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RNA In situ imaging through DNA self-assembly is advantaged in illustrating its structures and functions with high-resolution, while the limited reaction efficiency and time-consuming operation hinder its clinical application. Here, we first proposed a new strand displacement reaction (SDR) model (Cas12a thrusting SDR, CtSDR), in which Cas12a could overcome the inherent reaction limitation and dramatically enhance efficiency through energy replenishment and by-product consumption. The target-initiated CtSDR amplification was established for RNA analysis, with order of magnitude lower limit of detection (LOD) than the Cas13a system. The CtSDR-based RNA in situ imaging strategy was developed to monitor intra-cellular microRNA expression change and delineate the landscape of oncogenic RNA in 66 clinic tissue samples, possessing a clear advantage over classic in situ hybridization (ISH) in terms of operation time (1 h versus 14 h) while showing comparable sensitivity and specificity. This work presents a promising approach to developing advanced molecular diagnostic tools.
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Técnicas Biosensibles , ARN , ARN/genética , Sistemas CRISPR-Cas , ADN/genética , ADN/química , Sensibilidad y Especificidad , Hibridación in Situ , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas Biosensibles/métodosRESUMEN
BACKGROUND: PD-1/PD-L1 blockade has become a powerful method to treat malignant tumors. However, a large proportion of patients still do not benefit from this treatment, due to low tumor immunogenicity and low tumor penetration of the agents. Recently, neutrophil elastase has been shown to induce robust tumor immunogenicity, while the insufficient enzyme activity at the tumor site restricted its anti-tumor application. Here, we designed polyethyleneimine-modified neutrophil elastase (PEI-elastase) loaded with PD-L1small interfering RNA (PD-L1 siRNA) for improving enzymatic activity and delivering siRNA to tumor, which was expected to solve the above-mentioned problems. RESULTS: We first demonstrated that PEI-elastase possessed high enzymatic activity, which was also identified as an excellent gene-delivery material. Then, we synthesized anti-tumor lipopolymer (P-E/S Lip) by encapsulating PEI-elastase and PD-L1siRNA with pH-responsive anionic liposomes. The P-E/S Lip could be rapidly cleaved in tumor acidic environment, leading to exposure of the PEI-elastase/PD-L1 siRNA. Consequently, PEI-elastase induced powerful tumor immunogenicity upon direct tumor killing with minimal toxicity to normal cells. In parallel, PEI-elastase delivered PD-L1siRNA into the tumor and reduced PD-L1 expression. Orthotopic tumor administration of P-E/S Lip not only attenuated primary tumor growth, but also produced systemic anti-tumor immune response to inhibit growth of distant tumors and metastasis. Moreover, intravenous administration of P-E/S Lip into mice bearing subcutaneous tumors leaded to an effective inhibition of established B16-F10 tumor and 4T1 tumor, with histological analyses indicating an absence of detectable toxicity. CONCLUSIONS: In our study, a protease-based nanoplatform was used to cooperatively provoke robust tumor immunogenicity and down-regulate PD-L1 expression, which exhibited great potential as a combination therapy for precisely treating solid tumors.
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Antígeno B7-H1 , Inmunoterapia , Polietileneimina , ARN Interferente Pequeño , Animales , Polietileneimina/química , ARN Interferente Pequeño/química , Antígeno B7-H1/metabolismo , Ratones , Inmunoterapia/métodos , Línea Celular Tumoral , Femenino , Humanos , Ratones Endogámicos BALB C , Liposomas/química , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Neoplasias/inmunología , Ratones Endogámicos C57BL , Silenciador del GenRESUMEN
Nowadays, infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have constituted a new challenge for anti-infective treatment. Precise identification and rapid clinical diagnostics of MRSA from other methicillin-sensitive strains entail assays with robust diagnostic efficiency and simple operation steps. Sensitive detection of MecA gene is promising to indicate MRSA infection, but it is challenged by the lack of isothermal and simple strategies. A visual assay based on isothermal rolling circular amplification and G-quadruplex/hemin (G4/hemin) DNAzyme proximity assembly was proposed for the immediate, efficient, and cost-effective detection of MecA in simple operation steps and in a single tube. The presence of MecA specifically drove the formation of circular templates, which further triggered isothermal amplification. The amplified product offered abundant binding sites for DNA-grafted hemin probes to form a novel proximity-assembled G4/hemin DNAzyme structure for colorimetric changing diagnosis. This tandem-repeated novel DNAzyme possessed higher catalytic activity and a lower background signal than traditional G4/hemin DNAzyme, ensuring sensitive discrimination of MRSA (limit of detection: 9.6 pM). Assay stability and antimatrix interference capability enable clinical application, which shows compared diagnostic ability with classic methods (100% sensitivity and 100% specificity) but possesses more simplified procedures and shorter turnaround time (<6 h). This colorimetric strategy in a nonsite-specific and hypersensitive manner holds foreseeable prospects in clinical diagnostic and research applications.
