RESUMEN
Ether-based electrolytes are promising for secondary batteries due to their good compatibility with alkali metal anodes and high ionic conductivity. However, they suffer from poor oxidative stability and high toxicity, leading to severe electrolyte decomposition at high voltage and biosafety/environmental concerns when electrolyte leakage occurs. Here, we report a green ether solvent through a rational design of carbon-chain regulation to elicit steric hindrance, such a structure significantly reducing the solvent's biotoxicity and tuning the solvation structure of electrolytes. Notably, our solvent design is versatile, and an anion-dominated solvation structure is favored, facilitating a stable interphase formation on both the anode and cathode in potassium-ion batteries. Remarkably, the green ether-based electrolyte demonstrates excellent compatibility with K metal and graphite anode and a 4.2â V high-voltage cathode (200 cycles with average Coulombic efficiency of 99.64 %). This work points to a promising path toward the molecular design of green ether-based electrolytes for practical high-voltage potassium-ion batteries and other rechargeable batteries.
RESUMEN
Klebsiella pneumoniae (K. pneumoniae) is a common bacterium whose drug-resistant can cause surgical failures and incurable infections in hospital patients. Thus, how to reverse or delay the resistance induction has become a great challenge for development antiresistant drug. Recently, the combination of nanomaterial-loaded antibiotics with photothermal therapy showed the efficient antibacteria ability under a low dosage of antibiotics. In this study, a nanocomposite of HMPB NPs with inherent photothermal therapy capability was used to eradicate K. pneumoniae after loading with Ofloxacin, an antibiotic against K. pneumoniae in vitro and in vivo. The nanocomplexes named as Ofloxacin@HMPB@HA NPs showed a higher effect against K. pneumoniae by destroying cell integrity and inducing ATP leakage with the assistance of laser irradiation, compared with sole Ofloxacin@HMPB@HA NPs or laser irradiation. Surgical wound infection assay further demonstrated the efficient killing K. pneumoniae and promoting the formation of new tissues, as well, which was reflected by the rapid healing of surgical wound. In summary, these results indicate the great potential of this combinational tactic based on Ofloxacin@HMPB@HA NPs for preventing the failure caused by K. pneumoniae infection.
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Infecciones por Klebsiella , Herida Quirúrgica , Antibacterianos/farmacología , Humanos , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae , Ofloxacino/farmacología , Ofloxacino/uso terapéutico , Herida Quirúrgica/tratamiento farmacológicoRESUMEN
Cysteinyl aspartate-specific protease 8 (caspase-8) plays a key role in various biological processes by regulating apoptosis. Therefore, this makes accurate detection and intracellular imaging of caspase-8 of great importance for drug screening, disease diagnosis, and prognostication. Here, by designing a reduced graphene oxide (rGO) quenched peptide probe, we constructed a new biosensing system for monitoring in vitro and intracellular caspase-8 activity. In this system, a fluorophore-labeled peptide and rGO were used as the substrate of caspase-8 and the fluorophore quencher, respectively. The hydrolysis of caspase-8 on the polypeptide probe substrate can generate two fragments with different lengths. The release of the short fragment labeled with the fluorophore causes recovery of the fluorescence signal (Ex/Em = 520/576 nm). Under the optimized conditions, the proposed fluorescence method exhibited a linear response range of 0.2 to 5 U·mL-1 for caspase-8 with a limit of detection (LOD) of 0.2 U·mL-1 in vitro. Furthermore, this method has been successfully applied to monitoring the upregulation of intracellular caspase-8 activity caused by tert-butyl hydroperoxide (TBHP) and fluorouracil. Flow cytometry assay indicated the positive relation between the upregulation of intracellular caspase-8 activity and cell apoptosis rate. In summary, the above results demonstrated the practical application of this method for apoptosis-related cell imaging.
