RESUMEN
Bursaphelenchus xylophilus, the pine wood nematode (PWN), is the causal agent of pine wilt disease (PWD), which causes enormous economic loss annually. According to our previous research, fomepizole, as a selective inhibitor of PWN alcohol dehydrogenase (ADH), has the potential to be a preferable lead compound for developing novel nematicides. However, the underlying molecular mechanism is still unclear. The result of molecular docking showed that the stronger interactions between fomepizole and PWN ADH at the active site of ADH were attributed to hydrogen bonds. Low-dose fomepizole had a substantial negative impact on the egg hatchability, development, oviposition, and lifespan of PWN. Transcriptome analysis indicated that 2,124 upregulated genes and 490 downregulated genes in fomepizole-treated PWN were obtained. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes indicated that fomepizole could be involved in controlling PWN vitality mainly by regulating key signaling pathways, such as the ribosome, hippo signaling pathway, and lysosome. Remarkably, the results of RNA interference indicated that the downregulated serine/threonine-protein phosphatase gene (stpp) could reduce the egg hatchability, development, oviposition, and lifespan of PWN, which was closely similar to the consequences of nematodes with low-dose fomepizole treatment. In addition, the silencing of stpp resulted in weakness of PWN pathogenicity, which indicated that stpp could be a potential drug target to control PWN.
Asunto(s)
Pinus , Tylenchida , Animales , Virulencia , Transcriptoma , Fomepizol , Xylophilus , Simulación del Acoplamiento Molecular , Enfermedades de las Plantas , Pinus/genética , Fosfoproteínas Fosfatasas/genética , Treonina/genética , Serina/genética , Tylenchida/genéticaRESUMEN
Plant-parasitic nematodes cause severe economic losses annually which has been a persistent problem worldwide. As current nematicides are highly toxic, prone to drug resistance, and have poor stability, there is an urgent need to develop safe, efficient, and green strategies. Natural active polysaccharides such as chitin and chitosan with good biocompatibility and biodegradability and inducing plant disease resistance have attracted much attention, but their application is limited due to their poor solubility. Here, we prepared 6-oxychitin with good water solubility by introducing carboxylic acid groups based on retaining the original skeleton of chitin and evaluated its potential for nematode control. The results showed that 6-oxychitin is a better promoter of the nematicidal potential of Purpureocillium lilacinum than other water-soluble chitin derivatives. After treatment, the movement of J2s and egg hatching were obviously inhibited. Further plant experiments found that it can destroy the accumulation and invasion of nematodes, and has a growth-promoting effect. Therefore, 6-oxychitin has great application potential in the nematode control area.
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Antinematodos/farmacología , Quitina/análogos & derivados , Hypocreales/química , Tylenchoidea/efectos de los fármacos , Animales , Antinematodos/química , Cucumis sativus/parasitología , Locomoción , Reproducción , Tylenchoidea/patogenicidad , Tylenchoidea/fisiologíaRESUMEN
Pine wilt disease, which is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has caused huge damage to pine forests around the world. In this study, we analysed the PWN transcriptome to investigate the expression of genes related to the associated bacterial species Pseudomonas fluorescens and found that the gene adh-1 encoding alcohol dehydrogenase (ADH) was upregulated. The open reading frame of adh-1, which encoded a protein of 352 amino acid residues, was cloned from B. xylophilus. Recombinant ADH with a relative molecular weight of 39 kDa, was present mainly in inclusion bodies and was overexpressed in Escherichia coli BL21 (DE3) and purified after refolding. The biochemical assay revealed that recombinant ADH could catalyse the dehydrogen reaction of eight tested alcohols including ethanol in the presence of NAD+. Quantitative real-time RT-PCR analysis indicated that ethanol upregulated adh-1 expression in PWN. Results of RNA interference and inhibition of ADH treatment indicated that downregulating expression of adh-1 or inhibition of ADH could reduce ethanol tolerance and the vitality and reproduction ability of B. xylophilus, suggesting that adh-1 is involved in pathogenicity of PWN.
