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It has long been known that T cells participate in wound healing, however, it is still enigmatic about the landscape of the signaling derived from T cells in the process of wound healing. With the advantages of scRNA-seq, in combination of immunofluorescent imaging, we identified activated T cells, cytotoxic T cells (CTLs), exhausting T cells and Tregs existing in inflammation phase of wound healing. Further analysis revealed each T cell population possess distinguished signals contributed to wound healing, some are critical for improving the wound healing quality. Besides, this study discovered and validated the exhistance of exhausting T cells among the T cells accumulated in skin duing wound healing, and the molecular mechanism(s) and contribution of exhausting T cells to wound healing deserves extensive studies in the future.
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Background: Tryptophan (Trp) metabolism is closely related to tumor immunity, and its disorder can cause an immunosuppressive microenvironment, promoting the occurrence and development of hepatocellular carcinoma (HCC). The aim of this study is to explore and validate the independent prognostic genes in patients suffered from HCC. Methods: The transcriptome data of GSE87630 from GEO database were downloaded to analyze differentially expressed genes (DECs) which were intersected with the gene sets of Trp metabolism from MsigDB database. Univariate/multivariate COX regression was performed to identify the genes with independent prognostic significance. TCGA, GTEx, UALCAN, and GEPIA2 databases were applied to analyze DEGs for prognosis. RNA seq data of HCC from TCGA database were collected for Lasso regression analysis. The ssGSEA algorithm was used to perform the analysis of TCGA data. The effects of the candidate differential gene on HCC cells proliferation and migration were evaluated using EdU immunofluorescence and transwell assays. Results: Trp metabolism-related DECs for HCCs were obtained, including MAOB, CYP1A2, KYNU, CYP2E1, ALDH2, CYP2C18, TDO2, AOX1, CYP3A4 and INMT. Moreover, multivariate COX regression results showed that ALDH2 can serve as an independent prognostic molecule and its transcriptional and translational levels were significantly reduced in the tumor tissues. The low expression of ALDH2 was associated with poor prognosis. Overexpression of ALDH2 dramatically reduced the HCC cells proliferation and migration. Conclusion: ALDH2 is associated with Trp metabolism and its downregulation in HCC has a potential value on prognosis. Overexpression of ALDH2 can reduce the proliferation and migration of HCC cells.
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Background: Hepatocellular carcinoma (HCC) poses a global threat to life; however, numerical tools to predict the clinical prognosis of these patients remain scarce. The primary objective of this study is to establish a clinical scoring system for evaluating the overall survival (OS) rate and cancer-specific survival (CSS) rate in HCC patients. Methods: From the Surveillance, Epidemiology, and End Results (SEER) Program, we identified 45,827 primary HCC patients. These cases were randomly allocated to a training cohort (22,914 patients) and a validation cohort (22,913 patients). Univariate and multivariate Cox regression analyses, coupled with Kaplan-Meier methods, were employed to evaluate prognosis-related clinical and demographic features. Factors demonstrating prognostic significance were used to construct the model. The model's stability and accuracy were assessed through C-index, receiver operating characteristic (ROC) curves, calibration curves, and clinical decision curve analysis (DCA), while comparisons were made with the American Joint Committee on Cancer (AJCC) staging. Ultimately, machine learning (ML) quantified the variables in the model to establish a clinical scoring system. Results: Univariate and multivariate Cox regression analyses identified 11 demographic and clinical-pathological features as independent prognostic indicators for both CSS and OS using. Two models, each incorporating the 11 features, were developed, both of which demonstrated significant prognostic relevance. The C-index for predicting CSS and OS surpassed that of the AJCC staging system. The area under the curve (AUC) in time-dependent ROC consistently exceeded 0.74 in both the training and validation sets. Furthermore, internal and external calibration plots indicated that the model predictions aligned closely with observed outcomes. Additionally, DCA demonstrated the superiority of the model over the AJCC staging system, yielding greater clinical net benefit. Ultimately, the quantified clinical scoring system could efficiently discriminate between high and low-risk patients. Conclusions: A ML clinical scoring system trained on a large-scale dataset exhibits good predictive and risk stratification performance in the cohorts. Such a clinical scoring system is readily integrable into clinical practice and will be valuable in enhancing the accuracy and efficiency of HCC management.
