By engineering the subsite +1 of cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans, we improved its maltodextrin specificity for 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) synthesis. Specifically, we conducted site-saturation mutagenesis on Leu194, Ala230, and His233 in subsite +1 separately and gained 3 mutants L194N (leucine --> asparagine), A230D (alanine --> aspartic acid), and H233E (histidine --> glutamic acid) produced higher AA-2G yield than the wild-type and the other mutant CGTases. Therefore, the 3 mutants L194N, A230D, and H233E were further used to construct the double and triple mutations. Among the 7 obtained combinational mutants, the triple mutant L194N/A230D/H233E produced the highest AA-2G titer of 1.95 g/L, which was increased by 62.5% compared with that produced by the wild-type CGTase. Then, we modeled the reaction kinetics of all the mutants and found a substrate inhibition by high titer of L-AA for the mutants. The optimal temperature, pH, and reaction time of all the mutants were also determined. The structure modeling indicated that the enhanced maltodextrin specificity may be related with the changes of hydrogen bonding interactions between the side chain of residue at the three positions (194, 230 and 233) and the substrate sugars.
Ascorbic Acid ; analogs & derivatives ; chemistry ; Glucosyltransferases ; genetics ; metabolism ; Hydrogen Bonding ; Kinetics ; Mutagenesis, Site-Directed ; Paenibacillus ; enzymology ; Polysaccharides ; chemistry ; Protein Engineering ; Substrate Specificity ; Temperature

Objective To evaluate the precision of image guided radiotherapy (IGRT) for vertebra metastasis.Methods Cone-beam computed tomography (CBCT, ELEKTA SynergyTM) scanning was performed to 15 patients with vertebra metastasis treated with three dimensional conformal radiation therapy (3DCRT) or intensity modulated radiation therapy (IMRT).CBCT images were then compared with corresponding planning images to calculate the position errors.The isocenter was adjusted based on the errors calculated , CBCT scanning was re - performed , and the new - errors were then calculated .Results Compared to the firstly collected CBCT images, the average errors of 4 cases of cervical bone metastases in x (left-right), y (cervical-caudal), and z (anterior-posterior) directions were 1.8 mm, 0 mm and 3.6 mm respectively.After adjusting the isocenter, the new-errors were reduced to 0.1 mm, 0.4 mm and 0.3 mm.For 11 cases of thoracic and lumbar bone metastases, the average errors in x, y, and z directions were 1.9 mm, 0.1 mm, and -2.1 mm, respectively.While the new-errors were reduced to 0.9 mm, 0.5 mm and -0.3 mm.Conclusions IGRT can improve the precision of radiotherapy for vertebra metastasis to less than 2 mm, which provides a possibility of dose escalation in GTV while reduce the dose in the spinal cord.

Objective To study the changes of activities of phase Ⅰ and Ⅱ drug-metabolic enzymes in the spleen of rats with spleen-kidney yang deficiency syndrome.Methods The rat model of spleen-kidney yang deficiency syndrome was established by gastric gavage of Radix et Rhizoma Rhei decoction combined with injection of hydrocortisone for 17 continuous days.And then we detected the activities of 6 kinds of phase Ⅰ drug-metabolic enzymes of CYP2C19,CYP2D6,CYP2C9,CYP1A2,CYP2C8,CYP3A4,and 4 kinds of phase Ⅱ drugmetabolic enzymes of phenol sulfotransferase (PST),uridine diphosphate glucuronosyl transferase 1 (UGT1),glutathione transferase (GST),estrogen sulfotransferase (SULT1E1) in the spleen.Results Compared with the normal control group,the activities of PST,UGT1,GST and SULT1E1 in the model group were significantly decreased (P ＜ 0.05 or P ＜ 0.01),and the activity of CYP1A2 was significantly increased (P ＜ 0.01),while CYP2C19,CYP2D6,CYP2C8,CYP3A4,CYP2C9 enzymes showed no obvious changes(P ＞ 0.05).Conclusion The activities of splenic drug-metabolic enzymes,in particular the phase Ⅱ enzymes,are significantly varied under the state of spleen-kidney yang deficiency.

