RESUMO
Reliable multiple single nucleotide polymorphisms (SNPs) detection at low abundance is of great significance for disease diagnosis and biomedical research. Herein, we have developed a novel and simple method for multiple SNPs detection combining solid-phase capture by specific hybridization with online preconcentration of capillary gel electrophoresis-laser-induced fluorescence (CGE-LIF). The method presents an excellent performance due to its favorable traits: the solid-phase short-chain hybridization ensures the high specificity of SNP detection; the effective separation ability of CGE can easily achieve multiplex detection; the simple online preconcentration significantly improves the detection sensitivity of fluorescent probe by nearly 100-fold. For a single SNP target, the assay achieves a limit of detection as low as 0.01-0.02% for three different NRAS mutations in the same codon. For multiple SNP targets, as low as 0.05% abundance can be easily realized. Our method is simple, efficient, ultrasensitive, and universal for multiple SNPs detection without complex enzymatic or chemical ligation reaction, which shows great potential in early clinical diagnosis.
Assuntos
Eletroforese Capilar/métodos , Limite de Detecção , Polimorfismo de Nucleotídeo Único , Códon/genética , Hibridização de Ácido NucleicoRESUMO
BACKGROUND: Aging and the chronic non-communicable diseases (NCDs) challenge the Chinese government in the process of providing hospitalization services fairly and reasonably. The Chinese government has developed the basic medical insurance system to solve the problem of "expensive medical cost and difficult medical services" for vulnerable groups and alleviate the unfair phenomenon. However, few studies have confirmed its effect through longitudinal comparison. This study aimed to explore the trend in the inequity of inpatient use among middle-aged and elderly individuals with NCDs in China. METHODS: This longitudinal comparative study was based on CHARLS data in 2011, 2013 and 2015. Concentration index (CI) was used to measure the variation trend of inequity of inpatient services utilization, while the decomposition method of the CI was applied to measure the factors contributing to inequity in inpatient services utilization. The effect of each factor on the change of inequity in inpatient services utilization was divided into the change of the elasticity and the change of inequality using the Oaxaca-type decomposition method. RESULTS: The affluent middle-aged and elderly patients with NCDs used more inpatient services than poor groups. The per capita household consumption expenditure (PCE) and Urban Employee Basic Medical Insurance (UEBMI) contributed to the decline in pro-rich inequality of inpatient use, while the New Rural Cooperative Medical Scheme (NRCMS) contributed to the decline in pro-poor inequality of inpatient use. CONCLUSIONS: There was a certain degree of pro-rich unfairness in the probability and frequency of inpatient services utilization for middle-aged and elderly individuals with NCDs in China. The decrease of pro-wealth contribution of PCE and UEBMI offset the decrease of pro-poor contribution of NRCMS, and improved the equity of inpatient services utilization, favoring poor people.
Assuntos
Utilização de Instalações e Serviços/estatística & dados numéricos , Disparidades em Assistência à Saúde/estatística & dados numéricos , Hospitalização/estatística & dados numéricos , Doenças não Transmissíveis/terapia , Idoso , China , Doença Crônica , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fatores SocioeconômicosRESUMO
A G-triplex, a new kind of DNA structure, has been identified as an intermediate in the folding of G-quadruplexes. However, the studies on G-triplexes are still very limited, and the functions and applications of G-triplexes need to be further developed. In this paper, a new G-triplex sequence (5'-CTGGGAGGGAGGGA-3', G3), obtained by truncating four bases (GGGA) from the 3' end of an 18-base G-quadruplex sequence (G4), was found to significantly decrease the diffusion current of methylene blue (MB). In particular, we proved that (a) MB stabilized the structure of G3 and increased the Tm of G3 considerably based on circular dichroism; and (b) MB formed a 1:1 noncovalent complex with G3 based on electrospray ionization mass spectrometry. Moreover, molecular dynamics simulations established reliable speculation in the folding topology of G3 and interaction sites between G3 and MB. Based on the strong affinity of G3 with MB, we further developed a novel function of G3 as an electrochemical signal read-out and applied it in the fabrication of a sensitive homogeneous electrochemical aptasensor for cocaine. The features we observed in the G3/MB complex will serve as a new inspiring guideline for developing functional short G-rich ligands.
