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MicroRNAs (miRNAs) play indispensable roles in post-transcriptional gene regulation. The identification of target mRNAs is essential for dissecting the recognition basis, dynamics, and regulatory mechanism of miRNA-mRNA interactions. However, the lack of an unbiased method for detecting weak miRNA-mRNA interactions remains a long-standing obstacle for miRNA research. Here, we develop and provide proof-of-concept evidence demonstrating a chemical G-clamp-enhanced photo-cross-linking strategy for covalent capture of intracellular miRNA targets in different cell lines. This approach relies on an aryl-diazirine-G-clamp-modified-nucleoside (ARAGON) miRNA probe containing an alkynyl group that improves the thermal stability of miRNA-target mRNA duplex molecules and can rapidly cross-link with the complementary strand upon UV 365 nm activation, enhancing the transient capture of mRNA targets. After validating the accuracy and binding properties of ARAGON-based miRNA probes through the successful enrichment for the known targets of miR-106a, miR-21, and miR-101, we then extend ARAGON's application to screen for previously unknown targets of different miRNAs in various cell lines. Ultimately, results in this study uncover GAB1 as a target of miR-101 in H1299 lung cancer cells and show that miR-101 silencing of GAB1 can promote apoptosis in H1299 cells, suggesting an oncogenic mechanism of GAB1. This study thus provides a powerful and versatile tool for enhanced screening of global miRNA targets in cells to facilitate investigations of miRNA functions in fundamental cellular processes and disease pathogenesis.
Assuntos
MicroRNAs , MicroRNAs/metabolismo , MicroRNAs/química , MicroRNAs/genética , Humanos , Processos Fotoquímicos , RNA Mensageiro/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Diazometano/química , Reagentes de Ligações Cruzadas/química , Linhagem Celular Tumoral , Raios UltravioletaRESUMO
N6-methyladenosine (m6A) is the most abundant posttranscriptional chemical modification in mRNA, involved in regulating various physiological and pathological processes throughout mRNA metabolism. Recently, we developed GLORI, a sequencing method that enables the production of a globally absolute-quantitative m6A map at single-base resolution. Our technique utilizes the glyoxal- and nitrite-based chemical reaction, which selectively deaminates unmethylated adenosines while leaving m6A intact. The RNA library can then be prepared using a modified library construction protocol from enhanced UV crosslinking and immunoprecipitation (eCLIP) or commercial kits. Here we provide a detailed protocol for proper RNA sample handling and provide further guidelines for the use of a tailored bioinformatics pipeline (GLORI-tools) for subsequent data analysis. Compared with current methods, this new method is both exceptionally sensitive and robust, capable of identifying ~80,000 m6A sites with 50 Gb sequencing data in mammalian cells. It also provides a quantitative readout for m6A sites at single-base resolution. We hope the technique will provide a precise and unbiased tool for investigating m6A biology across various fields. Basic expertise in molecular biology and knowledge of bioinformatics are required for the protocol. The entire procedure can be completed within a week, with the library construction process taking ~4 d.
Assuntos
RNA , Transcriptoma , Animais , RNA/genética , RNA Mensageiro/metabolismo , Metilação , Biblioteca Gênica , Mamíferos/genéticaRESUMO
N6-methyladenosine (m6A) is the most abundant RNA modification in mammalian cells and the best-studied epitranscriptomic mark. Despite the development of various tools to map m6A, a transcriptome-wide method that enables absolute quantification of m6A at single-base resolution is lacking. Here we use glyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI) to develop an absolute m6A quantification method that is conceptually similar to bisulfite-sequencing-based quantification of DNA 5-methylcytosine. We apply GLORI to quantify the m6A methylomes of mouse and human cells and reveal clustered m6A modifications with differential distribution and stoichiometry. In addition, we characterize m6A dynamics under stress and examine the quantitative landscape of m6A modification in gene expression regulation. GLORI is an unbiased, convenient method for the absolute quantification of the m6A methylome.
