RESUMO
Nanopore sequencers can enrich or deplete the targeted DNA molecules in a library by reversing the voltage across individual nanopores. However, it requires substantial computational resources to achieve rapid operations in parallel at read-time sequencing. We present a deep learning framework, NanoDeep, to overcome these limitations by incorporating convolutional neural network and squeeze and excitation. We first showed that the raw squiggle derived from native DNA sequences determines the origin of microbial and human genomes. Then, we demonstrated that NanoDeep successfully classified bacterial reads from the pooled library with human sequence and showed enrichment for bacterial sequence compared with routine nanopore sequencing setting. Further, we showed that NanoDeep improves the sequencing efficiency and preserves the fidelity of bacterial genomes in the mock sample. In addition, NanoDeep performs well in the enrichment of metagenome sequences of gut samples, showing its potential applications in the enrichment of unknown microbiota. Our toolkit is available at https://github.com/lysovosyl/NanoDeep.
Assuntos
Aprendizado Profundo , Sequenciamento por Nanoporos , Nanoporos , Humanos , Biblioteca Gênica , Genoma BacterianoRESUMO
Rapid and sensitive RNA detection is of great value in diverse areas, ranging from biomedical research to clinical diagnostics. Existing methods for RNA detection often rely on reverse transcription (RT) and DNA amplification or involve a time-consuming procedure and poor sensitivity. Herein, we proposed a CRISPR/Cas12a-enabled amplification-free assay for rapid, specific, and sensitive RNA diagnostics. This assay, which we termed T7/G4-CRISPR, involved the use of a T7-powered nucleic acid circuit to convert a single RNA target into numerous DNA activators via toehold-mediated strand displacement reaction and T7 exonuclease-mediated target recycling amplification, followed by activating Cas12a trans-cleavage of the linker strands inhibiting split G-Quadruplex (G4) assembly, thereby inducing fluorescence attenuation proportion to the input RNA target. We first performed step-by-step validation of the entire assay process and optimized the reaction parameters. Using the optimal conditions, T7/G4-CRISPR was capable of detecting as low as 3.6 pM target RNA, obtaining â¼100-fold improvement in sensitivity compared with the most direct Cas12a assays. Meanwhile, its excellent specificity could discriminate single nucleotide variants adjacent to the toehold region and allow species-specific pathogen identification. Furthermore, we applied it for analyzing bacterial 16S rRNA in 40 clinical urine samples, exhibiting a sensitivity of 90% and a specificity of 100% when validated by RT-quantitative PCR. Therefore, we envision that T7/G4-CRISPR will serve as a promising RNA sensing approach to expand the toolbox of CRISPR-based diagnostics.
Assuntos
Sistemas CRISPR-Cas , Quadruplex G , Sistemas CRISPR-Cas/genética , Humanos , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/química , RNA/análise , RNA/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Proteínas Associadas a CRISPR/metabolismo , Proteínas de Bactérias , EndodesoxirribonucleasesRESUMO
Spinal cord injury (SCI), following explosive oxidative stress, causes an abrupt and irreversible pathological deterioration of the central nervous system. Thus, preventing secondary injuries caused by reactive oxygen species (ROS), as well as monitoring and assessing the recovery from SCI are critical for the emergency treatment of SCI. Herein, an emergency treatment strategy is developed for SCI based on the selenium (Se) matrix antioxidant system to effectively inhibit oxidative stress-induced damage and simultaneously real-time evaluate the severity of SCI using a reversible dual-photoacoustic signal (680 and 750 nm). Within the emergency treatment and photoacoustic severity assessment (ETPSA) strategy, the designed Se loaded boron dipyrromethene dye with a double hydroxyl group (Se@BDP-DOH) is simultaneously used as a sensitive reporter group and an excellent antioxidant for effectively eliminating explosive oxidative stress. Se@BDP-DOH is found to promote the recovery of both spinal cord tissue and locomotor function in mice with SCI. Furthermore, ETPSA strategy synergistically enhanced ROS consumption via the caveolin 1 (Cav 1)-related pathways, as confirmed upon treatment with Cav 1 siRNA. Therefore, the ETPSA strategy is a potential tool for improving emergency treatment and photoacoustic assessment of SCI.
