Aged, 80 and over ; Chordoma ; complications ; Humans ; Male ; Pharyngeal Neoplasms ; complications ; Respiration Disorders ; etiology

Aim To investigate the effect of vaccarin on mouse atherosclerosis in vivo and the underlying mechanism. Methods AopE mice aged 6 to 8 weeks old were used to establish the atherosclerosis model. Oil red O staining was used to determine the lipid levels in aorta and aortic root. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of serum inflammatory factors. Results Vaccarin could effectively reduce the levels of blood glucose and blood pressure in AopE

Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields, whereas their applications are limited by the low water solubility. Glycosylation is regarded as one of the effective approaches to improve water solubility. In this paper, the glycosylation of sophoricoside (genistein monoglucoside) was investigated using a cyclodextrin glucosyltransferase from Penibacillus macerans (PmCGTase). Saturation mutagenesis of D182 from PmCGTase was carried out. Compared with the wild-type (WT), the variant D182C showed a 13.42% higher conversion ratio. Moreover, the main products sophoricoside monoglucoside, sophoricoside diglucoside, and sophoricoside triglucoside of the variant D182C increased by 39.35%, 56.05% and 64.81% compared with that of the WT, respectively. Enzymatic characterization showed that the enzyme activities (cyclization, hydrolysis, disproportionation) of the variant D182C were higher than that of the WT, and the optimal pH and temperature of the variant D182C were 6 and 40℃, respectively. Kinetics analysis showed the variant D182C has a lower K_m value and a higher k_cat/K_m value than that of the WT, indicating the variant D182C has enhanced affinity to substrate. Structure modeling and docking analysis demonstrated that the improved glycosylation efficiency of the variant D182C may be attributed to the increased interactions between residues and substrate.
Cyclodextrins ; Genistein ; Glucosyltransferases/metabolism* ; Glycosylation ; Kinetics

Aim To investigate the effects of TRPV4-Nox2 complex on ROS production and aortic vasodilatory function in mice fed with high-fat diet.Methods Male C57 BL/6J mice and TRPV4 KO mice were randomly divided into seven groups, with 10 mice in each group: normal diet group(ND), high-fat diet group(HFD), TRPV4 KO mice fed with high-fat diet group(TRPV4 KO-HFD), HFD+AAV-Flt1-Vector/Nox2 ▵3 group, TRPV4 KO-HFD+AAV-Flt1 -Vector/Nox2 ▵3 group.Body weight and blood pressure were recorded.14 weeks later primary aortic endothelial cells were isolated for CM-H2DCFDA staining and immuno-FRET assay, and aortic rings were isolated for vascular tone assay.Results ① Obesity significantly increased ROS production, triggered vasodilatory dysfunction and increased the strength of physical coupling between TRPV4-Nox2 complex(P<0.05); ② Decreasing the physical association of TRPV4-Nox2 complex could help reduce obesity-induced increased ROS production and vasodilatory dysfunction(P<0.05); ③ Entrectinib had no effect on the expression and function of TRPV4 and Nox2, but only decreased the physical association of the TRPV4-Nox2, which in turn improved obesity-induced oxidative stress and restored vasodilatory function.Conclusions Reducing the physical association of TRPV4 and Nox2 through Entrectinib can help reduce obesity-induced increase in ROS production and improve vasodilatory function of obese mice.

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGAD^{S24R/D88R/Y309K} was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and Lpgad^{S24R/D88R/Y309K} genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD₆₀₀) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.
Glutamate Decarboxylase/genetics* ; Lactobacillus plantarum/genetics* ; Catalysis ; gamma-Aminobutyric Acid ; Hydrogen-Ion Concentration ; Glutamic Acid

