation results showed that gene abundances of tetA and tetW correlated significantly with their respective expression levels, indicating that they can evaluate expression levels to a certain extent, which can further mirror functional activities and environmental risks.

Long-term impact of a tetracycline concentration gradient on the bacterial resistance in anaerobic-aerobic sequential bioreactors.

Wastewater treatment systems are considered as hotspots for release of antibiotic resistance genes (ARGs) into the environment. Anaerobic-aerobic sequential (AAS) bioreactors now are intensively used for wastewater treatment worldwide. However, the occurrence of ARGs in wastewater treatment systems exposed to low-level (i.e., sub-inhibitory) antibiotic is poorly known. Here, we studied the distribution patterns of seven tetracycline resistance genes (tet genes) including tet(A), tet(C), tet(G), tet(X), tet(M), tet(O), and tet(W), as well as one mobile element [class 1 integron (intI1)] in AAS bioreactors under exposure to tetracycline from 50 µg/L to 500 µg/L. Additionally, effect on the removal performance of nutrients and tetracycline in both anaerobic and aerobic units was also investigated. A tetracycline concentration gradient selected for bacterial resistance in the anaerobic reactor, with the exception of tet(A) and tet(W), and the tetracycline removal deteriorated by 47%. However, the abundance of tet and intI1 genes reduced in the subsequent aerobic unit, and the removal of tetracycline, soluble COD, and NH4+-N maintained at average efficiencies of 91%, 90%, and 93%, respectively. The level of tet(X) was largely unaffected by AAS treatment. It is notable that intI1 genes probably played a crucial role on the horizontal dissemination of tet genes. The tetracycline levels and intI1 genes appear to be the primary factors influencing the occurrence of tet genes in AAS bioreactors. Nonetheless, AAS treatments still show promise for reducing antibiotics, ARGs and mobile elements without affecting nutrient removal, and need further research for practical applications.