Assuntos
Selênio , Traumatismos da Medula Espinal , Ratos , Camundongos , Animais , Antioxidantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/diagnóstico por imagem , Traumatismos da Medula Espinal/tratamento farmacológico , Estresse Oxidativo , Tratamento de EmergênciaRESUMO
Bacteria infection is a significant obstacle in the clinical treatment of exposed wounds facing widespread pathogens. Herein, we report a DNA origami-based bactericide for efficient anti-infection therapy of infected wounds in vivo. In our design, abundant DNAzymes (G4/hemin) can be precisely organized on the DNA origami for controllable generation of reactive oxygen species (ROS) to break bacterial membranes. After the destruction of the membrane, broad-spectrum antibiotic levofloxacin (LEV, loaded in the DNA origami through interaction with DNA duplex) can be easily delivered into the bacteria for successful sterilization. With the incorporation of DNA aptamer targeting bacterial peptidoglycan, the DNA origami-based bactericide can achieve targeted and combined antibacterial therapy for efficiently promoting the healing of infected wounds. This tailored DNA origami-based nanoplatform provides a new strategy for the treatment of infectious diseases in vivo.
Assuntos
Aptâmeros de Nucleotídeos , Infecção dos Ferimentos , Humanos , Antibacterianos/uso terapêutico , DNA/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Infecção dos Ferimentos/tratamento farmacológicoRESUMO
OBJECTIVE: Stromal barriers, such as the abundant desmoplastic stroma that is characteristic of pancreatic ductal adenocarcinoma (PDAC), can block the delivery and decrease the tumour-penetrating ability of therapeutics such as tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), which can selectively induce cancer cell apoptosis. This study aimed to develop a TRAIL-based nanotherapy that not only eliminated the extracellular matrix barrier to increase TRAIL delivery into tumours but also blocked antiapoptotic mechanisms to overcome TRAIL resistance in PDAC. DESIGN: Nitric oxide (NO) plays a role in preventing tissue desmoplasia and could thus be delivered to disrupt the stromal barrier and improve TRAIL delivery in PDAC. We applied an in vitro-in vivo combinatorial phage display technique to identify novel peptide ligands to target the desmoplastic stroma in both murine and human orthotopic PDAC. We then constructed a stroma-targeted nanogel modified with phage display-identified tumour stroma-targeting peptides to co-deliver NO and TRAIL to PDAC and examined the anticancer effect in three-dimensional spheroid cultures in vitro and in orthotopic PDAC models in vivo. RESULTS: The delivery of NO to the PDAC tumour stroma resulted in reprogramming of activated pancreatic stellate cells, alleviation of tumour desmoplasia and downregulation of antiapoptotic BCL-2 protein expression, thereby facilitating tumour penetration by TRAIL and substantially enhancing the antitumour efficacy of TRAIL therapy. CONCLUSION: The co-delivery of TRAIL and NO by a stroma-targeted nanogel that remodels the fibrotic tumour microenvironment and suppresses tumour growth has the potential to be translated into a safe and promising treatment for PDAC.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/patologia , Humanos , Camundongos , Nanogéis , Óxido Nítrico , Neoplasias Pancreáticas/patologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
The degranulation of mast cells accounts for the development of neuroinflammation following intracerebral hemorrhage (ICH). Inhibition of IRE1α, a sensor signaling protein related to endoplasmic reticulum stress, has been shown to exert anti-inflammatory effects in several neurological diseases. The objective of this study was to investigate the effects of IRE1α inhibition on mast cells degranulation in an ICH mouse model and to explore the contribution of miR-125/Lyn pathway in IRE1α-mediated mast cells degranulation. Male mice were subjected to ICH by intraparenchymal injection of autologous blood. STF083010, an inhibitor of IRE1α, was administered intranasally at 1 h after