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Alcohol Deshidrogenasa/genética , Etanol/farmacología , Proteínas del Helminto/genética , Rabdítidos/genética , Regulación hacia Arriba/genética , Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas del Helminto/química , Proteínas del Helminto/metabolismo , Pinus/parasitología , Pseudomonas fluorescens/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Esophageal squamous cell carcinoma (ESCC) is one of the most common types of malignancies in China. Most ESCC patients are diagnosed at middle to late stages with poor prognosis due to the lack of an effective method for early diagnosis. MicroRNAs (miRNAs) are a family of endogenous small non-coding RNAs that can regulate ESCC development and progression by repressing their specific target genes' expression. Compared to traditional biomarkers (e.g., mRNAs and proteins), miRNAs are more stable and can be readily screened and accurately quantitated and analyzed, making them ideal new-generation of biomarkers for early cancer detection and prognostic evaluation. Recent studies have shown that the changes of the expression levels of some serum miRNAs from ESCC patients significantly correlate with their diagnostic and prognostic outcome. In this review, we summarize the trend of the expression changes of miRNAs in ESCC patients' serum and discuss the possibility of detecting these miRNAs' expression changes as a novel method for ESCC early diagnosis and prognostic evaluation. Notably, the results of serum miRNAs from different detection methods are not completely consistent. Thus, we also discuss several possible reasons for such inconsistency.
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Biomarcadores de Tumor/sangre , Carcinoma de Células Escamosas/diagnóstico , Neoplasias Esofágicas/diagnóstico , MicroARNs/sangre , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/sangre , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago , Humanos , MicroARNs/genética , PronósticoRESUMEN
Chitosan oligosaccharide (COS) modification is a feasible way to develop novel green nematicides. This study involved the synthesis of various COS sulfonamide derivatives via hydroxylated protection and deprotection, which were then characterized using NMR, FTIR, MS, elemental analysis, XRD, and TG/DTG. In vitro experiments found that COS-alkyl sulfonamide derivatives (S6 and S11-S13) exhibited high mortality (>98 % at 1 mg/mL) against Meloidogyne incognita second-instar larvaes (J2s) among the derivatives. S6 can cause vacuole-like structures in the middle and tail regions of the nematode body and effectively inhibit egg hatching. In vivo tests have found that S6 has well control effects and low plant toxicity. Additionally, the structure-activity studies revealed that S6 with a high degree of substitution, a low molecular weight, and a sulfonyl bond on the amino group of the COS backbone exhibited increased nematicidal activity. The sulfonamide group is a potential active group for developing COS-based nematicides.
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Antinematodos , Quitosano , Oligosacáridos , Sulfonamidas , Tylenchoidea , Quitosano/química , Quitosano/farmacología , Animales , Tylenchoidea/efectos de los fármacos , Antinematodos/farmacología , Antinematodos/química , Oligosacáridos/química , Oligosacáridos/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Relación Estructura-Actividad , Larva/efectos de los fármacosRESUMEN
Chemical nematicide is the most common method of controlling plant-parasitic nematodes (PPN). Given the negative impact of chemical nematicides on the environment and ecosystem, it is necessary to seek their alternatives and novel modes of application. Chitin oligo/polysaccharide (COPS), including chitosan and chitosan oligosaccharide, has unique biological properties. By producing ammonia, encouraging the growth of antagonistic bacteria, and enhancing crop tolerance, COPSs help suppress PPN growth during soil remediation. COPS is also an effective sustained-release carrier that can be used to overcome the shortcomings of nematicidal substances. This review summarizes the advancements of COPS research in nematode control from three perspectives of action mechanism as well as in slow-release carrier-loaded nematicides. Further, it discusses potential agricultural applications for nematode disease management.