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Grafito , Caspasa 8 , Péptidos , Colorantes FluorescentesRESUMEN
Human apurinic/pyrimidine endonuclease 1 (APE1) played a critical role in the occurrence, progress and prognosis of tumors through overexpression and subcellular localization. Thus, it has become an important target for enhancing the sensitivity of tumor cells to radiotherapy and chemotherapy. Therefore, detecting and imaging its intracellular activity is of great significance for inhibitor discovery, cancer diagnosis and therapy. In this work, using DNA-based nanoprobe, we developed a new method for monitor intracellular APE1 activity. The detecting system was consisted by single fluorophore labeled hairpin probe and reduced graphene oxide (rGO). The in vitro result showed that a liner response of the detection method ranged from 0.02 U/mL to 2 U/mL with a limit of detection of 0.02 U/mL. Furthermore, this strategy possessing high specificity was successfully applied for APE1-related inhibitor screening using intracellular fluorescence imaging. Panaxytriol, an effective inhibitor of APE1 activity, was screened from traditional Chinese medicine (TCM) and its effect on APE1 activity was monitored in real time in A549 cells. In summary, this sensitive and specific APE1 detection technology is expected to provide an assistance for APE1-related inhibitor screening and diseases diagnosis.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , ADN/química , Grafito/química , Nanopartículas/química , Células A549 , ADN-(Sitio Apurínico o Apirimidínico) Liasa/antagonistas & inhibidores , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Evaluación Preclínica de Medicamentos , Enediinos/farmacología , Alcoholes Grasos/farmacología , Humanos , Imagen Óptica , Factores de TiempoRESUMEN
To reduce the corrosion of Q235 steel, environment-friendly and efficient N-doped carbon dots (N-CDs) were synthesized using 4-amino salicylic acid (4-ASA) and l-histidine (l-His) as precursors. The corrosion inhibition behavior of N-CDs for Q235 steel in 1 M HCl solution was systematically investigated using a weight-loss experiment, an electrochemical test, and corrosion morphology. Results showed that N-CDs could effectively inhibit the corrosion of Q235 steel, and the inhibitory efficiency reached 93% at 50 mg L-1. Quantum chemistry and molecular dynamics were used to study the inhibition mechanism of N-CDs. The results demonstrated that N-CDs exhibited a strong adsorption force on metal and the adsorption process followed the Langmuir adsorption isotherm, indicating physical/chemical mixed adsorption.
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Carbono , Acero , Adsorción , Corrosión , MetalesRESUMEN
Ribonuclease H is essential for the research and development of complex pathema. The high rigidity and versatility of DNA tetrahedrons means they are often used in biosensing systems. Inspired by "radar" technology, we proposed a radar-like monitor to detect RNase H activity in vitro and in situ by integrating DNA tetrahedral elements. The structure of a radar-like monitor was self-assembled from five customized single nucleic acid strands. Four DNA strands were assembled as DNA tetrahedrons with a long strand labeled by Dabcyl (quencher) at one of the apexes, while the fifth strand (DNA-RNA heterozygous strand) was labeled with a FAM (Fluorophore) hybrid with a long strand. The fluorescence was quenched because the fluorophore and the quencher were very close. In the presence of RNase H, the RNA chain was hydrolyzed and the fluorophore released, resulting in fluorescence recovery. The radar-like monitor was used to detect the RNase H activity in vitro with a detection limit of 0.01 U mL-1. Based on the RNase H activity detection and the inhibitory effect of natural-compounds-targeting RNase H, three inhibitors were obtained among 35 compounds extracted from Panax japonicus. Therefore, the radar-like monitor was successfully used to detect RNase H activity in situ due to the long-term anti-DNase I effect of the RNA/DNA hybrid structure and DNA tetrahedrons structure. Overall, this radar-like monitor can effectively avoid false-positive signals and significantly improve the accuracy, precision, and reliability of detection. It is expected that the development of such an intelligent nano-platform will open the door to cancer diagnosis and treatment in clinical systems.