ICH induction. AntimiR-125 was delivered by intracerebroventricular (i.c.v.) injection prior to ICH induction to elucidate the possible mechanisms. Western blot analysis, immunofluorescence staining, neurological test, hematoma volume, brain water content, toluidine blue staining and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) were performed. Endogenous phosphorylated IRE1α (p-IRE1α), tryptase, interleukin-17A (IL-17A), tumor necrosis factor α (TNF-α) and tryptase mRNA were increased in time dependent manner while miR-125b-2-3p was decreased after ICH. Inhibition of IRE1α, with STF083010, remarkably reduced brain water content, improved neurological function, decreased hematoma volume, upregulated the expression of miR-125b-2-3p, decreased the number of mast cells, and downregulated the protein expression of Lyn kinase, XBP1s (spliced X-box binding protein-1), tryptase, IL-17A and TNF-α. The downregulation of Lyn kinase, tryptase, IL-17A, TNF-α, and decreased mast cells number were reversed by antimiR-125. The present findings demonstrate that IRE1α inhibition attenuates mast cells degranulation and neuroinflammation, at least partially, through IRE1α/miR-125/Lyn signaling pathway after ICH.
Assuntos
Endorribonucleases , MicroRNAs , Animais , Hemorragia Cerebral/metabolismo , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Hematoma , Interleucina-17 , Masculino , Mastócitos/metabolismo , Mastócitos/patologia , Camundongos , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases , Triptases , Fator de Necrose Tumoral alfa , Água , Quinases da Família src/metabolismoRESUMO
Two new trimethoxyl A2B triaryl corroles 10-(2,4,6-trimethoxyphenyl)-5,15-bis(pentafluorophenyl)- corrole (1) and 10-(3,4,5-trimethoxyphenyl)-5,15-bis(pentafluorophenyl)-corrole (2) and their gallium(III) and phosphorus(V) (1-Ga, 1-P, 2-Ga and 2-P) complexes had been prepared and well characterized by UV-vis, NMR and HR-MS. Among all compounds, 2-Ga, 1-P and 2-P showed excellent in vivo photodynamic activity against the MDA-MB-231, A549, Hela and HepG2 cell lines upon light irradiation at 625 nm. And 2-P even exhibited higher phototoxicity than the clinical photosensitizer temoporfin. Also, 2-P exhibited the highest singlet oxygen quantum yield and photostability. The preliminary investigation revealed that 2-P could be rapidly absorbed by tumor cells and mainly located in the cytoplasm. After photodynamic therapy (PDT) treatment with 2-P, mitochondrial membrane potential destruction, intracellular ROS level increasing and nuclear fragmentation of cancer cells could be observed. Cell cycle analysis demonstrated that the 2-P PDT may cause tumor cell arrest at sub-G1 stage and induce early and late apoptosis of cells. These results suggest that 2-P is a promising candidate as a photosensitizer for photodynamic therapy.
Assuntos
Gálio , Fotoquimioterapia , Humanos , Gálio/farmacologia , Gálio/química , Fármacos Fotossensibilizantes/farmacologia , Fósforo/farmacologia , Linhagem Celular TumoralRESUMO
A series of halogenated gallium corroles were synthesized and characterized by UV-vis, HRMS, NMR, and FT-IR. The interaction between these gallium corroles and calf thymus DNA had been investigated by spectroscopic methods. These gallium corroles would interact with CT-DNA via an outside binding mode. The photodynamic antitumor activity in vitro of these gallium corroles toward different cell lines had also been tested. 3-Ga displayed low cytotoxicity to normal cells under both light and dark conditions but high phototoxicity to liver cancer cells HepG2. The vitro experiment results showed that 3-Ga could be efficiently absorbed by tumor cells. After light illumination, it may induce reactive oxygen species (ROS) and cause destruction of the mitochondrial membrane potential, which may finally trigger tumor cell apoptosis. Flow cytometry results showed that HepG2 cells were mainly distributed in the sub-G0 phase, which corresponds to cells with highly fragmented DNA or dead cells generally. This suggests that 3-Ga could lead to tumor cell apoptosis after light illumination.
