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Methanol-gasoline blends have emerged as a promising and environmentally friendly bio-fuel option, garnering widespread attention and promotion globally. The methanol content within these blends significantly influences their quality and combustion performance. This study explores the qualitative and qualitative analysis of methanol-gasoline blends using Raman spectroscopy coupled with machine learning methods. Experimentally, methanol-gasoline blends with varying methanol concentrations were artificially configured, commencing with initial market samples. For qualitative analysis, the partial least squares discriminant analysis (PLS-DA) model was employed to classify the categories of blends, demonstrating high prediction performance with an accuracy of nearly 100% classification. For the quantitative analysis, a consensus model was proposed to accurately predict the methanol content. It integrates member models developed on clustered variables, using the unsupervised clustering method of the self-organizing mapping neural network (SOM) to accomplish the regression prediction. The performance of this consensus model was systemically compared to that of the PLS model and uninformative variable elimination (UVE)-PLS model. Results revealed that the unsupervised consensus model outperformed other models in predicting the methanol content across various types of methanol gasoline blends. The correlation coefficients for prediction sets consistently exceeded 0.98. Consequently, Raman spectroscopy emerges as a suitable choice for both qualitative and quantitative analysis of methanol-gasoline blend quality. This study anticipates an increasing role for Raman spectroscopy in analysis of fuel composition.
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BACKGROUND: Exosomes, a special subtype of extracellular vesicles derived from human cells, serve as vital mediators of intercellular communication by transporting diverse bioactive cargos, including proteins and enzymes. However, the underlying mechanisms governing exosome secretion and regulation remain poorly understood. In this study, we employed a dual-reporter system consisting of bioluminescent Gaussia luciferase and fluorescent proteins to investigate the dynamics and regulation of exosome secretion in cultured human cells. RESULTS: Our results demonstrated that the engineered dual-reporters effectively monitored both exosome-mediated and ER-Golgi-mediated secretory pathways in a specific and quantitative manner. Notably, we observed distinct characteristics of exosome-mediated protein secretion, including significantly lower capacity and different dynamics compared to the ER-Golgi pathway. This phenomenon was observed in human kidney 293T cells and liver HepG2 cells, emphasizing the conserved nature of exosome-mediated secretion across cell types. Furthermore, we investigated the impact of brefeldin A (BFA), an inhibitor of ER-to-Golgi membrane trafficking, on protein secretion. Interestingly, BFA inhibited protein secretion via the ER-Golgi pathway while stimulating exosome-mediated protein secretion under same experimental conditions. CONCLUSIONS: Collectively, our study highlights the utility of the dual-reporter system for real-time monitoring and quantitative analysis of protein secretion through conventional ER-Golgi and unconventional exosome pathways. Moreover, our findings unveil distinct features of exosome-mediated protein secretion, shedding light on its differential capacity, dynamics, and regulatory mechanisms compared to ER-Golgi-mediated proteins in human cells.
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By rationally adjusting the weaving modes of point-star tiles, the curvature inherent in the tiles can be changed, and various DNA nanostructures can be assembled, such as planar wireframe meshes, perforated wireframe tubes, and curved wireframe polyhedra. Based on the weaving and tiling architectures for traditional point-star tiles with the core arm length at two DNA half-turns, we improved the weaving modes of our newly reported four-point-star tiles with the core arm length at three half-turns to adjust their curvature and rigidity for assembling 2D arrays of DNA grids and tubes. Following our previous terms and methods to analyze the structural details of E-tiling tubes, we used the chiral indices (n,m) to describe the most abundant tube of typical assemblies; especially, we applied both one-locus and/or dual-locus biotin/streptavidin (SA) labelling strategies to define the configurations of two specific tubes, along with the absolute conformations of their component tiles. Such structural details of the DNA tubes composed of tiles with addressable concave and convex faces and packing directions should help us understand their physio-chemical and biological properties, and therefore promote their applications in drug delivery, biocatalysis, biomedicine, etc.