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Técnicas Biosensibles , ADN Catalítico , G-Cuádruplex , Staphylococcus aureus Resistente a Meticilina , ADN Catalítico/química , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/metabolismo , Hemina/química , ADN , Técnicas Biosensibles/métodosRESUMEN
Exosomal microRNAs (exomiRNAs) have emerged as ideal biomarkers for early clinical diagnostics. The accurate detection of exomiRNAs plays a crucial role in facilitating clinical applications. Herein, an ultrasensitive electrochemiluminescent (ECL) biosensor was constructed using three-dimensional (3D) walking nanomotor-mediated CRISPR/Cas12a and tetrahedral DNA nanostructures (TDNs)-modified nanoemitters (TCPP-Fe@HMUiO@Au-ABEI) for exomiR-155 detection. Initially, the 3D walking nanomotor-mediated CRISPR/Cas12a strategy could effectively convert the target exomiR-155 into amplified biological signals for improving the sensitivity and specificity. Then, TCPP-Fe@HMUiO@Au nanozymes with excellent catalytic performance were used to amplify ECL signals because of the enhanced mass transfer and increased catalytic active sites, originating from its high surface areas (601.83 m2/g), average pore size (3.46 nm), and large pore volumes (0.52 cm3/g). Meanwhile, the TDNs as the scaffold to fabricate "bottom-up" anchor bioprobes could improve the trans-cleavage efficiency of Cas12a. Consequently, this biosensor achieved the limit of detection down to 273.20 aM ranging from 1.0 fM to 1.0 nM. Furthermore, the biosensor could discriminate breast cancer patients evidently by analyzing exomiR-155, and these results conformed to that of qRT-PCR. Thus, this work provides a promising tool for early clinical diagnostics.
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Técnicas Biosensibles , MicroARNs , Humanos , MicroARNs/análisis , Sistemas CRISPR-Cas , ADN/química , Fotometría , Técnicas Biosensibles/métodosRESUMEN
Rapid and accurate imaging of the BCR/ABL fusion gene isoforms (e.g., e13a2, e14a2 and co-expression type) of chronic myeloid leukemia (CML) is of vital importance to first-line drug selection, but there is no assay that meets clinical needs (e.g., clinical kits > 18 h without isoforms information). Herein, an in situ imaging platform is developed for the rapid and accurate detection of CML fusion gene isoforms using asymmetric sequence-enhanced hairpins DNA encapsulated silver nanoclusters (ADHA) and catalyzed hairpin assembly (CHA). The specific detection of e13a2 and e14a2 fusion gene isoforms with detection limits of 19.2 am (11.558 copies µL-1 ) and 32.56 am (19.601 copies µL-1 ) in one-pot is achieved. The feasibility of the developed assay for real-world applications are demonstrated by one-step fluorescence imaging (40 min) of e13a2, e14a2 and co-expression type in bone marrow quantitatively (International Standard: 15.66%-168.878%) and further validated by cDNA-sequencing. This work suggests that the developed imaging platform holds great potential for rapid identification of the fusion gene isoforms and isoform related treatment monitoring.