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DNA , Transcriptoma , Animais , Humanos , Metilação , Transcriptoma/genética , Regulação da Expressão Gênica , Metilação de DNA/genética , MamíferosRESUMO
With the rapid development of infrastructure construction, it is an inevitable trend to replace natural sand in short supply with manufactured sand to meet sustainable development. In this paper, the relationship between the particle shape characteristics of manufactured sand and concrete performance is discussed using a morphological analysis and concrete experiments. The particle shape parameters of five types of manufactured sand were obtained by using the aggregate image measurement system (AIMS) and digital image processing (DIP) techniques, and the correlations between different parameters were analyzed. Moreover, the properties of concrete with the five kinds of manufactured sand were tested. The results show that particle size and type have a significant impact on particle shape parameters. Particle shape parameters, especially angularity, correlate well with the workability and compressive strength of concrete while having little effect on the durability of concrete. An accurate understanding of the morphological characteristics of manufactured sand is conducive to the optimization of concrete mix designs. Therefore, it is suggested that a manufactured-sand shape test be included in aggregate specification.
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Carbon dots (CDs) have drawn much attention in recent decades due to their outstanding biocompatibility and environmental friendliness. In this work, the surface modifications of peroxidase-like carbon dots were carried out to show the influence of surface structure on the catalytic activity. Poly(ethyleneimine) and citric acid modified CDs, i.e. PEI-CDs and CA-CDs, were obtained and used in the catalytic oxidation of two different substrates 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The catalytic activity of CA-CDs for TMB is greater than that of unmodified CDs while the catalytic activity of PEI-CDs for ABTS is greater than that of unmodified CDs. These results may be ascribed to that TMB shows stronger affinity to negatively charged CA-CDs and ABTS shows stronger affinity to positively charged PEI-CDs. These results clearly suggested that the surface charge plays an important role in the catalytic activity of nanoenzyme. Furthermore, the mechanism of peroxidase-like activity of all CDs was investigated and both the generation of hydroxyl radical and enhanced electron transfer were involved in the catalytic process.
Assuntos
Carbono/química , Peroxidases/metabolismo , Benzidinas/química , Benzotiazóis/química , Oxirredução , Ácidos Sulfônicos/química , Propriedades de SuperfícieRESUMO
The liquid-liquid phase equilibria of {water/PEG200/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/n-decane} with the molar ratio of water to AOT being 37.9 and various concentrations cPEG of PEG in water were measured in this study. The critical exponent ß corresponding to the width of the coexistence curve was determined, which showed good agreement with the 3D-Ising value and supported the proposal of the pseudo binary droplet solution for these multiple microemulsions. A previously developed thermodynamic approach based on the Carnahan-Starling-van der Waals type equation was improved and used to analyze the coexistence curve data of {water/PEG200/AOT/n-decane} microemulsions to deduce the interaction properties between droplets and further to investigate the effect of the additive PEG200 on these interactions. It was found that the addition of PEG200 into the {water/AOT/n-decane} microemulsion resulted in the decrease in the critical temperature and the interaction enthalpy and entropy. Both the interaction enthalpy and entropy decreased first, then increased with an increase of cPEG and had minimum values at cPEG = 25 g L-1, which showed the same tendency as the isothermal titration microcalorimetric results for the {water/PEG200/AOT/isooctane} microemulsion we reported very recently. The effects of the interaction enthalpy and the entropy on the phase separation and their dependences on cPEG were discussed and related to the effects of the additive on the rigidity of the interface layer of the microemulion droplet.