Assuntos
Técnicas Biossensoriais , DNA/química , Técnicas Eletroquímicas , Quadruplex GRESUMO
Single nucleotide polymorphisms (SNPs) have been proven to be important biomarkers for disease diagnosis, prognosis and disease pathogenesis. Here, taking the advantages of a self-assembled oligonucleotide sandwich structure and robust chemical reactions, we have developed a simple, high-throughput and effective colorimetric analytical technique termed CuAAC-based ligation-assisted assays (CuAAC-LA) for SNP detection using a DNA-BIND 96-well plate. With the 5'-azide and 3'-alkyne groups labelled on two oligonucleotide probes, the target DNA can direct a Cu(i)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction. Since the small difference in duplex stability caused by a single-nucleotide mismatch was amplified by the steric effects of these reactive groups for the ligation reaction of an unstable duplex, CuAAC-LA exhibited an ultra-sensitive discrimination ability for a mutant type target in the presence of large amounts of wild type targets. As low as 0.05% SNP could be clearly detected, which was better than most previously reported methods by various DNA ligases, indicating that a simple and rapid synthetic method i.e., the DNA template-directed click reaction held the potential to replace the ligase for SNP detection.
Assuntos
Colorimetria/métodos , DNA/análise , Polimorfismo de Nucleotídeo Único , Alcinos/química , Armoracia/enzimologia , Azidas/química , Benzidinas/química , Química Click , Corantes/química , Reação de Cicloadição , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Peroxidase do Rábano Silvestre/química , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , OxirreduçãoRESUMO
It is known that selenium (Se) enhances plant growth and arsenic (As) accumulation in As-hyperaccumulator Pteris vittata, but the associated mechanisms are unclear. In this study, P. vittata was exposed to 50 µM arsenate (AsV) under hydroponics plus 25 or 50 µM foliar selenate. After 3-weeks of growth, the plant biomass, As and Se contents, As speciation, malondialdehyde (MDA) and glutathione (GSH and GSSG) levels, and important genes related to As-metabolism in P. vittata were determined. Foliar-Se increased plant biomass by 17 - 30 %, possibly due to 9.1 - 19 % reduction in MDA content compared to the As control. Further, foliar-Se enhanced the As contents by 1.9-3.5 folds and increased arsenite (AsIII) contents by 64 - 136 % in the fronds. The increased AsV reduction to AsIII was attributed to 60 - 131 % increase in glutathione peroxidase activity, which mediates GSH oxidation to GSSG (8.8 -29 % increase) in the fronds. Further, foliar-Se increased the expression of AsIII antiporters PvACR3;1-3;3 by 1.6 - 2.1 folds but had no impact on phosphate transporters PvPht1 or arsenate reductases PvHAC1/2. Our results indicate that foliar-Se effectively enhances plant growth and arsenic accumulation by promoting the GSH-GSSG cycle and upregulating gene expression of AsIII antiporters, which are responsible for AsIII translocation from the roots to fronds and AsIII sequestration into the fronds. The data indicate that foliar-Se can effectively improve phytoremediation efficiency of P. vittata in As-contaminated soils.
Assuntos
Arsênio , Arsenitos , Glutationa , Folhas de Planta , Pteris , Selênio , Arseniatos/metabolismo , Arsênio/metabolismo , Arsenitos/metabolismo , Biodegradação Ambiental , Glutationa/metabolismo , Malondialdeído/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Pteris/metabolismo , Pteris/crescimento & desenvolvimento , Pteris/genética , Pteris/efeitos dos fármacos , Selênio/metabolismo , Selênio/farmacologia , Poluentes do Solo/metabolismoRESUMO
Arsenic-hyperaccumulator Pteris vittata exhibits remarkable absorption ability for chromium (Cr) while beneficial element selenium (Se) helps to reduce Cr-induced stress in plants. However, the effects of Se on the Cr uptake and the associated mechanisms in P. vittata are unclear, which were investigated in this study. P. vittata plants were grown for 14 days in 0.2-strength Hoagland solution containing 10 (Cr10) or 100 µM (Cr100) chromate (CrVI) and 1 µM selenate (Se1). The plant biomass, malondialdehyde contents, total Cr and Se contents, Cr speciation, expression of genes associated with Cr uptake, and Cr subcellular distribution in P. vittata were determined. P. vittata effectively accumulated Cr by concentrating 96-99% in the roots under Cr100 treatment. Further, Se substantially increased its Cr contents by 98% to 11,596 mg kg-1 in the roots, which may result from Se's role in reducing its oxidative stress as supported by 27-62% reduction in the malondialdehyde contents. Though supplied with CrVI, up to 98% of the Cr in the roots was reduced to insoluble chromite (CrIII), with 83-89% being distributed on root cell walls. Neither Cr nor Se upregulated the expression of sulfate transporters PvSultr1;1-1;2 or phosphate transporter PvPht1;4, indicating their limited role in Cr uptake. P. vittata effectively accumulates Cr in the roots mainly as CrIII on cell walls and Se effectively enhances its Cr uptake by reducing its oxidative stress. Our study suggests that Se can be used to enhance P. vittata Cr uptake and reduce its oxidative stress, which may have application in phytostabilization of Cr-contaminated soils.