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Brazo , Nanoestructuras , Conformación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Nanoestructuras/química , ADN/química , Nanotecnología/métodosRESUMEN
BACKGROUND: Staphylococcus arlettae is a rarely reported coagulase-negative staphylococcus (CoNS) isolated from infected humans and livestock. Observing phage-bacteria interaction could improve the understanding of bacterial pathogenetic mechanisms, providing foundational evidence for phage therapy or phage detection. Herein, we aimed to characterise and annotate a novel bacteriophage, vB_SarS_BM31 (BM31), specific to S. arlettae. This bacteriophage was isolated from a milk sample associated with bovine mastitis and collected in the Sichuan Province, China. RESULTS: The BM31 genome comprised a linear double-stranded DNA of 42,271 base pair in length with a G + C content of 34.59%. A total of 65 open reading frames (ORFs) were assembled from phage DNA, of which 29 were functionally annotated. These functional genes were divided into four modules: the structural, DNA packing and replication, lysis, and lysogeny modules. Holin (ORF25), lysin (ORF26), and integrase (ORF28) were located closely in the entire BM31 genome and were important for lyse or lysogeny cycle of BM31. The phage was identified as a temperate phage according to whole genome analysis and life cycle assay, with basic biological characteristics such as small burst size, short latency period, and narrow host range, consistent with the characteristics of the family Siphoviridae, subcluster B14 of the Staphylococcus bacteriophage. CONCLUSIONS: The present isolation and characterisation of BM31 contributes to the Staphylococcus bacteriophage database and provides a theoretical foundation for its potential applications. To the best of our knowledge, BM31 is the only shared and completely reported phage against S. arlettae in the entire public database.
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ADN Viral , Genoma Viral , Animales , Bovinos , Femenino , Humanos , Análisis de Secuencia de ADN , ADN Viral/genética , ADN Viral/química , Staphylococcus/genética , Fagos de Staphylococcus/genéticaRESUMEN
PURPOSE: To friendly predict a reference prognostic outcome for idiopathic sudden sensorineural hearing loss (ISSNHL) patients with or without anxiety, we identified independent prognostic factors and developed practical predictive tools without invasive tests. METHODS: Patients with ISSNHL in our center were enrolled from June 2013 to December 2018. Univariate and multivariate logistic regression analyses were performed to identify independent prognostic factors of the complete recovery and the overall recovery for ISSNHL, which were subsequently utilized to develop the web nomograms. The discrimination, calibration, and clinical benefit were used to evaluate the performance of nomograms for ISSNHL. RESULTS: 704 ISSNHL patients were finally enrolled in this study. Multivariate logistic regression analysis showed that age, time of onset, gender, affected ear, degree, and type of hearing loss were independent prognostic factors of complete recovery. Age, time of onset, affected ear, and type of hearing loss were independent prognostic factors of overall recovery. Web predictive nomograms were developed with excellent discrimination, calibration, and clinical value. CONCLUSION: Based on the patients' data with a considerable size, independent noninvasive prognostic factors of complete recovery and overall recovery of ISSNHL were identified. Integrating these prognostic factors without invasive tests, practical web predictive nomograms were developed. Using web nomograms, clinical doctors could provide reference data (the predicted recovery rate) for supporting prognostic consultation of ISSNHL patients, especially those with anxiety.
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Sordera , Pérdida Auditiva Sensorineural , Pérdida Auditiva Súbita , Humanos , Pronóstico , Estudios Retrospectivos , Pérdida Auditiva Sensorineural/diagnóstico , Pérdida Auditiva Súbita/diagnósticoRESUMEN
The overwinding and underwinding of DNA duplexes between junctions have been used in designing left- and right-handed DNA origami nanostructures, respectively. For DNA tubes obtained from self-assembled tiles, only a theoretical approach of the intrinsic curvature of the tiles has been previously used to explain their formation. Details regarding the quantitative and structural descriptions of the tile's intrinsic curvature in DNA nanostructures have so far never been addressed. In this work, we designed three types of tile cores built around a circular scaffold using three- and four-branched junctions. Joining the tile cores with arms having two kinds of inter-tile distances, an odd and an even number of DNA half-turns, tended to form planar 2D lattices and tubes, respectively. Streptavidin bound to biotin was used as a labeling technique to characterize the inside and outside surfaces of the tubes and thereby the tile conformation of dihedrals with addressable faces. DNA tubes with either right- or left-handed chirality were obtained by the coupling of the intrinsic curvature of the tiles with the arm twist. We were able to assign the chiral indices (n,m) to a tube with its structure resolved by AFM at the single-tile level and therefore to estimate the global curvature of the tube (or its component tile) using a regular polygon model that approximated its transverse section. A deeper understanding of the integrated actions of different types of twisting forces on DNA tubes will be extremely helpful in engineering more elaborate DNA nanostructures in the future.
