Construction and efficacy evaluation of Rb luciferase reporter gene detection system / 西安交通大学学报(医学版)

【Objective】 To construct the Rb luciferase reporter gene assay system and detect the activation ability of Rb gene for screening the targeted drugs. 【Methods】 The synthetic Rb gene sequence was annealed to form a double-stranded DNA structure and then inserted into the polyclonal site of pGL6-TA. The junction product was transformed into E.coli DH5α competent cells for expanded culture, and the constructed pGL6-Rb-Luc plasmid and pGL6-TA plasmid were transfected into HEK293 cells. The monoclonal cell line HEK293-Rb-Luc with stable expression was screened by G418, and the activation and inhibition of Rb in HEK293-Rb-Luc were tested by serum and CDK4/6 inhibitor Palbociclib. 【Results】 The sequence of Rb reaction elements in pGL6-Rb-Luc was completely correct. The recovery of serum culture significantly increased the luciferase activity in HEK293-Rb-Luc (P<0.001). Compared with 0 nmol/L, 25, 50, 75 and 100 nmol/L, CDK4/6 inhibitor Palbociclib made the inhibition rate of Rb activity rise to 6.90%, 40.23%, 50.57% and 52.07%, respectively (P<0.05). 【Conclusion】 The Rb luciferase reporter gene detection system HEK293-Rb-Luc was successfully constructed, which can effectively detect the activation level of Rb.

Application of MAO-A/B subtype activity detection in diagnosis and treatment of neurological diseases / 中国临床药理学与治疗学

The abnormal expression of monoamine oxidases (MAOs), which distributed on the outer mitochondrial membrane can cause the dysfunction of neurotransmitter delivery and related to mental and neurodegenerative diseases. Therefore, the specific detection of MAOs (MAO-A/B) and the regulation of their activities will contribute to the diagnosis and treatment of neurological diseases. In order to distinguish the two subtypes of MAO-A/B, fluorescence detection and imaging techniques with highly specific and sensitive properties have important biological relevance. Herein, our research group based on the concept of "spatial configuration conversion" have designed the two-photon small molecule fluorogenic probes of U1 and F1, which capable of specific fluorescence "Switch-ON" behaviour, and combined with two-photon fluorescence microscopic imaging technology to realize the activity detection of endogenous MAO-B/A in cells and tissues. It is hoped that this works will benefit the understanding of physiological function about MAOs and developing innovative inhibitors, as well as diagnosing and treating in neurological diseases.

A simplified and miniaturized glucometer-based assay for the detection of β-glucosidase activity / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873–1.5498 U/mL and 0.4076–2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

Modulation of nuclear receptor-binding factor 2 on the activity of autophagy initiation complex PI3KC3-C1 / 上海交通大学学报（医学版）

Objective • To study the effects of different phosphorylation levels of nuclear receptor-binding factor 2 (NRBF2) on the activity of class III phosphatidylinositol 3-kinase complex (PI3KC3-C1), and find a type of PI3KC3-C1 with the highest kinase activity as a target for the screening of autophagy-specific inhibitors. Methods • First, in vitro protein purification system was established to obtain quaternary NRBF2-free PI3KC3-C1 and quinary PI3KC3-C1 containing different phosphorylation levels of NRBF2. The integrity of the complex was determined by gel filtration chromatography. The different kinase activity of purified PI3KC3-C1 was detected in vitro, and thus the effect of phosphorylation of NRBF2 on PI3KC3-C1 activity was observed. Results • The purified PI3KC3-C1 had good purity and yield. The five-membered PI3KC3-C1 containing NRBF2 showed higher kinase activity than the quaternary protein complex, and PI3KC3-C1 containing the continuous dephosphorylated NBRF2 had the highest kinase activity. Conclusion • NRBF2 does exist as one of the components of PI3KC3-C1. The presence of NRBF2 promotes the kinase activity of PI3KC3-C1, and PI3KC3-C1 containing continuous dephosphorylation of NRBF2 may serve as a new regulatory target for the screening of autophagy-specific inhibitor.

