Site-directed mutagenesis enhances the activity of benzylidene acetone synthase of polyketide synthase from Polygonum cuspidatum / 生物工程学报

Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.

Morphological changes of mitral valve geometry caused by repair procedure / 上海交通大学学报（医学版）

Objective · To assess the morphological changes of mitral valve geometry after mitral valve repair by using real-time 3D transesophageal echocardiography. Methods · The clinical data including 3D echocardiography of 36 patients undergoing mitral valve repair for mitral valve prolapse and 56 patients without mitral valve diseases were collected. Parameters of mitral annular and leaflet geometry were acquired and analyzed. Results · The ellipse index of the two-dimensional view (E2D), and non-planar leaflet angle (θNPA) were decreased, while other parameters were increased significantly in patients with mitral valve prolapse compared with controls before mitral valve repair. After repair, patients displayed larger θNPA, and still smaller E2D.Some parameters also get smaller, such as the anterior to posterior diameter of the mitral annulus, the anterolateral to posteromedial coaptation diameter,the minimum circumference of the three-dimensional view of the annulus, the minimum area of the two-dimensional view of the annulus, the exposed area of the anterior leaflet, inter-commissural diameter. Other parameters were not changed significantly. All parameters showed no significant difference between respect group and resect group in posterior valve prolapse before and after mitral valve repair. Conclusion · The repair procedure can restore the function of the mitral valve effectively. In view of the morphology, the geometry of the mitral valve annulus is still different from the normal apparently after the mitral valve repair, but the normal morphology of the leaflets can be regained. It seems to have similar curative effect morphologically for patients with posterior leaflet prolapse to have respect or resect strategy.

Preparation and crystallization of Polygonum cuspidatum benzalacetone synthase / 生物工程学报

The chalcone synthase (CHS) superfamily of the type III polyketide synthases (PKSs) generates backbones of a variety of plant secondary metabolites. Benzalacetone synthase (BAS) catalyzes a condensation reaction of decarboxylation between the substrates of 4-coumaric coenzyme A and malonyl coenzyme A to generate benzylidene acetone, whose derivatives are series of compounds with various biological activities. A BAS gene Pcpks2 and a bifunctional CHS/BAS PcPKSI were isolated from medicinal plant P. cuspidatum. Crystallographic and structure-based mutagenesis studies indicate that the functional diversity of the CHS-superfamily enzymes is principally derived from small modifications of the active site architecture. In order to obtain an understanding of the biosynthesis of polyketides in P. cuspidatum, which has been poorly described, as well as of its activation mechanism, PcPKS2 was overexpressed in Escherichia coli as a C-terminally poly-His-tagged fusion protein, purified to homogeneity and crystallized, which is helpful for the clarification of the catalytic mechanism of the enzyme and lays the foundation for its genetic engineering manipulation.

Imaging features of split cord malformation associated with scoliosis and its correlation with neurologic symptoms / 中华骨科杂志

Objective To analyze the imaging features of congenital spinal deformity (CSD) associated with split cord malformation (SCM) and other intraspinal abnormalities, and to investigate the relationship to neurological symptoms. Methods 105 cases CSD with SCM were retrospectively studied. Analysis the imaging features of SCM (including type of SCM, location of SCM, location and apical vertebrae, symmetry of divided cord) and other intraspinal abnormalities. To investigate the relationship of the factors and neurological symptoms using Chi?square test of one factor and multiple factors logistic regression analysis. Re?sults 28 cases (26.7%) were formation failure, 33 cases (31.4%) were segmentation failure, and 44 cases (41.9%) were combina?tion of 2 disorders. 41 cases had neurological symptoms, 64 cases were asymptomatic. The distribution of SCM combined with spi?nal deformities:thoracic (11 cases), thoracolumbar (18 cases) and lumbar (20 cases) in type I SCM, thoracic (31 cases), thoracolum?bar (20 cases) and lumbar (5 cases) in type II, none was in cervical. The location of SCM upper than apical vertebrae 29 cases, on apical vertebrae 25 cases, lower than apical vertebrae 51 cases. Spinal cord was splitted symmetric 27 cases and asymmetric 78 cases. 66 cases combined with other intraspinal abnormalities, lower conus 42 cases, syringomyelia 38 cases, meningocele 10 cas?es and sakrale zyste 5 cases. Associated with intraspinal abnormalities, the rate of neural symptoms was different. According to Chi?square test of one factor and multiple factors logistic regression analysis, lumbar SCM, spinal cord asymmetric and lower conus were related with neurological symptoms. Conclusion The predilection spinal deformity of type I is combination, type II SCM is segmentation failure. When SCM patients associated with other intraspinal abnormalities, the incidence of neurologic symptoms is increased. The lumbar SCM, hemicords asymmetry and lower lying conus have significant relationship with neurologic symptoms.

Double-layer nanoparticles loaded with rhBMP-2 and SDF-1:Preparation,characterization and drug re-lease in vitro / 实用口腔医学杂志

Objective:To construct double-layered controlled release system containing SDF-1 and rhBMP-2 molecules and to study the release profile of the system in vitro.Methods:The polylactic acid/chitosan(PLA/CS)nanoparticles were prepared with “emulsification-solution evaporation method”,the preparation parameters were determined by orthogonal test design.The particle size was observed by nanoparticle size analyzer,the morphology of the nanoparticles was observed with electron microscope.Then rhBMP-2 and SDF-1 were loaded into the nanoparticles in the process of emulsification,the loading efficiency and encapsulation efficiency were calculated and in vitro release was observed.Results:The double-layer nanoparticles showed spherical geometry,smooth surface and complete separation. The average particle size of the nanoparticles was (542.33 ±14.38)nm;The drug loading and incorporation efficiency of rhBMP-2 were (82.41 ±1.05)% and (24.67 ±0.43)ng/mg,those of rhBMP-2 were (75.58 ±0.84)% and (22.63 ±0.41)ng/mg,respectively. The release time of the drug from the system sustained over at least 30 days,the release profile of both drugs showed “biphasic release”. The cumulative release rate of SDF-1 and rhBMP-2 was 72.85% and 91.01% in 30 days respectively.Conclusion:The SDF-1 and rh-BMP-2 loaded PLA/CS nanoparticles have excellent morphology,high entrapment and good sustained-release in vitro.