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Quitosano , Nematodos , Animales , Quitina/farmacología , Quitina/química , Quitosano/farmacología , Quitosano/química , Ecosistema , Agricultura , Antinematodos/farmacología , Antinematodos/química , Oligosacáridos/farmacologíaRESUMEN
To replace biohazardous nematicides, there is an ever-increasing need to identify natural product-based agents to contain root-knot nematodes (RKNs) in agriculture. In this chemical ecology study, an antagonistic fungus Aspergillus fumigatus 1T-2, which could cause the formation of withering of the gut and vacuole-like structures in the nematode body, was isolated based on the gradually increased antagonistic interactions between the soil fungi and RKNs. Based on these typical morphological characteristics, a potent nematode-antagonistic compound 2-furoic acid, which had a simple structure, was successfully identified from 1T-2 fermentation broth by liquid chromatography-mass spectrometry (LC-MS). 2-Furoic acid showed effective mortality activity in vitro, of which the LC50 value to Megalaima incognita at 24 h was 37.75 µg/mL. 2-Furoic acid had similar mortality activity to the positive control fosthiazate at 30 µg/mL. Continuous 2-furoic acid exposure had obvious negative influences on both nematode vitality and egg hatchability. Notably, significant variations were observed in nematodes and eggs with 2-furoic acid treatment, which might be induced by novel nematocidal mechanisms. Furthermore, the 1T-2 fermentation broth and 2-furoic acid had significant control efficacy on M. incognita under the greenhouse test-tube assay. Overall, these findings provide valuable insights into the use of 2-furoic acid with biocontrol potential as a preferable lead structure for the development of innovative nematicides.
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Tylenchoidea , Animales , Antinematodos/farmacología , Aspergillus fumigatus , Hongos , PlomoRESUMEN
Pine wilt disease (PWD) caused by Bursaphelenchus xylophilus is considered a major threat to pine forests worldwide. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze the conjugation of small lipophilic compounds with sugars and play crucial roles in the detoxification and homeostatic processes in all living organisms. We investigated the molecular characteristics and biological functions of the gene UGT440A1 that encodes UGTs in B. xylophilus. The in situ hybridization results indicated that UGT440A1 is expressed in all developmental stages of B. xylophilus, particularly in the head, intestine, and hypodermis of the second-stage of juveniles (J2), third-stage of juveniles (J3) and fourth-stage of juveniles (J4) females and in almost the whole body of J4 males and adults. Recombinant UGT440A1 was observed mainly in the inclusion bodies, and the enzyme activity assay revealed that UGT440A1 could catalyze the glycosylation reaction of two types of flavonols (kaempferol and quercetin). RNA interference (RNAi) of UGT440A1 suppressed motility, feeding, and reproduction of B. xylophilus. Furthermore, UGT440A1 knockdown caused a delay in the development of PWD symptoms in the pine seedlings inoculated with the nematodes. These results suggest that UGT440A1 is involved in the pathogenic process of B. xylophilus and the information may facilitate a better understanding of the molecular mechanism of PWD.
RESUMEN
Hymexazol (HYM) is irreplaceable for treating soil-borne diseases due to its high efficiency and low cost, as a broad-spectrum fungicide. However, when HYM is absorbed by plants, it is rapidly converted into two glycoside metabolites, and the antifungal activities of these glycosides are inferior to that of HYM. Therefore, in this study, to maintain strong antifungal activity in vitro and in vivo, HYM was glycosylated with amino sugars that have diverse biological activities to simulate plant glycosylation. The antifungal experiment proved that glycoside 15 has the highest antifungal activity, and N-acetyl glucosamine and HYM had obvious synergistic effects. According to the structure-activity relationship studies, glycoside 15 had greater numbers of active electron-rich regions and front-line orbital electrons due to the introduction of N-acetyl glucosamine. Moreover, glycoside 15 can significantly promote plant growth and induce an increase in plant defense enzyme activity. Additionally, compared to HYM, the results of electron microscopy and proteomics revealed that glycoside 15 has a unique antifungal mechanism. The promising antifungal activity and interactions with plants mean that glycoside 15 is a potential green fungicide candidate. Furthermore, this research conducted an interesting exploration of the agricultural applications of amino sugars.
Asunto(s)
Antifúngicos , Fungicidas Industriales , Amino Azúcares , Antifúngicos/farmacología , Biomimética , Fungicidas Industriales/farmacología , Glucosamina , Glicósidos/farmacología , Pruebas de Sensibilidad Microbiana , Oxazoles , Plantas/microbiología , Relación Estructura-ActividadRESUMEN
Pinewood releases ethanol and other volatile compounds after Bursaphelenchus xylophilus infection. In the current study, we examined the influence of different ethanol concentrations on B. xylophilus reproduction. Low-concentrations of ethanol (8.5, 17, and 34 mM) increased egg production in B. xylophilus, whereas higher-concentrations (156 and 312 mM) reduced egg production. Transcriptome analysis was conducted to explore the molecular response of a low concentration of ethanol on the nematodes. The results suggest that the nematodes use ethanol as an energy source, which may promote survival. Ethanol induced changes in the expression of genes involved in the biosynthesis and metabolism of fatty acids and amino acids. Furthermore, ethanol promoted the expression of detoxification-related, cell wall-degrading, and reproduction-related genes. Such responses might contribute to the pathogenicity of B. xylophilus.
