Objective To develop a clinically useful assay for detecting the mutations of HBV pre-C/BCP based on the pyrosequencing and accuracy, reproducibility and reliability of this assay was evaluated. Methods The pyrosequencing primers for HBV pre-C/BCP mutation were designed through the cluster analysis among one hundred HBV gene sequences. After the amplification of the fragment of pre-C/BCP with the template of pre-C/BCP mutation plasmids, the pyrosequencing method for pre-C/BCP detection was initially set up with this standard sample. The accuracy, reliability and reproducibility of the pre-C/BCP pyrosequencing were confirmed through the pre-C/BCP plasmids as a standard sample when compared with Sanger/genechip sequencing method pre-C/BCP pyrosequencing assay was applied for detecting pre-C/BCP mutation types of 60 chnical serum samples in HBV patients. Results The pre-C/BCP mutation detection assay based on pyrosequencing has been established in our study. The coincidence rate between pyrosequencing and Sanger squencing was 100%. The coincidence rate between the result of pyrosequencing and of genechip method was 91.7%. The reproducibility of this assay was 97. 8%. It indicates the pre-C/BCP pyrosequencing is a high-accurate method with, good-reproducibility and high-reliability. And multi-site detection can be achieved by pyrosequencing one time. A rare mutation T1758C was also detected. Conclusion Pyrosequencing for pre-C/BCP mutations assay is high-throughout method for simultaneous detection of multi-site mutation.

BACKGROUND:With the deepening of bone tissue engineering research and bone metabolism understanding, it is a hotspot to analyze the blood supply and nutritional status of tissue-engineered bone.
OBJECTIVE:To compare different methods for evaluating smal vascular network distribution around the knee joint in rats in order to provide a guideline for the study of microvascular network in tissue-engineered bone.
METHODS:Eighteen Sprague-Dawley rats were randomly divided into three groups, with six rats in each group. Three commonly methods were used to evaluate the smal vascular network around the knee joint in rats:immunohistochemistry analysis, angiography analysis, and CT scans and reconstruction analysis.
RESULTS AND CONCLUSION:The microstructure of vascular network could be observed by immunohistochemistry, but the spatial distribution of vessels could not be evaluated. The spatial distribution of vessels could be showed by angiography and CT scans. However, some of micro vessels were showed unclearly by CT scans. The number of blood vessels detected by immunohistochemistry was (26.50±3.02) vessels, significantly higher than those detected by angiography and CT scans that were (14.12±1.47) and (9.00±1.79) vessels, respectively. Combination of immunohistochemistry and angiography can evaluate the microvascular network at microscopic and macroscopic levels, which can provide the whole information of the vascular network.

A new phenethanol, (2'R)-4-(2', 3'-dihydroxy-3'-methyl-butanoxy)-phenethanol (1), along with other eleven known benzene derivatives (2-12) were isolated from the roots, stems and leaves of Clausena excavata (Rutaceae). Compounds 3 and 4 are new natural products, and compounds 5-8, 10-12 were isolated from C. excavata for the first time. Their structures were elucidated on the basis of MS, 1D and 2D NMR spectroscopic analyses including HSQC, COSY and HMBC experiments. 1 was tested for its cytotoxicities against A549, HeLa and BGC-823 cancer cell lines, and antimicrobial activities against Candida albicans and Staphylococcus aureus. The results showed that 1 did not exhibit cytotoxic and antimicrobial activities.
Benzene Derivatives ; chemistry ; Candida albicans ; drug effects ; Cell Line, Tumor ; Clausena ; chemistry ; HeLa Cells ; Humans ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Plant Leaves ; chemistry ; Plant Roots ; chemistry ; Plant Stems ; chemistry ; Staphylococcus aureus ; drug effects

OBJECTIVETo investigate clinical effects of minimally invasive fixation and bone grafting througn medial side for the treatment of Schatzker III tibial plateau fracture.

METHODSFrom April 2009 to August 2011, 18 patients with Schatzker III tibial plateau fracture were treated with minimally invasive fixation and bone grafting through medial side. There were 15 males and 3 females ranging in age from 64 to 73 years, with an average of (69.75 ± 1.22) years. Sixteen patients were caused by falling down, 2 cases were caused by traffic accident. Operative time and length of incision were be recorded. Clinical and radiological follow-up was performed after operation. Hospital for Special Surgery (HSS) score and Kellgren-Lawrence score were used to evaluate clinical effects.