Objective To investigate the CT and MRI findings of Coats’disease in comparison with pathology.Methods CT,MRI and ultrasonic features,FFA findings of eight patients of Coats’disease with histo-pathologically confirmed were analyzed retrospectively.CT scanning,routine MRI scanning and ultrasonic examination were performed in all eight patients.Results Unilateral eyeball was involved in all eight cases.On CT scanning,the density of the vitreous body was increased homogeneously which boundary was clear(n=8).The retina was thick(n=8).The anterior chamber depth was shallow(n=5).Multiple calcified foci occured in lens and vitreous body(n=1).The volume of affected eyeball increased(n=1).The affected eyeball shrinked(n=1).The difference of volume of bilateral eyeball was not obvious(n=6). Lens were thick and dislocation(n=5).On MRI scanning,the lesions in the vitreous body showed iso T1 and iso T2 signal (n=6),short T1 and long T2 signal(n=1),long T1 and long T2 signal(n=1).The retina showed short T2 signal(n=3).The vitreous body was filled with lesions(n=5).The lesions looked like‘V’sticked to retina(n= 3).On ultrasonic examination low echo was showed in the vitreous body(n=6),the ball wall bulged(n=8),retinal detachment(n=8).The echo of the ball wall was obviously enhanced,which indica-ted ossification(n=1).Strong echo calcified plaque was showed in one case.FFA showed retinal telangiectasia(n=8),retinal capillary zone(n=2),mutiple chestnut shaped aneurysms(n= 6),retinal neovascularization(n= 1 ).Pathological examination showed retinal telangiectasia with foam macrophages and lipid deposition.Conclusion Coats’disease carries some typical CT and MRI features.To summarize the radiologic features,the findings of FFA and ultrasonic inspection are helpful to diagnosis.

Objective To evaluate the semen quality and sperm morphology in the patients with different grades of varicocele. Methods Semen from 121 patients with varicocele which were divided into three groups, gradel, grade Ⅱ and grade Ⅲ, were studied and those of 23 normal male were taken as the control. Semen analysis was performed with the methods described in the WHO manual and sperm morphology was evaluated by WHO cri-teria. Results A significant reduction of semen quality and sperm morphology and an increase of small oval head, tapering head and amorphous head sperm were found in patients with different grades of varicocele compa-ring with those of the control. There was no difference in routine analysis between different groups. A reducetion of normal morphology percentage in grade Ⅲ were found comparing with grade Ⅱ (P<0.01). An increase of a-morphous head sperm in grade Ⅲ was found comparing with that of sperm in grade Ⅱ (P <0.01). Canclusions The routine semen analysis can not distinguish seminal damage between different grades of varicocele, but the sperm morphology can reflect the sperm state. Therefore, the patients with varicocele should not only get routine semen analysis but also check the sperm morphology.

Objective To evaluate the application of cough reflex testing with various concentrations of citric acid for dysphagia post stroke. Methods 20 normal controls (NC), 20 stroke patients with dysphagia (SD) and 20 stroke patients without dysphagia (SND) were tested with cough reflex inhalated 4 kinds of dosage of citric acid: 0.2 mol/L, 0.4 mol/L, 0.6 mol/L and 0.8 mol/L. Results The incidence of pass (coughed twice or more) decreased in the SD compared with those in the NC as inhalated citric acid of 0.2 mol/L and 0.4 mol/L (P< 0.05), and decreased under 0.4 mol/L compared with the SND (P<0.05). There was no significant difference between the SND and the NC (P>0.05). 90% of the NC passed as inhalated citric acid of 0.4 mol/L; however, it was 45% in the SD, and increased when they inhalated more dosage of citric acid (P<0.05). The incidence of pass decreased under 0.2 mol/L citric acid in the SND compared with other concentration (P<0.05). The result of the test was reliable interrater (κ=0.97). The incidence of cough was consistent of 96.8% with the same concentration. No asthma and asphyxia was observed. Conclusion Cough reflex testing with citric acid inhalation can be used to assess the defensive function of airway in lower concentration for dysphagia after stroke.

Objective To observe the clinical effect of a modified version of constraint-induced movement therapy(MCIMT)on motor recovery in the upper extremities of stroke patients. Methods Twenty-seven stroke patients were randomly divided into a treatment group(n=14)and a routine group(n=13).The Bobath approach and functional arm exercise were administered to the patients in the routine group(2 h/d,5 times a week)for ten weeks.The MCIMT administered to the treatment group involved restriction of the functional arm by a sling during 90%of waking time and during training(by shaping)of the affected extremity(1 hour per day,three times a week)in addition to activities of daily living exercise for at least 2 hours every day.The main outcome measures were the ten items of the simplified test for evaluating hand function(STEF)and Wolf's Motor Function Test(WMFT). Results No significant difference was observed between the 2 groups with regard to WMFT and STEF scores before treatment.After treatment,both groups had improved their WMFT and STEF scores significantly,but there was a significant difference between the treatment and the control groups with regard to WMFT scores and 7 items of the STEF test. Conclusion Modified constraint-induced movement therapy is effective not only for promoting motor recovery of the upper extremity,but also for improving the flexibility and velocity of movement instroke patients.