Asunto(s)
Pinus , Rabdítidos , Animales , Etanol , Perfilación de la Expresión Génica , Transcriptoma , XylophilusRESUMEN
This paper reviews an important topic within the broader framework of the use of ratoon cotton for the development of a cost-saving and efficient method for the perennial production of hybrid cotton seeds. Cotton has a botanically indeterminate perennial growth habit and originated in the tropics. However, cotton has been domesticated as an annual crop in temperate areas worldwide. Ratoon cultivation has an important application value and is important for cotton production, breeding, and basic research. In particular, ratooned male-sterile lines have four advantages: an established root system, an indeterminate flowering habit, ratooning ability, and perennial maintenance of sterility in the absence of a matched maintainer. These advantages can help reduce the costs of producing F1 hybrid cotton seeds and can help breed high-yielding hybrid combinations because ratooning is a type of asexual reproduction that allows genotypes to remain unchanged. However, ratooning of cotton is highly complex and leads to problems, such as the accumulation of pests and diseases, decreased boll size, stand loss during severe winters, and harmful regrowth during mild winters, which need to be resolved. In summary, ratoon cotton has advantages and disadvantages for the production of hybrid cotton seeds, and future prospects of ratooning annual cotton for the perennial utilization of heterosis are promising if the mechanization of seed production can be widely applied in practice.
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A novel and efficient approach for stepwise recycling of valuable metals from Ni-rich cathode material is developed. First, the spent cathode materials are leached by H2SO4â¯+â¯H2O2 solution. The leaching efficiencies of lithium, nickel, manganese and cobalt reach almost 100%, 100%, 94% and 100%, respectively, under the conditions of 2â¯M sulfuric acid, 0.97â¯M hydrogen peroxide, 10â¯ml·g-1 liquid-solid ratio, 30â¯min and 80⯰C. Then, manganese and cobalt are co-extracted from the leaching liquor with PC88A, while almost 99% nickel and 100% lithium remain in the raffinate followed by being separated from each other by solvent extraction with neodecanoic acid (Versatic 10). The results show that 98% manganese and over 90% cobalt are co-extracted at pHâ¯=â¯5, 30â¯vol% PC88A and volume ratio of oil to water (O:A)â¯=â¯2:1, while 100% nickel is separated from lithium under the optimum extraction conditions of initial pHâ¯=â¯4, O:Aâ¯=â¯1:3 and 30â¯vol% Versatic 10. Finally, cobalt and manganese in the strip liquor of co-extraction are separated by selective precipitation method. Over 90% manganese is separated from cobalt under the conditions of pHâ¯=â¯0.5, 0.076â¯M KMnO4, 80⯰C and 60â¯min.
Asunto(s)
Peróxido de Hidrógeno , Litio , Cobalto , Suministros de Energía Eléctrica , Electrodos , Metales , ReciclajeRESUMEN
Since the Gravity Recovery and Climate Experiment (GRACE) satellite mission was started in 2002, a variety of spatial products have been made available to further understanding of mass redistribution in the Earth system. Two such mascon (mass concentration) solutions were developed by the Center for Space Research (CSR-M) and the NASA Jet Propulsion Laboratory (JPL-M), which offers significantly improved spatial localization and more accurate amplitude measurements of changes in recovered terrestrial Total Water Storage (TWS). However, it is difficult to validate GRACE-derived TWS mascons due to the lack of independent measurements of water storage in various forms at larger scales.In this study, we present a simple framework to evaluate GRACE mascon products based on in-situ GPS measurements from the Yangtze River Basin (YRB) in China. We found that the mascons show a more pronounced spatial difference in TWS distribution and highlight more details as compared to smoother results from empirical post-processing filtering applied to spherical harmonics (SH) data. The prediction of vertical displacements from CSR-M and JLP-M is closer to GPS than that from SH. The residual analysis showed the reductions in WRMS (weighted root-mean-squares) from the GPS minus the CSR-M average were greater than those for JPL-M in 41 GPS stations, and the scaling factors from CLM4.0 used in JPL-M-sf had few improvements with respect to agreement with GPS measurements. Our findings indicated CSR-M solutions were more consistent with in-situ observations and more in line with actual surface mass transport in the YRB. These findings also suggested that when using GRACE mascons to detect local TWS changes or when combining GRACE-derived data with GPS-observed displacement to estimate crustal response to loadings, users should note the contributions from effects of load signal sources from atmospheric, non-tidal ocean, and difference sensitivity kernels on differences between TWS from satellite-based and in-situ observations.