Assuntos
Cromo , Raízes de Plantas , Pteris , Selênio , Poluentes do Solo , Pteris/metabolismo , Pteris/efeitos dos fármacos , Cromo/metabolismo , Cromo/toxicidade , Selênio/metabolismo , Selênio/farmacologia , Poluentes do Solo/metabolismo , Poluentes do Solo/toxicidade , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Malondialdeído/metabolismo , Arsênio/metabolismo , Arsênio/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Biodegradação Ambiental , Cromatos/toxicidade , Cromatos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacosRESUMO
AIMS: Exploring the neurobiological mechanisms of early AD damage Background: The early diagnosis of Alzheimer's disease (AD) has a very important impact on the prognosis of AD. However, the early symptoms of AD are not obvious and difficult to diagnose. Existing studies have rarely explored the mechanism of early AD. AMPARs are early important learning memory-related receptors. However, it is not clear how the expression levels of AMPARs change in early AD. OBJECTIVE: We explored learning memory abilities and AMPAR expression changes in APP/PS1 mice at 4 months, 8 months, and 12 months. METHOD: We used the classic Morris water maze to explore the learning and memory impairment of APP/PS1 mice and used western blotting to explore the changes in AMPARs in APP/PS1 mice. RESULT: We found that memory impairment occurred in APP/PS1 mice as early as 4 months of age, and the impairment of learning and memory gradually became serious with age. The changes in GluA1 and p-GluA1 were most pronounced in the early stages of AD in APP/PS1 mice. CONCLUSION: Our study found that memory impairment in APP/PS1 mice could be detected as early as 4 months of age, and this early injury may be related to GluA1.
RESUMO
We developed a simple and highly-selective method for 5-methylcytosine detection of specific gene sequence based on binary-probe DNA hybridization. The sequence complementary to the target was designed into two probes, and each fragment of binary probes bound to a relatively short sequence of the target, which made it sensitive to the base mismatches introduced by bisulfite treatment. The advantages of a low detection limit of methylation abundance of 0.1% for the fully methylated target and high sensitivity of 10 pM have been proved by the successful design of binary-probe hybridization. The successful design of the binary probes makes it possible to quantify the average methylation levels of five CpG sites. Thirty-two DNA strands containing 5, 4, 3, 2, 1 and 0 CpG sites were successfully analyzed with the same pair of binary probes. The higher the average methylation level of the target was, the higher the degree of the hybridization reaction. Based on the simple construction of the binary-probe hybridization, the developed biosensor exhibited signals proportional to the average methylation level of the vimentin gene and could evaluate the average methylation level of artificial mixtures. Furthermore, the method has been used to detect vimentin methylation in a genomic context with good specificity, which indicated its potential in the pre-diagnosis of methylation related disease.
Assuntos
Técnicas Biossensoriais , Metilação de DNA , DNA , Sondas de DNA/genética , Hibridização de Ácido NucleicoRESUMO
Single nucleotide polymorphisms (SNPs) are emerging as important biomarkers for disease diagnosis, prognostics and disease pathogenesis. As one type of disease is always connected to several SNP sites, there is great demand for a reliable multiple SNP detection method. Herein, we mimicked a ligation reaction based on DNA ligase and originally utilized an enzyme-free DNA template-directed click reaction for SNP detection. With 5'-alkyne and 3'-azide groups labelled on two oligonucleotide probes, the target DNA-directed Cu(i)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction produced a new DNA strand with a triazole backbone, as a mimic of a DNA phosphodiester linkage. Trace amounts of the target (as low as 25 fmol in 50 µL) could be sensitively detected using capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF). Meanwhile, SNP caused an obvious difference in the efficiency of the click reaction, and 0.5% SNP could be easily detected. More importantly, multiplexed SNP detection in a one tube reaction was successfully achieved only by encoding different lengths of the DNA probes for the different SNP sites.