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Radar , Ribonucleasa H , ADN/genética , Reproducibilidad de los Resultados , InvestigaciónRESUMEN
Stress is known to induce retrograde tRNA translocation from the cytoplasm to the nucleus but translocation kinetics and tRNA-spatial distribution have not been characterized previously. We microinject fluorescently-labeled tRNA into living cells and use confocal microscopy to image tRNA spatial distribution in single cells at various levels of starvation and to determine translocation rate constants. Retrograde tRNA translocation occurs reversibly, within minutes after nutrition depletion of the extracellular medium. Such nutritional starvation leads to down-regulation of tRNA nuclear import and nearly complete curtailment of its nuclear export. Nuclear tRNA accumulation is suppressed in cells treated with the translation inhibitor puromycin, but is enhanced in cells treated with the microtubule inhibitor nocodazole. tRNA in the cytoplasm exhibits distinct spatial distribution inconsistent with diffusion, implying that such distribution is actively maintained. We propose that tRNA biological complexes and/or cytoplasmic electric fields are the likely regulators of cytoplasmic tRNA spatial distribution.
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Transporte de ARN/genética , ARN de Transferencia/genética , Inanición/genética , Estrés Fisiológico/genética , Transporte Activo de Núcleo Celular/genética , Animales , Núcleo Celular/genética , Citoplasma/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Ratones , Análisis de la Célula IndividualRESUMEN
An electrochemical sensor constructed by intercalated composites was developed for determination of heavy metal ions. The intercalated composites were composed of hydrosulphonyl functional covalent organic frameworks (COF-SH) and graphene (G). The presence of numerous adsorption sites, such as 18 sulfur atoms and 30 nitrogen atoms per big circle of COFs on COF-SH, was beneficial for the accumulation of heavy metals, while the graphene enhanced the electrical conductivity. The obtained sensor under the optimal conditions successfully detected the presence of heavy metal ions in coastal water samples at concentrations ranging from 1 to 1000 µg L-1. The detection limits of Cd (II), Pb (II), Cu (II), and Hg (II) were 0.3, 0.2, 0.2, and 1.1 µg L-1, respectively. Furthermore, the sensor still exhibited good stability after multiple uses less than 5%. When it is used in the analysis of actual samples, the recovery of standard addition is higher than 95%. In sum, the combination of hydrosulphonyl functional COFs with graphene looks very promising for the assembly of sensors with high sensitivity toward the determination of heavy metal ions for coastal environmental monitoring.
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Grafito/química , Iones/química , Metales Pesados/química , Nanocompuestos/química , Humanos , Estructuras MetalorgánicasRESUMEN
Glutathione (GSH) levels are closely related to the homeostasis of redox state which directly affects human disease occurrence by regulating cell apoptosis. Hence, real-time monitoring of dynamic changes in intracellular GSH levels is urgently needed for disease early diagnosis and evaluation of therapy efficiency. In this study, an endogenous cysteine (Cys)-assisted detection system based on GSH@AgNCs and reduced graphene oxide (rGO) with high sensitivity and specificity was developed for GSH detection. Compared with GSH, GSH@AgNCs with weaker affinity and bonding force was quite easier to extrude from the rGO surface when competing against GSH, leading to the obvious change in fluorescence signal. This phenomenon was termed as "a crowding out effect". Furthermore, the presence of Cys can improve GSH assay sensitivity by enhancing the quenching efficiency of rGO on the GSH@AgNCs. In vitro assay indicated that the efficiency of fluorescence recovery was positively related with GSH concentration in the range from 0 to 10 mM. In addition, the method was employed for real-time monitoring of the dynamic changes in GSH levels regulated by natural drugs. The imaging results showed that the natural compound 3 (C3) can downregulate GSH levels in HepG2 cells, which was accompanied by reactive oxygen species (ROS) release and apoptosis induction. Finally, the method was used to monitor the change of GSH levels in serum samples with chronic hepatitis B (CHB) infection. The results demonstrated that the occurrence and development of CHB may be positively correlated with GSH levels to some extent. Overall, the above results demonstrate the potential application of this new nanosystem in anticancer natural drug screening and clinical assay regarding GSH levels.