A simplified and miniaturized glucometer-based assay for the detection of β-glucosidase activity / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

Detección Automática y Clasificación de Eventos en Sonidos Cardiopulmonares de Sujetos Saludables / Automatic Detection and Classification of Cardiopulmonary Events

Resumen: La auscultación de señales basada en un estetoscopio estándar y/o electrónico no solo incluye sonidos internos del cuerpo, también incluye frecuentemente ruido externo de interferencia con componentes en el mismo rango. Esta forma de examinar es incluso afectada por los umbrales auditivos variantes de los profesionales de la salud y el grado de experiencia en reconocimiento de indicadores peculiares. Además, los resultados son a menudo caracterizados en términos cualitativos descriptivos sujetos a interpretaciones individuales. Para direccionar esta preocupación, los estudios presentados en este artículo contienen un procesamiento concurrente de las componentes dominantes de sonidos del corazón (HS) y del pulmón (HS), y una etapa de acondicionamiento que incluye la reducción de HS presente en señales LS. Específicamente, la transformada de Hilbert fue una técnica de caracterización para HS. En el caso de señales enfocadas a LS, las técnicas de detección de actividad de voz y el cálculo de umbrales de algunos componentes de los vectores acústicos de Coeficientes Cepstrales en Frecuencia Mel (MFCC), fueron útiles en la caracterización de eventos acústicos asociados. Las fases de inspiración y expiración fueron diferenciadas por medio de la sexta componente de MFCC. Con el fin de evaluar la eficiencia de esta aproximación, proponemos los Modelos Ocultos de Markov con Modelos Mesclados Gaussianos (HMM-GMM). Los resultados utilizando esta forma de detección son superiores cuando se desarrolla la clasificación con modelos HMM-GMM, la cual refleja las ventajas de la forma de detección cuantificable y clasificación sobre la aproximación clínica tradicional.

Abstract: A standard and/or electronic stethoscope based auscultatory signals include not only the internal sounds of the body but also interfering external noise often with similar frequency components. This form of examination is also affected by varying thresholds of clinical practitioner's hearing and degree of experience in recognition of peculiar auscultatory indicators. Further, the results are often characterized in qualitative descriptive terms subject to individual's interpretation. To address these concerns, presented studies include concurrent processing of dominant heart (HS) and lung (LS) sounds components and a conditioning stage involving HS presence reduction within LS focused signals. Specifically as determined, the Hilbert transform was a technique of choice in HS characterization. In the case of LS focused signals, the speech activity detection techniques (VAD) and the thresholds calculation of some components of acoustic vectors of Cepstral Coefficients in Mel Frequency (MFCC), were useful in characterization of associated acoustic events. The phases of inspiration and expiration were differentiated by means of the sixth component of MFCC. In order to evaluate the efficiency of this approach, we propose Hidden Markov Models with Mixed Gaussian Models (HMM-GMM). The results utilizing this form of detection are superior when performing classification with HMM-GMM models, which reflect the advantages of presented form of quantifiable detection and classification over traditional clinical approach.

Classification of Flying Insects with high performance using improved DTW algorithm based on hidden Markov model

ABSTRACT Insects play significant role in the human life. And insects pollinate major food crops consumed in the world. Insect pests consume and destroy major crops in the world. Hence to have control over the disease and pests, researches are going on in the area of entomology using chemical, biological and mechanical approaches. The data relevant to the flying insects often changes over time, and classification of such data is a central issue. And such time series mining tasks along with classification is critical nowadays. Most time series data mining algorithms use similarity search and hence time taken for similarity search is the bottleneck and it does not produce accurate results and also produces very poor performance. In this paper, a novel classification method that is based on the dynamic time warping (DTW) algorithm is proposed. The dynamic time warping algorithm is deterministic and lacks in modeling stochastic signals. The dynamic time warping (DTW) algorithm is improved by implementing a nonlinear median filtering (NMF). Recognition accuracy of conventional DTW algorithms is less than that of the hidden Markov model (HMM) by same voice activity detection (VAD) and noise-reduction. With running spectrum filtering (RSF) and dynamic range adjustment (DRA). NMF seek the median distance of every reference of time series data and the recognition accuracy is much improved. In this research work, optical sensors are used to record the sound of insect flight, with invariance to interference from ambient sounds. The implementation of our tool includes two parts, an optical sensor to record the "sound" of insect flight, and a software that leverages on the sensor information, to automatically detect and identify flying insects.