To develop a magnetic nanoparticle chemiluminescence immunoassay (CLIA) for the determination of type Ⅰ procollagen N-terminal peptide (PINP) in human serum, we expressed a recombinant PINP-α1 protein in Corynebacterium glutamicum and used it as an immunogen to immunize BALB/c mice. We obtained three hybridoma cell lines that stably secret antibody against PINP-α1 protein. After further pairing and screening, we chose a monoclonal antibody 8C12 coupled with biotin as the capture antibody, and a monoclonal antibody 1F11 labeled horseradish peroxidase as the detection antibody. The antibodies combined with the serum samples, forming a sandwich complex which was used to detect the concentration of PINP in serum. After optimizing the conditions, we determined that the best working concentration of the capture antibody and the detection antibody were 3 μg/mL, and the incubation time was 30 minutes. The quantitative assay had a detection range of 5-1 100 ng/mL, with recovery rates between 93%-107% and the minimum detection limit of 1.22 ng/mL achieved. The intra-and inter-assay precisions were lower than 10%. The correlation coefficient of PINP results between this CLIA method and the Roche electrochemiluminescence immunoassay system was 0.906 2. Therefore, this CLIA method is specific and can be used to quantitatively detect the content of PINP in serum, which has the potential to become an auxiliary approach for bone disease examination.
Animals ; Humans ; Immunoassay ; Luminescence ; Mice ; Mice, Inbred BALB C ; Peptide Fragments/isolation & purification* ; Procollagen/isolation & purification*

Trehalase is widely used in industrial fermentation, food, medicine and other fields. There is a lack of industrial varieties of trehalase with excellent performance in China. Moreover, the applied research on trehalase was not well conducted. In this study, a strain of Pectobacterium cypripedii was screened from nature, and the gene PCTre encoding an acidic trehalase was cloned and expressed in E. coli BL21(DE3). The highest enzyme activity reached 4130 U/mL after fermenting in a 5 L fermenter for 28 h. The enzymatic properties study showed that PCTre hydrolyzed trehalose specifically. The optimum pH and temperature were 5.5 and 35 ℃, respectively. 80% of the enzyme activity was retained after being treated at pH 4.0, 4.5, and 5.0 for 8 h, showing good acid tolerance. Moreover, it has good tolerance to organic solvents, 60% enzyme activity was retained after being treated with 20% (V/V) ethanol solution for 24 h. Furthermore, trehalose could be completely hydrolyzed within 16 h in a simulated fermentation system containing 20% (V/V) ethanol and 7.5% trehalose, with 500 U/L PCTre added. This indicated a good application potential for industrial ethanol fermentation.
Trehalase/metabolism* ; Trehalose/metabolism* ; Escherichia coli/metabolism* ; Ethanol/metabolism* ; Cloning, Molecular

OBJECTIVE: To establish a 3D finite element model of normal knee joint involved its meniscus, which can be used to simulate the anatomical morphology and characteristics of human knee joint, to verify the validity of the model by preliminary FEA mechanical analysis, and explain partially biomechanical mechanisms of meniscus. METHODS: CT and MRI data were harvested by scanning the knee joint of a healthy male volunteer, and then these data were imported into Mimics 10.01 software and Geomagic Studio software to constructed the 3D models of tissue structures of knee joint. These models were combined to constructed the 3D model of intact knee joint and meshed in ANSA software. Therefore the finite element model of intact knee joint was established. Finally, after the definitionof its material behavior, boundary conditions and loading. The finite element model of knee joint was analyzed and verified using ANSYS software. Meanwhile The biomechanical properties of meniscus were analyzed. RESULTS: The complete knee finite element model composed of bone, meniscus, articular cartilage, and major ligaments was established. It could effectively simulate the anatomical morphology and characteristics of knee joint and its meniscus. The contact area of medial meniscus was 771.05 mm, while the contact area of lateral meniscus was 634.31 mm, and the ratio was 1.216. The stress distribution was uniform, but the stress of the medial meniscus was higher than that of the lateral meniscus, and the peak stress located in the posterior horn of the medial meniscus and the anterior horn of the lateral meniscus, respectively, and the peak stress value was 4.11 MPa. The maximum displacement of the meniscus was located in body, and the displacement of the medial meniscus was more remarkable than that of the lateral meniscus, and the maximum displacement value is 0.33 mm. The obtained finite element analysis results corresponded to that reported in the literature, which mean the model's reliability. CONCLUSION The established finite element model of knee joint are proved to be have validity, and is a useful model for finite element analysis of meniscus tear and menisectomy. The results of finite element analysis can explain partially biomechanical mechanisms of meniscus which can provide theoretical guidance for clinical treatment of meniscus injury.
Biomechanical Phenomena ; Finite Element Analysis ; Humans ; Knee Joint ; Male ; Menisci, Tibial ; Meniscus ; Reproducibility of Results ; Stress, Mechanical