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Proteínas de Fusión bcr-abl , Leucemia Mielógena Crónica BCR-ABL Positiva , Humanos , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/uso terapéutico , Médula Ósea , Plata/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Isoformas de Proteínas/genética , ADN Complementario , Imagen ÓpticaRESUMEN
The emergence of multidrug-resistant microbial pathogens poses a significant threat, severely limiting the options for effective antibiotic therapy. This challenge can be overcome through the photoinactivation of pathogenic bacteria using materials generating reactive oxygen species upon exposure to visible light. These species target vital components of living cells, significantly reducing the likelihood of resistance development by the targeted pathogens. In our research, we have developed a nanocomposite material consisting of an aqueous colloidal suspension of graphene oxide sheets adorned with nanoaggregates of octahedral molybdenum cluster complexes. The negative charge of the graphene oxide and the positive charge of the nanoaggregates promoted their electrostatic interaction in aqueous medium and close cohesion between the colloids. Upon illumination with blue light, the colloidal system exerted a potent antibacterial effect against planktonic cultures of Staphylococcus aureus largely surpassing the individual contributions of the components. The underlying mechanism behind this phenomenon lies in the photoinduced electron transfer from the nanoaggregates of the cluster complexes to the graphene oxide sheets, which triggers the generation of reactive oxygen species. Thus, leveraging the unique properties of graphene oxide and light-harvesting octahedral molybdenum cluster complexes can open more effective and resilient antibacterial strategies.
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Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Molibdeno/farmacología , Especies Reactivas de Oxígeno , Antibacterianos/farmacologíaRESUMEN
BACKGROUND: Previous observational studies have found an association between gastroesophageal reflux disease (GERD) and chronic respiratory diseases, but it remains uncertain whether GERD causally influences these diseases. In this study, we aimed to estimate the causal associations between GERD and 5 chronic respiratory diseases. METHODS: 88 GERD-associated single nucleotide polymorphisms (SNPs) identified by the latest genome-wide association study were included as instrumental variables. Individual-level genetic summary data of participants were obtained from corresponding studies and the FinnGen consortium. We applied the inverse-variance weighted method to estimate the causality between genetically predicted GERD and 5 chronic respiratory diseases. Furthermore, the associations between GERD and common risk factors were investigated, and mediation analyses were conducted using multivariable MR. Various sensitivity analyses were also performed to verify the robustness of the findings. RESULTS: Our study demonstrated that genetically predicted GERD was causally associated with an increased risk of asthma (OR 1.39, 95%CI 1.25-1.56, P < 0.001), idiopathic pulmonary fibrosis (IPF) (OR 1.43, 95%CI 1.05-1.95, P = 0.022), chronic obstructive disease (COPD) (OR 1.64, 95%CI 1.41-1.93, P < 0.001), chronic bronchitis (OR 1.77, 95%CI 1.15-2.74, P = 0.009), while no correlation was observed for bronchiectasis (OR 0.93, 95%CI 0.68-1.27, P = 0.645). Additionally, GERD was associated with 12 common risk factors for chronic respiratory diseases. Nevertheless, no significant mediators were discovered. CONCLUSIONS: Our study suggested that GERD was a causal factor in the development of asthma, IPF, COPD and chronic bronchitis, indicating that GERD-associated micro-aspiration of gastric contents process might play a role in the development of pulmonary fibrosis in these diseases.
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Asma , Bronquitis Crónica , Reflujo Gastroesofágico , Fibrosis Pulmonar Idiopática , Trastornos Respiratorios , Humanos , Bronquitis Crónica/complicaciones , Estudio de Asociación del Genoma Completo , Análisis de la Aleatorización Mendeliana , Reflujo Gastroesofágico/complicaciones , Reflujo Gastroesofágico/genética , Asma/epidemiología , Asma/genética , Asma/complicaciones , Trastornos Respiratorios/complicacionesRESUMEN
Background: As an important supplementary approach to clinical in orthotopic lung transplantation (LTx), lobar LTx currently lacks a stable animal model and in the orthotopic left LTx model, the right lung of the donor mouse is completely removed and discarded. We introduce a novel mouse lobar LTx model that potentially provides a mouse model for clinical lobar LTx and increase the utilization rate of the experimental donor. Methods: Lobar and orthotopic left LTx were performed in syngeneic strain combinations. We performed micro-computed tomography and tested arterial blood gases to assess the graft function 28 days after transplantation. Hematoxylin-eosin and Masson's trichrome staining were used to evaluate pathological changes. Results: We performed ten lobar LTx with an operation success rate of 90%, accompanied by ten orthotopic left LTx from the same donors with an operation success rate of 100%. The graft preparation for lobar LTx was longer than that of the orthotopic left LTx (42.11±3.79 vs. 30.10±3.14 minutes, P<0.001). The recipient procedure for lobar LTx was nearly equivalent to the orthotopic left LTx. The graft function and histopathological changes for lobar LTx were comparable to those of orthotopic left LTx 28 days after transplantation. Conclusions: We describe a lobar LTx model in the mouse, which potentially provides a model for clinical lobar LTx and effectively addresses the issue of resource wastage in the orthotopic left LTx model.
