Dynamic tracing of intracellular telomerase activity plays a crucial role in cancer cell recognition and correspondingly in earlier cancer diagnosis and personalized precision therapy. However, due to the complexity of the required reaction system and insufficient loading of reaction components into cells, achieving a high-fidelity determination of telomerase activity is still a challenge. Herein, an Aptamer-Liposome mediated Telomerase activated poly-Molecular beacon Arborescent Nanoassembly(ALTMAN) approach was described for direct high-fidelity visualization of telomerase activity. Briefly, intracellular telomerase activates molecular beacons, causing their hairpin structures to unfold and produce fluorescent signals. Furthermore, multiple molecular beacons can self-assemble, forming arborescent nanostructures and leading to exponential amplification of fluorescent signals. Integrating the enzyme-free isothermal signal amplification successfully increased the sensitivity and reduced interference by leveraging the skillful design of the molecular beacon and the extension of the telomerase-activated TTAGGG repeat sequence. The proposed approach enabled ultrasensitive visualization of activated telomerase exclusively with a prominent detection limit of 2 cells center dot mu L-1 and realized real-time imaging of telomerase activity in living cancer cells including blood samples from breast cancer patients and urine samples from bladder cancer patients. This approach opens an avenue for establishing a telomerase activity determination and in situ monitoring technique that can facilitate both telomerase fundamental biological studies and cancer diagnostics.

BACKGROUND Atherosclerosis is a chronic lipid-driven inflammatory disease, largely influenced by hemodynamics. Neutrophil extracellular traps (NETs)-mediated inflammation plays an important role in atherosclerosis. However, little is known about the mechanism of the generation of NETs under different shear stress and subsequent damage to endothelial cells. We sought to identify a novel mechanical signal provokes NETs generation and to investigate its potential role in atherosclerosis. METHODS ApoE −/− mice were fed with high-fat diet (HFD) to induce atherosclerosis. The model of lower shear stress (LSS) with a partial ligation of the left carotid artery was established to assess the role of LSS in NETs generation and atherosclerotic lesions development. Furthermore, the underlying mechanism of LSS promoting NETs generation and injuring endothelial cells was deciphered in neutrophil-like human promyelocytic leukemia (HL-60) cells in parallel-plate flow chamber. RESULTS We found that LSS correlated spatially with both NETs and atherosclerosis, while inhibition of NETosis could significantly reduce plaque formation in ApoE −/− mice. In vitro , LSS could promote NETs generation directly through down-regulation of Piezo1, a mechanosensitive ion channel. downexpression of Piezol could activate neutrophils and promote NETosis in static. Conversely, Yoda1-evoked activation of Piezo1 attenuated LSS-induced NETosis. Mechanistically, the downexpression of Piezo1 resulted in decreased Ca 2+ influx and increased histone deacetylase 2 (HDAC2), which increase reactive oxygen species levels, then led to NETosis. LSS-induced NETs generation promoted the apoptosis and adherence of endothelial cells. CONCLUSIONS LSS directly promotes NETosis through piezo1-HDAC2 axis in atherosclerosis progression. This study uncovers the essential role of Piezo1-mediated mechanical signaling in NETs generation and plaque formation, which provides a promising therapeutic strategy for atherosclerosis. Graphic Abstract Proposed mechanism for lower shear stress LSS exacerbating atherosclerosis. LSS stimuli decrease Piezo1 expression in the neutrophils, resulting in decreased intracellular Ca 2+ concentration, as well as the higher expression level of HDAC2, which could activate oxidative stress and promote intracellular reactive oxygen species formation, and ultimately lead to NETs generation. NETs could aggravate endothelial cells injury and exasperate atherosclerosis. Highlights ■ Lower shear stress (LSS) promotes Neutrophil extracellular traps (NETs) formation, which is critical for lipid deposits and plaque formation in Atherosclerosis. ■ Atherosclerotic plaque formation was significantly reduced in the aorta of high fat diet fed ApoE −/− mice intraperitoneal injected with NETs inhibitor, GSK484, especially in the lower shear stress regions. ■ Piezo1 is a key molecule in the process of neutrophils sense lower shear stress. ■ lower shear stress inhibits the activation of Piezo1 and promotes NETosis through piezo1-HDAC2 axis. ■ LSS-induced NETs promote the apoptosis and adhesion of endothelial cells.

Accurate analysis of S-phase fraction is crucial for the assessment of cell proliferation levels, tumor malignancy and prognostic effects of treatment. Most of the currently developed methods for S-phase cell analysis rely on flow cytometric analysis of DNA content determination. However, the lack of standardized procedures for sample analysis and interpretation of cell cycle fitting graphs poses a significant limitation in clinical practice for utilizing flow cytometry to measure the cell cycle based on DNA content. Herein, we developed an approach for analyzing S-phase cells based on telomerase activity determination. Briefly, this approach distinguishes S-phase cells in cell populations via direct fluorescence tracking of telomerase activity within individual cells. The dynamic analysis of telomerase activity in different cell cycles was made possible by the ALTMAN strategy developed in our previous studies, which has been successfully employed to distinguish S-phase cells in cultured cells. This method offers a novel avenue for the assessment of cell cycle status and the evaluation of the proliferation status of tumor cells and the prognosis effect of tumor patients via analyzing the differences in telomerase activity during different cell cycle processes.

Cancer-derived small extracellular vesicles (csEVs) are crucial liquid biopsy indicators that reflect the presence and progression of many malignancies. However, reliable discrimination of csEVs remains a great challenge owing to the interference from normal sEVs (nsEVs) and low abundance in the early stages of cancer. In this work, we developed a Two-Elements Selectively Triggered csEVs Recognization (TESTER) strategy for selective identification of csEVs from the complex clinical body fluid samples. This method was based on the MNAzyme-controlled synchronous recognition to EpCAM and CD63 proteins on the membrane of csEVs. Efficient recognition to csEVs via EpCAM aptamer and CD63 aptamer prompted the release of Partzyme A and Partzyme B probes to induce a MNAzyme structure formation, resulting in the cyclic cleavage of substrate chain to produce cascade fluorescence signal amplification. The detection threshold of the developed TESTER approach for csEVs in complicated biological samples was 72 particles μL-1, accomplishing the highly sensitive and selective quantification of csEVs. At the same time, we successfully constructed a new platform for bimolecular simultaneous recognition, which provides a good idea for the construction of bimolecular-activated detection switch in the future.

肿瘤是危害人类健康的严重疾病,探索新型肿瘤治疗方式、提高其靶向性和药物递送效率、避免肿瘤细胞免疫逃逸、降低副反应是目前肿瘤治疗研究的热点问题.细菌既可以依赖天然的特性靶向缺氧、低pH、高渗透性和免疫抑制的肿瘤微环境,又可以通过免疫调节、细菌毒素促凋亡、抑制血管新生等机制发挥抗肿瘤作用.同时,研究者可以利用基因工程技术改造细菌,使之成为靶向性更高、穿透性更好的工程菌.此外,利用细菌外膜囊泡装载化疗药物或结合纳米材料抗肿瘤以及细菌联合化疗、放疗、光热/光动疗法的出现,使得细菌治疗肿瘤方面研究进展迅速.本文综述了近年来细菌介导肿瘤治疗的最新研究进展,并展望了其未来的发展方向.