ANALYTICAL CHEMISTRY
·
2020
·
IF 6.785
Jing Lei,Yuqin Huang,Wenjun Zhong,Dan Xiao,Cuisong Zhou
With the aim of detecting low frequency of drug resistant mutation T790M against wild-type sequences, we reported a two-dimensional signal analysis strategy by combining a three locked nucleic acids (LNAs)-modified probe (LP15-3t) and an α-HL nanopore. The specific hybridization of the LP15-3t probe with the T790M generated unique long two-level signals, including characteristic blocking current and characteristic dwell time. Due to the significant dwell time difference (114.2-fold) and the blocking current difference ranging from 81% to 96%, this two-dimensional signal analysis strategy can simultaneously distinguish T790M sequences with a sensitivity of 0.0001% against wild-type sequences. The LOD of T790M was 0.1 pM. This high discrimination capability would have great potential in the detection of rare mutation sequences and the early monitoring of clinical outcome of NSCLC patients with TKI drug resistance.
研究领域:
分子生物学
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医学诊断
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纳米科技
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DOI
高影响因子
EUROPEAN JOURNAL OF MEDICAL RESEARCH
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2024
·
IF 2.8
Shaoyi Cao,Huaqiu Li,Xiaoyan Ye,Xinxing Xing,Yonghuan Xie,Xiangfeng Zeng,Hongjiao Liu,Xing Zhong,Xinyi Yang,Wenxiu Xing,Cairong Zhu,Xiaoping Wu
Background The T790M mutation in the epidermal growth factor receptor ( EGFR ) gene is the primary cause of resistance to EGFR-tyrosine kinase inhibitor (TKI) therapy in non-small cell lung cancer (NSCLC) patients. Previous research demonstrated that certain traditional Chinese medicine (TCM) monomers exhibit anti-tumor effects against various malignancies. This study aims to investigate the potentials of shikonin screened from a TCM monomer library containing 1060 monomers in killing EGFR -T790M drug-resistant NSCLC cells and elucidate the underlying mechanisms. Methods MTT method was used to screen for the TCM monomers with significant killing effects on H1975 cells carrying the EGFR -T790M mutation. The influences of the identified monomer shikonin on cell growth were determined by the colony formation assay. Annexin-V/PI staining and JC-1 staining were applied to detect the effects of shikonin on cell apoptosis. The influences of shikonin on cell membrane integrity were detected by lactate dehydrogenase (LDH) release assay. Reactive oxygen species (ROS) generation was analyzed using DCFH-DA as probe. The mechanisms of shikonin affecting the stability of cyclooxygenase-2 (COX-2) were evaluated by using specific inhibitors for protein degradation pathways. Western blotting was performed to assess the effects of the alteration of COX-2 expression or enzymatic activity on the related signal pathways as well as the apoptotic and pyroptotic markers. Results Shikonin was identified as a potent cytotoxic compound against EGFR -T790M-mutant NSCLC cells. Shikonin induced cell apoptosis and pyroptosis by triggering the activation of the caspase cascade and cleavage of poly (ADP-ribose) polymerase and gasdermin E by elevating intracellular ROS levels. Further investigations revealed that shikonin induced the degradation of COX-2 via the proteasome pathway, thereby decreasing COX-2 protein level and enzymatic activity and subsequently inhibiting the downstream PDK1/Akt and Erk1/2 signaling pathways through the induction of ROS production. Notably, COX-2 overexpression attenuated shikonin-induced apoptosis and pyroptosis, whereas COX-2 inhibition with celecoxib enhanced the cytotoxic effects of shikonin. Conclusions Combination treatment with shikonin and COX-2 inhibitor may be a suitable therapeutic strategy for EGFR -T790M-mutant NSCLC treatment. Supplementary Information The online version contains supplementary material available at 10.1186/s40001-024-02187-7.