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Background: PLAUR has been found upregulated in various tumors and closely correlated with the malignant phenotype of tumor cells. The aim of this study was to investigate the relationship between PLAUR and clear cell renal cell carcinoma (ccRCC) and its potential mechanism of promoting tumor progression. Methods: The expression levels and clinical significance of PLAUR, along with the associated signaling pathways, were extensively investigated in ccRCC samples obtained from The Cancer Genome Atlas (TCGA). PLAUR expression in 20 pairs of ccRCC tumor tissues and the adjacent tissues was assessed using qRT-PCR and IHC staining. Additionally, a series of in vitro experiments were conducted to investigate the impact of PLAUR suppression on cellular proliferation, migration, invasion, cell cycle progression, and apoptosis in ccRCC. The Western blot analysis was employed to investigate the expression levels of pivotal genes associated with the PI3K/AKT/mTOR signaling pathway. Results: The expression of PLAUR was significantly upregulated in ccRCC compared to normal renal tissues, and higher PLAUR expression in ccRCC was associated with a poorer prognosis than low expression. The in-vitro functional investigations demonstrated that knockdown of PLAUR significantly attenuated the proliferation, migration, and invasion capabilities of ccRCC cells. Concurrently, PLAUR knockdown effectively induced cellular apoptosis, modulated the cell cycle, inhibited the EMT process, and attenuated the activation of the PI3K/AKT/mTOR signaling pathway. PLAUR may represent a key mechanism underlying ccRCC progression. Conclusions: The involvement of PLAUR in ccRCC progression may be achieved through the activation of the PI3K/AKT/mTOR signaling pathway, making it a reliable biomarker for the identification and prediction of ccRCC.
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Carcinoma de Células Renais , Proliferação de Células , Progressão da Doença , Neoplasias Renais , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Serina-Treonina Quinases TOR , Humanos , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Neoplasias Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proliferação de Células/genética , Linhagem Celular Tumoral , Masculino , Feminino , Apoptose , Movimento Celular/genética , Pessoa de Meia-Idade , Regulação Neoplásica da Expressão Gênica , Prognóstico , Regulação para CimaRESUMO
BACKGROUND: MicroRNAs (miRNAs) are important non-coding RNA entities that affect gene expression and function by binding to target mRNAs, leading to degradation of the mRNAs or inhibiting their translation. MiRNAs are widely involved in a variety of biological processes, such as cell differentiation, development, metabolism, and apoptosis. In addition, miRNAs are associated with many diseases, including cancer. However, conventional detection techniques often suffer from shortcomings such as low sensitivity, so we need to develop a rapid and efficient detection strategy for accurate detection of miRNAs. RESULTS: We have developed an innovative homogeneous electrochemiluminescence (ECL) biosensor. This biosensor employs CRISPR/Cas12a gene editing technology for accurate and efficient detection of microRNA (miRNA). Compared to conventional technologies, this biosensor employs a unique homogeneous detection format that eliminates laborious probe fixation steps and greatly simplifies the detection process. By using two amplification techniques - isothermal amplification and T7 RNA polymerase amplification - the biosensor improves the sensitivity and specificity of the assay, providing excellent detection performance in the assay. This makes it possible to evaluate miRNA directly from a variety of biological samples such as cell lysates and diluted human serum. Experimental results convincingly demonstrate the extraordinary performance of this biosensor, including its extremely low detection limit of 1.27 aM, high sensitivity, reproducibility and stability. SIGNIFICANCE: The application of our constructed sensor in distinguishing between cancerous and non-cancerous cell lines highlights its potential for early cancer detection and monitoring. This innovative approach represents a major advancement in the field of miRNA detection, providing a user-friendly, cost-effective, and sensitive solution with broad implications for clinical diagnosis and patient care, especially in point-of-care settings.