RESUMEN
Although many studies describe ionic liquids (ILs) as potentially greener solvents, few studies address their relationship with the environment. Recent researches suggest that some ILs have toxicity. 1-Butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]) was reported to have the lowest toxicity among ILs. In this work, we studied the toxicity of this IL on wheat seedlings. It was shown that wheat germination was reduced to 38.0% in the presence of 4.4 mmol/L [C4mim][BF4] compared with 100% germination for the control. Similarly, the root and shoot length of wheat seedlings decreased with increasing concentrations of [C4mim][BF4]. The activity of amylase increased in shoots and roots, but it decreased significantly in germinating seeds when the [C4mim][BF4] concentration exceeded 1.8 mmol/L. Peroxidase (POD) activity and soluble protein content in shoots treated with [C4mim][BF4] changed similarly, but chlorophyll content tended to decrease with increasing concentration of [C4mim][BF4], except for at 1.8 mmol/L. Thus, [C4mim][BF4] at the concentration of 0.9 mmol/L or more was toxic to wheat seedlings. Some remedial measures are suggested to deal with IL pollution in the environment.
Asunto(s)
Imidazoles/toxicidad , Líquidos Iónicos/toxicidad , Plantones/efectos de los fármacos , Triticum/efectos de los fármacos , Amilasas/metabolismo , Clorofila/análisis , Peroxidasa/metabolismo , Proteínas de Plantas/análisis , Raíces de Plantas/química , Raíces de Plantas/crecimiento & desarrollo , Brotes de la Planta/química , Brotes de la Planta/crecimiento & desarrollo , Plantones/enzimología , Plantones/crecimiento & desarrollo , Semillas/química , Semillas/efectos de los fármacos , Semillas/enzimología , Pruebas de Toxicidad , Triticum/enzimología , Triticum/crecimiento & desarrolloRESUMEN
INTRODUCTION: MicroRNA373 was highly expressed in many tumors including esophageal cancer. However, its molecular mechanism is still unclear, especially epigenetic modification, in esophageal squamous cell carcinoma (ESCC). METHODS: In this study, we investigated serum levels of the miR-371-373 cluster in ESCC patients before and after surgical removal, and further focused on the expression level of miR-373-3p in tumor tissues of ESCC patients and its target genes. In addition, the epigenetic alterations of miR-373-3p promoter was analyzed. RESULTS: The expression levels of miR-371-5p and miR-373-3p were significantly increased in preoperative serum of ESCC patients compared with that of healthy volunteers (P<0.01); however, they dropped significantly after surgical removal (P<0.01). Compared with adjacent normal tissues, miR-373-3p also showed significant up-regulation in cancer tissues (P<0.05). The methylation levels of miR-373-3p promoter were 42.86% in ESCC cancer tissue and 66.67% in adjacent normal tissues. The low methylation of the miR-373-3p promoter may promote the expression of miR-373-3p. Large tumor suppressor 2 (LATS2) and oxidation resistance 1(OXR1) are predicted to be targets of miR-373-3p by the bioinformatics method. They are the genes in the Hippo and the p53 signaling pathway, respectively. Their respective upstream genes, neurofibromatosis type 2 (NF2) and Jun Kinase, and the downstream genes, transcriptional co-activator with PDZ-binding motif (TAZ) and caspase 9, were also detected. The expression of all these genes were significantly decreased in ESCC cancer tissues compared with adjacent normal tissues. CONCLUSIONS: This study shows that DNA epigenetic modification in the miR-373-3p promoter region and the Hippo and p53 signaling pathways play important roles during the miR-373-3p mediating ESCC development process.