研究领域:
肿瘤学
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药理学
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分子生物学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Xuelan Wu,Ting Ju,Zeyang Li,Jingwen Li,Xingwei Zhai,Kun Han
Circulating tumor DNA (ctDNA) is a noninvasive biomarker which offer valuable information for cancer diagnosis and prognosis. In this study, a target-independent fluorescent signal system, Hybridization chain reaction-Fluorescence resonance energy transfer (HCR-FRET) system, is designed and optimized. Combined with CRISPR/Cas12a system, a fluorescent biosensing protocol was developed for sensing assay of T790 M. When the target is absent, the initiator remains intact, opens the fuel hairpins and triggers the following HCR-FRET. At presence of the target, the Cas12a/crRNA binary complex specifically recognizes the target, and the Cas12a trans -cleavage activity is activated. As a result, the initiator is cleaved and subsequent HCR responses and FRET processes are attenuated. This method showed detection range from 1 pM to 400 pM with a detection limit of 316 fM. The target independent property of the HCR-FRET system endows this protocol a promising potential to transplant to the assay of other DNA target in parallel.
研究领域:
分子生物学
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医学诊断
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生物化学
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产品关联
DOI
PubMed
高影响因子
Analytical Methods
·
2021
·Vol. 13
·期 8
·1013-1018
·
IF 2.896
Ting Ju,Xingwei Zhai,Xinfeng Liu,Kun Han
Sensitive assay of EGFR T790M, a circulating tumor DNA marker in non-small-cell carcinoma, provides critical information for the decision of clinical treatments, evaluation of radiotherapy effect, and monitoring the progress of recurrence and metastasis. In this report, a novel flow cytometry-based sensing method is proposed for detecting T790M. The toehold-sequence hybridizes with the biotin-labeled initiator sequence and forms IT-dsDNA. The presence of a target induces the displacement of initiator-sequence from IT-dsDNA. The targets are continuously set free with the aid of a helper hairpin sequence for the next cycle. In tandem, the free initiator sequence starts the hybridization chain reaction, which binds the serial of fluorescence-labeled probe sequences. The products of the hybridization chain reaction are captured and separated by magnetic beads, which are finally assayed via flow cytometry. The capability to distinguish single-nucleotide polymorphism and the tolerance of complex matrix in blood serum indicate that this strategy has the promising potential to be applied in the liquid biopsy of clinical samples.
研究领域:
分子生物学
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肿瘤学
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临床诊断
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DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
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2025
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IF 5.1
Ming Li,Junhu Zhao,Meiyan Liao,Heng Zheng,Fei Cheng,Youhang Zhang,Hanfei Zhang,Pier-Luc Tremblay,Tian Zhang
The accurate detection of circulating tumor DNA (ctDNA) in patients’ biological fluids can facilitate cancer diagnosis and prognosis. This study reports the development of a differential pulse voltammetric (DPV) biosensor for the detection of ctDNA coding for the key T790M lung cancer mutation of the epidermal growth factor receptor (EGFR). The biosensor was built with a working electrode (WE) made of optimized electroconductive particles comprising covalent organic frameworks (COF) displaying Au nanoparticles (Au NPs) modified with polyethyleneimine (COF@Au-PEI). Au NPs accelerated electron transfer while COFs and PEI maximized the architecture of the WE to improve electrochemical reactions and molecular probe binding. Next, hairpin DNA probes (haiDNA) specific to the EGFR T790M ctDNA analyte were bonded to COF@Au-PEI. Lastly, Au@Pt nanohybrids were attached to the WE to enhance its electrochemical performance. In this system, the long polynucleotide EGFR T790M ctDNA analyte interferes with electrochemical reactions upon binding to the haiDNA probes, resulting in a proportional decrease of the DPV signal. The device detected EGFR T790M ctDNA in blood plasma and exhibited good sensitivity, accuracy, stability, and reproducibility. The sensor was also highly selective with no DPV signal for the EGFR wildtype allele harboring a difference of only one base pair.