Detection ofα1 antitrypsin activity by chromogenic substrate assay with initial veloci-ty of enzymatic reaction / 军事医学

Objective To detect the activity of α1 antitrypsin(AAT) with initial velocity of enzymatic reaction in order to detect the activity of samples in the process of separating and purifying plasma protein ,chromogenic substrate assay was optimized.Methods The effect of trypsin concentration and reaction time on enzymatic reaction was acquired by the kinetic monitoring mode of the microplate reader .Initial velocity was calculated to confirm the largest concentration of trypsin which was saturated by substrate .AAT was incubated with trypsin and absorbance produced by enzymatic reaction of remaining trypsin and substrate could reflect the activity of AAT .A standard curve was established with △D fitting with the activity of AAT standard.The activity of related samples was detected and the precision and accuracy of the method were evaluated . Results Trypsin concentration was 0.0625 mg/ml.Within 20 minutes, enzymatic reaction was with initial velocity .The range of the standard curve was 200-1200 IU/ml.Correlation coefficient was more than 0.99.The activity of Cohn Ⅳ, samples of pre-processing and elution were (720.59 ±18.63), (601.84 ±19.18),and (568.09 ±24.83)IU/ml, respec-tively.The relative standard deviation was less than 10%. Sample recovery rate was 90%-110%.Conclusion The optimized chromogenic substrate assay greatly improves accuracy and precision .The method can be used for the detec-tion of AAT activity of samples in laboratories and workshops .

Bio-evaluation methods and optimization for Isatidis Radix quality control based on antiviral activity detection / 中草药

Objective: To establish and optimize a bio-evaluation method based on antiviral activity detection for Isatidis Radix quality control. Methods: According to the fact that antivirus activity is the main pharmacological effect of Isatidis Radix, the means of hemagglutination activity detection and the means of influenza virus neuraminidase (NA) activity detection were used for establishing two antiviral activity detection methods for the bio-evaluation of Isatidis Radix. In addition, contrastive analyzation and optimization of the two methods were carried out in this study. Results: The two methods could be used to evaluate and distinct the biological activities of different batches of Isatidis Radix samples and both methods have good repeatabilities (RSD = 7.0% and 5.78%). The result shows a good correlation (P<0.01, r = -0.81) between the neuraminidase inhibitory activity and the hemagglutination activity of Isatidis Radix. The relationship between agglutination activity and antiviral effect of Isatidis Radix was further verified. Agglutination detection method has the superiority to its safety, inexpensiveness, easiness, and practicality. Conclusion: This novel method could reflect the pharmacodynamic activity of Isatidis Radix on anti-influenza virus and be helpful for the quality control of Isatidis Radix. This study provides reference for the bio-evaluation on the quality control of Chinese materia medica.

Clearing, amplification and activity detection of the recombinant adeno-associated virus vector2/1with adiponectin / 中华微生物学和免疫学杂志

Objective To clear, amplify and detect the activity of the recombinant adeno-assoeiated virus vector with adiponectin( rAAV2/1-Aerp30 ). Methods Recombinant plasmid pSNAV2.0-Acrp30 was obtained. The recombinant plasmid was then transfected into BHK21 cells using LipofectAMINETM 2000. The G418 resistant cells were obtained consequently. These cells were infected with HSVI-rc/△UL2 which has the function of packaging and copying recombinant AAV. After purification, the construction of recombinant rAAV2/1-Aerp30 was collected. Results The construction of recombinant pSNAV2.0-Acrp30 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequencing showed that the recombinant pSNAV2.0-Acrp30 was correct. The virus titer was about 1.0×1012 μg/ml. The purity f the recombinant AAV2/1 was fairly high using the SDS-PAGE method. Conclusion With this method, rAAV2/-Aerp30 with high virus titers and purity can be acquired successfully and it can meet the demands of the experimental study of Acrp30 gene therapy of GK rats.

CTL activity detection of immune response to hepatitis Cviral nucleic acid vaccine / 中国免疫学杂志

Objective:To discuss mice cellular immune response to the hepatitis C viral nucleic acid vaccine.Methods:Hepatitis C viral nucleic acid vaccine pSVL HCV/C+E 1 was inoculated BALB/C mice by subcutaneous injection.To detect the CTL activity of immune mice,Made the standard 4 hours lysis test by labeling the target cells with (Na) 2 51 CrO 4,which was prepared from the syngentic BALB/C mouse myeloma derived cell strain SP2/0 transfected by the eukaryotic expression recombinant plasmid pEGFP N 1 HCV/C+E 1.Results:The kill ratio of CTL is up to 40.7%.Conclusion:The result suggested hepatitis C viral nucleic acid vaccine could induce strong cellular immune response