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Cisteína/química , Medicamentos Herbarios Chinos/farmacología , Colorantes Fluorescentes/química , Glutatión/sangre , Grafito/química , Nanopartículas del Metal/química , Doxorrubicina/farmacología , Etilmaleimida/farmacología , Glutatión/química , Glutatión/efectos de los fármacos , Células Hep G2 , Humanos , Límite de Detección , Especies Reactivas de Oxígeno/metabolismo , Plata/química , Espectrometría de Fluorescencia/métodosRESUMEN
As a highly conserved damage repair protein, Fpg can specifically recognize and digest 8-oxoG from a damaged DNA backbone. Meanwhile, DNAzyme, a single-stranded DNA with enzymatic activity, can cleave RNA in the presence of cofactors. In this study, we established a highly sensitive method for Fpg assay using a DNAzyme-mediated signal cascade amplification strategy. Based on the Fpg-dependent fluorescence response of the "turn-on" manner, we could reliably determine Fpg activity down to 0.14 U mL-1 with a linear response from 0.10 to 40 U mL-1 under optimal conditions. In addition, this strategy was successfully applied to analyze the kinetic parameter of Fpg with Km of 0.061 µM. Furthermore, the developed sensing system was used to screen the regulators of Fpg from natural compounds and antibiotics. These results indicated that all of the 14 natural compounds and 6 kinds of antibiotics deferentially showed an active effect on Fpg in vitro. In summary, these results show that the method not only provides an alternative for monitoring Fpg activity but also shows great potential for drug screening in the future.
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ADN Catalítico/genética , ADN-Formamidopirimidina Glicosilasa/sangre , ADN-Formamidopirimidina Glicosilasa/química , ADN/genética , Proteínas de Escherichia coli/sangre , Técnicas de Amplificación de Ácido Nucleico/métodos , Secuencia de Bases , Productos Biológicos/química , ADN/química , ADN Catalítico/química , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Fluorescencia , Colorantes Fluorescentes/química , Humanos , Cinética , Simulación del Acoplamiento MolecularRESUMEN
T4 polynucleotide kinase (PNK) is the primary member of the 5'-kinase family that can transfer the γ-phosphate residue of ATP to the 5'-hydroxyl group of oligonucleotides. In this article, using the differential quenching ability of reduced graphene oxide (rGO) towards the fluorophore-labeled DNA probe, we propose a novel method for detecting T4 PNK activity assisted by ligase reaction. Under the optimized conditions, the detection limit of T4 PNK was estimated to be 0.0002 U µL-1 in the linear region of 0.001 U µL-1-0.1 U µL-1. Additionally, the developed method was used to screen regulators of T4 PNK from natural compounds. The compound f isolated from the root of Kadsura coccinea (Lem.) A.C. Smith was found to stimulate T4 PNK activity in a concentration-dependent manner in vitro. Finally, the method was used to monitor the relation of T4 PNK activity with pelvic inflammatory disease (PID). The results demonstrated that the development of this disease could inhibit T4 PNK activity to some extent. In summary, the above data indicate that the method not only provides a universal platform for monitoring T4 PNK activity, but also shows great potential to be used in drug screening and clinic diagnosis.
