Aim To purify Phospholipid-binding anticoagulation protein(PBAP) from Agkistrodon halys Brevicaudus Venom and study the biochemical characterization.Methods The Phospholipid-binding anticoagulation protein was purified from Agkistrodon halys Brevicaudus Venom by Cation ion exchange chromatography on CM Sephadex C-25 and negative ion exchange chromatography on DEAE Sepharose CL-6B,gel filtration on Sephacryl S-200 and Sephadex G-75 chromatography.Its anticoagulant activities in vitro were assayed by activated partial thromboplastin time(APTT);its molecular weight was calculated by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and its isoelectric point was estimated by the isoelectric focusing electrophoresis.Binding experiments of anticoagulation protein to phospholipids vesicles were performed with thinlayer chromatography.Results A kind of protein was purified from Agkistrodon halys Brevicaudus Venom which was able to prolong APTT.The SDSPAGE showed that it was dimer and its molecular weight was 24.0?10~3 under non-reducing condition and 14.6?10~3 under reducing condition.The isoelectric point was pH 5.2 by the isoelectric focusing electrophoresis.Having arginine ester-hydrolyzing enzyme and binding Phospholipid activify,its effect on APTT was activity stronger with concentration increasing.Conclusion It is a successful method of the purification of Phospholipid-binding anticoagulation protein from the Agkistrodon halys Brevicaudus Venom.

Objective To investigate the clinical feature and antibiotic resistance profile of K.pneumoniae isolates from patients for better management of K.pneumoniae infections.Methods Nonduplicate K.pneumoniae strains were collected from January to December in 2015.K.pneumoniae strains were identified by VITEK 2-Compact 60 and tested for antimicrobial susceptibility by KirbyBauer method.Results A total of 753 strains ofK.pneumoniae were included,most (40.9％,308/753) of which were isolated from sputum,followed by urine (18.2％,137/753).Most of the strains were from old patients at least 60 years of age (40.8％,307/753),and primarily from intensive care units (16.7％,126/753) and Department of Respiratory Medicine (13.7％,103/753).Respiratory tract infection was found in 144 patients,of which 71.5％ (103/144) were due to K.pneumoniae.More than half of the K.pneumoniae strains were resistant to piperacillin (66.3 ％),cefazolin (60.8 ％) and cefitroxime (59.4 ％).Only a few strain were resistant to imipenem (2.4 ％) and meropenem (2.0).ESBLs were produced in 410 (54.4 ％) of the 753 strains,and 29 (3.9 ％) strains were carbapenem-resistant,492 (65.3 ％) strains were resistant to multiple antimicrobial agents.Conclusions Clinical K.pneumoniae isolates are highly resistant to most of the antimicrobial agents tested.The strains were mostly isolated from sputum and urine,and positive for ESBLs.MDR K.pneumoniae sWains are emerging.K.pneumoniae isolates are still very susceptible to carbapenems in vitro.

Objective To analyze the correlation between antibiotic susceptibility andbiofilm formation of Staphylococcus aureus. Methods According to Standardized Operational Guidance for Clinical Microbiological Testing, fifty-eight non-repetitive pathogenic Staphylococcus aureus isolates were collected from blood, urine, sputum and purulent secretions of inpatients in the Laboratory Department of the First Affiliated Hospital of Guangxi Medical University in January 2018. The antibiotics susceptibility of strains was characterized by disk diffusion method and VITEK-2.96-hole culture. The biofilm formation ability was detected bythe crystal violet assay and Congo redplate methods.The in vitro dynamic forming process of S.aureus′sbiofilm was analyzed by crystal violet staining combined with bacterial culture in 96 wells.Besides, the rate differences of drug resistance between biofilm producers and non-producers was performed by Chi-square test. The diameter of bacteriostasis zone of biofilm producers and non-producers was compared by t test. The drug resistance among strains with different biofilm formation ability was analyzed withnonparametric rank sum test. Results The positive ratio of biofilm producing S. aureus was higher in MRSA (68.42％) than in MSSA(20.00％)(χ2=12.304,P=0.001. Antibiotic resistance rates of biofilm producers were higher than non-producers.The resistance rates of biofilm positive strains to oxacillin and clindamycin were 73.33％ and 53.33％, respectively. The antibiotic resistance of the strain was higher along with the biofilm forming ability was increasing. (χ2=9.099, P=0.008). Depths of the S. aureus′s biofilm on the 96-well plates increased significantly over time and reached biofilm maturation after 72 hours′ incubation. Light microscopic observation revealed that the mature biofilm was compact and growing with many layers. Conclusions For the clinical isolates of S. aureus, the antibiotic resistance of biofilm producers is significantly higher than non-producers. Strains with stronger biofilm forming ability had higher antibiotics resistance. Depths of the S. aureus′s biofilm on the 96-well plates increased significantly over time and reached biofilm maturation after 72 hours′incubation.