Objective To investigate the changes in function of liver and kidney of the rabbits bearing VX2 liver tumor after transarterial embolization and hyperthermia with magnetic nanoparticles suspended in lipiodol(MN-L) and its therapeutic effect Methods Thirty-two rabbits bearing VX2 liver tumor were randomly divided into four groups and each group contained 8 rabbits The four groups were MN-L embolization hyperthermia group (Group A), MN-L embolization group(Group B),Lipiodol embolization group(Group C), and Control group (Group D), Each rabbit in Group A and B was embolized with 0.5-0.8 ml MN-L through hepatic artery, while each rabbit in Group C was embolized with 0.5-0.8 ml lipiodol.Hyperthermia in alternating magnetic field was performed in Group A after embolization.The remaining groups did not undergo hyperthermia.The rabbits in control group were not treated.The function of liver and kidney of all the animals was measured 1d before embolization,and 1,7,and 14 d after embolization/hyperthermia respectively.Alanine aminotransferase (ALT) and aspartate aminotransaminase (AST) were used to reflect the function of liver,and blood urea nitrogen(BUN) and creatinine (Cr) were used to reflect the function of kidney.CT was performed on all of subjects before and after embolization to determine the embolization effect and the tumor size, and follow-up CT was performed weekly.All of subjects were sacrificed 14 days after embolization/hyperthermia, and their livers, spleens, kidneys and lungs were removed for histopathology examination.The data from every group were analyzed using analysis of variance of repeated measure data.Results On 1 day before embolization and 1,7, and 14 d after embolization/hyperthermia, the function of liver of the rabbits was as follows:Group A:ALT was (43.9±19.0),(795.1±327.1),(67.0±9.3), and(41.9±10.8) U/L respectively,and AST was (50.2±13.6),(1011.2±655.9),(62.4±24.1),and(51.6±7.9) U/L respectively; Group B: ALT was(45.0±19.1),(580.8±160.4),(67.2±31.0),and(47.6±7.8) U/L respectively, and AST was (52.9±20.3),(735.2±186.1),(57.9±24.8),and (50.9±9.8) U/L respectively; Group C: ALT was (47.4±14.6),(558.5±167.8),(63.5±21.9),and (48.0±9.3) U/L respectively, and AST was (51.8±9.5),(752.5±112.0),(56.5±20.6),and(51.4±8.6) U/L respectively.Both ALT and AST mean values of the rabbits were significantly elevated 1 d after embolization/hyperthermia in Group A, B and C, and the data showed statistically significant difference comparing with that before therapy and that of Group D 1 d after therapy (P<0.01).The function of liver showed no statistically significant difference between 7 or 14 days after embolization and 1 day before embolization in Group A,B and C. BUN and Cr mean values in pre-embolization and post-embolization rabbits revealed no statistically significant difference in group A, B, C and D.The MN-L /lipiodol were deposited in the tumor when it was injected, which was validated by CT.To compare with immediate CT after embolization, the MN-L deposited in tumors was not significantly different on CT 7 d after embolization .On the 14 th day after treatment,the MN-L deposited in tumors became concentrative and compact in Group A, while the MN-L/lipiodol deposited at the rim of tumors disappeared on CT in five rabbits of Group B and C.And the tumor size decreased by 21.7% compared to that before treatment in Group A [from (7.8±1.4)cm~3 to(6.1±0.6) cm~3,F=17.56, P<0.01], but tumor size increased by 16.2% and 18.9% in Group B and C respectively [from (7.9±1.1)and (7.8±0.9)cm~3 to (9.1±0.8) and (9.3±1.0)cm~3, F =25.23,55.50, P<0.01].Histopathologically, the tumor of Group A was necrotic for at least 80% 14 day after embolization, while the tumor of Group B and C was necrotic for 30% to 50% .Conclusion Transarterial embolization and hyperthermia with MN-L is safe, effective and feasible on the rabbits bearing VX2 liver tumor.

OBJECTIVETo study the effect of Percoll selection technique on normal morphology and acrosin activity of human spermatoza.

METHODSThe sperm morphology and sperm acrosin activity were analyzed by automated sperm morphology analyzer(ASMA) and spectrocolorimetry.

RESULTSThe normal morphology sperm rate and acrosin activity were significantly increased after Percoll selection technique (P < 0.001).

CONCLUSIONPercoll selection technique could affect normal morphology sperm ratio and acrosin activity.

Acrosin ; metabolism ; Adult ; Centrifugation, Density Gradient ; Fertilization in Vitro ; Humans ; Male ; Spermatozoa ; cytology ; enzymology

Calorie restriction is one of the diet therapies for people with diabetes.Calorie restriction can reduce weight and improve glycemic control,but the long-term effects are controversial.The effects can be influenced by the degree of calorie restriction,the study duration,and the types of food used for intervention.The mechanism includes the improvement of β cell function and insulin sensitivity.The existing problems are compliance of patients,long-term effects,possible adverse effects,and the lack of high-quality studies.In this review,we introduced the current research progress of calorie restriction on glucose metabolism in patients with type 2 diabetes.