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Técnicas Biosensibles/métodos , ADN Ligasas/química , Sondas de ADN/química , Grafito/química , Polinucleótido 5'-Hidroxil-Quinasa/antagonistas & inhibidores , Polinucleótido 5'-Hidroxil-Quinasa/análisis , Bacteriófago T4/enzimología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Femenino , Colorantes Fluorescentes/química , Humanos , Simulación del Acoplamiento Molecular , Enfermedad Inflamatoria Pélvica/enzimología , Espectrometría de Fluorescencia , Células THP-1RESUMEN
Hepatitis C virus (HCV) is a major cause of chronic liver disease, which affects 2-3% of the world population. Until now, the early detection of HCV has been a great challenge, especially for those who live in developing countries. In this study, we developed a novel and ultrasensitive assay for the detection of HCV RNA based on the reduced graphene oxide nanosheets (rGONS) and hybridization chain reaction (HCR) amplification technique. This detection system contains a pair of single fluorophore-labeled hairpin probes that can freely exist in the solution in the absence of target RNA. The introduction of target RNA can robustly trigger a HCR with the two probes and produce long nanowires containing a double-stranded structure. The weak adsorption to rGONS makes the long nanowires emit a strong fluorescence. Using this enzyme-free amplification strategy, we developed a new method for the HCV RNA assay with a detection limit of 10 fM, which is far more sensitive than the common GO-based fluorescence method. Furthermore, the new method exhibits high selectivity for the discrimination of perfectly complementary and mismatched sequences. Finally, the new method was successfully used as a HCV RNA assay in biological samples with a strong anti-interference capability in complicated environments. In summary, these remarkable characteristics of the new method highlight its potential use in a clinical sample primary screening.
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Bioensayo/métodos , Técnicas Biosensibles/métodos , Grafito/química , Hepacivirus/aislamiento & purificación , ARN Viral/análisis , Línea Celular Tumoral , ADN/química , ADN/genética , Sondas de ADN/química , Sondas de ADN/genética , Fluoresceínas/química , Fluorescencia , Colorantes Fluorescentes/química , Grafito/síntesis química , Células HEK293 , Humanos , Límite de Detección , Técnicas de Amplificación de Ácido Nucleico/métodos , Hibridación de Ácido Nucleico , Oxidación-Reducción , Prueba de Estudio Conceptual , ARN Viral/genética , Espectrometría de Fluorescencia/métodosRESUMEN
A new fluorometric method is delineated for the detection of RNase H activity by combining DNAzyme with reduced graphene oxide (rGO). In the absence of RNase H, the fluorescence of FAM-labeled probe is quenched due to the strong adsorption on the rGO. The presence of RNase H can release the active DNAzyme from the DNA-RNA chimeric strand. This triggers the cleavage of the signal probe at the rA site with the help of the cofactor Mg2+. The recycle cleavage can directly result in the amplified signal emitted by the FAM-labeled short fragment. The method allows the activity of RNase H to be detected in a linear range of 0.01 to 5 U·mL-1. The detection limit of 0.018 U·mL-1 is calculated by the principle of three-time standard deviation over the blank signal. Then, RNase H-targeting natural compounds were screened for their inhibitory action. Among the investigated compounds, five were screened as RNase H inhibitors in a concentration-dependent manner, and 4 compounds were identified as activators. Finally, the method was reliably used for discriminating the difference of RNase H activity in human serum. It is found that RNase H activity was upregulated in patients with hepatitis C virus infection. Graphical abstract The schematic presentation of rGO-DNAzyme-based RNase H detection. RNase H triggers the active DNAzyme releasing from the DNA-RNA chimeric strand, which can cleavage probes to FAM-labeled short fragments and make the fluorescence signal cycle amplified.
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Sondas de ADN/química , ADN Catalítico/química , Grafito/química , Ribonucleasa H/sangre , Espectrometría de Fluorescencia/métodos , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Fluoresceínas/química , Colorantes Fluorescentes/química , Humanos , Límite de Detección , Ribonucleasa H/antagonistas & inhibidoresRESUMEN
In addition to being an important object in theoretical and experimental studies in enzymology, RNase A also plays an important role in the development of many kinds of diseases by regulating various physiological or pathological processes, including cell growth, proliferation, differentiation, and invasion. Thus, it can be used as a useful biomarker for disease theranostics. Here, a simple, sensitive, and low-cost assay for RNase A was constructed by combining a fluorogenic substrate with reduced graphene oxide (rGO). The method with detection limit of 0.05 ng/mL was first applied for RNase A targeted drug screening, and 14 natural compounds were identified as activators of this enzyme. Then, it was applied to detect the effect of drug treatment and Hepatitis B virus (HBV) infection on RNase A activity. The results indicated that RNase A level in tumor cells was upregulated by G-10 and Chikusetsusaponin V in a concentration-dependent manner, while the average level of RNase A in the HBV infection group was significantly inhibited compared with that in the control group. Furthermore, the concentration-dependent inhibitory effect of heavy metal ions on RNase A was observed using the method and the results indicated that Ba2+, Co2+, Pb2+, As3+, and Cu2+ inhibited RNase A activity with IC50 values of 93.7 µM (Ba2+), 90.9 µM (Co2+), 110.6 µM (Pb2+), 171.5 µM (As3+), and 165.1 µM (Cu2+), respectively. In summary, considering the benefits of rapidity and high sensitivity, the method is practicable for RNase A assay in biosamples and natural compounds screening in vitro and in vivo.
