Metabolic Characterization of the Anthocyanidin Reductase Pathway Involved in the Biosynthesis of Flavan-3-ols in Elite Shuchazao Tea (Camellia sinensis) Cultivar in the Field.

Anthocyanidin reductase (ANR) is a key enzyme in the ANR biosynthetic pathway of flavan-3-ols and proanthocyanidins (PAs) in plants. Herein, we report characterization of the ANR pathway of flavan-3-ols in Shuchazao tea (Camellia sinesis), which is an elite and widely grown cultivar in China and is rich in flavan-3-ols providing with high nutritional value to human health. In our study, metabolic profiling was preformed to identify two conjugates and four aglycones of flavan-3-ols: (-)-epigallocatechin-gallate [(-)-EGCG], (-)-epicatechin-gallate [(-)-ECG], (-)-epigallocatechin [(-)-EGC], (-)-epicatechin [(-)-EC], (+)-catechin [(+)-Ca], and (+)-gallocatechin [(+)-GC], of which (-)-EGCG, (-)-ECG, (-)-EGC, and (-)-EC accounted for 70-85% of total flavan-3-ols in different tissues. Crude ANR enzyme was extracted from young leaves. Enzymatic assays showed that crude ANR extracts catalyzed cyanidin and delphinidin to (-)-EC and (-)-Ca and (-)-EGC and (-)-GC, respectively, in which (-)-EC and (-)-EGC were major products. Moreover, two ANR cDNAs were cloned from leaves, namely CssANRa and CssANRb. His-Tag fused recombinant CssANRa and CssANRb converted cyanidin and delphinidin to (-)-EC and (-)-Ca and (-)-EGC and (-)-GC, respectively. In addition, (+)-EC was observed from the catalysis of recombinant CssANRa and CssANRb. Further overexpression of the two genes in tobacco led to the formation of PAs in flowers and the reduction of anthocyanins. Taken together, these data indicate that the majority of leaf flavan-3-ols in Shuchazao's leaves were produced from the ANR pathway.

Development of a one-step immunocapture real-time RT-PCR assay for detection of tobacco mosaic virus in soil.

Tobacco mosaic virus (TMV) causes significant losses in many economically important crops. Contaminated soils may play roles as reservoirs and sources of transmission for TMV. In this study we report the development of an immunocapture real-time RT-PCR (IC-real-time RT-PCR) assay for direct detection of TMV in soils without RNA isolation. A series of TMV infected leaf sap dilutions of 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (w/v, g/mL) were added to one gram of soil. The reactivity of DAS-ELISA and conventional RT-PCR was in the range of 1:102 and 1:103 dilution in TMV-infested soils, respectively. Meanwhile, the detection limit of IC-real-time RT-PCR sensitivity was up to 1:106 dilution. However, in plant sap infected by TMV, both IC-real-time RT-PCR and real-time RT-PCR were up to 1:106 dilution, DAS-ELISA could detect at least 1:103 dilution. IC-real-time RT-PCR method can use either plant sample extracts or cultivated soils, and show higher sensitivity than RT-PCR and DAS-ELISA for detection of TMV in soils. Therefore, the proposed IC-real-time RT-PCR assay provides an alternative for quick and very sensitive detection of TMV in soils, with the advantage of not requiring a concentration or RNA purification steps while still allowing detection of TMV for disease control.

[Evaluation of occlusal reconstruction and cone-beam CT measurement of temporomandibular joint in patients with severe tooth wear].