研究领域:
生物传感器技术
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癌症诊断
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分子生物学
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纳米材料科学
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产品关联
DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2024
·
IF 6.7
Yaping Li,Yongqi Yang,Chunmeng Zhong,Dan Xiao,Cuisong Zhou
Sensitive detection of resistance mutation T790 M is of great significance for early diagnosis and prognostic monitoring of non-small-cell lung cancer (NSCLC). In this paper, we showed a highly sensitive detection strategy for T790 M using a three-level characteristic current signal pattern in an α-hemolysin nanopore. A probe was designed that formed a C–T mismatched base pair with wild-type/P and a T–T mismatched with the T790M/P. The T790M/P produced a unique three-level characteristic current signal in the presence of mercury ions(II): first, T790M–Hg2+–P entering the vestibule of α-HL under the transmembrane potential and overhang of probe occupying the β-barrel, then probe unzipping from the T790M/P, T790 M temporally residing inside the nanocavity due to the interaction with Hg(II), and finally T790 M passing through the β-barrel. The blocking current distribution was concentrated with a small relative standard deviation of about 3%, and the signal peaks of T790 M and wild-type can be completely separated with a high separation resolution of more than 2.5, which achieved the highly sensitive detection of T790 M down to 0.001 pM (confidence level P 95%) with a linear range from 0.001 pM to 1 nM in human serum samples. This highly sensitive recognition strategy enables the detection of low abundance T790 M and provides a method for prognostic monitoring in NSCLC patients.
研究领域:
生物物理学
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医学诊断
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分子生物学
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产品关联
DOI
PubMed
高影响因子
LIFE SCIENCES
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2026
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IF 5.1
Zhongquan Song,Yanmei Zhu,Surong Fang,Shuhua Han
Aims The EGFR-C797S mutation is the main cause of Osimertinib resistance. No effective treatment regimen is currently available for patients harboring EGFR-activation mutation/T790M/cis-C797S triple mutations. Materials and methods Nine patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR-activation mutation/T790M/cis-C797S mutation were investigated. After Osimertinib resistance, these patients were treated with Endostar combination with Osimertinib. Network pharmacology and molecular dynamics simulations were used to clarify active sites of Endostar and the effect on protein conformation. In vivo and in vitro , EGFR L858R-T790M-C797S/H1975 cells were used to investigate the efficacy of co-treatment. Key findings Two patients achieved partial response (PR), six achieved stable disease (SD) and one had progressive disease (PD) at the 6-month efficacy assessment of co-treatment, resulting in an objective response rate (ORR) of 66.7% and a disease control rate (DCR) of 88.9%. These patients achieved a median progression-free survival (PFS) of 8.9 months and a median overall survival (OS) of 18.5 months, and no patients experienced grade III or IV adverse events. Endostatin, the active ingredient of Endostar, bound EGFR to “pseudo” mediate EGFR signaling pathway, and the introduction of Endostar enhanced the binding of Osimertinib to EGFR L858R/T790M/C797S protein. In vitro and in vivo experiments have found the co-treatment restored sensitivity to Osimertinib. Moreover, the combination therapy significantly inhibited the activation of the EGFR signaling pathway and tumor neovascularization. Significance This work demonstrated for the first time that Endostar enhanced the antitumor efficacy of Osimertinib, offering a potential therapeutic approach for patients harboring triple mutations.
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产品关联
DOI
PubMed
高影响因子
BIOMEDICAL CHROMATOGRAPHY
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2022
·Vol. 37
·期 1
·e5525
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IF 1.911
Yuhao Fu,Ying Li,Yinling Ma,Xueru He,Xuejiao Xun,Yanjun Cui,Liju Fan,Zhanjun Dong
Almonertinib was included in the first-line treatment of non-small cell lung cancer with EGFR T790M mutations by the Chinese Society of Clinical Oncology in 2021. Considering that immunocompromised lung cancer patients are prone to opportunistic fungal infections, and most triazole antifungal drugs are moderate or strong inhibitors of CYP3A4, this study was conducted to develop and validate an accurate and rapid ultra-performance liquid chromatography tandem mass spectrometry method for quantifying almonertinib in plasma and for investigating the pharmacokinetic changes of almonertinib caused by voriconazole and fluconazole in rats. After liquid–liquid extraction with tert -butyl methyl ether, an XSelect HSS T3 column (2.1 × 100 mm, 2.5 μm, Waters) was used for the chromatographic separation of almonertinib and sorafenib-D 3 (internal standard). The analytes were detected using an AB Sciex Triple Quad 5,500 mass spectrometer in the positive ionization mode. The method exhibited great linearity (0.5–200 ng/ml, r > 0.997) and stability under the established experimental conditions. All validation experiments were in accordance with the guidelines, and the results were all within the acceptable limits. This method was successfully applied to the researches of pharmacokinetics and drug interactions for almonertinib in rats. Voriconazole and fluconazole significantly altered the pharmacokinetic profiles of almonertinib and increased the systemic exposure of almonertinib in rats to different degrees, but further human trials should be conducted to validate the results.
