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This review addresses the role of tight junction proteins at the blood-brain barrier (BBB). Their expression is described, and their role in physiological and pathological processes at the BBB is discussed. Based on this, new approaches are depicted for paracellular drug delivery and diagnostics in the treatment of cerebral diseases. Recent data provide convincing evidence that, in addition to its impairment in the course of diseases, the BBB could be involved in the aetiology of CNS disorders. Further progress will be expected based on new insights in tight junction protein structure and in their involvement in signalling pathways.
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Barrera Hematoencefálica , Proteínas de Uniones Estrechas , Uniones Estrechas , Barrera Hematoencefálica/metabolismo , Humanos , Proteínas de Uniones Estrechas/metabolismo , Animales , Uniones Estrechas/metabolismo , Enfermedades del Sistema Nervioso Central/metabolismo , Transducción de SeñalRESUMEN
Since RNA is an important biomarker of many infectious pathogens, RNA detection of pathogenic organisms is crucial for disease diagnosis and environmental and food safety. By simulating the base mismatch during DNA replication, this study presents a novel three-way junction structure-mediated reverse transcription-free exponential amplification reaction (3WJ-RTF-EXPAR) for the rapid and sensitive detection of pathogen RNA. The target RNA served as a switch to initiate the reaction by forming a three-way junction (3WJ) structure with the ex-trigger strand and the ex-primer strand. The generated trigger strand could be significantly amplified through EXPAR to open the stem-loop structure of the molecular beacon to emit fluorescence signal. The proofreading activity of Vent DNA polymerase, in combination with the unique structure of 2+1 bases at the 3'-end of the ex-primer strand, could enhance the role of target RNA as a reaction switch to reduce non-specific amplification and ensure excellent specificity to differentiate target pathogen from those causing similar symptoms. Furthermore, detection of target RNA showed a detection limit of 1.0×104 copies/mL, while the time consumption was only 20 min, outperforming qRT-LAMP and qRT-PCR, the most commonly used RNA detection methods in clinical practice. All those indicates the great application prospects of this method in clinical diagnostic.
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Límite de Detección , Técnicas de Amplificación de Ácido Nucleico , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/análisis , ARN Viral/genética , ARN Bacteriano/análisis , ARN Bacteriano/genética , HumanosRESUMEN
Since microRNAs (miRNAs) are predictors of tumorigenesis, accurate identification and quantification of miRNAs with highly similar sequences are expected to reflect tumor diagnosis and treatment. In this study, a highly selective and sensitive electrochemiluminescence (ECL) biosensor was constructed for miRNAs determination based on Y-shaped junction structure equipped with locked nucleic acids (LNA), graphene oxide-based nanocomposite to enrich luminophores, and conductive matrix. Specifically, two LNA-modified probes were designed for specific miRNA recognition, that is, a dual-amine functionalized hairpin capture probe and a signal probe. A Y-shaped DNA junction structure was generated on the electrode surface upon miRNA hybridizing across the two branches, so as to enhance the selectivity. Carbon quantum dots-polyethylene imine-graphene oxide (CQDs-PEI-GO) nanocomposites were developed to enrich luminophores CQDs, and thus enhancing the ECL intensity. For indirect signal amplification, an electrochemically activated poly(2-aminoterephthalic acid) (ATA) film decorated with gold nanoparticles was prepared on electrode as an effective matrix to accelerate the electron transfer. The fabricated ECL biosensor achieved sensitive determination of miRNA-222 with a limit-of-detection (LOD) as low as 1.95 fM (S/N = 3). Notably, Y-shaped junction structures equipped with LNA probes endowed ECL biosensor with salient single-base discrimination ability and anti-interference capacity. Overall, the proposed Y-shaped ECL biosensor has considerable promise for clinical biomarker determination.