OBJECTIVE: To investigate the clinical outcome of single or double-segment pedicle subtraction osteotomy (PSO) for the treatment of old thoracolumbar osteoporotic compression fractures with kyphosis. METHODS: The clinical data of 26 patients with old thoracolumbar osteoporotic compression fractures with kyphosis who underwent surgery from January 2015 to June 2017 were retrospectively analyzed. There were 12 males and 14 females, aged from 58 to 72 years old with an average of 65.6 years. The time interval from fracture to surgery was (8.2±1.5) years. According to different surgical methods, the patients were divided into single-segment PSO group (group A) and double-segment PSO group(groupB). The perioperative data, preoperative and postoperative imaging data and postoperative complications were recorded, and the Oswestry Disability Index (ODI) and visual analogue scale (VAS) were used before and after surgery to evaluate the clinical effect. RESULTS: All patients were followed up for 6-24 months with an average of 14.5 months. There was a statistically significant difference in operation time and intraoperative blood loss between the two groups (<0.05). There was no significant difference in total drainage volume and length of hospital stay (>0.05). The postoperative correction angle of the single segment PSO group was (33.3± 9.4) ° with the correction rate of 85.3% and the double segment PSO group was (41.0±13.5) ° with the correction rate of 92.7%, and there was statistical significance between two groups (<0.05). There were no significant differences in postoperative lumbar lordosis (LL), sagittal vertical axis (SVA), and ODI, VAS between two groups (>0.05). One case of cone injury occurred in the single-segment PSO group and recovered after treatment. There were no complications of spinal cord injury in the double segment group. Loose screws occurred during surgery in three patients in the single-segment PSO group and one patient in the double-segment PSO group (<0.05). All 4 patients were re inserted with screws after using bone cement to reinforce the nail pathway. There were no complications such as anterior spinal vascular and nerve damage or osteotomy unfusion. CONCLUSION Both single-segment PSO and double-segment PSO can achieve good orthopedic and clinical effects in the treatment of old thoracolumbar osteoporotic fractures with kyphosis. For patients with correction angles greater than 40° , the double-segment PSO provides a better correction angle and is safer, but it is with longer operation time and more intraoperative bleeding.
Aged ; Female ; Humans ; Kyphosis ; Lumbar Vertebrae ; Male ; Middle Aged ; Osteoporotic Fractures ; surgery ; Osteotomy ; Retrospective Studies ; Thoracic Vertebrae ; Treatment Outcome

OBJECTIVE: To explore the safety and effectivity of ultrasonic bone knife in osteotomy of degenerative kyphosis. METHODS: The clinical data of 32 patients with degenerative kyphosis treated from February 2014 to May 2016 were retrospectively analyzed. There were 12 males and 20 females, aged 50 to 71 years with an average of (62.1±12.3) years. Preoperative Cobb angle was 25.3° to 36.7° with an average of (28.6±10.2) °. All patients underwent multi-segment Ponte osteotomy combined with posterior long segmental internal fixation. According to different osteotomy tools, the patients were divided into traditional tool group (group A, 18 cases) and ultrasonic bone knife group (group B, 14 cases). The operation time, laminectomy time, number of osteotomy segments, intraoperative blood loss, postoperative drainage, hospitalization time and postoperative complications were recorded.Visual analogue scale (VAS) and Oswestry Disability Index (ODI) were used to evaluate clinical outcomes before and 1 month after surgery. RESULTS: All the patients were followed up from 8 to 24 months with an average of 16.4 months. There were no significant differences in operative time and hospitalization time between two groups (>0.05). Intraoperative single laminectomy time of group A was more than that of group B (<0.05). The number of osteotomy segments in group A was less than that in group B (<0.05). Intraoperative blood loss and postoperative drainage in group A were more than that in group B (<0.05). There were no significant differences in VAS and ODI between two groups (>0.05). There were no dural, nerve and spinal cord injuries in both groups, and there were no complications such as improper operation of the instrument and nonunion of the osteotomy. CONCLUSION The use of ultrasonic bone knife for Ponte osteotomy is safe and effective. It can effectively save the time of single laminectomy while reducing the amount of intraoperative blood loss and postoperative drainage. The safety and clinical efficacy of ultrasonic bone knife are no less than traditional tools.