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Biomarcadores de Tumor/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Movimiento Celular , Proliferación Celular , Metilación de ADN , Epigénesis Genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Estudios de Seguimiento , Humanos , Proteínas Mitocondriales , Pronóstico , Proteínas Serina-Treonina Quinasas/genética , Proteínas/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Esophageal squamous cell carcinoma (ESCC) is one of the most widespread malignancies in China. MicroRNAs (miRNAs/miRs) are endogenous evolutionarilyconserved small noncoding RNAs that are able to regulate ESCC formation and deterioration by negatively regulating specific target genes. In the present study, the expression levels of miR4835p and its associated mRNAs were measured by quantitative polymerase chain reaction (PCR) analysis, and the methylation levels of the insulinlike growth factor 2 (Igf2) promoter were detected via the methylationspecific PCR method in serum and tissues from patients with ESCC. The results demonstrated that the expression level of miR4835p was significantly upregulated in preoperative serum and cancer tissues from patients with ESCC (P<0.01), and the miR4835p expression levels were correlated with the tumor, node, metastasis stage (P<0.05) and lymph node metastasis (P<0.05). In addition, the mRNA levels of miR4835p target genes (Rho GDP dissociation inhibitor α, activated leukocyte cell adhesion molecule, and suppressor of cytokine signaling 3) in cancer tissues were significantly decreased compared with adjacent noncancerous tissues. These results indicated that miR4835p and its target genes may be involved in the developmental process of ESCC. The Igf2 levels in cancer tissues were significantly increased compared with adjacent noncancerous tissues (P<0.01). Additionally, the methylation levels of the Igf2 promoter region were 31.82 and 54.55% in cancer tissues and adjacent noncancerous tissues, respectively, suggesting that low methylation of the Igf2 gene promoter region may promote the expression of Igf2 and miR4835p; this, in turn, induces the degradation of miR4835p target genes, and leads to the upregulation of oncogenes and the downregulation of tumor suppressors, which promotes the development of ESCC.
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Carcinoma de Células Escamosas/genética , Epigénesis Genética , Neoplasias Esofágicas/genética , Regulación Neoplásica de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina/genética , MicroARNs/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Metilación de ADN , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago , Perfilación de la Expresión Génica , Humanos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Interferencia de ARNRESUMEN
Development of new strategies for the sensitive and selective detection of ultra-low concentrations of specific cancer markers is of great importance for assessing cancer therapeutics due to its crucial role in early clinical diagnoses and biomedical applications. In this work, we have developed two types of fluorescence polarization (FP) amplification assay strategies for the detection of biomolecules by using TiS2 as a FP enhancer and Zn(2+)-dependent self-hydrolyzing deoxyribozymes as catalysts to realize enzyme-catalyzed target-recycling signal amplification. One approach is based on the terminal protection of small-molecule-linked DNA, in which biomolecular binding to small molecules in DNA-small-molecule chimeras can protect the conjugated DNA from degradation by exonuclease I (Exo I); the other approach is based on the terminal protection of biomolecular bound aptamer DNA, in which biomolecules directly bound to the single strand aptamer DNA can protect the ssDNA from degradation by Exo I. We select folate receptor (FR) and thrombin (Tb) as model analytes to verify the current concept. It is shown that under optimized conditions, our strategies exhibit high sensitivity and selectivity for the quantification of FR and Tb with low detection limits (0.003 ng mL(-1) and 0.01 pM, respectively). Additionally, this strategy is a simple "mix and detect" approach, and does not require any separation steps. This biosensor is also utilized in the analysis of real biological samples, the results agree well with those obtained by the enzyme-linked immunosorbent assay (ELISA).