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Técnicas Biosensibles , Nanopartículas del Metal , MicroARNs , Puntos Cuánticos , MicroARNs/genética , Carbono/química , Puntos Cuánticos/química , Oro/química , Mediciones Luminiscentes , Nanopartículas del Metal/química , ADN/química , Sondas de Ácido Nucleico , Polietileneimina/química , Técnicas ElectroquímicasRESUMEN
Lens gap junctions (GJs) formed by Cx46 and Cx50 are important to keep lens transparency. Functional studies on Cx46 and Cx50 GJs showed that the Vj-gating, single channel conductance (γj), gating polarity, and/or channel open stability could be modified by the charged residues in the amino terminal (NT) domain. The role of hydrophobic residues in the NT on GJ properties is not clear. Crystal and cryo-EM GJ structures have been resolved, but the NT domain structure has either not been resolved or has showed very different orientations depending on the component connexins and possibly other experimental conditions, making it difficult to understand the structural basis of the NT in Vj-gating and γj. Here, we generated missense variants in Cx46 and Cx50 NT domains and studied their properties by recombinant expression and dual whole-cell patch clamp experiments on connexin-deficient N2A cells. The NT variants (Cx46 L10I, N13E, A14V, Q15N, and Cx50 I10L, E13N, V14A, N15Q) were all able to form functional GJs with similar coupling%, except Cx46 N13E, which showed a significantly reduced coupling%. The GJs of Cx46 N13E, A14V and Cx50 E13N, N15Q showed a reduced coupling conductance. Vj-gating of all the variant GJs were similar to the corresponding wild-type GJs except Cx46 L10I. The γj of Cx46 N13E, A14V, Cx50 E13N, and N15Q GJs was reduced to 51%, 82%, 87%, and 74%, respectively, as compared to their wild-type γjs. Structural models of Cx46 L10I and A14V predicted steric clashes between these residues and the TM2 residues, which might be partially responsible for our observed changes in GJ properties. To verify the importance of hydrophobic interactions, we generated a variant, Cx50 S89T, which also shows a steric clash and failed to form a functional GJ. Our experimental results and structure models indicate that hydrophobic interactions between the NT and TM2 domain are important for their Vj-gating, γj, and channel open stability in these and possibly other GJs.
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Uniones Comunicantes , Activación del Canal Iónico , Conexinas/metabolismo , Uniones Comunicantes/genética , Uniones Comunicantes/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Canales Iónicos/metabolismoRESUMEN
The bond graph methodology for modelling an integrated energy distillation column is applied in this paper. The distillation column is built by five trays for a binary mixture. However, due to its modular construction in a bond graph, the number of trays can be increased. In order to link the analysis tools of systems modeled in the bond graph to the mathematical model given to a distillation column, a junction structure of the proposed bond graph is presented. Hence, this junction structure is a way to obtain the state space representation of the modeled column in bond graphs. Likewise, it is well known that distillation columns determine a class of nonlinear systems, so throughout this paper, these systems in a bond graph approach can be analyzed. In order to learn the behavior of the distillation column in the physical domain, simulation results using 20-Sim software are shown. In addition, with the simulation of two case studies consisting of two mixtures with different relative volatilities, the versatility of the column model in a bond graph is presented. In both cases, the increase in the feed flow, the mole fraction of the light component in the feed or the distillate reflux that enriches the concentration of light in the column determine an increase in the mole fraction of light in the distillate and in the bottom reflow. Further, the control design for a distillation column in the physical domain can be extended.
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This research work presents a thorough analysis of Traditional Organic Solar Cell (TOSC) and novel designed Inverted OSC (IOSC) using Bulk Hetero-Junction (BHJ) structure. Herein, 2D photovoltaic device models were used to observe the results of the semiconducting Single Wall Carbon Nanotube (s-SWCNT):C60-based organic photovoltaic. This work has improved the BHJ photodiodes by varying the active layer thickness. The analysis has been performed at various active layer thicknesses from 50 to 300 nm using the active material s-SWCNT:C60. An analysis with various parameters to determine the most effective parameters for organic photovoltaic performance has been conducted. As a result, it has been established that IOSC has the maximum efficiency of 10.4%, which is higher than the efficiency of TOSC (9.5%). In addition, the active layer with the highest efficacy has been recorded using this material for both TOSC and IOSC Nano Photodiodes (NPDs). Furthermore, the diode structure and geometrical parameters have been optimized and compared to maximize the performance of photodiodes.