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Técnicas Biossensoriais , Sistemas CRISPR-Cas , Técnicas Eletroquímicas , Medições Luminescentes , MicroRNAs , Humanos , Técnicas Biossensoriais/métodos , MicroRNAs/análise , MicroRNAs/sangue , MicroRNAs/genética , Sistemas CRISPR-Cas/genética , Técnicas Eletroquímicas/métodos , Limite de Detecção , Proteínas Associadas a CRISPR/genética , Proteínas de Bactérias , EndodesoxirribonucleasesRESUMO
Bone Marrow mesenchymal Stem Cells (BMSCs) are considered as an important source of cells for regenerative medicine, In particular, Bone Marrow mesenchymal Stem Cells Exosomes (BMSCs-EXO) have the most significant effect in the treatment of Spinal Cord Injury (SCI), but the mechanism of action is still unknown. This study found that compared with other SCI groups, BMSCs-EXO loaded with miR-146a could significantly improve the functional recovery of the hind limbs of SCI rats. Hematoxylin and eosin (H&E) indicated that the lesion area of spinal cord injury was less, nissl staining indicated that the number of nissl bodies remained more; the mechanism may be through inhibiting the expression of IRAK1 and TRAF6, blocking the activation of NF-κB p65, reducing the expression of TNF-α, IL-1ß and IL-6 inflammatory factors and oxidative stress, improving the SCI microenvironment, and promoting the repair of neural function. In general, we found that BMSCs-EXO loaded with miR-146a could reduce the inflammatory response and oxidative stress in SCI by inhibiting the activation of IRAK1/TRAF6/NF-κB p65 signaling pathway, and promote the recovery of neurological function in SCI rats.
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The article details a groundbreaking platform for detecting microRNAs (miRNAs), crucial biomolecules involved in gene regulation and linked to various diseases. This innovative platform combines the CRISPR-Cas13a system's precise ability to specifically target and cleave RNA molecules with the amplification capabilities of the hybridization chain reaction (HCR). HCR aids in signal enhancement by creating branched DNA structures. Additionally, the platform employs electrochemiluminescence (ECL) for detection, noted for its high sensitivity and low background noise, making it particularly effective. A key application of this technology is in the detection of miR-17, a biomarker associated with multiple cancer types. It exhibits remarkable detection capabilities, characterized by low detection limits (14.38 aM) and high specificity. Furthermore, the platform's ability to distinguish between similar miRNA sequences and accurately quantify miR-17 in cell lysates underscores its significant potential in clinical and biomedical fields. This combination of precise targeting, signal amplification, and sensitive detection positions the platform as a powerful tool for miRNA analysis in medical diagnostics and research.
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Sistemas CRISPR-Cas , Técnicas Eletroquímicas , Medições Luminescentes , MicroRNAs , Hibridização de Ácido Nucleico , MicroRNAs/análise , MicroRNAs/genética , Humanos , Sistemas CRISPR-Cas/genética , Técnicas Eletroquímicas/métodos , Técnicas Biossensoriais/métodos , Limite de DetecçãoRESUMO
B-type natriuretic peptide (BNP) is a biomarker for heart failure, a serious and prevalent disease that requires rapid and accurate diagnosis. In this study, we developed a novel electrochemical biosensor for BNP detection based on CRISPR/Cas13a and chain substitution reaction. The biosensor consists of a DNA aptamer that specifically binds to BNP, a T7 RNA polymerase that amplifies the signal, a CRISPR/Cas13a system that cleaves the target RNA, and a two-dimensional DNA nanoprobe that generates an electrochemical signal. The biosensor exhibits high sensitivity, specificity, and stability, with a detection limit of 0.74 aM. The biosensor can also detect BNP in human serum samples with negligible interference, demonstrating its potential for clinical and point-of-care applications. This study presents a novel strategy for integrating CRISPR/Cas13a and chain substitution reaction into biosensor design, offering a versatile and effective platform for biomolecule detection.
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Técnicas Biossensoriais , Sistemas CRISPR-Cas , Técnicas Eletroquímicas , Peptídeo Natriurético Encefálico , Técnicas Biossensoriais/métodos , Peptídeo Natriurético Encefálico/sangue , Peptídeo Natriurético Encefálico/química , Humanos , Sistemas CRISPR-Cas/genética , Limite de Detecção , Aptâmeros de Nucleotídeos/químicaRESUMO
α-synuclein oligomer is a marker of Parkinson's disease. The traditional enzyme-linked immunosorbent assay for α-synuclein oligomer detection is not conducive to large-scale application due to its time-consuming, high cost and poor stability. Recently, DNA-based biosensors have been increasingly used in the detection of disease markers due to their high sensitivity, simplicity and low cost. In this study, based on the DNAzyme-driven DNA bipedal walking method, we developed a signal-on electrochemical sensor for the detection of α-syn oligomers. Bipedal DNA walkers have a larger walking area and faster walking kinetics, providing higher amplification efficiency compared to conventional DNA walkers. The DNA walker is driven via an Mg2+-dependent DNAzyme, and the binding-induced DNA walker will continuously clamp the MB, resulting in the proliferation of Fc confined near the GE surface. The linear range and limit of detection were 1 fg/mL to 10 pg/mL and 0.57 fg/mL, respectively. The proposed signal-on electrochemical sensing strategy is more selective. It will play a significant role in the sensitive and precise electrochemical analysis of other proteins.