Objective:To investigate correlation between neutrophil extracellular traps(NETs) formation and T cell subsets in mice with experimental autoimmune thyroiditis(EAT) and the impact of active vitamin D intervention.Methods:Six-week-old female BALB/c mice were randomly divided into Control group, EAT group and 1, 25 dihydroxy vitamin D 3[1, 25(OH) 2D 3] treatment group(VitD group; n=6/group). HE staining was used to observe thyroid pathology. Plasma thyroglobulin antibody(TGAb), thyroid peroxidase antibody(TPOAb), and 1, 25(OH) 2D 3 were measured by ELISA. Peripheral NETs formation, Th1, Th2, and Th17 cell ratio from spleen were measured by flow cytometry. Correlation between NETs formation rate and Th1, Th2, and Th17 cell ratio was analyzed. Results:Compared with Control group, mice in EAT group had significantly increased thyroid inflammation scores, thyroiditis morbidity, TPOAb, TGAb levels, NETs formation rate, Th2(CD4 + IL-4 + or CD4 + IL-13 + )and Th17 cell proportions( P were <0.001, 0.002, 0.007, <0.001, <0.001, 0.003, 0.001, and 0.002, respectively), and significant decreased 1, 25(OH) 2D 3, Th1 cell proportions, Th1/Th2(CD4 + IL-4 + ), Th1/Th2(CD4 + IL-13 + ), and Th1/Th17 ratios( P were 0.010, 0.018, 0.010, 0.005, and 0.007, respectively). Compared with the EAT group, the VitD group had lower thyroid inflammation scores, TPOAb, TGAb levels, NETs formation rate, Th2(CD4 + IL-4 + or CD4 + IL-13 + ) and Th17 cell proportions( P were 0.044, 0.007, <0.001, 0.001, 0.014, 0.008, and 0.001, respectively), and significant higher Th1 cell ratio, Th1/Th2(CD4 + IL-13 + ) and Th1/Th17 ratio( P were 0.011, 0.009, and 0.003, respectively). The Th1/Th2(CD4 + IL-4 + ) was not significantly increased in VitD group compared with EAT group( P=0.174). NETs formation rate was positively correlated with Th2(CD4 + IL-4 + or CD4 + IL-13 + ) and Th17 cell proportion( r were 0.65, 0.59, and 0.61; and P were 0.004, 0.010, and 0.007, respectively), but not with Th1 cell proportion( r=-0.47, P=0.051). Conclusion:EAT mice were more prone to NETs formation. Active vitamin D may relieve immune imbalance with increased Th2 and Th17 cell ratio and decreased Th1 cell ratio by reducing the formation of NETs in EAT mice. Vitamin D played the protective role in thyroid by reducing thyroid pathological damage and thyroid autoantibody levels, and relived overall lymphocyte imbalance.

Objective:To establish a model of patellar dislocation by femoral osteotomy or surgical release of medial retinaculum in immature rabbits, and observe morphological and trabecular microarchitectural changes in the trochlea.Methods:Forty rabbits at 3 months of age were included. All right knees underwent surgery, 20 knees were treated with femoral osteotomy and internal rotation of distal femur to increase femoral anteversion angle (Osteotomy group, OS group), and another 20 knees were treated with surgical release of medial retinaculum and overlap suture of lateral retinaculum (Soft tissue group, ST group). All left knees were acting as internal controls. Micro-CT scans for distal femur were acquired after 4 months post surgery. the height of Medial, central, and lateral trochlear, sulcus angle, and lateral and medial trochlear slope were measured to describe the trochlear morphology, and bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.Sp), and bone mineral density (BMD) were calculated to evaluate the microarchitectural structure. All parameters were compared between groups.Results:In OS group, one rabbit sustained a hip dislocation without patellar dislocation. Three knees developed complete patellar dislocation in daily flexion position, and the remaining 16 patellae were dislocated when the knee was placed in the maximal extension position. In ST group, 15 knees were complete patellar dislocation in daily flexion position, and 5 knees were without dislocation. A local boss was formed proximal to the entrance of the groove and the articular cartilage was smooth, and no obvious osteoarthritis was observed in OS group. In ST group no boss was formed, while obvious cartilage degeneration and defect was seen. Compared to the control group, the central trochlear height and sulcus angle were greater in both groups, but without significant difference between the two groups. The Tb.Th was increased in both medial and lateral condyle, and Tb.N was decreased in medial condyle compared with its control knees in OS group. The BV/TV, Tb.Th, Tb.N and BMD were decreased and Tb.Sp was increased in both medial and lateral condyle compared with its control knees in ST group. Compared to the OS group, the BV/TV, Tb.Th, Tb.N and BMD were smaller and Tb.Sp was greater in both medial and lateral condyle in ST group, with significant differences.Conclusion:The model of patellar dislocation could be successfully achieved by femoral rotational osteotomy to increase femoral anteversion or surgical release of medial retinaculum and overlap suture of lateral retinaculum, and subsequent morphological and trabecular microarchitectural changes in the trochlea are different. Different bony and soft tissue factors should be addressed for different patients with patellar dislocation in clinical practice.