研究领域:
药理学
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肿瘤学
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分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Fan Yang,JinBo Gong,Ming Li,Xiangyang Jiang,Jiawen Zhang,Meiyan Liao,Hanfei Zhang,Pier-Luc Tremblay,Tian Zhang
PCR-based techniques routinely employed for the detection of mutated linear DNA molecules, including circulating tumor DNA (ctDNA), require large nucleotide sections on both sides of the mutation for primer annealing. This means that DNA fragments with a mutation positioned closer to the extremities are unlikely to be detected. Thus, sensors capable of recognizing linear DNA with characteristic mutations closer to the ends would be advantageous over the state-of-the-art approaches. Here, an electrochemiluminescence-resonance energy transfer (ECL-RET) biosensor comprising capped CdS quantum dots and hairpin DNA probes labeled with Au nanoparticles was developed for the detection of epidermal growth factor receptor (EGFR) ctDNA carrying the critical T790M lung cancer mutation. The ECL-RET system detected different DNA molecules including single-stranded 18-nucleotides (nt) and 40-nt as well as double-stranded 100-nt with the single nucleotide polymorphism (SNP) coding for T790M located either in the middle or only 7 nt from one end. For all target DNA, the sensor’s limits of detection (LODs) were in the aM range, with excellent selectivity. It was the case of 100-nt target linear ctDNA fragments with LODs of 8.1 and 3.4 aM when the EGFR T790M SNP was either in the middle or at the end, respectively. These results show that ECL-RET systems can sense mutations in DNA fragments that would remain undetected by standard techniques.
研究领域:
生物传感器
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纳米技术
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癌症研究
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分子生物学
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分析化学
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产品关联
DOI
PubMed
高影响因子
ANALYST
·
2019
·Vol. 144
·期 16
·4757-4771
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IF 4.019
Xiurui Zhu,Shisheng Su,Mingzhu Fu,Zhiyong Peng,Dong Wang,Xiao Rui,Fang Wang,Xiaobin Liu,Baoxia Liu,Lingxiang Zhu,Wenjun Yang,Na Gao,Guoliang Huang,Gaoshan Jing,Yong Guo
Droplet digital PCR (ddPCR) is a single-molecule amplification technology with broad applications in precision medicine and clinical diagnosis. Dual-fluorescence and four-cluster ddPCR (two/four-ddPCR) assay is an effective way to quantify copy numbers. Currently, two/four-ddPCR data are usually classified with manual thresholds. For clinical applications, automatic and accurate methods are required to avoid subjectivity in diagnosis. Although there are some automatic classification algorithms, their accuracy and robustness still need to be improved to meet the needs of clinical diagnosis. Therefore, a new method is in high demand to automatically classify two/four-ddPCR data in an accurate and robust way. Here, a novel density-watershed algorithm (DWA) method was developed for the accurate, automatic and unsupervised classification of two/four-ddPCR data. First, data gridding was applied to a scatter plot of the fluorescence signal intensity to calculate data densities. Based on the data densities, the watershed algorithm was used to divide the gridded scatter plot into isolated regions automatically. Next, an optimal cluster pattern was determined based on these isolated regions, and excess regions were merged. Finally, the two/four-ddPCR data were classified based on the merged regions, and DNA template copy numbers were calculated accordingly. Using the DWA method for the quantification of both wild types and mutants of epidermal growth factor receptor (EGFR) L858R and T790M, the classification results were highly consistent with expectations, and significantly better than commonly-used automatic algorithms for now. The computed template copy numbers scaled proportionally to the relative concentration of input templates (r2 > 0.998) in four orders of magnitude with a good reproducibility, and achieved a limit of detection over 40 times lower than the commonly-used automatic algorithms. Furthermore, the DWA method was validated on 254 clinical DNA samples derived from frozen tissues, formalin-fixed paraffin-embedded tissues and peripheral blood. In most cases, the DWA method derived accurate and valid classification results. This highly effective DWA method may be widely used for automatically classifying two/four-ddPCR data, and it will greatly promote the application of ddPCR in clinical diagnosis.