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Antivirales/farmacología , Productos Biológicos/farmacología , Colorantes Fluorescentes/química , Grafito/química , Ribonucleasa Pancreática/antagonistas & inhibidores , Ribonucleasa Pancreática/análisis , Antivirales/química , Antivirales/aislamiento & purificación , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Colorantes Fluorescentes/metabolismo , Grafito/metabolismo , Hepatitis B/tratamiento farmacológico , Hepatitis B/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/metabolismo , Humanos , Juglandaceae/química , Metales Pesados/química , Metales Pesados/farmacología , Pruebas de Sensibilidad Microbiana , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Hojas de la Planta/química , Ribonucleasa Pancreática/metabolismo , Espectrometría de FluorescenciaRESUMEN
Rice blast caused by Magnaporthe oryzae severely impacts global rice yield stability. The rice endophyte Streptomyces sporocinereus OsiSh-2, with strong antagonistic activity towards M. oryzae, has been reported in our previous study. To decipher the model of the antagonistic action of OsiSh-2 towards M. oryzae, we compared the iron-capturing abilities of these two strains. The cultivation of OsiSh-2 and a M. oryzae strain under iron-rich and iron-starved conditions showed that M. oryzae depended more on iron supplementation for growth and development than did OsiSh-2. Genomic analysis of the S. sporocinereus and M. oryzae species strains revealed that they might possess different iron acquisition strategies. The actinobacterium OsiSh-2 is likely to favor siderophore utilization compared to the fungus M. oryzae. In addition, protein annotations found that OsiSh-2 contains the highest number of the siderophore biosynthetic gene clusters among the 13 endophytic actinomycete strains and 13 antifungal actinomycete strains that we compared, indicating the prominent siderophore production potential of OsiSh-2. Additionally, we verified that OsiSh-2 could excrete considerably more siderophores than Guy11 under iron-restricted conditions and displayed greater Fe3+-reducing activity during iron-supplemental conditions. Measurements of the iron mobilization between the antagonistic OsiSh-2 and Guy11 showed that the iron concentration is higher around OsiSh-2 than around Guy11. In addition, adding iron near OsiSh-2 could decrease the antagonism of OsiSh-2 towards Guy11. Our study revealed that the antagonistic capacity displayed by OsiSh-2 towards M. oryzae was related to the competition for iron. The highly efficient iron acquisition system of OsiSh-2 may offer valuable insight for the biocontrol of rice blast.
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Endófitos/fisiología , Hierro/metabolismo , Magnaporthe/metabolismo , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Sideróforos/metabolismo , Streptomyces/metabolismo , Resistencia a la Enfermedad , Relación Dosis-Respuesta a DrogaRESUMEN
As an essential phosphate group hydrolase, alkaline phosphatase (ALP), whose level in serum is correlated with bone disease, liver dysfunction, and cancer, could be used as a biomarker for clinical diagnosis and biomedical studies. Hence, developing a convenient and sensitive method for ALP assay has importance in disease diagnosis, drug treatment, and prognosis assessment. In this work, using a hairpin DNA strand as the substrate, we developed an ultrasensitive and simple fluorescence method for quantitative ALP assay based on the binding difference of reduced graphene oxide (rGO) with different DNA strands coupled with λ exonuclease (λ exo) cleavage. Under the optimal conditions, the limit of detection (LOD) of ALP is estimated to be 0.01 U/L with the linear region from 0.5 U/L to 70 U/L. Furthermore, the proposed assay was used to detect ALP in complicated cell-free extracts and evaluate the inhibitory effects of two well-known inhibitors of ALP activity. Finally, the method was used to investigate the effect of natural compounds on ALP activity and five compounds with different inhibitory capability were screened. In summary, we propose that the new method for ALP assay can be applied for therapeutic drug monitoring (TDM) and high-throughput compound screening in combination with multiwell plate technology.
