OBJECTIVE:To establish a method for content determination of baicalin in Scutellaria baicalensis Georgi extract and to optimize the extraction technique.METHODS:The content of baicalin was adopted as index and was deter?mined by HPLC;Single factor test and orthogonal test were employed to optimize the extraction technique.RESULTS:The detected concentration of baicalin showed a good linear relation in the range of0.05912～0.02956mg/ml(r=0.99974),the average recovery was99.5%,RSD=1.10%(n=5);The optimal technique was twice recirculation in water solvent14times the amount of Scutellaria baicalensis Georgi flour,each time for1hour.CONCLUSION:The established method is conve?nient,and the extraction technique is stable,reasonable and practicable.

OBJECTIVETo investigate the clinical value of pleural biopsy in the etiological diagnosis of pleurisy in children.

METHODTotally 213 cases with pleurisy, who underwent pleural biopsy and hospitalized in Children's Hospital of Chongqing Medical University from January 2007 to April 2014 were enrolled into this study. Clinical symptoms, imaging manifestations, pleural fluid characteristics, the results of pleural biopsy and postoperative complications were retrospectively analyzed to evaluate the clinical value and security of pleural biopsy in making the etiological diagnosis of pleurisy.

RESULT(1) Of the 213 cases, 144 were boys and 69 were girls, their mean age was (6. 5 ± 4. 1) years. (2) Two hundred and thirteen patients had a surgical pleural biopsy under general anesthesia, the cause of 97 cases (45. 5%) were made clear by histopathological examination, including 35 purulent pleurisy, 55 tuberculous pleurisy and 7 paragonimus infection. For the remaining 83 (41. 3%) cases a final diagnosis was made based on the full analysis of clinical data, including 63 cases of purulent pleurisy, 3 cases of tuberculous pleurisy and 17 cases of paragonimiasis pleurisy but for 33 patients no exact cause was found at the end. (3) The mean operating time of the biopsy was (1. 4 ± 0. 6) hours. Seventy one (33. 3%) patients required blood transfusion during or after the operation. Thirty one (14. 6%) cases used the ventilator after surgery, and the ventilator supporting time was (6. 6 ± 5. 8) hours on average. The wound healing reached grade A in 200 cases (93. 9%), grade B in 13 cases (14. 6%). Postoperative complications included pneumothorax in 92 cases (43. 2%), subcutaneous emphysema in 18 cases (8. 5%), bronchopleural fistula in 3 cases(1. 4%). The average days of hospitalization was (17. 7 ± 7. 1) d.

CONCLUSIONPleural biopsy is of great diagnostic value in the etiological diagnosis and differential diagnosis of pleurisy in children, and it is considered reasonable to be used in the clinical practice when appropriate.

Biopsy ; Child ; Diagnosis, Differential ; Female ; Humans ; Infection ; diagnosis ; Male ; Pleura ; Pleurisy ; diagnosis ; etiology ; Retrospective Studies ; Tuberculosis, Pleural ; complications ; diagnosis

OBJECTIVE:To establish mathematical model of liquid volume added of TCM medicinal broth decoction machine to accurately calculate liquid volume added in the process of medicinal herb decocting,so as to guarantee the quality of medicinal herb decocting. METHODS:The water absorption rate of representative TCM decoction piece with high use frequency were deter-mined,and cluster analysis of water absorption rate of TCM decoction piece was conducted according to closely related index as density,size,shape,moisture. TCM decoction piece with similar water absorption rate were bracketed together,so that of single ingredient TCM decoction piece can be estimated by water absorption of representative TCM decoction piece;the quantity of water evaporation and liquid extrusion were determined among different types of decoction machine (powered by electric and gas);ac-cording to the above parameters,mathematical model of liquid volume of TCM medicinal broth prepared by different types of de-coction machine had been established,and validated with TCM formula. RESULTS:Factors that affected the liquid volume added included the water absorption of each ingredient,the quantity of water evaporation and extrusion function. The mathematical model was liquid volume added=water absorption of each ingredient × quality of decoction piece+the quantity of water evaporation+re-quired amount of liquid-parameters of extrusion function×total weight of decoction piece;in validation test,the percentage of the practical amount of liquid to required amount was within ±5%. CONCLUSIONS:Established model can promote the accuracy li-quid volume added and guarantee the quality of TCM decoction when using TCM decoction machine.