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The heat capacities of nine binary room temperature ionic solutions {[C4mim][BF4] + 1,2-butandiol}, {[C8mim][BF4] + 1-pentanol}, {[C8mim][BF4] + 2-pentanol}, {[C8mim][BF4] + 1-hexanol}, {[C8mim][BF4] + 1-heptanol}, {[C8mim][PF6] + 1-propanol}, {[C8mim][PF6] + 1-butanol}, {[C8mim][PF6] + 2-butanol} and {[C8mim][PF6] + tert-butanol} are reported herein. The combination of the data obtained with the corresponding measured coexistence curves infers that the critical asymmetry parameter of the coexistence curves linearly varies with the molar volume ratio of the two components for each of the studied binary ionic solutions after the heat capacity contribution is considered. This indicates the importance of the heat capacity contribution to the critical asymmetry. For further analysis of the critical characteristics of the ionic solutions, a large amount of experimental data was collected and systematically discussed in detail regarding which critical character is important in the binary ionic solutions. A general increasing tendency for the RPM (restricted primitive model)-rescaled critical parameters with the relative permittivity εr,c of the solvent at the critical temperature of the corresponding system was found. This is attributed not only to the screening effect of the solvent medium, but also to solvophobic interactions, which both increase with εr,c. This study also demonstrates that the critical amplitudes increased, while the relative contribution of the heat capacity to the asymmetry of the coexistence curve decreased with an increase in εr,c.
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The heats of mixing for a series of DTAB/TX100 mixed surfactant aqueous solutions were measured by flow-mixing calorimetry and isothermal titration calorimetry (ITC) at 298.15 K and 85 kPa, which were used to calculate the inter-micellar interaction enthalpies (-ΔHC). The signs of -ΔHC for pure DTAB and TX100 micellar systems were contrary to the inter-micellar interaction parameters reported for the same systems in the literature, suggesting that these interaction parameters might have a Gibbs free energy character dominated by entropy changes. It was found that the inter-micellar interaction enthalpies varied with the total surfactant concentration and the mixed ratio of the two surfactants, and characterized different structures of the mixed micelles. These phenomena were discussed based on the effects of structural water around the micellar interface, the hydration of counterions, and the repulsive Coulombic interaction.
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The liquid-liquid coexistence curve, heat capacity in the critical and noncritical regions, and the turbidity in critical one-phase and two-phase regions of the binary solution {iodobenzene + 1-decyl-3-methylimidazolium bis(trifluorosulfonyl)imide ([C10mim][NTf2])} have been precisely measured. From the data collected in the critical region, the critical exponents α, ß, γ and ν, as well as the universal critical amplitude ratios RB and X were obtained and were shown to agree well with their theoretical values for the 3D-Ising universality class, which further confirmed the 3D-Ising criticality of ionic solutions even in a very low relative permittivity solvent. The coulombic character of the studied system was suggested by the small values of the RPM reduced critical temperature and density. Furthermore, it was found that both the asymmetric behaviors of the diameter of the coexistence curve and the osmotic compressibility could be well described using the complete scaling theory.
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A mild and transition-metal-free protocol is herein presented for chloro-, bromo- and trifluoromethylthiotrifluoromethylation of unactivated alkenes. The easy-handling Langlois reagent, as well as N-halophthalimide and N-trifluoromethylthiosaccharin, is used in this method. In the presence of an organic photoredox catalyst N-methyl-9-mesityl acridinium, a broad range of desired products were afforded in satisfactory yields upon visible-light irradiation via a radical process.
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The liquid-liquid phase equilibria of [water/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/n-decane] with the molar ratio w0 of water to AOT being 37.9 and [water/AOT/ethoxylated-2,5,8,11-tetramethyl-6-dodecyne-5,8-diol(Dynol-604)/n-decane] with w0 = 37.9 and the mole fraction α of Dynol-604 in the total surfactants being 0.158 were measured in this study. From the data collected in the critical region, the critical exponent ß corresponding to the width of the coexistence curve was determined, which showed good agreement with the 3D-Ising value. A thermodynamic approach based on the Carnahan-Starling-van der Waals type equation was proposed to describe the coexistence curves and to deduce the interaction properties between droplets in the microemulsions. The interaction enthalpies were found to be positive for the studied systems, which evidenced that the entropy effect dominated the phase separations as the temperature increased. The addition of Dynol-604 into the (water/AOT/n-decane) microemulsion resulted in the decrease in the critical temperature and the interaction enthalpy. Combining the liquid-liquid equilibrium data for (water/AOT/n-decane) microemulsions with various w0 values determined previously, it was shown that the interaction enthalpy decreased with w0 and tended to change its sign at low w0, which coincided with the results from the isothermal titration calorimetry investigation. All of these behaviors were interpreted by the effects of entropy and enthalpy and their competition, which resulted from the release of solvent molecules entrapped in the interface of microemulsion droplets and were dependent on the rigidity of the surfactant layers and the size of the droplet.