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Técnicas Biossensoriais , DNA Catalítico , DNA Catalítico/química , alfa-Sinucleína/química , DNA/química , Hibridização de Ácido NucleicoRESUMO
A novel electrochemical biosensor that combines the CRISPR-Cas12a system with a gold electrode is reported for the rapid and sensitive detection of microphthalmia-associated transcription factor (MITF). The biosensor consists of a gold electrode modified with DNA1, which contains the target sequence of MITF and is labeled with ferrocene, an electroactive molecule. The biosensor also includes hairpin DNA, which has a binding site for MITF and can hybridize with helper DNA to form a double-stranded complex that activates CRISPR-Cas12a. When MITF is present, it binds to hairpin DNA and prevents its hybridization with helper DNA, thus inhibiting CRISPR-Cas12a activity and preserving the DPV signal of ferrocene. When MITF is absent, hairpin DNA hybridizes with helper DNA and activates CRISPR-Cas12a, which cleaves DNA1 and releases ferrocene, thus reducing the DPV signal. The biosensor can detect MITF with high sensitivity (with an LOD of 8.14 fM), specificity, and accuracy in various samples, such as cell nuclear extracts and human serum. The biosensor can also diagnose and monitor melanocyte-related diseases and melanin production. This work provides a simple, fast, sensitive, and cost-effective biosensor for MITF detection and a valuable tool for applications in genetic testing, disease diagnosis, and drug screening.
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Sistemas CRISPR-Cas , Fator de Transcrição Associado à Microftalmia , Humanos , Fator de Transcrição Associado à Microftalmia/genética , Metalocenos , Ouro , DNA/genéticaRESUMO
BACKGROUND: Escherichia coli (E.coli) is both a commensal and a foodborne pathogenic bacterium in the human gastrointestinal tract, posing significant potential risks to human health and food safety. However, one of the major challenges in E.coli detection lies in the preparation and storage of antibodies. In traditional detection methods, antibodies are indispensable, but their instability often leads to experimental complexity and increased false positives. This underscores the need for new technologies and novel sensors. Therefore, the development of a simple and sensitive method for analyzing E.coli would make significant contributions to human health and food safety. RESULTS: We constructed an electrochemical biosensor based on triple-helical DNA and entropy-driven amplification reaction (EDC) to inhibit the cleavage activity of Cas12a, enabling high-specificity detection of E.coli. Replacing antibodies with nucleic acid aptamers (Apt) as recognition elements, we utilized the triple-helical DNA generated by the binding of DNA2 and DNA5/DNA6 double-helical DNA through the entropy-driven amplification reaction to inhibit the collateral cleavage activity of clustered regularly interspaced short palindromic repeats gene editing system (CRISPR) and its associated proteins (Cas). By converting E.coli into electrical signals and recording signal changes in the form of square wave voltammetry (SWV), rapid detection of E.coli was achieved. Optimization of experimental conditions and data detection under the optimal conditions provided high sensitivity, low detection limits, and high specificity. SIGNIFICANCE: With a minimal detection limit of 5.02 CFU/mL and a linear range of 1 × 102 - 1 × 107 CFU/mL, the suggested approach was successfully verified to analyze E.coli at various concentrations. Additionally, after examining E.coli samples from pure water and pure milk, the recoveries ranged between 95.76 and 101.20%, demonstrating the method's applicability. Additionally, it provides a feasible research direction for the detection of pathogenic bacteria causing other diseases using the CRISPR/Cas gene editing system.