PURPOSE: To explore the correlation between increased vertical dimensions and temporomandibular joint (TMJ) spaces of patients with severe tooth wear after occlusal reconstruction treatment. METHODS: The TMJ spaces and the condylar positions of 30 patients were measured by cone-beam CT (CBCT) before and after treatment. SPSS 19.0 software package was used to analyze the anterior, superior and posterior space of TMJ. Independent sample t test was utilized to evaluate the correlation between vertical dimension and TMJ spaces. RESULTS: There was no significant difference between TMJ spaces of two sides, the data of two sides were combined for further analysis. The mean anterior interspace, superior interspace, posterior interspace was (2.01±0.52)mm, (3.15±0.94)mm, (2.70±0.98)mm before treatment, and (2.03±0.35)mm, (3.46±0.74)mm, (2.79±0.58)mm after treatment, respectively. Based on calculating the In(P/A) and linear ratio(LR), condylar position moved backward for 52% patients before treatment. After occlusal reconstruction, the reduction rate of anterior displacement of condyle was 86.7%. CONCLUSIONS: In addition to restoring its vertical dimensions of patients with severe tooth wear, the condyle should be induced to appropriate position to prevent TMJ symptoms.

[Comparison of clinical effects of Co-Cr alloy cast post-core and everStick fiber post in restoration of labially or lingually inclined maxillary central incisor].

PURPOSE: To compare the clinical effect of Co-Cr alloy cast post-core and everStick fiber post in restoration of maxillary central incisor with labial or lingual inclination, and provide theoretical basis for clinical application. METHODS: Ninety-seven labially or lingually inclined maxillary central incisors were treated in our hospital from March 2012 to March 2014. The patients were randomly divided into group A (n=49) and group B (n=48), and received post -core and crown restoration. Patients in group A underwent Co-Cr alloy cast post and core restoration and patients in group B underwent everStick fiber post and core restoration. After two-year of follow-up, root fracture, post break, crown or post dislodgment and gingival marginal discoloration were recorded and analyzed using SPSS 19.0 software package. RESULTS: Chi-square test showed that the success rate of restoration was significantly different between 2 groups (P<0.05). The incidence of root fracture and gingival marginal discoloration of Co-Cr alloy cast post-core was higher than that of everStick fiber post, but there was no significant difference in the incidence of post break, crown or post dislodgment. CONCLTSIONS: EverStick fiber post is better than Co-Cr alloy cast post and core to prevent root fracture and gingival marginal discoloration. Its fracture pattern is repairable and favorable for preserving tooth.

Design, synthesis, and biological evaluation of chrysin derivatives as potential FabH inhibitors.

New series of chrysin derivatives (4a-4t) were designed and synthesized by introducing different substituted piperazines at C-7 position. Their inhibitory effects on FabH were evaluated using two Gram-negative bacterial strains, Escherichia coli and Pseudomonas aeruginosa, and two Gram-positive bacterial strains, Bacillus subtilis and Staphylococcus aureus. To our delight, most of these compounds exhibited a dramatic increase in inhibitory potency, compared with the control positive drugs. Among them, compound 4s exhibited the most potent inhibitory activity with IC50 values of 5.78 ± 0.24 µm inhibiting E. coli FabH and potent antibacterial activity against S. aureus and E. coli with MIC of 1.25 ± 0.01, 1.15 ± 0.12 µg/mL, respectively, comparing to the control positive drugs penicillin G (7.56 ± 0.30 µm). Docking simulation was performed to position compound 4s into the FabH active site, and the result showed that compound 4s could bind well with the FabH as potent FabH inhibitor.

[Reconstruction of three-dimensional numerical model and numerical simulation of airflow in a human upper airway].

PURPOSE: To rapidly reconstruct a three-dimensional numerical model of the human upper airway and investigate the relationship between anatomical structures with airflow distribution by using the computational fluid dynamics. METHODS: A three-dimensional model of the human upper airway was reconstructed based on computed tomographic images of a healthy volunteer's skull. Numerical simulation of the upper airway airflow was performed by using computational fluid dynamics (CFD) method. RESULTS: A three-dimensional model of the human upper airway including nasal cavity, pharynx and larynx was reconstructed rapidly. A detailed anatomical structure and velocity distribution characteristics of airflow was obtained and a large velocity gradient in nasal valve area, nasopharynx, up and downstream of epiglottis was found. CONCLUSIONS: The model has good simulation of upper airway. Numerical simulation results provide the basic trend of airflow of the upper respiratory tract.The numerical model meets the needs of computational fluid dynamics analysis, and provide data control and research foundation for pathologic upper airway airflow numerical simulation.