Assuntos
DNA/química , Gálio/química , Halogenação , Neoplasias/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/química , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Fármacos Fotossensibilizantes/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Análise Espectral/métodos , Testes de Toxicidade AgudaRESUMO
Dentin matrix protein 1 (DMP1) is an extracellular matrix phosphoprotein that is known to facilitate mineralization of collagen in bone and promote osteoblast/odontoblast differentiation. Blood-brain barrier (BBB) disruption is the major pathogenesis in secondary brain injury after intracerebral hemorrhage (ICH). This study aimed to investigate the expression pattern of DMP1 in the mouse brain and explore the role of DMP1 in BBB disruption and brain injury in a mouse model of ICH. Mice were subjected to autologous blood injection-induced ICH. Immunofluorescence staining, western blot analysis, neurobehavioral tests, brain water content measurements, Evans blue permeability assay, and transmission electron microscopy were performed. Small interfering RNA targeting DMP1 (DMP1 siRNA) was administered at 72 h prior to ICH. Results showed that DMP1 is expressed extensively in the mouse brain, and is upregulated in the ICH model. Administration of DMP1 siRNA effectively ameliorated BBB disruption, attenuated brain edema, and improved neurological function after ICH. Moreover, the expression of zonula occludens-1 (ZO-1) and occludin were upregulated, and matrix metalloproteinase-9 (MMP-9) was downregulated in the ICH model. DMP1 siRNA administration reversed the expression of ZO-1, occludin, and MMP-9. These results demonstrated that DMP1 upregulation plays an essential role in inducing BBB disruption and brain injury after ICH. The inhibition of DMP1 could be a potential therapeutic strategy for ICH treatment.
Assuntos
Barreira Hematoencefálica/metabolismo , Edema Encefálico/prevenção & controle , Hemorragia Cerebral/terapia , Proteínas da Matriz Extracelular/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Animais , Barreira Hematoencefálica/ultraestrutura , Edema Encefálico/genética , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Hemorragia Cerebral/genética , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/patologia , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Ocludina/genética , Ocludina/metabolismo , RNA Interferente Pequeno/genética , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Ribonuclease A (RNase A) is increasingly considered as a biomarker for tumor diagnosis, and it is of great significance to develop an ultrasensitive, cost-effective assay for RNase A detection. Electrochemiluminescence (ECL) technology has distinctive advantages in the development of biosensors for diverse targets. However, most of the ECL biosensors require the complex process of electrode modification, which is laborious and time consuming. In this work, an immobilization-free homogeneous ECL assay was developed for the highly sensitive detection of RNase A activity for the first time. On the basis of the fact that RNase A can specifically hydrolyze RNA at the site of ribonucleotide uracil (rU), a rU-containing chimeric DNA probe is designed and labeled with Ru(bpy)32+ (act as ECL indicator). The chimeric DNA probe hardly diffuses to the surface of negatively charged indium tin oxide (ITO) electrode due to the strong electrostatic repulsion between the negatively charged DNA and ITO electrode, resulting in a weak ECL signal detected. When the RNase A is present, the chimeric DNA probe is hydrolyzed into small fragments, which contains little negative charge and can diffuse easily to the ITO electrode surface due to the decreased electrostatic repulsion. In this case, an enhanced ECL signal can be detected. Under the optimal conditions, there is a linear relationship between the ECL signal and the concentration of RNase A in the range of 0.001-0.10 ng/mL, and the detection limit is 0.2 pg/mL. In addition, the proposed ECL sensing system is also applied to detect the RNase A inhibitor, taking As3+ as an example. The proposed homogeneous ECL sensing system provides a new approach for the highly sensitive and convenient detection of RNase A as well as other ribonucleases only by redesigning a responding chimeric DNA probe.