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A novel polypyrrole (PPy) and graphene quantum dots (GQDs) @ Prussian Blue (PB) nanocomposite has been grafted on a graphite felt (GF) substrate (PPy/GQDs@PB/GF), and has been proven to be an efficient electrochemical sensor for the determination of l-cysteine (l-cys). GQDs, which were fabricated by carbonization of citric acid and adsorbed on GF surface ultrasonically, played an important role for promoting the synthesis process of PB via a spontaneous redox reaction between Fe(3+) and [Fe(CN)6](3-). The PPy film has been electro-polymerized to improve the electrochemical stability of the PPy/GQDs@PB/GF electrode. The as-prepared electrode was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), infrared spectroscopy (IR), X-ray diffraction (XRD) and electrochemical methods. It exhibited an excellent activity for the electrocatalytic oxidation of l-cys, with a detection sensitivity equal to 0.41 Amol(-1) L for a concentration range of 0.2-50 µmolL(-1), and equal to 0.15 Amol(-1) L for a concentration range of 50-1000 µmolL(-1). A low detection limit of 0.15 µmolL(-1), as well as a remarkable long-time stability and a negligible sensitivity to interfering analytes, were also ascertained.
Assuntos
Condutometria/instrumentação , Cisteína/análise , Ferrocianetos/química , Grafite/química , Polímeros/química , Pirróis/química , Pontos Quânticos , Desenho de Equipamento , Análise de Falha de Equipamento , Microeletrodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Rates of SN1 hydrolysis reactions for 2-chloro-2-methylbutane in the critical solution of isobutyric acid + water and for 2-bromo-2-methylpropane in the critical solution of triethylamine + water in the one-phase region and at various temperatures have been determined respectively by conductance measurements. It was found that the reaction rates at different temperatures for those two SN1 reactions were well described by the Arrhenius equation in the noncritical region, whereas near the critical points the critical slowing down was clearly detected. These results are inconsistent with a previous report in the literature (J. Phys. Chem. A 2003, 107, 8435 - 8443). Reanalyzing the literature data, we found that if an Arrhenius equation appropriate to either the one-phase region or the two-phase region being examined was used as the background, a critical slowing down rather than a speeding up was detected for the reaction in both the one-phase region and the two-phase region. The experimental data from different sources were fitted with a simplified crossover formalism characterizing the critical effect on the reactions to determine the critical slowing down exponents, which were found to be about 0.04, showing that only a dynamic critical slowing down could be detected for these reaction systems. This phenomenon was attributed to the fact that the SN1 hydrolysis reaction is neither a first-order nor a pseudo-first-order reaction in the reverse direction and the kinetic measurements were carried out in a region quite far from the equilibrium of the reactions.
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In this article, a new application of the room temperature ionic liquid, tetramethylammonium tetrafluoroborate, is described. This ionic liquid is used as a stabilizing agent and the reaction medium for the synthesis of CoFe Prussian blue analogue nanoparticles using N,N-dimethyl formamide as a complexing agent. The as-synthesized Prussian blue analogues were characterized by various techniques and were used to catalyze the reaction of epoxidation of styrene. The catalytic activity of Prussian blue analogues prepared in ionic liquid was superior to that of Prussian blue analogues prepared in aqueous solution.
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The aggregation behaviors of surface active ionic liquid 1-butyl-3-methylimidazolium bis(2-ethylhexyl) sulfosuccinate in aqueous solutions were investigated by conductometry, densimetry, fluorimetry, (1)H nuclear magnetic resonance ((1)H NMR) spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM), which confirmed two distinguished critical concentrations. The first critical concentration was believed to be the critical aggregation concentration (CAC), where two different types of aggregates were formed, namely, micelles with the size of 10 nm and vesicles with the size of 100 nm. The second critical concentration at two-fold CAC was suggested to be resulting from the insertion of the imidazolium cations into aggregates.