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Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Productos Biológicos/química , Técnicas Biosensibles , Línea Celular Tumoral , ADN/química , Monitoreo de Drogas/métodos , Fluorescencia , Grafito/química , Humanos , Sensibilidad y EspecificidadRESUMEN
As a highly conserved damage repair protein, RNase H can specifically hydrolyze RNA in DNA-RNA chimeric strands. DNAzyme, a synthetic single-stranded DNA consisting of binding and catalytic sites, can cleave RNA in the presence of cofactors. In this study, we establish a highly sensitive RNase H assay assisted with DNAzyme's cleavage property. A DNA-RNA chimeric strand, which contains DNAzyme sequences, is used as the hydrolysis substrate of RNase H. The RNase H hydrolysis of the chimeric substrate results in the release of DNAzyme. Subsegment DNAzyme digest, a molecular beacon, causes a "turn-on" fluorescence signal by disrupting its hairpin structure. Furthermore, the fluorescence signal is amplified by cyclic digestion of DNAzyme to the substrate of molecular beacon. Under the optimal conditions, the detection limit of RNase H is 0.01 U/mL, which is superior to those of several alternative approaches. Additionally, the method was further used for RNase H detection in heterogeneous biological samples as well as to investigate the effects of natural compounds on this enzyme. In summary, these results show that the method not only provides a universal platform for monitoring RNase H activity but also shows great potential in biomedical studies and drug screening.
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ADN Catalítico/metabolismo , ADN de Cadena Simple/metabolismo , Colorantes Fluorescentes/química , ARN/metabolismo , Ribonucleasa H/metabolismo , Espectrometría de Fluorescencia , Línea Celular Tumoral , ADN de Cadena Simple/química , Pruebas de Enzimas , Humanos , ARN/química , Ribonucleasa H/sangre , Especificidad por SustratoRESUMEN
BACKGROUND: Biocontrol is a promising strategy in the control of rice blast disease. In the present study, we isolated and characterized a novel antagonist to the pathogen Magnaporthe oryzae from rice endophytic actinomycetes. RESULTS: Out of 482 endophytic actinomycetes isolated from rice blast infected and healthy rice, Streptomyces endus OsiSh-2 exhibited remarkable in vitro antagonistic activity. Scanning electron microscopy observations of M. oryzae treated by OsiSh-2 revealed significant morphological alterations in hyphae. In 2-year field tests, the spraying of OsiSh-2 spore solution (107 spores mL-1 ) is capable of reducing rice blast disease severity by 59.64%. In addition, a fermentation broth of OsiSh-2 and its cell-free filtrates could inhibit the growth of M. oryzae, suggesting the presence of active enzymes and secondary metabolites. OsiSh-2 tested positive for polyketide synthase-I and nonribosomal peptide synthetase genes and can produce cellulase, protease, gelatinase, siderophore, indole-3-acetic acid and 1-amino-cyclopropane-1-carboxylate deaminase. A preliminary separation indicated that the methanol extract of OsiSh-2 could suppress the growth of pathogens. The major active component was identified as nigericin. CONCLUSION: Endophytic S. endus OsiSh-2 has potential as a biocontrol agent against rice blast in agriculture. © 2016 Society of Chemical Industry.