Assuntos
Arsênio/análise , Técnicas Eletroquímicas/métodos , Inibidores Enzimáticos/análise , Medições Luminescentes/métodos , Ribonuclease Pancreático/análise , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/química , Técnicas Biossensoriais/métodos , Sondas de DNA/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Corantes Fluorescentes/química , Hidrólise , Limite de Detecção , Luminescência , Compostos Organometálicos/química , Reprodutibilidade dos Testes , Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/química , Compostos de Estanho/químicaRESUMO
Exosomes serve as significant information carriers that regulate important physiological and pathological processes. Herein, functionalized DNA is engineered to be a hinge that anchors quantum dots (QDs) onto the surface of exosomes, realizing a moderate and biocompatible labeling strategy. The QDs-labeled exosomes (exosome-DNA-QDs complex) can be swiftly engulfed by tumor cells, indicating that exosome-DNA-QDs can be applied as a specific agent for tumor labeling. Furthermore, the engineered artificial vesicles of M1 macrophages (M1mv) are constructed via a pneumatic liposome extruder. The results reveal that the individual M1mv can kill tumor cells and realize desirable biological treatment. To reinforce the antitumor efficacy of M1mv and the specificity of drug release, a target-triggered drug delivery system is constructed to realize a specific microRNA-responded delivery system for visual therapy of tumors. These strategies facilitate moderate labeling and functionalization of exosomes/vesicles and construct artificial drug-delivery vesicles that simultaneously possess biological treatment and chemotherapy functions, and thus have the potential to serve as a new paradigm for tumor labeling and therapy.
Assuntos
DNA/metabolismo , Exossomos/metabolismo , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diagnóstico por Imagem , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Exossomos/efeitos dos fármacos , Exossomos/ultraestrutura , Humanos , Células MCF-7 , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Distribuição Tecidual/efeitos dos fármacosRESUMO
Exosome-containing microRNAs (exomiRs) can be employed as potential biomarkers for tumor diagnosis and have drawn much attention in the past few years. However, the separation of exosomes and the detection of exomiRs are still inconvenient or even difficult to implement. Thus, it is important to develop a simple, accurate, and reliable strategy for the separation of exosomes and the biomedical analysis of exomiRs. Herein, a novel exosome-specific tumor diagnosis strategy was constructed by integrating the rapid magnetic exosome-enrichment platform and the Ru(bpy)32+-polymer amplified electrochemiluminescence (ECL) strategy. This strategy realized the rapid and efficient capture of tumor-derived exosomes through a biological-affinity identification platform of the EpCAM antibody. The biomedical analysis of exomiRs achieved a preferable specificity and high sensitivity of 103 particles. Furthermore, we investigated the performance index for clinical blood samples from tumor patients; the results indicated that the exosome-specific tumor diagnosis strategy readily and consistently responded to exomiRs. These results indicated that the exosome-specific tumor diagnosis strategy provided new opportunities for the sensitive and efficient analysis of tumor-derived exomiRs. This strategy greatly simplified the biomedical analysis process and established the non-destructive detection mode of fluid biopsy for tumors.
Assuntos
Biomarcadores Tumorais/sangue , Exossomos/metabolismo , MicroRNAs/sangue , Neoplasias/diagnóstico , Células A549 , Biomarcadores Tumorais/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Células Hep G2 , Humanos , Luminescência , Células MCF-7 , Nanopartículas de Magnetita/química , MicroRNAs/química , Neoplasias/ultraestrutura , Compostos Organometálicos/química , Polímeros/química , Sensibilidade e EspecificidadeRESUMO
A series of iron(III), manganese(III) and copper(III) mono-hydroxyl corrole complexes had been prepared and well characterized by UV-vis, 1H NMR, 19F NMR and HR-MS. These metallocorroles may bind to CT-DNA through external binding mode. Metallocorrole Fe-2c exhibited significant phototoxicity and low toxicity toward A549 tumor cells. While manganese (III) and copper (III) corroles showed hypotoxicity to A549, MCF-7 and HepG-2 tumor cells, whether under dark or illumination conditions. All tested metallocorroles exhibited non-toxicity to human normal cells (GES-1) with or without irradiation at 625â¯nm. Cell cycle analysis indicated that metallocorrole Fe-2c arrested the cell cycle at G2/M phase and increased the Sub-G1 phase in A549 cell lines. It was mainly localized at mitochondria and could significantly reduce mitochondrial membrane potential after photodynamic treatment, which would further induce tumor cell apoptosis.