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The concentration-dependent enthalpies of mixing for water/sodium bis(2-ethylhexyl)-sulfosuccinate (AOT)/n-alkane microemulsions with different water contents ω0 and chain lengths n of n-alkane were determined by isothermal titration microcalorimetry (ITC) and flow-mixing microcalorimetry at 298.15 K and used to calculate the interaction enthalpies (-ΔH(C)) between the droplets. It was found that -ΔH(C) increased with ω0, and changed from negative to positive at about ω0 = 10. The investigation of the dependence of -ΔH(C) on n revealed that the values of -ΔH(C) were negative and had a minimum for ω0 = 5; while they were positive and had a maximum for ω0 = 15. These phenomena were discussed based on the competition of the overlapping contribution of the surfactant tails between two neighbouring droplets and the penetration contribution of the solvent molecules into the surfactant tails. These results indicated the important role of entropy in the stability of the microemulsion systems.
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The densities of AOT/isooctane micelles and water/AOT/isooctane microemulsions with the molar ratios R of water to AOT being 2, 8, 10, 12, 16, 18, 20, 25, 30, and 40 were measured at 303.15 K. The apparent specific volumes of AOT and the quasi-component water/AOT at various concentrations were calculated and used to estimate the volumetric properties of AOT and water in the droplets and in the continuous oil phase, to discuss the interaction between the droplets, and to determine the critical micelle concentration and the critical microemulsion concentrations. A thermodynamic model was proposed to analysis the stability boundary of the microemulsion droplets, which confirms the maximum value of R being about 65 for the stable AOT/water/isooctane microemulsion droplets.
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In this work, we studied the kinetics of the oxidation of iodide ion by persulfate ion in the critical water/bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/n-decane microemulsions with the molar ratios of water to AOT being 35.0 and 40.8 via the microcalorimetry at various temperatures. It was found that the Arrhenius equation was valid for correlating experimental measurements in the noncritical region, but the slowing down effect existed significantly in the near critical region. We determined the values of the critical slowing down exponent and found it to be 0.187 ± 0.023 and 0.193 ± 0.032, respectively, which agreed well with the theoretical value of 0.207 predicted by the Griffiths-Wheeler rule for the singularity of the dimer/monomer droplet equilibrium in the critical AOT/water/n-decane microemulsions.
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In this paper, we investigated the dilution enthalpies of the droplets in water/AOT/oil microemulsions with oil being isooctane, decane, or cyclohexane by isothermal titration microcalorimetry (ITC). Combining with the results obtained from the study of the water/AOT/toluene system in our previous work, it was found that the enthalpy interactions between droplets for isooctane and decane systems were repulsive, while the enthalpy interactions were attractive for cyclohexane and toluene systems. The repulsive droplet interaction for the isooctane system was also confirmed by static light scattering. The solvents appear to play important roles in varying the droplet enthalpy interactions from positive to negative, and the entropy contribution seems to be dominant for the stability of these microemulsion droplet systems.
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The liquid-liquid coexistence curves for binary solutions {1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]) + 1-propanol} and {1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]) + 2-propanol} have been precisely measured. The values of the critical exponents ß obtained from the liquid-liquid equilibrium data in the critical region confirmed the 3D-Ising universality. The isobaric heat capacities per unit volume were measured for {[C4mim][BF4] + 1-propanol (or 2-propanol, 1,3-propanediol, 1,4-butanediol)} in both critical and non-critical regions. The experimental results indicate a major solvophobic contribution to the criticality for the studied ionic solutions. The complete scaling theory was applied to well represent the asymmetric behavior of the diameter of the coexistence curves with the consideration of the heat capacity contribution. It was found that the contribution of the heat capacity related term in the ionic solution decreased with the increase of the permittivity of alcohol and was more important in the description of the asymmetry of the coexistence curve of the ionic solutions than that of the molecular solutions.