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Twenty-one human genes encode connexins, a family of homologous proteins making gap junction (GJ) channels, which mediate direct intercellular communication to synchronize tissue/organ activities. Genetic variants in more than half of the connexin genes are associated with dozens of different Mendelian inherited diseases. With rapid advances in DNA sequencing technology, more variants are being identified not only in families and individuals with diseases but also in people in the general population without any apparent linkage to Mendelian inherited diseases. Nevertheless, it remains challenging to classify the pathogenicity of a newly identified connexin variant. Here, we analyzed the disease- and Genome Aggregation Database (gnomAD, as a proxy of the general population)-linked variants in the coding region of the four disease-linked α connexin genes. We found that the most abundant and position-sensitive missense variants showed distinct domain distribution preference between disease- and gnomAD-linked variants. Plotting missense variants on topological and structural models revealed that disease-linked missense variants are highly enriched on the structurally stable/resolved domains, especially the pore-lining domains, while the gnomAD-linked missense variants are highly enriched in the structurally unstable/unresolved domains, especially the carboxyl terminus. In addition, disease-linked variants tend to be on highly conserved residues and those positions show evolutionary co-variation, while the gnomAD-linked missense variants are likely on less conserved residue positions and on positions without co-variation. Collectively, the revealed distribution patterns of disease- and gnomAD-linked missense variants further our understanding of the GJ structure-biological function relationship, which is valuable for classifying the pathogenicity of newly identified connexin variants.
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Conexinas/genética , Bases de Datos Genéticas , Uniones Comunicantes/genética , Enfermedades Genéticas Congénitas/patología , Genética de Población , Mutación Missense , Secuencia de Aminoácidos , Enfermedades Genéticas Congénitas/genética , Humanos , Dominios Proteicos , Homología de SecuenciaRESUMEN
Glycine is an amino acid with unique properties because its side chain is composed of a single hydrogen atom. It confers conformational flexibility to proteins and conserved glycines are often indicative of protein domains involving tight turns or bends. All six beta-type connexins expressed in human epidermis (Cx26, Cx30, Cx30.3, Cx31, Cx31.1 and Cx32) contain a glycine at position 12 (G12). G12 is located about halfway through the cytoplasmic amino terminus and substitutions alter connexin function in a variety of ways, in some cases altering protein interactions and leading to cell death. There is also evidence that alteration of G12 changes the structure of the amino terminus in connexin- and amino acid- specific ways. This review integrates structural, functional and physiological information about the role of G12 in connexins, focusing on beta-connexins expressed in human epidermis. The importance of G12 substitutions in these beta-connexins is revealed in two hereditary skin disorders, keratitis ichthyosis and erythrokeratodermia variabilis, both of which result from missense mutations affecting G12.
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Conexinas/metabolismo , Epidermis/metabolismo , Eritroqueratodermia Variable/metabolismo , Uniones Comunicantes/metabolismo , Ictiosis/metabolismo , Mutación Missense , Sustitución de Aminoácidos , Conexinas/genética , Epidermis/patología , Eritroqueratodermia Variable/genética , Eritroqueratodermia Variable/patología , Uniones Comunicantes/genética , Uniones Comunicantes/patología , Glicina/genética , Glicina/metabolismo , Humanos , Ictiosis/genética , Ictiosis/patologíaRESUMEN
We investigated the flash light sintering process to effectively reduce electrical resistance in silver nanowire networks. The optimum condition of the flash light sintering process reduces the electrical resistance by ~20%, while the effect of the conventional thermal annealing processes is rather limited for silver nanowire networks. After flash light sintering, the morphology of the junction between the silver nanowires changes to a mixed-phase structure of the two individual nanowires. This facile and fast process for silver nanowire welding could be highly advantageous to the mass production of silver nanowire networks.
