Journal of Materials Chemistry B
·
2019
·Vol. 7
·期 11
·1926-1932
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IF 5.047
Fanyu Meng,Hua Chai,Xiaoyi Ma,Yuguo Tang,Peng Miao
Telomerase catalyzes the elongation of telomeres, which is closely associated with tumorigenesis. Therefore, development of reliable and convenient methods for telomerase activity analysis is critical for clinical diagnosis of cancers. However, most of the current probes for intracellular telomerase activity assay suffer from inevitable false-positive interferences. In this study, we have proposed a novel fluorescence resonance energy transfer (FRET)-based strategy for ultrasensitive monitoring intracellular telomerase activity with an amplification-free procedure. DNA nanoprobes constructed using a DNA tetrahedron and Flare DNA are designed to circumvent the problem of false-positive interference. Generally, telomerase catalyzes DNA extension and changes the configuration of the DNA nanoprobe, which finally leads to the increase of the distance between two labelled fluorophores. Based on the notably decreased FRET efficiency, a simple ratiometric sensor is established for the detection of telomerase activity at the single-cell level, which is much suitable for clinical applications. In addition, this method can be further applied in the detection of telomerase inhibitors, which is important for anti-cancer drug discovery.
研究领域:
分子生物学
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生物技术
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癌症研究
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纳米技术
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生物化学
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DOI
PubMed
高影响因子
JOURNAL OF CELLULAR PHYSIOLOGY
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2018
·Vol. 233
·期 12
·9763-9776
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IF 3.923
Xing Xie,Yuan Gan,Maoda Pang,Guoqing Shao,Lei Zhang,Beibei Liu,Qi Xu,Haiyan Wang,Yanyan Feng,Yanfei Yu,Rong Chen,Meng Wu,Zhenzhen Zhang,Lizhong Hua,Qiyan Xiong,Maojun Liu,Zhixin Feng
Primary porcine bronchial epithelial cells (PBECs) are an ideal model to study the molecular and pathogenic mechanisms of various porcine respiratory pathogens. However, the short lifespan of primary PBECs greatly limit their application. Here, we isolated and cultured primary PBECs and established immortalized PBECs by transfecting primary PBECs with the pEGFP-hTERT recombinant plasmid containing human telomerase reverse transcriptase (hTERT). Immortalized PBECs (hTERT-PBECs) retained the morphological and functional features of primary PBECs as indicated by cytokeratin 18 expression, telomerase activity assay, proliferation assays, karyotype analysis, and quantitative reverse-transcriptase polymerase chain reaction. Compared to primary PBECs, hTERT-PBECs had higher telomerase activity, extended replicative lifespan, and displayed enhanced proliferative activity. Moreover, this cell line is not transformed in vitro and does not exhibit a malignant phenotype in vivo, suggesting that it can be safely used in further studies. Besides, hTERT-PBECs were susceptible to swine influenza virus of H3N2 subtype and porcine circovirus type 2. In conclusion, the immortalized hTERT-PBECs represent a valuable in vitro model, which can be widely used in the study of porcine respiratory pathogenic infections.
研究领域:
细胞生物学
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病毒学
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分子生物学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2019
·
IF 5.256
Xiaoxia Liu,Xia Li,Jin Li,Bingying Jiang,Ruo Yuan,Yun Xiang
The development of methods that can detect telomerase with high selectivity and sensitivity is critical for early diagnosis and treatment of telomerase-related cancers. In this regard, we describe in this work the establishment of a telomerase-initiated and nicking endonuclease-assisted cascade recycling signal amplification approach for non-label and highly sensitive fluorescent detection of telomerase from cancer cells. The target telomerase triggers the elongation of one strand in a partial dsDNA duplex with a pre-designed sequence to induce the release of a ssDNA, which can initiate three cascaded recycling cycles for the generation of many G-quadruplex sequences by cleaving two hairpin signal probes with the assistance of the Nt.AlwI endonuclease . The thioflavin dye further binds these G-quadruplex sequences to exhibit substantial fluorescence enhancement for sensitive detection of telomerase at 8.93 × 10 −11 IU. Moreover, this developed method is capable of differentiating telomerase activity among different cancer cells and screening telomerase inhibitors for anticancer drugs .