RESULTSThe mean operative time was (45.32 ± 1.58) min, and the mean length of incision was (5.21 ± 0.65) cm. Postoperative X-ray showed excellent reduction. Eighteen patients were followed up for 10 to 13 months with an average of (11.5 ± 1.35) months. The mean HSS score was 86.51 ± 2.71, 12 cases got excellent results,4 good and 2 fair. Three patients were developed mild osteoarthritis according to the Kellgren-Lawrence system.

CONCLUSIONMinimally invasive fixation and bone grafting through medial side, not only could reduce surgical invasive, but also guarantee early function activities. It has advantages of keeping well after reduction. So it has the favorable future in clinic.

Aged ; Bone Transplantation ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Male ; Middle Aged ; Minimally Invasive Surgical Procedures ; methods ; Retrospective Studies ; Tibial Fractures ; surgery

OBJECTIVE:To investigate the compatible stability of Levetiracetam(Lev)injection with 3 injections. METHODS:Each Lev injection 1000 mg mixed with 0.9% Sodium chloride injection 100 mL,5% Glucose injection 100 mL or Sodium lactate Ringer's injection 100 mL respectively. Under the light condition,at 25 ℃,the color and clarification degree of mixtures were ob-served at different time points within 24 h after mixing;pH value and the number of insoluble particles were determined. The contents of related impurities(impurity A,B,C,D,2-hydroxypyridine)and Lev in mixtures were determined by HPLC. RESULTS:Under above condition,all mixtures were colorless clear liquid within 24 h;pH value had no significant change (RSD<1%,n=7);the number of insoluble particles was no more than the range stated in Chinese Pharmacopeia(2015 edition). Impurity B and C were not detected;the contents of other impurities were in line with the requirements of foreign pharmacopeia. No marked change was noted for relative content of Lev(RSD<1%,n=7). CONCLUSIONS:After mixing with 0.9% Sodium chloride injection,5% Glucose injec-tion or Sodium lactate Ringer's injection,Lev injection keep stable at 25℃within 24 h under the light condition.

ObjectiveTo detect the changes of gastroesophageal reflux disease (GERD) with high resolution endoscopy,and to explore its diagnostic value for nonerosive reflux disease (NERD). Methods From April 2007 to January 2008, consecutive out-patients visiting the Department of Gastroenterology due to continuous or recurrent symptoms of acid reflux, heartburn, cardiac-like chest pain for at least three months and volunteer healthy controls were recruited to the study. The subjects were classified into the normal group ( n =48 ), the NERD group ( n =70), the erosive esophagitis (EE) group ( n =70), and the Barrett esophagus (BE) group ( n =48). All subjects underwent endoscopy, and the shape of Z-lines, the shape of mucosal pits, mucosa roughness above Z-line and the shape of mucosal blood vessels were observed. The NERD changes were analyzed with optimal scale. ResultsZ-line shape of NERD group showed a significant difference from that of EE and BE groups ( P ＜ 0. 01 ), which was not different from that of normal controls ( P ＞0. 01 ). The shape of mucosal pits of NERD group was different from that of EE and BE (P ＜0. 01 ), which also was not different from that of control (P＞0. 01 ). Roughness of the mucosa above Z-line of NERD group was different from other 3 groups (P ＜0. 05). And the shape of mucosal blood vessels of NERD was different from the control (P＜0. 05), but was not from two others. Features of NERD relative images were thin,straight and spiral blood vessels, bar-like pits, plat mucosa, round, smooth and wide tooth-like Z-line and mucosa of white particle hypertrophy. ConclusionHigh resolution endoscopic features of most NERD patients are cloudy, white and rough mucosal surface, with white particular hypertrophy, spiral vascular dilation, extending to Z-line. These features can be indicators of NERD.