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Proteínas Bacterianas/metabolismo , Agentes de Control Biológico/farmacología , Fungicidas Industriales/farmacología , Magnaporthe/efectos de los fármacos , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Streptomyces , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Agentes de Control Biológico/química , Medios de Cultivo , Endófitos/química , Endófitos/enzimología , Endófitos/aislamiento & purificación , Fermentación , Filtración , Fungicidas Industriales/química , Genes Bacterianos , Hifa , Ácidos Indolacéticos/metabolismo , Magnaporthe/patogenicidad , Nigericina/análisis , Nigericina/farmacología , Sideróforos/metabolismo , Esporas Bacterianas , Streptomyces/química , Streptomyces/enzimología , Streptomyces/aislamiento & purificaciónRESUMEN
As a waste-management endonuclease, DNase I has been suggested to be one of the deoxyribonucleases responsible for DNA fragmentation during apoptosis. We report here an alternative fluorescence method for DNase I assay with high accuracy and sensitivity by applying a DNA/GO (graphene oxide) probe. The method with a detection limit of 1 U mL(-1) was then applied to investigate the effects of external factors including antibiotics and heavy metal ions on DNase I. The results demonstrated that gentamicin sulfate was a strong inhibitor with an IC50 value of 0.57 ± 0.12 mM. The investigated heavy metal ions showed an inhibitory effect on DNase I activity in a concentration dependent manner with IC50 values of 0.04 µg/mL (Hg(2+)), 0.10 µg/mL (Pb(2+)), 1.35 µg/mL (Cd(2+)), 1.20 µg/mL (As(2+)), and 1.80 µg/mL (Cu(2+)). Finally, the new method was applied to detect DNase levels in complicated tumor tissue and cell samples and the results showed that DNase levels increased in tumor tissues compared with that of adjacent tissue. From the above results, we conclude that the method can be widely used for high - throughput assay of DNase I in biological samples as well as drug screening in vitro. Graphical Abstract The schematic of real-time monitoring of DNase I using GO - quenched hairpin probe as the substrate. The process of nucleotide digestion catalyzed by DNase I produces short fragments of hairpin probe and accordingly causes a significant increase in fluorescence. At first, GO can absorb the hairpin probes and quenched their fluorescence. When there is DNase I, the DNase can cleave the double strands of DNA. Fluorescence is restored due to the significantly weaker binding ability of small DNA fragments to GO compared with long DNA fragments. So, we can detect the increase in fluorescence to study the activity of DNase.
Asunto(s)
Desoxirribonucleasa I/metabolismo , Sondas Moleculares , Antibacterianos/química , Técnicas In Vitro , Concentración 50 Inhibidora , Cinética , Límite de Detección , Neoplasias/enzimología , Espectrometría de FluorescenciaRESUMEN
Chronic wound infections, particularly multidrug-resistant microbe-caused infections, have imposed severe challenges in clinical administration. The therapeutic effectiveness of the current strategy using conventional antibiotics is extremely unsatisfactory. The development of novel treatment strategies to inhibit the infections caused by multidrug-resistant bacteria is highly desired. In this work, based on the combination of nanocompounds with the assistance of NIR laser, an antibacterial strategy was designed for MRSA-infected abscesses in diabetic mice. The nanocompounds named Ag@Chi-PB were prepared by using chitosan-coated Prussian blue (PB) as a nanocarrier for silver nanoparticles anchoring. Combined with near-infrared (NIR) laser, the nanocompounds were more efficient at killing Escherichia coli (E. coli) and Methicillin-resistant staphyllococcus aureus (MRSA) in vitro. Notably, MRSA was significantly removed in vivo and promoted diabetic abscess healing by the combined therapy of this nanocompound and NIR laser, owing to the synergistic antibacterial effect of photothermal therapy and release of Ag+. Meanwhile, the nanocompound showed satisfactory biocompatibility and superior biosafety. Collectively, the combination therapy of this nanocompound with the assistance of NIR laser may represent a promising strategy for clinical anti-infection.