研究领域:
生物信息学
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分子生物学
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临床诊断
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精准医疗
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产品关联
DOI
PubMed
高影响因子
Scientific Reports
·
2015
·Vol. 5
·期 1
·1-17
·
IF 5.578
Cao Wei,Liu Ying,Zhang Ran,Zhang Bo,Wang Teng,Zhu Xianbing,Mei Lin,Chen Hongbo,Zhang Hongling,Ming Pinghong,Huang Laiqiang
Tyrosine kinase inhibitors (TKIs) are mostly used in non-small cell lung cancer (NSCLC) treatment. Unfortunately, treatment with Gefitinib for a period of time will result in drug resistance and cause treatment failure in clinic. Therefore, exploring novel compounds to overcome this resistance is urgently required. Here we investigated the antitumor effect of homoharringtonine (HHT), a natural compound extracted from Cephalotaxus harringtonia , on Gefitinib-resistant NSCLC cell lines in vitro and in viv o. NCI-H1975 cells with EGFR T790M mutation are more sensitive to HHT treatment compared with that of A549 cells with wild type EGFR. HHT inhibited cells growth, cell viability and colony formation, as well as induced cell apoptosis through mitochondria pathway. Furthermore, we explored the mechanism of HHT inhibition on NSCLC cells. Higher level of interleukin-6 (IL-6) existed in lung cancer patients and mutant EGFR and TGFβ signal requires the upregulation of IL-6 through the gp130/JAK pathway to overactive STAT3, an oncogenic protein which has been considered as a potential target for cancer therapy. HHT reversiblely inhibited IL-6-induced STAT3 Tyrosine 705 phosphorylation and reduced anti-apoptotic proteins expression. Gefitinib-resistant NSCLC xenograft tests also confirmed the antitumor effect of HHT in vivo . Consequently, HHT has the potential in Gefitinib-resistant NSCLC treatment.
研究领域:
肿瘤学
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药理学
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分子生物学
·
细胞生物学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2020
·
IF 5.977
Fang Hu,Wancun Zhang,Wei Meng,Yuxiang Ma,Xianwei Zhang,Ying Xu,Peng Wang,Yueqing Gu
Single nucleotide polymorphisms (SNPs) are crucial during the early diagnosis of a given disease as well as in evaluating their response to certain drugs. Thus, this study sought the development of ferrocene (Fc)-labeled electrochemical biosensor for SNP detection. This proposed system involves the ligation of four short probes (e.g., A, B, A′, and B′, where B′ is labeled with an Fc-tag) in the presence of target DNA via ligase chain reaction (LCR), resulting in the formation of Fc-tagged duplex AB-A′B′ in 2 n . Subsequently, immobilization of the Fc-tagged duplex AB-A′B′ on a single-stranded DNA capture probe (SC-DNA)-carboxyl multi-wall carbon nanotube (MWCNT-COOH) modified glassy carbon electrode (GCE) was accomplished through hybridization. Owing to the specificity of hybridization, and the use of Fc as electrochemical probe for detection of duplex AB-A′B′, such strategy realized directly analysis of LCR products without the need for purification . By taking advantage of the thermal stability and high-discrimination ability of HiFi Taq DNA ligase for single-base differences, the specificity of hybridization, the EGFR T790 M mutant DNA (MT-DNA) biosensor was developed to offer a low limit of detection (0.75 aM), a high discrimination of single-base mismatches [as low as 0.01% (molar fraction)], a wide linear range of more than 7 orders of magnitude (1 aM–10 pM), and the recovery rates (95.3%–107.8%) from human serum samples. Thus, the biosensor under development was found to be economical, highly-sensitive, and exceptionally selective for detection of SNPs, and as well as extending the versatile applications of LCR to offer great potential for diagnosis and individual clinical regimens.
研究领域:
生物化学
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生物技术
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分析化学
·
分子生物学
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医学诊断
✓ 已验证
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）