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Nanoestructuras , ADN/química , Microscopía de Fuerza Atómica , Nanoestructuras/química , Nanotecnología/métodos , Conformación de Ácido Nucleico , InvestigaciónAsunto(s)
Recurrencia , Tonsilectomía , Tonsilitis , Humanos , Tonsilitis/cirugía , Niño , Resultado del TratamientoRESUMEN
OBJECTIVE: To study the role of gamma-delta T (γδâ T) cells and its subsets in the immunopathogenesis of Henoch-Schönlein purpura (HSP) in children, and to provide new ideas for the treatment of HSP in children from the aspect of γδ T cell regulation. METHODS: A total of 33 children with HSP were enrolled as the HSP group, and 21 healthy children were enrolled as the healthy control group. The percentages of γδâ T cells and its subsets Vδ1+â T and Vδ2+â T cells among peripheral blood mononuclear cells (PBMCs) were measured, as well as the apoptosis rate of γδâ T cell and plasma level of interleukin-17 (IL-17). RESULTS: Compared with the healthy control group, the HSP group had significantly lower percentages of lymphocytes in PBMCs and Vδ2+â T cells in γδâ T cells (P<0.05). The HSP group had significantly higher percentage of Vδ1+â T cells in γδâ T cells and plasma level of IL-17 than the healthy control group. The HSP group had a significantly higher overall apoptosis rate of γδâ T cells than the healthy control group (P<0.05), especially early apoptosis. The percentage of Vδ2+â T cells was positively correlated with overall apoptosis rate (rs=0.615, P<0.05) and was negatively correlated with IL-17 level (rs=-0.398, P<0.05). CONCLUSIONS: Vδ1+/Vδ2+â T cell immune imbalance mediated by γδâ T cells and over-activation of IL-17 may be involved in the development of HSP, among which the disturbance of immune tolerance induced by Vδ2+â T cells plays an important role in the pathophysiology of the disease.
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Vasculitis por IgA , Linfocitos T , Niño , Humanos , Leucocitos Mononucleares , Receptores de Antígenos de Linfocitos T gamma-deltaRESUMEN
A novel series of benzodihydrofuran derivatives was developed as potent MEK inhibitors through scaffold hopping based on known clinical compounds. Further SAR exploration and optimization led to another benzofuran series with good oral bioavailability in rats. One of the compounds EBI-1051 (28d) demonstrated excellent in vivo efficacy in colo-205 tumor xenograft models in mouse and is suitable for pre-clinical development studies for the treatment of melanoma and MEK associated cancers. Compared to AZD6244, EBI-1051 showed superior potency in some cancer cell lines such as colon-205, A549 and MDA-MB-231.
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Benzofuranos/química , Benzofuranos/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Benzofuranos/administración & dosificación , Benzofuranos/farmacocinética , Benzofuranos/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Trasplante HeterólogoAsunto(s)
Complicaciones Posoperatorias , Tonsilectomía , Niño , Humanos , Método Doble Ciego , Dolor Postoperatorio , Náusea y Vómito Posoperatorios/etiología , Periodo Posoperatorio , Tonsilectomía/efectos adversos , Tonsilectomía/mortalidad , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/mortalidadRESUMEN
Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell-cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.
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Técnicas de Visualización de Superficie Celular/métodos , Exosomas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Nanopartículas , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de la Célula Individual/métodos , Tetraspanina 30/química , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Vesículas Transportadoras/metabolismo , Proteína Fluorescente RojaRESUMEN
BACKGROUND: A number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance efficiency of different mutants on virus packaging and gene expression has not been done. RESULTS: Site-directed mutagenesis was used to generate five integrasedeficient mutants for non-integrative lentiviral packaging (NILVP). The five mutants were then individually incorporated to make different integrase defective lentivirus plasmid packaging mix, keeping other packaging factors constant. CD511B-1, a lentivectorexpressing GFP from an EF1 promoter, was packaged with each of the five different lentivirus packaging mix to make pseudotypedviral particles. The performance and packaging efficiency of each of the integrase deficient mutants was evaluated based on GFP expression in HT1080 cells, while the wild type lentivirus packaging mix was used as a control. Of the five integrase mutant candidates, one with the highestGFP transgene expression level was chosen for further characterization. The non-integrative nature of this candidate was confirmed by quantitative polymerase chain reaction and characterized using both dividing and non-dividing cells. Finally, a detailed standard protocol for NILVP using this integrase defective mutant was developed. CONCLUSIONS: An efficient lentiviral packaging system for producing on-integrative lentivirus was established. This system is compatible with most existing lentivectors and can be used to transduce both dividing and non-dividing cells.