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Existing RNA in situ imaging strategies mostly utilize parallel repetitive nucleic acid self-assembly to achieve multiple analysis, with limitations of complicated systems and cumbersome steps. Here, a Cas9 code key system with key probe (KP) encoder and CRISPR/Cas9 signal exporter is developed. This system triggers T-protospacer adjacent motif (T-PAM structural transitions of multiple KP encoders to form coding products with uniform single-guide RNA (sgRNA) target sequences as tandem nodes. Only single sgRNA/Cas9 complex is required to cleave multiple coding products, enabling efficient "many-to-one" tandem signaling, and non-collateral cleavage activity-dependent automatic signaling output through active introduction of mismatched bases. Compared with conventional parallel multiple signaling analysis model, the proposed system greatly simplifies reaction process and enhances detection efficiency. Further, a rapid multiple RNA in situ imaging system is developed by combining the Cas9 code key system with a T-strand displacement amplification (T-SDA) signal amplifier. The constructed system is applied to tumor cells and clinicopathology slices, generating clear multi-mRNA imaging profiles in less than an hour with just one step. Therefore, this work provides reliable technical support for clinical tumor typing and molecular mechanism investigation.
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Dyes that are released into the environment may have negative effects on living organisms. To address this issue, a biomass-derived carbon adsorbent made from Enteromorpha was tested for its ability to remove methyl orange (MO) from wastewater. The adsorbent was found to be effective in removing MO, with a 1:4 impregnation ratio producing an adsorbent that could remove 96.34% of MO from a 200 mg/L solution using only 0.1 g of adsorbent. At higher concentrations, the adsorption capacity increased up to 269.58 mg/g. Through molecular dynamics simulations, it was discovered that after mono-layer adsorption reached saturation, the remaining MO molecules in solution formed hydrogen bonds with the adsorbed MO, which led to further aggregation on the adsorbent surface and increased adsorption capacity. Additionally, theoretical investigations revealed that the adsorption energy of anionic dyes increased with Nitrogen-doped carbon materials, with the pyrrolic-N site having the highest adsorption energy for MO. The carbon material derived from Enteromorpha showed promise in treating wastewater containing anionic dyes, thanks to its high adsorption capacity and strong electrostatic interaction with the sulfonic acid groups of MO.
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Aguas Residuales , Contaminantes Químicos del Agua , Carbono , Adsorción , Contaminantes Químicos del Agua/análisis , Nitrógeno , Biomasa , Colorantes/químicaRESUMEN
An isothermal, one-pot toolbox (called OPT-Cas) based on CRISPR-Cas12a collateral cleavage capability is proposed for highly sensitive and selective determination of terminal deoxynucleotidyl transferase (TdT) activity. Oligonucleotide primers with 3'-hydroxyl (OH) terminal were randomly introduced for TdT-induced elongation. In the presence of TdT, dTTP nucleotides polymerized at the 3' terminals of the primers to generate abundant polyT-tails, which function as triggers for the synchronous activation of Cas12a proteins. Finally, the activated Cas12a trans-cleaved FAM and BHQ1 dual-labeled single-stranded DNA (ssDNA-FQ) reporters, producing significantly amplified fluorescence signals. This one-pot assay, that is primer, crRNA, Cas12a protein and ssDNA-FQ reporter are all in one tube, allows simple but high-sensitive quantification of TdT activity with a low detection limit of 6.16 × 10-5 U µL-1 in the concentration scope from 1 × 10-4 U µL-1 to 1 × 10-1 U µL-1, and achieves extraordinary selectivity with other interfering proteins. Furthermore, the OPT-Cas was successfully used to detect TdT in complex matrices and accurate determination of TdT activity in acute lymphoblastic leukemia cells, which might be a reliable technique platform for the diagnosis of TdT-related diseases and biomedical research applications.