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Técnicas Biossensoriais , Ácidos Nucleicos , Humanos , Sistemas CRISPR-Cas , Edição de Genes , DNA/genética , Oligonucleotídeos , Anticorpos , Escherichia coli/genéticaRESUMO
In the current study, a novel electrochemiluminescence biosensor based on the entropy-driven DNA tetrahedron for the detection of matrix metalloproteinase 2 (MMP2), an enzyme that regulates extracellular matrix remodeling and affects aging was reported. The biosensor utilizes an inverted DNA tetrahedron structure, which exposes three vertices to the solution, as molecular recognition units for capturing specific biomolecules. The biosensor also employs a ratiometric method and an entropy-driven reaction, which enhance the response rate and sensitivity of the detection. The biosensor can detect MMP2 with a detection limit of 55.2 fM, which is lower than that of conventional sensors. The biosensor also exhibits excellent stability and reproducibility, and can accurately measure MMP2 levels in complex samples, such as human serum. The paper demonstrates the feasibility and effectiveness of using the "inverted" DNA tetrahedron structure and the entropy-driven process to construct interfacial biosensors. The paper also discusses the potential applications of the biosensor in clinical diagnosis and anti-aging research, where MMP2 plays a crucial role in tissue damage and repair. The paper provides a valuable contribution to the field of biosensor development, and opens up new possibilities for using DNA nanotechnology for sensitive and reliable detection of various biomolecules.
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Envelhecimento , Metaloproteinase 2 da Matriz , Humanos , Reprodutibilidade dos Testes , DNA , EntropiaRESUMO
Apolipoprotein A4 (Apo-A4) is considered as a prospective molecular biomarker for diagnosis of depression due to its neurosynaptic toxicity. Here, we propose a neighboring hybridization induced catalyzed hairpin assembly (CHA) driven bipedal DNA walker that mediates hybridization of Ag nanoparticles (Ag NPs) with DNA probes for highly sensitive electrochemical quantitative detection of Apo-A4. Driven by CHA, this bipedal DNA walker can spread all over the surface of the sensor, induce the HP1-HP2 double chain structure, make the surface of the sensor negatively charged, and adsorb a large number of Ag ions. After chemical reduction with hydroquinone, the Ag NPs formed provide signal tracers for electrochemical dissolution analysis of the target. The Ag NPs formed by chemical reduction of hydroquinone can provide signal traces for electrochemical stripping analysis of target thrombin. The linear range of this method is from 10 pg mL-1 to 1000 ng mL-1, and the detection limit is 5.1 pg mL-1. This enzyme-free and labeling detection method provides a new strategy for rapid clinical detection of Apo-A4 and accurate identification of depression.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Hidroquinonas , Nanopartículas Metálicas/química , Técnicas Biossensoriais/métodos , Prata/química , DNA/química , Técnicas Eletroquímicas/métodos , Limite de Detecção , Ouro/químicaRESUMO
By merging DNA entropy-driven technology with triple-stranded nucleic acids in an electrochemical biosensor to detect the SARS-CoV-2 RdRp gene, we tackled the challenges of false negatives and the high cost of SARS-CoV-2 detection. The approach generates a CRISPR-Cas 13a-activated RNA activator, which then stimulates CRISPR-Cas 13a activity using an entropy-driven mechanism. The activated CRISPR-Cas 13a can cleave Hoogsteen DNA due to the insertion of two uracil (-U-U-) in Hoogsteen DNA. The DNA tetrahedra changed on the electrode surface and can therefore not construct a three-stranded structure after cleaving Hoogsteen DNA. Significantly, this DNA tetrahedron/Hoogsteen DNA-based biosensor can regenerate at pH = 10.0, which keeps Hoogsteen DNA away from the electrode surface, allowing the biosensor to function at pH = 7.0. We could use this technique to detect the SARS-CoV-2 RdRp gene with a detection limit of 89.86 aM. Furthermore, the detection method is very stable and repeatable. This technique offers the prospect of detecting SARS-CoV-2 at a reasonable cost. This work has potential applications in the dynamic assessment of the diagnostic and therapeutic efficacy of SARS-CoV-2 infection and in the screening of environmental samples.