Objective:To construct Streptococcus suis type 2 Δ0948 complementary strain and verify its effect on suilysin (SLY) secretion and virulence. Methods:The SSU05_0948 gene sequence with promoter was amplified by PCR and ligated to pAT18 vector to construct complementary strain and verify its expression through Western blot. Growth curve was drawn to compare the growth of complementary strain against the wild-type strain and mutant strain in different periods. CD1 mice challenge model was used to verify whether complementary strain could restore the virulence of mutant. SLY hemolytic activity and Western blot were compared the effect of complementary strain and wild-type strain and mutant strain on SLY protein secretion at different time points.Results:The complementary strain was successfully constructed, but the expression of SSU05_0948 was lower than the wild-type strain. The growth rate of the complementary strain was significantly slower than the wild-type strain and mutant strain in the logarithmic growth phase, but the same in the platform phase. The CD1 mice challenge model showed the complementary strain could basically restore the virulence of the mutant strain. The hemolytic activity of SLY and Western blot showed that SSU05_0948 could inhibit the secretion of SLY protein in the early and middle logarithmic phase, but did not affect the secretion of SLY in the late logarithmic and platform phase, while the complementary strain could restore the secretion of SLY protein.Conclusions:The complementary strain CΔ0948 of Streptococcus suis can restore the virulence of mutant strain Δ0948, and SSU05_0948 affects the virulence of Δ0948, which provides a new idea for the prevention and treatment of Streptococcus suis.

OBJECTIVETo explore the value of pleural biopsy in the diagnosis of tuberculous pleurisy in children.

METHODFifty-one cases with tuberculous pleurisy, whose diagnosis was established according to the clinical diagnostic criteria of the child pulmonary tuberculosis formulated by the Chinese Medical Association (CMA) in 2006, after pleural biopsy hospitalized in Children's Hospital of Chongqing Medical University from Jan. 1, 2007 to Jan. 1, 2013 were enrolled into this study. Clinical symptoms, history traits, laboratory examination, imaging tests, pleural fluid characteristics and the results of pleural biopsy were retrospectively analyzed. Medical records of the cases who were diagnosed with tuberculous pleurisy by histological examination were reviewed to assess tuberculosis detection rate of pleural biopsy and to get the percentage of cases with a preoperative diagnosis inconsistent with the final diagnosis.

RESULTThere were 35 boys and 16 girls, and the mean age was (9.7 ± 3.5) years. The common symptoms included fever (82%), cough (71%) , chest pain (23%), weakness (10%) and shortness of breath (10%); 27% (14/51) children had shown tuberculosis toxic symptoms; 76% (39/51) patients had BCG vaccination history; 12% (6/51) cases had a history of contact with tuberculosis patients. The positive rates of the tuberculin skin test, serum tuberculosis antibody detection, detection of Mycobacterium tuberculosis DNA by polymerase chain reaction, acid-fast bacillus test of sputum (or gastric juice) smear, acid-fast bacilli (AFB) smear and culture of pleural effusion were respectively 61% (20/33), 6% (3/46), 0 (0/12), 4% (1/27), 22% (7/32). Pleural effusion was found by using imaging tests in 50 cases, among whom 28 cases (55%) with encapsulated effusion, and the multilocular cysts separated by fibrous tissue in 12 patients (23%) . Other features included pleural thickening (53%) , hilar and mediastinal lymph-nodes enlargement (14%) and white nodules of calcification (10%) . Thoracocentesis was performed in 31 cases, and pleural effusion obtained from which were exudative. The cell count, mainly mononuclear cells, increased in 28 patients (90%) . Among the 51 children investigated, 47 (92%) were histologically diagnosed to be tuberculous pleurisy. The typical pathologic changes of tuberculosis (caseous necrosis, granulomas, Langhans' giant cells and inflammatory cell infiltration) were observed in 40 cases, granulomatous inflammation without caseous necrosis were the main manifestations in 7 other patients. The pathological changes of the remaining 4 cases were not consistent with the pathological characteristics of tuberculosis. All 47 cases were given a preoperative diagnosis of tuberculous pleurisy (32%), purulent pleurisy (51%) and pleural effusion of unknown origin (17%) respectively before pleural biopsy. Therefore, the tuberculosis detection rate of pleural biopsy was 92%, and the preoperative misdiagnosis rate was 68%.

CONCLUSIONPleural biopsy was of great diagnostic value for children with tuberculous pleurisy.

Biopsy, Needle ; methods ; Child ; DNA, Bacterial ; isolation & purification ; Diagnostic Errors ; Female ; Humans ; Lung ; microbiology ; pathology ; Male ; Mycobacterium tuberculosis ; isolation & purification ; Pleura ; microbiology ; pathology ; