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ß-Branched chiral amines with contiguous stereocenters are valuable building blocks for preparing various biologically active molecules. However, their asymmetric synthesis remains challenging. Herein, we report a highly diastereo- and enantioselective biocatalytic approach for preparing a broad range of ß-branched chiral amines starting from their corresponding racemic ketones. This involves a dynamic kinetic resolution-asymmetric reductive amination process catalyzed using only an imine reductase. Four rounds of protein engineering endowed wild-type PocIRED with higher reactivity, better stereoselectivity, and a broader substrate scope. Using the engineered enzyme, various chiral amine products were synthesized with up to >99.9% ee, >99:1 dr, and >99% conversion. The practicability of the developed biocatalytic method was confirmed by producing a key intermediate of tofacitinib in 74% yield, >99.9% ee, and 98:2 dr at a challenging substrate loading of 110 g L-1. Our study provides a highly capable imine reductase and a protocol for developing an efficient biocatalytic dynamic kinetic resolution-asymmetric reductive amination reaction system.
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The composition of protection monolayer exerts great influence on the molecular and electronic structures of atomically precise monolayer protected metal nanoclusters. Four isostructural Ag/cyanurate/phosphine metallamacrocyclic monolayer protected Ag22 nanoclusters are synthesized by kinetically controlled in-situ ligand formation-driven strategy. These eight-electron superatomic silver nanoclusters feature an unprecedented interfacial bonding structure with diverse E-Ag (E=O/N/P/Ag) interactions between the Ag13 core and metallamacrocyclic monolayer, and displays thermally activated delayed fluorescence (TADF), benefiting from their distinct donor-acceptor type electronic structures. This work not only unmasks a new core-shell interface involving cyanurate ligand but also underlines the significance of high-electron-affinity N-heterocyclic ligand in synthesizing TADF metal nanoclusters. This is the first mixed valence Ag0/I nanocluster with TADF characteristic.
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Linezolid is the first synthetic oxazolidone agent to treat infections caused by Gram-positive pathogens. Infected patients with liver dysfunction (LD) are more likely to suffer from adverse reactions, such as thrombocytopenia, when standard-dose linezolid is used than patients with LD who did not use linezolid. Currently, pharmacokinetics data of linezolid in patients with LD are limited. This study aimed to characterize pharmacokinetics parameters of linezolid in patients with LD, identify the factors influencing the pharmacokinetics, and propose an optimal dosage regimen. We conducted a prospective study and established a population pharmacokinetics model with the Phoenix NLME software. The final model was evaluated by goodness-of-fit plots, bootstrap analysis, and prediction corrected-visual predictive check. A total of 163 concentration samples from 45 patients with LD were adequately described by a one-compartment model with first-order elimination along with prothrombin activity (PTA) and creatinine clearance as significant covariates. Linezolid clearance (CL) was 2.68 liters/h (95% confidence interval [CI], 2.34 to 3.03 liters/h); the volume of distribution (V) was 58.34 liters (95% CI, 48.00 to 68.68 liters). Model-based simulation indicated that the conventional dose was at risk for overexposure in patients with LD or severe renal dysfunction; reduced dosage (300 mg/12 h) would be appropriate to achieve safe (minimum steady-state concentration [Cmin,ss] at 2 to 8 µg/ml) and effective targets (the ratio of area under the concentration-time curve from 0 to 24 h [AUC0-24] at steady state to MIC, 80 to 100). In addition, for patients with severe LD (PTA, ≤20%), the dosage (400 mg/24 h) was sufficient at an MIC of ≤2 µg/ml. This study recommended therapeutic drug monitoring for patients with LD. (This study has been registered in the Chinese Clinical Trial Registry under no. ChiCTR1900022118.).
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Monitoreo de Drogas , Hepatopatías , Área Bajo la Curva , Humanos , Linezolid , Hepatopatías/tratamiento farmacológico , Estudios ProspectivosRESUMEN
Microglia are the primary immune cells in the central nervous system with functional plasticity. They can be activated into M1 and M2 phenotypes when neuroinflammation-related diseases occur. M1 phenotype cells produce pro-inflammatory mediators that cause neuroinflammation and the M2 phenotype can secrete anti-inflammatory cytokines that protect neurons from damage. Therefore, inhibiting the M1 phenotype while stimulating the M2 phenotype has been suggested as a potential therapeutic approach for treating neuroinflammation-related diseases. Puerarin has been demonstrated to exert anti-inflammatory and neuroprotective effects. However, the role of puerarin in regulating microglia polarization and its reaction mechanism has not been fully elucidated. In this paper, a metabolomics approach with ultra-performance liquid chromatography-mass spectrometry was performed to investigate the metabolic changes of BV-2 cells in different phenotypes and test the effects of puerarin on polarization. Thirty-nine metabolites were identified as the biomarkers related to the polarization of BV-2 cells and puerarin intervention reverted the content of most of the biomarkers. Our study demonstrated that puerarin could play a key role in M1/M2 polarization of BV-2 cells from a perspective of metabolomics, and it could regulate the balance between promotion and suppression of inflammation.
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Polaridad Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión/métodos , Isoflavonas/farmacología , Espectrometría de Masas/métodos , Metaboloma/efectos de los fármacos , Aminoácidos/análisis , Aminoácidos/metabolismo , Animales , Línea Celular , Glicerofosfolípidos/análisis , Glicerofosfolípidos/metabolismo , Metabolómica , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Esfingolípidos/análisis , Esfingolípidos/metabolismoRESUMEN
γδ T cells are a unique population of lymphocytes that have regulatory roles in patients with chronic hepatitis B (CHB); however, their role in acute hepatitis B (AHB) infection remains unclear. Phenotype and function of γδ T cells were analyzed in 29 AHB patients, 28 CHB patients, and 30 healthy controls (HCs) using immunofunctional assays. Compared with HCs and CHB patients, decreased peripheral and increased hepatic γδ T cells were found in AHB patients. Increased hepatic γδ T cells in AHB patients were attributed to elevated hepatic chemokine levels. Peripheral γδ T cells exhibited highly activated and terminally differentiated memory phenotype in AHB patients. Consistently, peripheral γδ T cells in AHB patients showed increased cytotoxic capacity and enhanced antiviral activity which was further proved in longitudinal study. Activated γδ T cells in AHB patients exhibited increased cytotoxicity and capacity for viral clearance associated with liver injury and the control of infection.
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Hepatitis B/inmunología , Linfocitos Intraepiteliales/inmunología , Enfermedad Aguda , Adolescente , Adulto , Quimiocinas/inmunología , Enfermedad Crónica , ADN Viral/análisis , Femenino , Hepatitis B/virología , Virus de la Hepatitis B/genética , Humanos , Hígado/inmunología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Ischemic heart diseases (IHDs) cause great morbidity and mortality worldwide, necessitating effective treatment. Salvianic acid A sodium (SAAS) is an active compound derived from the well-known herbal medicine Danshen, which has been widely used for clinical treatment of cardiovascular diseases in China. This study aimed to confirm the cardioprotective effects of SAAS in rats with myocardial infarction and to investigate the underlying molecular mechanisms based on proteome and transcriptome profiling of myocardial tissue. The results showed that SAAS effectively protected against myocardial injury and improved cardiac function. The differentially expressed proteins and genes included important structural molecules, receptors, transcription factors, and cofactors. Functional enrichment analysis indicated that SAAS participated in the regulation of actin cytoskeleton, phagosome, focal adhesion, tight junction, apoptosis, MAPK signaling, and Wnt signaling pathways, which are closely related to cardiovascular diseases. SAAS may exert its cardioprotective effect by targeting multiple pathways at both the proteome and transcriptome levels. This study has provided not only new insights into the pathogenesis of myocardial infarction but also a road map of the cardioprotective molecular mechanisms of SAAS, which may provide pharmacological evidence to aid in its clinical application.
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Cardiotónicos/uso terapéutico , Lactatos/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Proteoma/metabolismo , Transcriptoma/efectos de los fármacos , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Perfilación de la Expresión Génica , Corazón/efectos de los fármacos , Masculino , Miocardio/patología , Mapeo de Interacción de Proteínas , Proteómica , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
In this paper,the design and development of sustained and controlled release preparations in traditional Chinese medicine( TCM) based on quality by design( QbD) was put forward,aiming at the current situation that the final product quality is affected by multiple factors during the development of sustained and controlled release preparations in TCM. The important development of sustained and controlled release preparations in western medicine in recent years was summarized. According to the complex process of TCM,the concept of QbD was proposed to design and develop sustained and controlled release preparations in TCM. QbD concept was used to analyze the complex factors affecting sustained and controlled release preparations in TCM,and determine the high risk factors affecting the quality of the final product. The high risk factors were controlled from the process to achieve the goal of quality control.This article could provide research ideas for the sustained and controlled release preparations with complex components in TCM,so as to promote the research and development of sustained and controlled release preparations in TCM.
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Medicamentos Herbarios Chinos , Medicina Tradicional China , Preparaciones de Acción Retardada , Control de CalidadRESUMEN
The stable quality of hospital preparations is the basis for their clinical efficacy. Gynecological antipruritic prescription is widely used in gynecology clinics of Chinese medicine hospitals. Therefore,in this study,the production process of gynecological antipruritic lotion was optimized based on the concept of quality by design( QbD). The production process of the gynecological antipruritic lotion was developed to ensure its process stability and reliable quality,and enhance its clinical applicability. With total amount of matrine and oxymatrine used as the critical quality attribute( CQA) of the production process,parameter levels were designed based on production practice of hospital preparations,and Plackett-Burman and Box-Behnken experiments were used to optimize the water extraction and alcohol precipitation process of antipruritic lotion based on CQA of intermediates and final product. The soaking time,the first extraction time,and the second extraction time were determined as the critical process parameters( CPPs) of the production process. The optimal preparation process was as follows: water volume of 8 times,soaking for 0. 5 h,extraction for 2 times,the first extraction for 30 min,the second extraction for 56 min,alcohol concentration of 50%,and alcohol precipitation for 3 h. Furthermore,the design space was established based on the binomial regress model between CPPs and CQA,so as to set the optimization target and risk range; and the control space was displayed by overlay plot. The results of three repeated experiments in the control space showed that the relative standard deviation( RSD) of CQA was 4. 70%,and the similarity of chromatogram for gynecological antipruritic lotion was 0. 978,0. 974,and 0. 998,respectively. The above results indicated that the operation in the control space can guarantee the quality and stability of gynecological antipruritic lotion,suitable for practical application.
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Antipruriginosos , Medicamentos Herbarios Chinos , AguaRESUMEN
Cadmium is considered one of the most harmful carcinogenic heavy metals in the human body. Although many scientists have performed research on cadmium toxicity mechanism, the toxicokinetic process of cadmium toxicity remains unclear. In the present study, the kinetic response of proteome in/and A549 cells to exposure of exogenous cadmium was profiled. A549 cells were treated with cadmium sulfate (CdSO4 ) for different periods and expressions of proteins in cells were detected by two-dimensional gel electrophoresis. The kinetic expressions of proteins related to cadmium toxicity were further investigated by reverse transcription-polymerase chain reaction and western blotting. Intracellular cadmium accumulation and content fluctuation of several essential metals were observed after 0-24 hours of exposure by inductively coupled plasma mass spectrometry. Fifty-four protein spots showed significantly differential responses to CdSO4 exposure at both 4.5 and 24 hours. From these proteins, four expression patterns were concluded. Their expressions always exhibited a maximum abundance ratio after CdSO4 exposure for 24 hours. The expression of metallothionein-1 and ZIP-8, concentration of total protein, and contents of cadmium, zinc, copper, cobalt and manganese in cells also showed regular change. In synthesis, the replacement of the essential metals, the inhibition of the expression of metal storing protein and the activation of metal efflux system are involved in cadmium toxicity.
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Cadmio/toxicidad , Proteínas de Transporte de Catión/metabolismo , Metalotioneína/metabolismo , Proteoma/metabolismo , Células A549 , Cadmio/metabolismo , Proteínas de Transporte de Catión/genética , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Humanos , Metalotioneína/genética , Proteoma/genética , Factores de Tiempo , ToxicocinéticaRESUMEN
Accurate identification of the molecular targets of bioactive small molecules is a highly important yet challenging task in biomedical research. Previously, a method named DPAL (DNA-programmed affinity labeling) for labeling and identifying the cellular targets of small molecules and nucleic acids was developed. Herein, DPAL is applied for the target identification of Alisertib (MLN8237), which is a highly specific aurora kinaseâ A (AKA) inhibitor and a drug candidate being tested in clinical trials for cancer treatment. Apart from the well-established target of AKA, several potential new targets of MLN8237 were identified. Among them, p38 mitogen-activated protein kinase (p38) and laminin receptor (LAMR) were validated to be implicated in the anticancer activities of MLN8237. Interestingly, these new targets were not identified with non-DNA-based affinity probes. This work may facilitate an understanding of the molecular basis of the efficacy and side effects of MLN8237 as a clinical drug candidate. On the other hand, this work has also demonstrated that the method of DPAL could be a useful tool for target identification of bioactive small molecules.
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Azepinas/química , ADN/química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química , Marcadores de Afinidad , Antineoplásicos/química , Antineoplásicos/metabolismo , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/metabolismo , Azepinas/metabolismo , Sitios de Unión , Línea Celular , Humanos , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Pirimidinas/metabolismo , Receptores de Laminina/antagonistas & inhibidores , Receptores de Laminina/metabolismo , Resonancia por Plasmón de Superficie , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Identification of bioactive compounds directly from complex herbal extracts is a key issue in the study of Chinese herbs. The present study describes the establishment and application of a sensitive, efficient, and convenient method based on surface plasmon resonance (SPR) biosensors for screening active ingredients targeting tumor necrosis factor receptor type 1 (TNF-R1) from Chinese herbs. Concentration-adjusted herbal extracts were subjected to SPR binding assay, and a remarkable response signal was observed in Rheum officinale extract. Then, the TNF-R1-bound ingredients were recovered, enriched, and analyzed by UPLC-QTOF/MS. As a result, physcion-8-O-ß-D-monoglucoside (PMG) was identified as a bioactive compound, and the affinity constant of PMG to TNF-R1 was determined by SPR affinity analysis (K D = 376 nM). Pharmacological assays revealed that PMG inhibited TNF-α-induced cytotoxicity and apoptosis in L929 cells via TNF-R1. Although PMG was a trace component in the chemical constituents of the R. officinale extract, it had considerable anti-inflammatory activities. It was found for the first time that PMG was a ligand for TNF receptor from herbal medicines. The proposed SPR-based screening method may prove to be an effective solution to analyzing bioactive components of Chinese herbs and other complex drug systems. Graphical abstract Scheme of the method based on SPR biosensor for screening and recovering active ingredients from complex herbal extracts and UPLC-MS for identifying them. Scheme of the method based on SPR biosensor for screening and recovering active ingredients from complex herbal extracts and UPLC-MS for identifying them.
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Técnicas Biosensibles/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Medicamentos Herbarios Chinos/química , Espectroscopía de Resonancia Magnética/instrumentación , Mapeo de Interacción de Proteínas/métodos , Receptores del Factor de Necrosis Tumoral/química , Resonancia por Plasmón de Superficie/instrumentación , Sitios de Unión , Técnicas Biosensibles/métodos , Descubrimiento de Drogas/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Ligandos , Espectroscopía de Resonancia Magnética/métodos , Extractos Vegetales/química , Plantas Medicinales/química , Unión Proteica , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
With the development of spectral imaging technology, hyperspectral anomaly detection is getting more and more widely used in remote sensing imagery processing. The traditional RX anomaly detection algorithm neglects spatial correlation of images. Besides, it does not validly reduce the data dimension, which costs too much processing time and shows low validity on hyperspectral data. The hyperspectral images follow Gauss-Markov Random Field (GMRF) in space and spectral dimensions. The inverse matrix of covariance matrix is able to be directly calculated by building the Gauss-Markov parameters, which avoids the huge calculation of hyperspectral data. This paper proposes an improved RX anomaly detection algorithm based on three-dimensional GMRF. The hyperspectral imagery data is simulated with GMRF model, and the GMRF parameters are estimated with the Approximated Maximum Likelihood method. The detection operator is constructed with GMRF estimation parameters. The detecting pixel is considered as the centre in a local optimization window, which calls GMRF detecting window. The abnormal degree is calculated with mean vector and covariance inverse matrix, and the mean vector and covariance inverse matrix are calculated within the window. The image is detected pixel by pixel with the moving of GMRF window. The traditional RX detection algorithm, the regional hypothesis detection algorithm based on GMRF and the algorithm proposed in this paper are simulated with AVIRIS hyperspectral data. Simulation results show that the proposed anomaly detection method is able to improve the detection efficiency and reduce false alarm rate. We get the operation time statistics of the three algorithms in the same computer environment. The results show that the proposed algorithm improves the operation time by 45.2%, which shows good computing efficiency.
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Metabolic changes have been linked to the development of inflammatory bowel disease (IBD), which includes colitis. Allulose, an endogenous bioactive monosaccharide, is vital to the synthesis of numerous compounds and metabolic processes within living organisms. Nevertheless, the precise biochemical mechanism by which allulose inhibits colitis remains unknown. Allulose is an essential and intrinsic protector of the intestinal mucosal barrier, as it maintains the integrity of tight junctions in the intestines, according to the current research. It is also important to know that there is a link between the severity of inflammatory bowel disease (IBD) and colorectal cancer (CRC), chemically-induced colitis in rodents, and lower levels of allulose in the blood. Mice with colitis, either caused by dextran sodium sulphate (DSS) or naturally occurring colitis in IL-10-/- mice, had less damage to their intestinal mucosa after being given allulose. Giving allulose to a colitis model starts a chain of reactions because it stops cathepsin B from ejecting and helps lysosomes stick together. This system effectively stops the activity of myosin light chain kinase (MLCK) when intestinal epithelial damage happens. This stops the breakdown of tight junction integrity and the start of mitochondrial dysfunction. To summarise, the study's findings have presented data that supports the advantageous impact of allulose in reducing the advancement of colitis. Its ability to stop the disruption of the intestinal barrier enables this. Therefore, allulose has potential as a medicinal supplement for treating colitis.
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Colitis , Enteritis , Fructosa , Enfermedades Inflamatorias del Intestino , Enfermedades Mitocondriales , Humanos , Ratones , Animales , Catepsina B/metabolismo , Células CACO-2 , Enfermedades Inflamatorias del Intestino/metabolismo , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/metabolismo , Mucosa Intestinal , Uniones Estrechas , Enfermedades Mitocondriales/metabolismo , Sulfato de Dextran/farmacología , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Epstein-Barr virus (EBV) is an etiological cause of many human lymphocytic and epithelial malignancies. EBV expresses different genes that are associated with three latency types. To date, as many as 44 EBV-encoded miRNA species have been found, but their comprehensive profiles in the three types of latent infection that are associated with various types of tumors are not well documented. METHODS: In the present study, we utilized poly (A)-tailed quantitative real-time RT-PCR in combination with microarray analysis to measure the relative abundances of viral miRNA species in a subset of representative lymphoid and epithelial tumor cells with various EBV latency types. RESULTS: Our findings showed that the miR-BHRF1 and miR-BART families were expressed differentially in a tissue- and latency type-dependent manner. Specifically, in nasopharyngeal carcinoma (NPC) tissues and the EBV-positive cell line C666-1, the miR-BART family accounted for more than 10% of all detected miRNAs, suggesting that these miRNAs have important roles in maintaining latent EBV infections and in driving NPC tumorigenesis. In addition, EBV miRNA-based clustering analysis clearly distinguished between the three distinct EBV latency types, and our results suggested that a switch from type I to type III latency might occur in the Daudi BL cell line. CONCLUSIONS: Our data provide a comprehensive profiling of the EBV miRNA transcriptome that is associated with specific tumor cells in the three types of latent EBV infection states. EBV miRNA species represent a cluster of non-encoding latency biomarkers that are differentially expressed in tumor cells and may help to distinguish between the different latency types.
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Perfilación de la Expresión Génica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , MicroARNs/genética , ARN Viral/genética , Latencia del Virus , Biopsia , Células Cultivadas , Humanos , Leucemia Linfoide/virología , MicroARNs/biosíntesis , Análisis por Micromatrices , Neoplasias Glandulares y Epiteliales/virología , ARN Viral/biosíntesis , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
An improved N-FINDR endmember extraction algorithm by combining manifold learning and spatial information is presented under nonlinear mixing assumptions. Firstly, adaptive local tangent space alignment is adapted to seek potential intrinsic low-dimensional structures of hyperspectral high-diemensional data and reduce original data into a low-dimensional space. Secondly, spatial preprocessing is used by enhancing each pixel vector in spatially homogeneous areas, according to the continuity of spatial distribution of the materials. Finally, endmembers are extracted by looking for the largest simplex volume. The proposed method can increase the precision of endmember extraction by solving the nonlinearity of hyperspectral data and taking advantage of spatial information. Experimental results on simulated and real hyperspectral data demonstrate that the proposed approach outperformed the geodesic simplex volume maximization (GSVM), vertex component analysis (VCA) and spatial preprocessing N-FINDR method (SPPNFINDR).
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OBJECTIVE: To analyze the chemical constituents of Gardenia jasminoides by UHPLC-Q-TOFMS. METHODS: A ACQUITY UPLC HSS T3 (100 mm x 2.1 mm, 1.8 microm) column was used. The mobile phase consisted of water containing 0.1% methane acid and acetonitrile was used in gradient elution. The flow rate was 0.4 mL/min. TOFMS was applied for qualitative analysis under negative ion mode. RESULTS: Under optimized condition, 18 major constituents in Gardenia jasminoides were identified by quadrupole-time of flight mass spectrometry and structure-relevant fragment ions. CONCLUSION: A simple and reliable method using UHPLC-Q-TOFMS is established and can be used to identify the chemical constituents of Gardenia jasminoides.
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Cromatografía Líquida de Alta Presión , Flavonas/química , Frutas/química , Gardenia/química , Iridoides/química , Espectrometría de Masa por Ionización de Electrospray , Ácido Clorogénico/química , Ácido Clorogénico/aislamiento & purificación , Flavonas/aislamiento & purificación , Iridoides/aislamiento & purificación , Estructura MolecularRESUMEN
OBJECTIVE: The purpose of this study was to explore the effect of night-shift work on the risk of hypertension for improving workers' health. METHODS: A total of 10,038 Chinese participants were constituted in the cross-sectional study. Logistic regression and restricted cubic spline were used to estimate the effect of night shift on hypertension. RESULTS: There were higher odds of having hypertension in any night-shift workers (odds ratio [OR], 1.16 [95% confidence interval, 1.03-1.30]) when compared with day workers. Having 5 to 10 night shifts per month were significantly more likely to be hypertensive (OR, 1.19 [95% confidence interval, 1.03-1.38]). The OR for hypertension increased as the number of night shifts increased as the result of the restricted cubic spline. CONCLUSIONS: Our results support the hypothesis that night shift is associated with an elevated risk of hypertension.
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Hipertensión , Tolerancia al Trabajo Programado , Humanos , Adulto , Estudios Transversales , Pueblos del Este de Asia , Factores de Riesgo , Hipertensión/epidemiología , Hipertensión/etiologíaRESUMEN
The rapid formation of a glial/fibrotic scar is one of the main factors hampering axon growth after spinal cord injury. The bidirectional EphB2/ephrin-B2 signaling of the fibroblast-astrocyte contact-dependent interaction is a trigger for glial/fibrotic scar formation. In the present study, a new in vitro model was produced by coculture of fibroblasts and astrocytes wounded by scratching to mimic glial/fibrotic scar-like structures using an improved slide system. After treatment with RNAi to downregulate EphB2, changes in glial/fibrotic scar formation and the growth of VSC4.1 motoneuron axons were examined. Following RNAi treatment, fibroblasts and astrocytes dispersed without forming a glial/fibrotic scar-like structure. Furthermore, the expression levels of neurocan, NG2 and collagen I in the coculture were reduced, and the growth of VSC4.1 motoneuron axons was enhanced. These findings suggest that suppression of EphB2 expression by RNAi attenuates the formation of a glial/fibrotic scar and promotes axon growth. This study was approved by the Laboratory Animal Ethics Committee of Jiangsu Province, China (approval No. 2019-0506-002) on May 6, 2019.
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OBJECTIVE: To establish a multiplex PCR point mutation screening technique for the genotyping of CYP2C19. METHODS: Deoxyinosine multiplex-polymerase chain reaction (PCR) primers (DMPs) were designed to detect simultaneously CYP2C19*1,*2,*3 alleles in one PCR tube. RESULTS: The above technique could detect the genotypes of CYP2C19*1, CYP2C19*2 and CYP2C19*3 successfully. And the results were completely consistent with those of DNA sequencing. CONCLUSION: A novel screening technique of multiplex PCR point mutation is successfully established. With the advantages of high specificity, convenient handling, fast completion and low cost, it provides a reasonable and reliable detection method for basic researches and personalized medicine.
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Hidrocarburo de Aril Hidroxilasas/genética , Técnicas de Genotipaje/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Citocromo P-450 CYP2C19 , Cartilla de ADN , Humanos , Mutación PuntualRESUMEN
OBJECTIVE: To investigate the role of dietary capsaicin in activating transient receptor potential vanilloid 1 (TRPV1) and thus influencing the vascular dysfunction mediated by high-fat diet and the potential mechanisms. METHODS: A total of 80 male C57BL/6J mice aged 10 weeks were equally divided into four groups, in which the mice were fed with normal diet (ND), normal diet plus capsaicin (NC), high-fat diet (HD), or high-fat diet plus capsaicin (HC) for 20 weeks. Tail-cuff blood pressure (BP), vascular function of mice aortic rings, expressions of voltage-gated potassium-channel Kv1.4, RhoA and Rho kinase in aorta were examined. RESULTS: Compared with ND group, both nitroglycerin [(18.9 +/- 13)% vs. 100%, P < 0.01] and acetylcholine [(26 +/- 12)% vs. 100%, P < 0.01] induced vasorelaxation of aortic rings were significantly reduced in HD group. Both endothelium dependent and independent aortic rings vasorelaxation in HC group were significantly improved compared with that in HD group [acetylcholine: (69 +/- 15)%; nitroglycerin: (46.5 +/- 6)%, P < 0.05], but still reduced compared with that in ND group (P < 0.05, P < 0.01). High fat diet induced the expression of RhoA and Rho kinase. Dietary capsaicin down-regulated the expression of RhoA and Rho kinase but up-regulated the expression of Kv1.4 in aorta in mice fed with normal or high fat diet (all P < 0.05). CONCLUSION: Dietary capsaicin can ameliorate vasorelaxation dysfunction mediated by high-fat diet. The potential mechanisms may be related with TRPV1 activation, which in turn stimulates potassium channel and inhibits RhoA and Rho kinase in the vasculature.