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Investigación Biomédica , Técnicas Biosensibles , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Sistemas CRISPR-Cas , ADN Nucleotidilexotransferasa , Bioensayo , ADN de Cadena Simple/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMEN
OBJECTIVES: Talaromyces marneffei is an emerging pathogen, and the number of infections in HIV-negative individuals is rapidly increasing. Nevertheless, there is no sufficient comprehensive report on this issue, and awareness needs to be raised among clinicians. METHODS: We analyzed the differences in the clinical data of patients who are HIV-negative and HIV-positive with Talaromyces marneffei infection (TMI) from 2018 to 2022. RESULTS: A total of 848 patients were included, among whom 104 were HIV-negative. The obvious differences between the HIV-positive and HIV-negative groups were as follows: (i) the patients who are HIV-negative were older and more likely to exhibit cough and rash, (ii) the time in days from symptom onset to diagnosis among patients who are HIV-negative was longer, (iii) the laboratory findings and radiological presentations seemed more severe in patients who are HIV-negative, (iv) differences were observed regarding the underlying conditions and co-infection pathogens, and correlation analysis showed that correlations existed for many indicators, (v) and persistent infection was more likely to occur in patients who are HIV-negative. CONCLUSION: TMI in patients who are HIV-negative differs from that in patients who are HIV-positive in many aspects, and more investigations are needed. Clinicians should be more aware of TMI in patients who are HIV-negative.
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Infecciones por VIH , Micosis , Talaromyces , Humanos , Micosis/complicaciones , Micosis/diagnóstico , Micosis/microbiología , Micosis/patología , Estudios Retrospectivos , Infecciones por VIH/complicaciones , Masculino , Femenino , Adulto , Persona de Mediana EdadRESUMEN
Exosomal PD-L1 plays a critical role in tumor progress and immunotherapy. However, accurately analyzing exosomal PD-L1 is greatly limited by the small-sized and free-floating nature of exosomes and the few proteins each exosome carries. We described herein a single-step and highly sensitive method, termed aptamer-triggered cascade primer exchange reaction (PER)-generated branched DNA nanostructures, for the quantification and imaging of exosomal PD-L1. The presence of exosomal PD-L1 converted the conformation of the recognition probe, accompanied by the exposure of primer 1. Then, primer 1 actuated the cascade PER, which generated branched DNA nanostructures containing numerous G-quadruplex for binding to thioflavin T (ThT) dye, leading to an amplified fluorescence signal. Profiting from directly growing branched DNA nanostructures on the surface of exosomes, the size of exosomes was enlarged and the movement of exosomes was limited, achieving the imaging of exosomal PD-L1 by conventional optical microscopy in a wash- and label-free fashion. Analyzing exosomal PD-L1 from serum samples of 15 cancer patients and 15 healthy volunteers demonstrated that this simple strategy could distinguish NSCLC patients from healthy donors with high clinical accuracy. Therefore, the developed assay has great potential as a transformative diagnostic toolkit for cancer detection and immunotherapy monitoring.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Nanoestructuras , Humanos , Antígeno B7-H1 , Oligonucleótidos , ADNRESUMEN
Activated carbon (ENAC) was prepared by NaOH activation, using macroalgae (Enteromorpha clathrate) as raw material. The prepared activated carbon has a large surface area (1238.491 m2 g-1) and its total pore volume and average pore size are 0.6823 cm3g-1 and 2.2038 nm, respectively. The ENAC was characterized by SEM, FTIR, BET and XPS. The effects of contact time (0-960 min), initial tetracycline (TC) concentration (50-500 mg L-1), temperature (30-50 °C) and initial pH (2-11) on TC adsorption were evaluated. The adsorption isotherm and adsorption kinetics were discussed. Results showed that the adsorption isotherm was the Langmuir model, and the adsorption process can be described by the pseudo-second-order model. The N2 adsorption-desorption isotherm was type IV, indicating that the activated carbon had mesoporous structure. Thermodynamic analysis showed that the adsorption process was endothermic and spontaneous. The maximum adsorption capacity of TC was 381.584 mg g-1. Density functional theory (DFT) was used to simulate and analyze the adsorption process, and the influence of different types of N on the adsorption was expounded. The results showed that there are electrostatic interactions, π-π interactions and hydrogen bonding between the adsorbent and TC. These results indicated that the prepared ENAC had a great application prospect in the removal of antibiotics from aqueous solution.