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A highly sensitive fluorometric method is described for the determination of let-7a microRNA. It is based on the use of target-triggered cascade signal amplification that involves the use of (a) catalytic hairpin assembly (CHA), (b) exonuclease-assisted signal amplification (EASA), and (c) DNA-templated fluorescent silver nanoclusters (DNA-AgNCs) having excitation/emission maxima at 535/616 nm. The CHA reaction is initiated by the hybridization of the microRNA with hairpin probe HP1. The opening of HP1 results in the assembly of HP1 and another hairpin probe (HP2) to form a duplex. This releases the microRNA that which initiates another CHA reaction. The HP1-HP2 duplex binds to hairpin probe HP3 to form a stable Y-shaped junction structure HP2-HP1-HP3. The Y-shaped junction structure is cleaved by λ exonuclease to recycle the HP1-HP2 duplex to initiate the EASA reaction. This generates a large number of single-stranded reporter sequences. These act as scaffolds for the synthesis of fluorescent AgNCs by reduction of Ag (I) ions. A remarkably amplified fluorescent signal is observed whose intensity increases linearly in the 1 fM to 10 nM let-7a microRNA concentration range. The detection limit is 0.89 fM. The method can well discriminate let-7a microRNA from other microRNAs of the same family. Graphical Abstract Schematic representation of a fluorometric method based on target-triggered cascade signal amplification and DNA-templated silver nanoclusters (DNA-AgNCs) for sensitive detection of microRNA (miRNA).
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ADN/química , Nanopartículas del Metal/química , MicroARNs/sangre , Sondas Moleculares/química , Técnicas de Amplificación de Ácido Nucleico , Plata/química , Técnicas Biosensibles , Humanos , MicroARNs/análisis , Espectrometría de FluorescenciaRESUMEN
Most of the early studies on gap junction (GJ) channel function and docking compatibility were on rodent connexins, while recent research on GJ channels gradually shifted from rodent to human connexins largely due to the fact that mutations in many human connexin genes are found to associate with inherited human diseases. The studies on human connexins have revealed some key differences from those found in rodents, calling for a comprehensive characterization of human GJ channels. Functional studies revealed that docking and formation of functional GJ channels between two hemichannels are possible only between docking-compatible connexins. Two groups of docking-compatible rodent connexins have been identified. Compatibility is believed to be due to their amino acid residue differences at the extracellular loop domains (E1 and E2). Sequence alignment of the E1 and E2 domains of all connexins known to make GJs revealed that they are highly conserved and show high sequence identity with human Cx26, which is the only connexin with near atomic resolution GJ structure. We hypothesize that different connexins have a similar structure as that of Cx26 at the E1 and E2 domains and use the corresponding residues in their E1 and E2 domains for docking. Based on the Cx26 GJ structure and sequence analysis of well-studied connexins, we propose that the E1-E1 docking interactions are staggered with each E1 interacting with two E1s on the docked connexon. The putative E1 docking residues are conserved in both docking-compatible and -incompatible connexins, indicating that E1 does not likely serve a role in docking compatibility. However, in the case of E2-E2 docking interactions, the putative docking residues are only conserved within the docking-compatible connexins, suggesting the E2 is likely to serve the function of docking compatibility. Docking compatibility studies on human connexins have attracted a lot of attention due to the fact that putative docking residues are mutational hotspots for several connexin-linked human diseases. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.
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Conexinas/química , Simulación del Acoplamiento Molecular , Secuencias de Aminoácidos , Animales , Conexina 26 , Conexinas/genética , Conexinas/metabolismo , Humanos , Mutación , Dominios ProteicosRESUMEN
MicroRNAs (miRNAs) are vital for many biological processes and have been regarded as cancer biomarkers. Specific and sensitive detection of miRNAs is essential for cancer diagnosis and therapy. Herein, a split recognition mode combined with cascade signal amplification strategy is developed for highly specific and sensitive detection of miRNA. The split recognition mode possesses two specific recognition processes, which are based on toehold-mediated strand displacement reaction (TSDR) and direct hybridization reaction. Two recognition probes, hairpin probe (HP) with overhanging toehold domain and assistant probe (AP), are specially designed. Firstly, the toehold domain of HP and AP recognize part of miRNA simultaneously, accompanied with TSDR to unfold the HP and form the stable DNA Y-shaped junction structure (YJS). Then, the AP in YJS can further act as primer to initiate strand displacement amplification, releasing numerous trigger sequences. Finally, the trigger sequences hybridize with padlock DNA to initiate circular rolling circle amplification and generate enhanced fluorescence responses. In this strategy, the dual recognition effect of split recognition mode guarantees the excellent selectivity to discriminate let-7b from high-homology sequences. Furthermore, the high amplification efficiency of cascade signal amplification guarantees a high sensitivity with the detection limit of 3.2 pM and the concentration of let-7b in total RNA sample extracted from Hela cells is determined. These results indicate our strategy will be a promising miRNA detection strategy in clinical diagnosis and disease treatment.