Spermatogonial transplantation (ST) is a novel technique which mechanism is similar to other transplantations. The donor testicular cells transferred into recipient testes by the microinjection can initiate spermatogenesis and produce sperm in the recipient testis. The homograft has got success. The xenograft in which (man, horse, bull or rat, can be the donor; immunodeficiency mouse can be the recipient) and cryopreserved germ cells can not clonize in the recipient testes. The reason may be immunoreaction. ST technique will be propitious to these fields, 1. to investigate fundamental aspects of spermatogenesis; 2. to regenerate spermatogenesis in infertile individuals; 3. to develop transgenic animals.
Animals ; Animals, Genetically Modified ; Humans ; Male ; Spermatogenesis ; physiology ; Spermatogonia ; transplantation ; Transplantation, Heterologous

BACKGROUNDAdrenocorticotrophin (ACTH)-secreting pituitary adenomas account for approximately 7% - 14% of all pituitary adenomas, but its pathogenesis is still enigmatic. This study aimed to explore mechanisms underlying the pathogenesis of ACTH-secreting pituitary adenomas.

METHODSWe used fiber-optic beadarray to examine gene expression in three ACTH-secreting adenomas compared with three normal pituitaries. Four differentially expressed genes from the three ACTH-secreting adenomas and three normal pituitaries were chosen randomly for validation by reverse transcriptase-real time quantitative polymerase chain reaction (RT-qPCR). We then analyzed the differentially expressed gene profile with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.

RESULTSFiber-optic beadarray analysis showed that the expression of 28 genes and 8 expressed sequence tags (ESTs) were significantly increased and the expression of 412 genes and 31 ESTs were significantly decreased. Bioinformatic and pathway analysis showed that the genes HIGD1B, EPS8, HPGD, DAPK2, and IGFBP3 and the transforming growth factor (TGF)-β signaling pathway and extracellular matrix (ECM)-receptor interaction pathway may play important roles in tumorigenesis and progression of ACTH-secreting pituitary adenomas.

CONCLUSIONSOur data suggest that numerous aberrantly expressed genes and several pathways are involved in the pathogenesis of ACTH-secreting pituitary adenomas. Fiber-optic beadarray combined with pathway analysis of differential gene expression appears to be a valid method of investigating tumour pathogenesis.

ACTH-Secreting Pituitary Adenoma ; etiology ; genetics ; Adenoma ; etiology ; genetics ; Adult ; Disease Progression ; Expressed Sequence Tags ; Extracellular Matrix Proteins ; physiology ; Female ; Fiber Optic Technology ; Gene Expression Profiling ; Humans ; Male ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; methods ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; physiology ; Transforming Growth Factor alpha ; physiology

Objective To evaluate the efficacy of naloxone used in the postoperation of cerebral tumor.Methods Eighty patients were randomly assigned to receive (treated group:40 patients) or not re- ceive (control group:40 patients) naloxone.Both the two groups accepted the conventional therapy.Re- sults After operation,the content of?-EP,ET decreased continuously but the one of the treated groups was more obviously than that of the control groups (P

Objective To prepare endo-beta-N-acetylglucosaminidase H (Endo-H) expressed in Pichia pastoris, and apply it to N-glycosylation analysis .Methods One complete gene was synthesized on the basis of the cDNA sequence encoding Streptomyces plicatus reported in GenBank .The gene was cloned into the expression vector pPIC 9.The expression vector pPIC9-Endo-H was transformed into P.pastoris(JC308).The expression products were induced by methanol , puri-fied by two-step chromatography , used to analyze the glycan structures of RNaseB by the DNA sequencer assisted fluoro-phore-assisted carbohydrate electrophoresis (DSA-FACE)methods, and finally compared with peptide-N-asparagine amidase F(PNGase F).Results This enzyme expressed in P.pastoris(JC308) had the ability to hydrolyze natural or denatured high-mannose type of oligosaccharide linked by β-1,4-glycosidic bonds , but not complex-type oligosaccharide .The result of DSA-FACE showed that carbohydrate chains of Man 5 GlcNAc-Man9 GlcNAc could be obtained when RNaseB was hydrolyzed by Endo-H, and that Man5 GlcNAc2-Man9 GlcNAc2 chains became available when RNaseB was hydrolyzed by PNGase F . Conclusion Endo-H expressed in P.pastoris has bioactivity which can be used to analyze N-glycosylation with the method of DSA-FACE.