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COVID-19 , Ácidos Nucleicos , Humanos , SARS-CoV-2/genética , Entropia , COVID-19/diagnóstico , DNA/genética , Tecnologia , RNA Polimerase Dependente de RNARESUMO
Background: Hepatoblastoma (HB), hepatocellular carcinoma (HCC), and embryonal sarcoma (ES) are the three main types of liver tumors in children and adolescents. At present, epidemiological knowledge and predictors of these three liver tumor types in multi-ethnic populations are limited. This study aimed to outline the clinical features and construct a prognostic nomogram for these tumors, which can contribute to the prediction of dynamic overall survival probability during the follow-up period. Methods: A total of 1,122 patients liver tumor patients between 2000 to 2019 in Surveillance, Epidemiology, and End Results (SEER) database were enrolled for the current study, and separated into 824 HB, 219 HCC, and 79 ES according to the type of pathology. Independent prognostic factors were screened by univariate and multivariate Cox regression analysis, and a prognostic nomogram was constructed for overall survival. The accuracy and discriminative abilities of the nomogram were evaluated by concordance index as well as time-dependent receiver operating characteristic curves and calibration curves. Results: Race (P=0.0016), surgery [hazard ratio (HR): 0.1021, P<0.001], and chemotherapy (HR: 0.27, P=0.00018) are independent prognostic factors for hepatoblastoma. Pathological tissue grading (P=0.00043), tumor node metastasis (TNM) staging (P=0.00061), and surgery are independent prognostic factors for hepatocellular carcinoma. Household income and surgery (HR: 0.1906, P<0.001) are independent prognostic factors for embryonal sarcoma. All of these prognostic factors are significantly associated with prognosis. A nomogram consisting of these variables was established, which showed a good concordance index (0.747, 0.775, and 0.828 in hepatoblastoma, hepatocellular carcinoma, and embryonal sarcoma, respectively). Also, the 5-year area under curve (AUC) of the nomogram were 0.738, 0.812, and 0.839 in hepatoblastoma, hepatocellular carcinoma, and embryonal sarcoma, respectively. In the calibration diagram, an optimal agreement between the nomogram-predicted and actual observed survival was evident. Conclusions: We developed an effective prognostic nomogram for overall survival prediction in hepatoblastoma, hepatocellular carcinoma, and embryonal sarcoma in children and adolescent patients, which will further benefit the assessment of long-term outcomes.
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In this study, we introduce an electrochemiluminescence (ECL) sensing platform based on the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). This platform combines a programmable entropy-driven cycling strategy, T7 RNA polymerase, and the CRISPR/Cas13a system to amplify the determination of the SARS-CoV-2 RdRp gene. The Ti3C2Tx-compliant ECL signaling molecule offers unique benefits when used with the ECL sensing platform to increase the assay sensitivity and the electrode surface modifiability. To obtain the T7 promoter, the SARS-CoV-2 RdRp gene may first initiate an entropy-driven cyclic amplification response. Then, after recognizing the T7 promoter sequence on the newly created dsDNA, T7 RNA polymerase starts transcription, resulting in the production of many single-stranded RNAs (ssRNAs), which in turn trigger the action of CRISPR/Cas13a. Finally, Cas13a/crRNA identifies the transcribed ssRNA. When it cleaves the ssRNA, many DNA reporter probes carrying -U-U- are cleaved on the electrode surface, increasing the ECL signal and allowing for the rapid and highly sensitive detection of SARS-CoV-2. With a detection limit of 7.39 aM, our method enables us to locate the SARS-CoV-2 RdRp gene in clinical samples. The detection method also demonstrates excellent repeatability and stability. The SARS-CoV-2 RdRp gene was discovered using the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). The developed ECL test had excellent recoveries in pharyngeal swabs and environmental samples. It is anticipated to offer an early clinical diagnosis of SARS-CoV-2 and further control the spread of the pandemic.
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Técnicas Biossensoriais , COVID-19 , Humanos , COVID-19/diagnóstico , Entropia , SARS-CoV-2/genética , RNA Polimerase Dependente de RNARESUMO
Amyloid-ß oligomer has been considered as a promising molecular biomarker for the diagnosis of Alzheimer's disease due to their significant neural synapse toxicity. Therefore, it is essential to create an easy approach for the selective detection of Amyloid-ß oligomer that has high sensitivity and cheap cost. In this work, we developed an innovative enzyme-free electrochemical aptasensor based on the DNAzyme-driven DNA bipedal walker tactics for sensing Amyloid-ß oligomer. Bipedal DNA walkers demonstrate a wider walking region, better walking kinetics, and higher amplification effectiveness than typical DNA walkers. The Mg2+-dependent DNAzyme drove the DNA walker, and the binding-induced DNA walker can sequentially shear MBs and form MB fragment structure. Finally, the detection probes modified AgNPs hybridized with the MB fragment structure, resulting in the multiplication of AgNPs on the electrode surface. Electrochemical stripping of AgNPs was used to test the performance of the obtained electrochemical sensor. In particular, a low detection limit of 5.94 fM and a wide linear range of 0.01 pM-0.1 nM were attained. The detection of Amyloid-ß oligomer in human serum was then carried out using this bipedal DNA walker biosensor, which shown good selectivity and outstanding reproducibility, indicating its usefulness in bioanalysis.