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Algas Marinas , Contaminantes Químicos del Agua , Adsorción , Antibacterianos , Carbón Orgánico , Concentración de Iones de Hidrógeno , Cinética , Hidróxido de Sodio , Tetraciclina , TermodinámicaRESUMEN
More than 405 million people have contracted coronavirus disease 2019 (COVID-19) worldwide, and mycotic infection may be related to COVID-19 development. There are a large number of reports showing that COVID-19 patients with mycotic infection have an increased risk of mortality. However, whether mycotic infection can be considered a risk factor for COVID-19 remains unknown. We searched the PubMed and Web of Science databases for studies published from inception to December 27, 2021. Pooled effect sizes were calculated according to a random-effects model or fixed-effect model, depending on heterogeneity. We also performed subgroup analyses to identify differences in mortality rates between continents and fungal species. A total of 20 articles were included in this study. Compared with the controls, patients with mycotic infection had an odds ratio (OR) of 2.69 [95% confidence interval (CI): 2.22-3.26] for mortality and an OR of 2.28 (95% CI: 1.65-3.16) for renal replacement therapy (RRT). We also conducted two subgroup analyses based on continent and fungal species, and we found that Europe and Asia had the highest ORs, while Candida was the most dangerous strain of fungi. We performed Egger's test and Begg's test to evaluate the publication bias of the included articles, and the p-value was 0.423, which indicated no significant bias. Mycotic infection can be regarded as a risk factor for COVID-19, and decision makers should be made aware of this risk.
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COVID-19 , Asia , COVID-19/epidemiología , Europa (Continente) , Humanos , Oportunidad Relativa , Factores de RiesgoRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based detection strategies with a fluorophore quencher-labeled ssDNA reporter or gold nanoparticle ssDNA reporter have been widely used in point-of-care (POC) molecular diagnostics. However, the potential of these CRISPR/Cas12a strategies for POC molecular diagnostics is often compromised due to the complex labeling, high cost, and low signal-to-noise ratio. Herein, we show a pre-folded G-quadruplex (G4) structure with tunable tolerance to CRISPR/Cas12a trans-cleavage and explore its mechanism. Two G4 structures (i.e., Tel22-10 and G16C) sensitive or tolerant to CRISPR/Cas12a trans-cleavage are designed and used as signal elements to fabricate a label-free visible fluorescent strategy or "signal-on" colorimetric strategy, respectively. These two strategies facilitate an ultrasensitive visual nucleic acid determination of Group B Streptococci with a naked-eye limit of detection of 1 aM. The feasibility of the developed G4-assisted CRISPR/Cas12a strategies for real-world applications is demonstrated in clinical vaginal/anal specimens and further verified by a commercial qPCR assay. This work suggests that the proposed G4 structures with tunable tolerance can act as promising signal reporters in the CRISPR/Cas12a system to enable ultrasensitive visible nucleic acid detection.