研究领域:
癌症生物学
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分子诊断学
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生物技术
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分析化学
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DOI
PubMed
高影响因子
TALANTA
·
2018
·
IF 4.244
Yingxin Ma,Guobin Mao,Yucheng Zhong,Guoqiang Wu,Wanjun Wu,Yonghao Zhan,Zhike He,Weiren Huang
Telomerase, as a valuable biomarker, is an important target in cancer diagnosis. Here, we report a ratiometric fluorescent probe for telomerase activity assay in urine and bladder cancer diagnoses based on the color change of Rox-DNA functionalized quantum dots (QDs). The green fluorescence of the QDs was sensitive to H 2 O 2 , but the red fluorescence of Rox showed no change. An HRP-mimicking hemin/G-quadruplex, which was formed with the help of telomerase activity, catalyzed H 2 O 2 into H 2 O and O 2 . This quadruplex effectively avoided H 2 O 2 interference with green fluorescence. In the presence of H 2 O 2 , the detected color changed from red to yellow-green by increasing the telomerase concentration. The detection limit (LOD) was 10 cells, and response time was within 60 min. More importantly, a paper sensor was developed based on this probe and used for the assay of telomerase activity in urine samples. The results were highly sensitive and reproducible, and visual semi-quantitative detection was realized using the naked eye.
研究领域:
生物化学
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医学诊断
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纳米技术
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
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2024
·
IF 10.7
Yi Chen,Hengyi Chen,Xiaohui Chen,Yufang Zhang,Wei Tu,Jiaxi Mo,Shanshan Tian,Chenyu Sun,Xiaoqin Meng,Zhizeng Wang,Jichun Yang,Yang Luo
Accurate intracellular visualization of human telomerase RNA (hTR) is imperative for early diagnosis and treatment monitoring of hepatocellular carcinoma (HCC). While isothermal amplification-based DNA cascade strategies are promising, challenges persist in achieving great intake efficiency of detection probes within tumor cells and enhancing intracellular reaction efficiency. This study introduces a SA@Comb-HCR nanosystem , a highly effective approach for in situ hTR detection in HCC cells. Sodium alginate-coated liposomes ensures efficient nanoprobe delivery, which are then combined with proximity effect-inspired signal amplification . The coating of sodium alginate facilitates receptor-mediated endocytosis , prevents serum protein adhesion, and mitigates cationic liposome cytotoxicity. The designed Comb-like consolidated hairpin probe enhances the concentration of the local reactant, resulting in cascade amplification upon hTR activation. This technique achieves precision detection of intracellularly overexpressed hTR in HCC cells with a remarkable detection limit of 0.7 pM. This approach holds great promise for advancing targeted and sensitive early clinical diagnosis of HCC.
研究领域:
分子生物学
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肿瘤学
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生物医学工程
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DOI
PubMed
高影响因子
BIOORGANIC CHEMISTRY
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2020
·
IF 4.831
Chao Liu,Hua Zhou,Xiao Bao Sheng,Xin Hua Liu,Fei Hu Chen
Telomerase has become one of the new popular targets for the development of anti-tumor drugs. Based on the structural characteristics of the BIBR1532 which has entered the stage of clinical research, six series total of 64 new compounds with diverse structural characteristics were designed and synthesized. The inhibitory activity against SGC-7901, MGC-803, SMMC-7721, A375 and GES cell lines and their telomerase inhibitory activity were tested. Among them, eight compounds showed good activity against cancer cells, among them compounds 56 , 57 and 59 also showed low toxicity. Some of them showed excellent telomerase inhibitory activity with IC 50 values ranging from 0.62 μM to 8.87 μM. Based on above, in depth structure-activity relationships were summarized, the compounds by replacing methyl group with cyanide and retaining amide moiety had good anti-tumor activity, moderate cytotoxicity, and better telomerase inhibitory activity. The results should be used for reference in BIBR1532-based structural optimization for further development of small molecule telomerase inhibitors.
研究领域:
药理学
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肿瘤学
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药物化学
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DOI
PubMed
高影响因子
JOURNAL OF CHEMICAL RESEARCH
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2023
·
IF 1.4
Zhe Chen,Yunxia Wang
A label- and enzyme-free fluorescence assay based on thioflavin T–induced G-quadruplexes is developed to sensitively and specifically detect telomerase activity. Thioflavin T has a dual role as an efficient inducer and fluorescent probe, and the incorporation of thioflavin T into the thioflavin T–induced G-quadruplexes results in an intense fluorescence enhancement. In the presence of thioflavin T and K+, G-quadruplexes are formed by elongation of the telomerase substrate primer that is catalyzed by telomerase extracted from cancer cells. Thus, the telomerase activity in cancer cell extracts can be evaluated by measuring the thioflavin T fluorescence. More importantly, thioflavin T can specifically recognize and bind to G-quadruplexes, whereas it cannot recognize single- and double-stranded DNAs, which leads to the thioflavin T–based fluorescence assay exhibiting a reduced background and improved signal-to-noise ratio. As a result, the proposed assay has the linear range from 5 to 200 HeLa cells and the detection limit is 34 HeLa cells, which holds great potential for use in the detection of telomerase activity and the diagnosis of cancer.