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Sondas de ADN/genética , MicroARNs/análisis , MicroARNs/genética , ARN Neoplásico/análisis , ARN Neoplásico/genética , Análisis de Secuencia de ARN/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Células HeLa , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ARN/métodosRESUMEN
Aberrant expression of micro RNA (miRNA) is associated with development of cancers and diseases, so miRNA has become a tissue-based biomarker for cancer prognosis and diagnosis. Herein, a novel trigger-assisted exponential enzymatic amplification (T-EXPEA) method for ultrasensitive miRNA detection based on three-way junction (3-WJ) structure driven has been reported, which can be used in potential applications in cancer prognosis and diagnoses. In this assay, target miRNA can unfold hairpin probe and start the reaction, and thus specifically form stable 3-WJ structure with helper. Then it can produce triggers under the synergetic polymerase and restriction endonucleases amplification. The produced triggers could be used to unfold molecular beacon (MB) and initiate T-EXPEA process. In the EXPEA part, the exponential triggers were generated to initiate new T-EXPEA and high enhancement fluorescence amplification efficiency was obtained. The feature of our strategy lies in the T-EXPEA combining with 3-WJ structure has been utilized for fluorescence miRNA detection. It is worth noting that the sequence of the triggers in T-EXPEA part is the same to that of triggers generated from the 3-WJ part. In addition, the design of restriction enzyme cutting sites using the same restriction enzyme (Nt.BbvCI) in hairpin probe and MB respectively, improved reaction efficiency cost-efficiently. This method can quantitatively detect sequence-specific miRNA in a dynamic range from 10 aM to 10 pM with a detection limit as low as 7.8 aM.
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Técnicas Biosensibles/métodos , MicroARNs/análisis , Técnicas de Amplificación de Ácido Nucleico/métodos , Enzimas de Restricción del ADN/química , ARN Polimerasas Dirigidas por ADN/química , Fluorescencia , Células Hep G2 , Humanos , Límite de Detección , Conformación de Ácido Nucleico , Espectrometría de Fluorescencia/métodosRESUMEN
Taking advantage of "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification, a dual-probe electrochemical DNA (DE-DNA) biosensor was designed to detect double-stranded DNA (dsDNA) of acute promyelocytic leukemia (APL) related gene. Two groups of detection probes were designed, and each group was composed of a biotinylated capture probe and an assisted probe. They were separately complementary with two strands of target dsDNA in order to prevent the reannealing of the two separate strands from target dsDNA. First, thiol functionalized capture probes (C1 and C2) were severally assembled onto two different gold electrodes, followed by hybridizing with target dsDNA (S1a-S1b) and assistant probes to form two Y-junction-structure ternary complexes. Subsequently, restriction sites on the ternary complexes were digested by Rsa I, which can release S1a, S1b and biotins from the electrode surfaces. Meanwhile, the released S1a and S1b can further hybridize with the unhybridized corresponding detection probes and then initiate another new hybridization-cleavage-separation cycle. Finally, the current signals were produced by the enzyme-catalyzed reaction of streptavidin-horse reddish peroxidase (streptavidin-HRP). The distinct difference in current signals between different sequences allowed detection of target dsDNA down to a low detection limit of 47 fM and presented excellent specificity with discriminating only a single-base mismatched dsDNA sequence. Moreover, this biosensor was also used for assay of polymerase chain reaction (PCR) samples with satisfactory results. According to the results, the power of the DE-DNA biosensor as a promising tool for the detection of APL and other diseases.