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Técnicas Biossensoriais , DNA Catalítico , Nanopartículas Metálicas , Humanos , DNA Catalítico/química , Peptídeos beta-Amiloides/análise , Prata/química , Nanopartículas Metálicas/química , Reprodutibilidade dos Testes , Limite de Detecção , Técnicas Eletroquímicas/métodos , DNA/química , Técnicas Biossensoriais/métodosRESUMO
As a common technique for detecting AßO, the enzyme-linked immunosorbent assay (ELISA) method is time-consuming, high in cost, and poor in stability. Therefore, it is necessary to develop a highly sensitive, method-simple and low-cost method for the selective detection of AßO. Here, we created a novel signal-on and label-free electrochemical aptamer sensor for the detection of AßO based on a DNAzyme-driven DNA bipedal walking strategy. Compared with common DNA walkers, bipedal DNA walkers exhibit larger walking areas and faster walking kinetics, and provide higher amplification efficiency. The DNAwalker is powered by an Mg2+-dependent DNAzyme, and the binding-induced DNAwalker continuously clamps the MB, unlocking several active G-quadruplex-forming sequences. These G-quadruplexes can be further combined by hemin to generate a G-quadruplex/heme complex, resulting in an amperometric signal, resulting in a broad proportional band from 0.1 pM to 1 nM and an excellent detection range of 46 fM. A bipedal DNA walker aptamer sensor can detect human serum AßO with remarkable specificity, high reproducibility and practical application value.
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Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA Catalítico , Quadruplex G , Humanos , DNA Catalítico/genética , Peptídeos beta-Amiloides/genética , Reprodutibilidade dos Testes , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , DNA/genética , Hemina , Limite de DetecçãoRESUMO
A disintegrin and metalloprotease 10 (ADAM-10), a member of the ADAM protease family, has biological activities related to TNF-α activation, cell adhesion, and migration, among other functions. Macrophages are important immune cells that are involved in the inflammatory response of the body. ADAM-10 is involved in inflammatory responses, but the specific regulatory mechanisms are not fully understood. In this study, we investigated the regulatory mechanism of ADAM-10 in the lipopolysaccharide-promoted proliferation (LPS) of the macrophage inflammatory response. Differentially expressed or regulated proteins were identified in interfered ADAM-10 (sh ADAM-10) macrophages using tandem mass tag (TMT) proteomics. The changes and regulatory role of ADAM-10 during LPS-induced inflammatory response in normal, interfering, and overexpressing ADAM-10 (EX ADAM-10) cells were determined. Results indicated that ADAM-10 interference affected inflammation-related pathways and reduced matrix metalloproteinase 12 (MMP-12) protein levels, as identified by TMT proteomics. In normal cells, LPS decreased ADAM-10 gene expression, but promoted ADAM-10 secretion, MMP-12 and TNF-α gene expression, and MMP-12, iNOS, IL-10, and cyclinD1 protein expression. Additionally, ADAM-10 knockdown decreased macrophage viability in sh-ADAM-10 cells. Moreover, an MMP-12 inhibitor had no impact on the viability effect of LPS on cells or the expression of ADAM-10. iNOS expression decreased, whereas IL-10 expression increased after ADAM-10 depletion. ADAM-10 knockdown decreased MMP-12, iNOS, TNF-α, IL-1ß, and FKN, while overexpression had an opposite effect. ADAM-10 overexpression further increased MMP-12, iNOS, and TNF-α gene expression in response to LPS. Cell viability was increased in EX ADAM-10 cells, and ADAM-10 secretion was further increased in the EX and LPS groups. Flow cytometry and immunofluorescence staining revealed that EX-ADAM 10 cells had increased iNOS expression, which acted as an IL-6 expression driver. In summary, we found that ADAM-10 is activated by LPS and positively participates in LPS-stimulated macrophage inflammatory responses by positively regulating MMP-12 during the inflammatory process.