研究领域:
生物化学
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分子生物学
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癌症研究
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DOI
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2015
·Vol. 99
·期 21
·9135-9146
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IF 3.337
Xie Xing,Pang Maoda,Liang Shan,Yu Lei,Zhao Yanbing,Ma Ke,Kalhoro Dildar Hussain,Lu Chengping,Liu Yongjie
Dogs are susceptible to infectious diseases that occur primarily in the respiratory tract. The airway epithelium acts as a first line of defense and is constantly exposed to microorganisms present in the environment. Respiratory epithelial cells have recently gained wide use as a cell model for studying the pathogenesis of human, murine or swine respiratory pathogen infections. However, studies of the pathogenic mechanisms of canine pathogens have been hindered by the lack of reliable respiratory cell lines. Here, we cultured primary canine bronchiolar epithelial cells (CBECs), whose characteristics were confirmed by their expression of the epithelial cell-specific marker cytokeratin 18, and have provided protocols for their isolation and ex vivo expansion. Further, we established immortalized CBECs containing the human telomerase reverse transcriptase (hTERT) gene via transfection of primary CBECs with the recombinant plasmid pEGFP-hTERT. Immortalized bronchiolar epithelial cells (hTERT-CBECs) retain the morphological and functional features of primary CBECs, as indicated by reverse transcriptase polymerase chain reaction, proliferation assays, karyotype analysis, telomerase activity assay, and Western blotting, which demonstrate that hTERT-CBECs have higher telomerase activity, an extended proliferative lifespan, and a diploid complement of chromosomes, even after Passage 50. Moreover, this cell line is not transformed, as evaluated using soft agar assays and tumorigenicity analysis in nude mice, and can therefore be safely used in future studies. The isolation and establishment of stable hTERT-CBECs is of great importance for use as an in vitro model for mechanistic studies of canine pathogenic infections.
研究领域:
兽医学
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细胞生物学
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分子生物学
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微生物学
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2020
·
IF 6.785
Li Zou,Xinghui Li,Ji Zhang,Liansheng Ling
Precise evaluation of telomerase activity is highly crucial for early cancer diagnosis. In this study, a sensitive catalytic hairpin assembly–dynamic light scattering (CHA–DLS) assay for telomerase activity detection is developed by using the diameter change of gold nanoparticle (AuNP) probes. The telomerase substrate primer can be extended in the presence of telomerase, producing a telomerase extension product (TEP) with telomeric repeat units (TTAGGG)n at its 3′-end. The TEP can specifically trigger the CHA process and form tremendous AuNPs-H1/H2 nanostructures, resulting in a significant increase in the diameter measured by DLS. Telomerase activity from different cancer cell lines (MCF-7, Huh7, and 5637) was detected using the proposed strategy, the diameter of AuNP probes increased with the number of cancer cells, and this method can accurately detect telomerase activity down to 6 MCF-7 cells, 10 Huh7 cells, and three 5637 cells. Moreover, the CHA–DLS biosensor was successfully applied in urine specimens from healthy individuals and different cancer patients, which can distinguish bladder cancer patients from healthy people and other cancer patients, indicating that the noninvasive method has a great potential for application in early diagnosis of bladder cancer.
研究领域:
生物医学
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癌症诊断
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纳米技术
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分子生物学
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产品关联
DOI
PubMed
高影响因子
VIEW
·
2023
·
IF 8.6
Chen Li,Hui Chen,Tingting Fan,Jingru Zhao,Zheng Ding,Zeyu Lin,Shuqing Sun,Chunyan Tan,Feng Liu,Hongtao Jiang,Ying Tan
The accurate evaluation of telomerase activity, a typical cancer biomarker, is vital for early cancer screening. In this study, we developed a dark-field microscopy (DFM) visual single-particle detection scheme to detect telomerase activity based on automatic counting gold nanoparticles (AuNPs). This method started with attaching the telomerase substrate (TS) primer to the magnetic beads (MBs) through streptavidin-biotin interaction. In the presence of telomerase and dNTPs, the TS primer was expanded with (TTAGGG) n repeat units to form the telomerase extension product (MBs-telomerase extension product), which could be hybridized with the complementary DNA (cDNA) modified with AuNPs through Au-S bonds (AuNPs-SH-cDNA). After magnetic separation and DNA double-strand unwinding, AuNPs were collected from the supernatant, and the telomerase activity was quantitatively measured by visually counting bright spots based on DFM. This strategy achieved a limit of detection as low as 1 HeLa cell and distinguished telomerase activity among different cell lines, thus verifying its excellent sensitivity and specificity. Further, two common telomerase inhibitors (BIBR1532 and curcumin) were screened with the consistent IC 50 values with other methods, respectively. It is worth mentioning that this strategy can clearly identify bladder cancer among various urinary diseases. Consequently, the visualized automatic particle counting strategy is potential as a powerful tool in early and noninvasive diagnosis of bladder cancer.