Assuntos
Antineoplásicos/farmacologia , Cobre/química , DNA/metabolismo , Compostos Férricos/química , Manganês/química , Neoplasias/terapia , Porfirinas/química , Antineoplásicos/química , Apoptose , Humanos , Radical Hidroxila , Potencial da Membrana Mitocondrial , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/patologia , Fotoquimioterapia , Células Tumorais CultivadasRESUMO
Electrochemiluminescence (ECL) has been engineered to perform various tasks in the area of immunoassays and molecular diagnosis. However, there is still substantial potential for developments of ECL assay with high efficiency to achieve trace analysis. Herein, we demonstrate a polymer-amplified ECL assay via construction of linear Ru(bpy)32+-polymer. This new polymer material compensates for the relatively low ECL intensity from single ECL luminophore and realizes a stable and controllable labeling process. The polymer-amplified ECL assay achieved a remarkable sensitivity of 100 amol. The wide-ranging applications of the polymer-amplified ECL assay for Hepatitis B virus, carcinoembryonic antigen, 16sRNA, and thrombin also demonstrate its superiority. Hence, the polymer-amplified ECL assay possesses the potential to create a new paradigm in amplified ECL assays that could provide outstanding performance for biomedical analysis.
Assuntos
Biomarcadores/análise , Complexos de Coordenação/química , Técnicas Eletroquímicas/métodos , Medições Luminescentes/métodos , Polímeros/química , Antígeno Carcinoembrionário/análise , Complexos de Coordenação/síntese química , Vírus da Hepatite B/isolamento & purificação , Polímeros/síntese química , RNA Ribossômico 16S/análise , Rutênio/química , Trombina/análiseRESUMO
Nucleic acids have been engineered to participate in a wide variety of tasks. Among them, the enzyme-free amplification modes, enzyme-free DNA circuits (EFDCs), and hybridization chain reactions (HCRs) have been widely applied in a series of studies of bioanalysis. We demonstrated here an ultrasensitive hairpin probe-based circulation for continuous assemble of DNA probe. This strategy improved the analyte stability-dependent amplification efficiency of EFDC and signal enhancement without being limited by the analyte's initial concentration, and it was used to produce a novel microRNA (miRNA) trace analysis assay with ultrasensitive amplification properties. Through the detection of standard miRNA substances, 1 amol-level sensitivity and satisfactory specificity were achieved. Compared with EFDCs and HCRs, the sensitivity of ultrasensitive hairpin probe-based circulation was higher by 3 or 4 orders of magnitude. Furthermore, the excellent performance of this platform was also demonstrated in the detection of miRNAs in tumor cells. The sensitivities for the detection of miRNAs in HepG2, A549 and MCF-7 tumor cells were 10, 10, and 100 cells, respectively. In addition, a high detection rate of 83% was achieved for tumor tissues. Thus, this ultrasensitive hairpin probe-based circulation possesses the potential to be a technological innovation in the field of tumor diagnosis.