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Nanopartículas del Metal , Ácidos Nucleicos , Femenino , Humanos , Sistemas CRISPR-Cas , Oro , Colorantes FluorescentesRESUMEN
As one of the crucial factors associated with human life span and cancer progression, telomerase is regarded as an emerging biomarker for cancer diagnosis. Therefore, a facile, rapid and sensitive approach for telomerase activity detection with point-of-care (POC) diagnosis potential is in great demands. Herein, an all-in-one telomerase activity detection assay was established based on the telomere synthesis activated CRISPR-Cas12a system. A telomerase extension reaction generated telomere repeats sequences (TTAGGG)n, which was recognized by a customized CRISPR-guided RNA (crRNA) simultaneously, and finally activated a typical trans-cleavage based CRISPR-Cas12a detection assay. With the inherent sensitivity of CRISPR-Cas12a, this approach achieved a great linear regression ranging from 100 to 2000 HeLa cells and a limitation of detection down to 26 HeLa cells. Moreover, by using the proposed method, telomerase can be detected in one pot under isothermal condition (37 °C) by a simple and fast workflow (one step within 1 h). Due to its excellent performance, this all-in-one method shows great potential in POC detection of the telomerase activity.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Telomerasa , Sistemas CRISPR-Cas , Células HeLa , Humanos , Telomerasa/genética , Telómero/genéticaRESUMEN
The BCR/ABLp210 fusion gene is a classic biomarker of chronic myeloid leukemia, which can be divided into e13a2 and e14a2 isoforms according to different breakpoints. These two isoforms showed distinct differences in clinical manifestation, treatment effect, and prognosis risk. Herein, a strategy based on nanocluster beacon (NCB) fluorescence was developed to identify the e13a2 and e14a2 isoforms in one-pot. Because the fluorescence of AgNCs can be activated when they are placed in proximity to the corresponding enhancer sequences, thymine-rich (T-rich) or guanine-rich (G-rich). In this work, we explored an ideal DNA-AgNCs template as an excellent molecular reporter with a high signal-to-noise ratio. After recognition with the corresponding isoforms, the AgNCs can be pulled closer to the T-rich or G-rich sequences to form a three-way junction structure and generate fluorescence with corresponding wavelengths. Therefore, by distinguishing the corresponding wavelengths of AgNCs, we successfully identified two isoforms in one tube with the limitation of 16 pM for e13a2 and 9 pM for e14a2. Moreover, this strategy also realized isoform identification in leukemia cells and newly diagnosed CML patients within 40 min, which provides a powerful tool to distinguish fusion gene subtypes at the same time.
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Proteínas de Fusión bcr-abl , Leucemia Mielógena Crónica BCR-ABL Positiva , Proteínas de Fusión bcr-abl/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Pronóstico , Isoformas de Proteínas/genéticaRESUMEN
In this work, the conversion of Enteromorpha clathrata into bio-oil through hydrothermal liquefaction (HTL) was investigated under different preparation conditions. A two-step reaction method was compared with single-step reaction. At a high temperature, bio-oil produced through the two-step hydrothermal reaction displayed slight changes in yield, but solid residue rate was low. The liquid-to-material ratio of the optimal preparation condition was 40/4 (mL/g). Bio-oil produced in each experiment at this ratio was further analyzed using GC/MS. Furthermore, density functional theory (DFT) quantitative calculation was used in analyzing and proving the possible reaction path of the conversion of furan compounds to aromatic compounds during a direct high-temperature liquefaction process. Results revealed that the two-step method can ensure a high bio-oil yield, while preventing the occurrence of side reactions caused by long-term high-temperature reactions, and improve the bio-oil quality.
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Algas Marinas , Biocombustibles , Biomasa , Aceites de Plantas , Polifenoles , Temperatura , AguaRESUMEN
A rapid and sensitive isothermal method is crucial for point-of-care (POC) nucleic acid testing. Recently, RNA-guided CRISPR/Cas12a proteins were discovered to exhibit target-triggered nonspecific single-stranded deoxyribonuclease (ssDNase) activity. Herein, the ssDNase cleavage capacity of the CRISPR/Cas12a system for interfacial hairpin DNA (hpDNA) and linear DNA was investigated in detailed. A novel electrochemical DNA biosensor was then developed via target-induced Cas12a cleaving interfacial hpDNA. In this strategy, the RNA-guided target DNA binding activates the robust Cas12a ssDNase activity. The immobilized hpDNA electrochemical reporters with a low surface coverage and incompact morphological structure present accessible substrates for highly efficient Cas12a cleavage, leading to a highly sensitive electrochemical DNA biosensor. Under the optimal conditions, as low as 30 pM target DNA was detected in about 60 min with 3.5 orders of magnitude dynamic range from 50 pM to 100 nM. Furthermore, the practical application ability of the established sensing method for detecting the target in complex matrices was also demonstrated. The proposed strategy enables rapid and sensitive DNA determination, providing a potential tool for POC molecular diagnostics.