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Significant progress in the functional understanding of microRNAs (miRNAs) has been made in mice, but a need remains to develop efficient tools for bi-allelic knockouts of miRNA in the human genome. Transcription activator-like effector nucleases (TALENs) provide an exciting platform for targeted gene ablation in cultured human cells, but bi-allelic modifications induced by TALENs alone occur at low frequency, making screening for double knockouts a tedious task. Here, we present an approach that is highly efficient in bi-allelic miRNA ablation in the human genome by combining TALENs targeting to the miRNA seed region with a homologous recombination donor vector and a positive selection strategy. A pilot test of this approach demonstrates bi-allelic miR-21 gene disruption at high frequency (â¼87%) in cultured HEK293 cells. Analysis of three independent clones showed a total loss of miR-21 expression. Phenotypical analysis revealed increased miR-21 target gene expression, reduced cell proliferation, and alterations of global miRNA expression profiles. Taken together, our study reveals a feasible and efficient approach for bi-allelic miRNA ablation in cultured human cells and demonstrates its usefulness in elucidating miRNA function in human cells.
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Esterasas/genética , Genoma Humano/genética , MicroARNs/genética , Activación Transcripcional/genética , Alelos , Proliferación Celular , Técnicas de Inactivación de Genes/métodos , Marcación de Gen/métodos , Vectores Genéticos/genética , Células HEK293 , Humanos , Proyectos Piloto , Transcriptoma/genéticaRESUMEN
Hollow Au-Cu2O core-shell nanoparticles were synthesized by using hollow gold nanoparticles (HGNs) as the plasmon-tailorable cores to direct epitaxial growth of Cu2O nanoshells. The effective geometry control of hollow Au-Cu2O core-shell nanoparticles was achieved through adjusting the HGN core sizes, Cu2O shell thicknesses, and morphologies related to structure-directing agents. The morphology-dependent plasmonic band red-shifts across the visible and near-infrared spectral regions were observed from experimental extinction spectra and theoretical simulation based on the finite-difference time-domain method. Moreover, the hollow Au-Cu2O core-shell nanoparticles with synergistic optical properties exhibited higher photocatalytic performance in the photodegradation of methyl orange when compared to pristine Cu2O and solid Au-Cu2O core-shell nanoparticles under visible-light irradiation due to the efficient photoinduced charge separation, which could mainly be attributed to the Schottky barrier and plasmon-induced resonant energy transfer. Such optical tunability achieved through the hollow cores and structure-directed shells is of benefit to the performance optimization of metal-semiconductor nanoparticles for photonic, electronic, and photocatalytic applications.
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A novel series of pyrazolo[3,4-c]isoquinoline derivatives was discovered as B-Raf(V600E) inhibitors through scaffold hopping based on a literature lead PLX4720. Further SAR exploration and optimization led to the discovery of potent B-Raf(V600E) inhibitors with good oral bioavailability in rats and dogs. One of the compounds EBI-907 (13g) demonstrated excellent in vivo efficacy in B-Raf(V600E) dependent Colo-205 tumor xenograft models in mouse and is under preclinical studies for the treatment of melanoma and B-Raf(V600E) associated cancers.
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Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Administración Oral , Animales , Sitios de Unión , Línea Celular Tumoral , Perros , Evaluación Preclínica de Medicamentos , Semivida , Humanos , Isoquinolinas/química , Isoquinolinas/farmacocinética , Isoquinolinas/uso terapéutico , Melanoma/tratamiento farmacológico , Ratones , Conformación Molecular , Simulación de Dinámica Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pirazoles/química , Pirazoles/farmacocinética , Pirazoles/uso terapéutico , Ratas , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
Luciferase assay has become an increasingly important technique to monitor a wide range of biological processes. However, the mainstay protocols require a luminometer to acquire and process the data, therefore limiting its application to specialized research labs. To overcome this limitation, we have developed an alternative protocol that utilizes a commonly available cooled charge-coupled device (CCCD), instead of a luminometer for data acquiring and processing. By measuring activities of different luciferases, we characterized their substrate specificity, assay linearity, signal-to-noise levels, and fold-changes via CCCD. Next, we defined the assay parameters that are critical for appropriate use of CCCD for different luciferases. To demonstrate the usefulness in cultured mammalian cells, we conducted a case study to examine NFκB gene activation in response to inflammatory signals in human embryonic kidney cells (HEK293 cells). We found that data collected by CCCD camera was equivalent to those acquired by luminometer, thus validating the assay protocol. In comparison, The CCCD-based protocol is readily amenable to live-cell and high-throughput applications, offering fast simultaneous data acquisition and visual and quantitative data presentation. In conclusion, the CCCD-based protocol provides a useful alternative for monitoring luciferase reporters. The wide availability of CCCD will enable more researchers to use luciferases to monitor and quantify biological processes.