Assuntos
Sondas de DNA/química , MicroRNAs/análise , Linhagem Celular Tumoral , Células Hep G2 , Humanos , Limite de Detecção , Células MCF-7 , Neoplasias/diagnóstico , Hibridização de Ácido Nucleico , Sensibilidade e EspecificidadeRESUMO
MicroRNAs (miRNAs) participate in important processes of life course. Because of their characters of small sizes, vulnerable degradabilities, and sequences similarities, the existing detection technologies mostly contain enzymatic amplification reactions for acquisition of high sensitivities and specificities. However, specific reaction conditions and time-dependent enzyme activities are caused by the accession of enzymes. Herein, we designed a target-triggered enzyme-free amplification platform that is realized by circulatory interactions of two hairpin probes and the integrated electrochemiluminescence (ECL) signal giving-out component. Benefiting from outstanding performances of the enzyme-free amplification system and ECL, this strategy is provided with a simplified reaction process, high sensitivity, and operation under isothermal conditions. Through detection of the miRNA standard substance, the sensitivity of this platform reached 10 fmol, and a splendid specificity was achieved. We also analyzed three tumor cell lines (human lung adenocarcinoma, breast adenocarcinoma, and hepatocellular liver carcinoma cell lines) through this platform. The sensitivities of 10(3) cells, 10(4) cells, and 10(4) cells were, respectively, achieved. Furthermore, clinical tumor samples were tested, and 21 of 30 experimental samples gave out positive signals. Thus, this platform possesses potentials to be an innovation in miRNA detection methodology.
Assuntos
MicroRNAs/análise , Neoplasias/genética , Linhagem Celular Tumoral , Enzimas/química , Humanos , Limite de Detecção , Neoplasias/patologiaRESUMO
BACKGROUND: Endoplasmic reticulum (ER) stress and oxidative stress are the major pathologies encountered after intracerebral hemorrhage (ICH). Inositol-requiring enzyme-1 alpha (IRE1α) is the most evolutionarily conserved ER stress sensor, which plays a role in monitoring and responding to the accumulation of unfolded/misfolded proteins in the ER lumen. Recent studies have shown that ER stress is profoundly related to oxidative stress in physiological or pathological conditions. The purpose of this study was to investigate the role of IRE1α in oxidative stress and the potential mechanism. METHODS: A mouse model of ICH was established by autologous blood injection. The IRE1α phosphokinase inhibitor KIRA6 was administrated intranasally at 1 h after ICH, antagomiR-25 and agomiR-25 were injected intraventricularly at 24 h before ICH. Western blot analysis, RT-qPCR, immunofluorescence staining, hematoma volume, neurobehavioral tests, dihydroethidium (DHE) staining, H2O2 content, brain water content, body weight, Hematoxylin and Eosin (HE) staining, Nissl staining, Morris Water Maze (MWM) and Elevated Plus Maze (EPM) were performed. RESULTS: Endogenous phosphorylated IRE1α (p-IRE1α), miR-25-3p, and Nox4 were increased in the ICH model. Administration of KIRA6 downregulated miR-25-3p expression, upregulated Nox4 expression, promoted the level of oxidative stress, increased hematoma volume, exacerbated brain edema and neurological deficits, reduced body weight, aggravated spatial learning and memory deficits, and increased anxiety levels. Then antagomiR-25 further upregulated the expression of Nox4, promoted the level of oxidative stress, increased hematoma volume, exacerbated brain edema and neurological deficits, whereas agomiR-25 reversed the effects promoted by KIRA6. CONCLUSION: The IRE1α phosphokinase activity is involved in the oxidative stress response through miR-25/Nox4 pathway in the mouse ICH brain.