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Aptámeros de Nucleótidos/química , Técnicas Biosensibles , ADN de Cadena Simple/química , Polarización de Fluorescencia , Receptor 1 de Folato/análisis , Límite de Detección , Trombina/análisisRESUMEN
An anionic peroxidase RsPrx1 was purified (RZ=3.0) and characterized from roots of Chinese red radish (Raphanus sativus var. niger, Brassicaceae). The specific activity of RsPrx1 (micromol mg(-1) min(-1)) is 413.5 (ferulic acid); 258.7 (ABTS); 177.3 (caffeic acid) and 10.0 (guaiacol acid). The optimum pH is 4.0 (citrate buffer) using ABTS as substrate. RsPrx1 can utilise the red pigment present in the root, pelargonidin, as substrate and the specific activity is 93.6 micromol mg(-1) min(-1). The molecular mass of RsPrx1 is 45 kDa (denatured) and 46 kDa (native) as determined by SDS-PAGE and gel filtration, respectively. The isoelectric point (pI) determined by native IEF is 4.7 and by chromatofocusing (Mono P) is 5.1. Analysis of tryptic peptides by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS) covered 27% of the RsPrx1 sequence and confirmed its identity. The gene encoding RsPrx1 was cloned by PCR and the amino acid sequence showed the highest identity (82%) to peroxidase AtPrx22 and AtPrx23 from Arabidopsis thaliana and to HRPC3 and HRPE5 from horseradish, respectively. Activity-stained IEF gels show that RsPrx1 is primarily expressed in the roots in agreement with the expression profile of the orthologous genes. These five orthologous peroxidases have three introns of variable length and sequence at conserved locations between the distal and proximal histidine. The results suggest that RsPrx1 orthologs are widespread in the Brassicaceae plant family with a 15-residue-long C-terminal propeptide in common. Based on the results, we propose that RsPrx1 and orthologs are targeted to the vacuoles to modify stored anthocyanins like pelargonidin.
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Antocianinas/metabolismo , Clonación Molecular , Peroxidasas/aislamiento & purificación , Raphanus/enzimología , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Armoracia/enzimología , Armoracia/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Datos de Secuencia Molecular , Peso Molecular , Peroxidasas/química , Peroxidasas/genética , Filogenia , Raíces de Plantas/enzimología , Raíces de Plantas/metabolismo , Raphanus/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TemperaturaRESUMEN
To observe the effects of peroxidase on hyperlipidemia, mice were fed a diet high in cholesterol and fat. At the same time, the mice were given different-purity peroxidase (radish juice, crude radish peroxidase, and horseradish peroxidase), and their serum cholesterol, triglyceride, blood glucose, amylase, and esterase activities, and malondialdehyde in the mouse small intestines and livers, were tested after 15 days on the test diets. The results showed that peroxidase decreased the levels of total serum cholesterol, triglyceride, blood glucose, and lipid peroxidation in the small intestines and livers of hyperlipidemic mice. This suggests that peroxidase may be a contributing factor in the prevention of hyperlipidemia.
Asunto(s)
Hiperlipidemias/tratamiento farmacológico , Peroxidasa/uso terapéutico , Amilasas/sangre , Animales , Bebidas , Glucemia/análisis , Colesterol/sangre , Colesterol en la Dieta/administración & dosificación , Esterasas/sangre , Femenino , Peroxidasa de Rábano Silvestre/uso terapéutico , Intestino Delgado/química , Intestino Delgado/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/química , Hígado/metabolismo , Malondialdehído/análisis , Ratones , Triglicéridos/sangre , Verduras/enzimologíaRESUMEN
The antioxidant and pro-oxidant potential of an extract from red radish, in which the major compounds were acylated pelargonidin derivatives, were assessed with a variety of assays in vitro. The extract appeared to form a complex with Fe(3+) or Cu(2+). It displayed a concentration-dependant reducing power (1.16OD(700 nm) at a concentration of 4mM) and scavenging effect against 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radicals (with IC(50)=1.74 +/- 0.03 mM). It could promote the cleavage of plasmid DNA with Cu(II)/H(2)O(2) or Cu(II) alone. This DNA damage could be inhibited by horseradish peroxidase, catalase, and EDTA, respectively. The extract also showed growth inhibition of Bel-7402 cells at lower concentration. The results suggested that the formation of reactive oxygen species might be involved in the mechanism of DNA damage. The acylated pelargonidin derivatives extracted from red radish could act as antioxidant and pro-oxidant and their antioxidant and pro-oxidant properties were relative to the reaction conditions. It might provide novel antioxidant and anticarcinogenic agents.