研究领域:
生物医学
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癌症诊断
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纳米技术
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产品关联
DOI
高影响因子
TALANTA
·
2020
·
IF 5.339
Huixian Jia,Ningzhao Shang,Xiaobo He,Anaclet Nsabimana,Danna Sun,Huan Wang,Yufan Zhang
Telomerase is regarded as a crucial biomarker for the early diagnosis of malignant tumors and a valuable therapeutic target. In this work, a telomerase-triggered amplification strategy was designed on the basis of a catalyzed hairpin assembly (CHA) for bridging a signal probe of platinum nanoparticles (Pt NPs) anchored on three-dimensional (3D) epoxy-functionalized macroporous carbon (Pt/MPC-COOH) in an ultrasensitive electrochemical biosensor. Pt/MPC-COOH nanomaterials with interconnected macroporous structure not only immobilized hairpin DNA probe 2 (H2) via an amide reaction (Pt/MPC-COOH-H2), but they also generated an obvious electrochemical signal in response to acetaminophen (AP) oxidation. After the introduction of telomerase, telomerase primer (TP) was extended to a telomerase extension product (TEP) with several hexamer repeats (TTAGGG)n to initiate the CHA cycle, leading to signal amplification. Subsequently, with the TEP-triggered CHA cycle amplification strategy, a large amount of Pt/MPC-COOH-H2 was introduced on the electrode surface for the construction of the electrochemical platform, which realized the sensitive detection of telomerase activity from 10 2 to10 7 cells mL −1 with a limit of detection (LOD) of 9.02 cells mL −1 . This strategy provides a sensitive method for the detection of biomolecules that could be useful for bioanalysis and early clinical diagnoses of diseases.
研究领域:
生物传感
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分子诊断
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纳米医学
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产品关联
DOI
PubMed
高影响因子
CANCER SCIENCE
·
2012
·Vol. 103
·期 8
·1434-1439
·
IF 3.325
Bing Xiu Ren,Fan Yang,Gao Hong Zhu,Zi Xing Huang,Hua Ai,Rui Xia,Xi Jiao Liu,Man Lu,Bin Song
To develop a molecular probe for MRI detection of human tumor telomerase reverse transcriptase (hTERT) mRNA expression. Uniformly phosphorothioate-modified hTERT antisense oligonucleotide (ASON) homing hTERT mRNA was labeled with gadolinium (Gd) through the bifunctional chelator 1,4,7, 10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA) stirred within 45 minutes at 60°C. The Gd labeled probes were characterized in vitro . The cellular uptake rate and biodistribution of 99m Tc-DOTA-ASON was measured instead of that of Gd-DOTA-ASON. A549 lung adenocarcinoma model was established in BALB/c nude mice and Gd-DOTA-ASON was injected intraperitoneally and MR images were acquired using 7.0T Micro-MRI (Bruker Biospec, Ettlingen, Germany) at different time points. Immunohistochemical analysis of telomerase activity of each xenograft was operated two days after in vivo imaging. The binding efficiency of Gd-DOTA-ASON reached as high as 71.7 ± 4.5% ( n = 6). Gd-DOTA-ASON displayed perfect stability in fresh human serum at 37°C for 24 h. Compared with normal lung cells, A549 cells showed an obviously higher uptake of 99m Tc-DOTA-ASON than that of lung cells (10.5 ± 2.7% vs. 4.8 ± 2.6%, P < 0.05). The signal intensity of A549 xenografts can be enhanced by Gd-DOTA-ASON and the signal to noise ratio (SNR) of tumor to muscle reached 2.37 and maintained a relatively high level within 6 h after injection. The activity of hTERT in A549 tumors can be suppressed by Gd-DOTA-ASON in pathological slices. The results of this study show that Gd-DOTA-ASON can be a promising intracellular MR contrast probe for targeting telomerase-positive carcinomas. ( Cancer Sci, doi: 10.1111/j.1349-7006.2012.02316.x, 2012)
研究领域:
医学影像学
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分子生物学
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肿瘤学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）