Assuntos
Edema Encefálico , Imidazóis , MicroRNAs , Naftalenos , Pirazinas , Camundongos , Animais , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Endorribonucleases/metabolismo , Antagomirs/metabolismo , Peróxido de Hidrogênio , Estresse Oxidativo , Hemorragia Cerebral/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Hematoma , Peso Corporal , NADPH Oxidase 4/genéticaRESUMO
Dual-mode readout platforms with colorimetric and electrochemiluminescence (ECL) signal enhancement are proposed for the ultrasensitive and flexible detection of the monkeypox virus (MPXV) in different scenes. A new nanotag, Ru@U6-Ru/Pt NPs is constructed for dual-mode platforms by integrating double-layered ECL luminophores and the nanozyme using Zr-MOF (UiO-66-NH2) as the carrier, which not only generates enhanced ECL and colorimetric signals but also provide greater stability than that of commonly used nanotags. Dual-mode platforms are used within 15 min from the "sample in" to the "result out" steps, without nucleic acid amplification. The colorimetric mode allows the screening of MPXV with the visual limit of detection (vLOD) of 0.1 pM (6 × 108 copies µL-1) and the ECL mode supports quantitative detection of MPXV with an LOD as low as 10 aM (6 copies·µL-1), resulting in a broad sensing range of 60 to 3 × 1011 copies·µL-1 (10 orders of magnitude). Validation is conducted using 50 clinical samples, which is 100% concordant to those of quantitative polymerase chain reaction (qPCR), indicating that Ru@U6-Ru/Pt NPs-based dual-mode sensing platforms showed great promise as rapid, sensitive, and accurate tools for diagnosis of the nucleic acid of MPXV and other infectious pathogens.
RESUMO
Molecular hybridization is a well-established strategy for developing new drugs. In the pursuit of promising photosensitizers (PSs) with enhanced photodynamic therapy (PDT) efficiency, a series of novel 5-fluorouracil (5FU) gallium corrole conjugates (1-Ga-4-Ga) were designed and synthesized by hybridizing a chemotherapeutic drug and PSs. Their photodynamic antitumor activity was also evaluated. The most active complex (2-Ga) possesses a low IC50 value of 0.185 µM and a phototoxic index of 541 against HepG2 cells. Additionally, the 5FU-gallium corrole conjugate (2-Ga) exhibited a synergistic increase in cytotoxicity under irradiation. Excitedly, treatment of HepG2 tumor-bearing mice with 2-Ga under irradiation could completely ablate tumors without harming normal tissues. 2-Ga-mediated PDT could disrupt mitochondrial function, cause cell cycle arrest in the sub-G1 phase, and activate the cell apoptosis pathway by upregulating the cleaved PARP expression and the Bax/Bcl-2 ratios. This work provides a useful strategy for the design of new corrole-based chemo-photodynamic therapy drugs.
Assuntos
Apoptose , Fluoruracila , Gálio , Fotoquimioterapia , Fármacos Fotossensibilizantes , Porfirinas , Fluoruracila/farmacologia , Fluoruracila/química , Fluoruracila/uso terapêutico , Humanos , Gálio/química , Gálio/farmacologia , Animais , Porfirinas/farmacologia , Porfirinas/química , Porfirinas/uso terapêutico , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/uso terapêutico , Camundongos , Apoptose/efeitos dos fármacos , Células Hep G2 , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
Study on corrole photosensitizers (PSs) for photodynamic therapy (PDT) has made remarkable progress. Targeted delivery of PSs is of great significance for enhancing therapeutic efficiency, decreasing the dosage, and reducing systemic toxicity during PDT. The development of PSs that can be specifically delivered to the subcellular organelle is still an attractive and challenging work. Herein, we synthesize a series of azide-modified corrole phosphorus and gallium complex PSs, in which phosphorus corrole 2-P could not only precisely target the endoplasmic reticulum (ER) with a Pearson correlation coefficient (PCC) up to 0.92 but also possesses the highest singlet oxygen quantum yields (ΦΔ = 0.75). This renders it remarkable PDT activity at a very low dosage (IC50 = 23 nM) towards HepG2 tumor cell line while ablating solid tumors in vivo with excellent biosecurity. Furthermore, 2-P exhibits intense red fluorescence (ΦF = 0.25), outstanding photostability, and a large Stokes shift (190 nm), making it a promising fluorescent probe for ER. This study provides a clinically potential photosensitizer for cancer photodynamic therapy and a promising ER fluorescent probe for bioimaging.