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Luciferasas de Luciérnaga/análisis , Luciferasas de Renilla/análisis , Sustancias Luminiscentes/análisis , Mediciones Luminiscentes/instrumentación , Animales , Luciérnagas/enzimología , Genes Reporteros , Células HEK293 , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Luciferasas de Luciérnaga/genética , Luciferasas de Renilla/genética , Sustancias Luminiscentes/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Renilla/enzimología , TransfecciónRESUMEN
OBJECTIVE: To evaluate the value of MDCT on diagnosis of congenital vascular rings on children. METHODS: Retrospective analysis on 43 cases of congenital vascular rings, which underwent MDCT during Oct 2008 to Dec 2014 in Beijing Anzhen hospital affiliated to capital medical university. 21 males, 22 females; age from 29 days to 8 years, mean age 1.46 years, 33 cases are not beyond 1 year. All the results were compared with that of the echocardiogram or record of the surgery. The CT data were read and reconstructed with multiplanar reconstruction (MPR), maximum intensity projection (MPR), minimum intensity projection (MinIP), volume rendering (VR). The image quality was evaluated and the diagnostic value and the standard diagnostic program were discussed. RESULTS: Of 43 cases of vascular rings:there were 6 cases of pulmonary artery sling (13.95%), 9 cases of right aortic arch /aberrant left subclavian artery(20.93%), 18 cases of left aortic arch/aberrant right subclavian artery (41.86%), 10 cases of double aortic arch (23.26%). Forty cases (93.02%) were combined with other cardiovascular or pulmonary malformations. Every malformation was revealed clearly and proved by echocardiogram. Of 3 cases (6.98%) without any other malformation, 2 cases were combined tracheal stenosis. A pulmonary artery sling was proved by surgery; the other 2 cases were double aortic arch. All the images of 43 cases could be reconstructed well. MPR and VR showed the origin, shape, and whole course of vascular rings directly; MinIP and VR could display the shape, width and development of trachea, revealed the relationship between vascular rings, trachea and esophagus. It was important to show and measure the component vascular of the ring. Attention should be paid to the whole course of trachea and esophagus, especially those segments which were close to the ring vascular. The tracheal stenosis as well as intra-cardio anatomy malformations should be measured on MPR images if existed. According to the segmental analysis method, comes the overall final diagnosis. A standard diagnostic program on vascular ring was proposed. CONCLUSION: MDCT axis images with various 3D post processing methods could reveal the compose of vascular rings and the relationship between vascular rings, trachea and esophagus.
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Síndromes del Arco Aórtico , Aorta Torácica , Beijing , Niño , Preescolar , Ecocardiografía , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Estudios Retrospectivos , Tomografía Computarizada por Rayos X , Tráquea , Estenosis TraquealRESUMEN
Despite their fundamental importance, the dynamics of signaling pathways in living cells remain challenging to study, due to a lack of non-invasive tools for temporal assessment of signal transduction in desired cell models. Here we report a dual-reporter strategy that enables researchers to monitor signal transduction in mammalian cells in real-time, both temporally and quantitatively. This is achieved by co-expressing green fluorescent protein and firefly luciferase in response to signaling stimuli. To display the versatility of this approach, we constructed and assessed eight unique signaling pathway reporters. We further validated the system by establishing stable NF-κB pathway reporter cell lines. Using these stable cell lines, we monitored the activity of NF-κB-mediated inflammatory pathway in real-time, both visually and quantitatively. Live visualization has the power to reveal individual cell responses and is compatible with single cell analysis, In addition, we provide evidence that this system is readily amenable to a high-throughput format. Together, our findings demonstrate the potential of the dual reporter system, which significantly improves the capacity to study signal transduction pathways in mammalian cells.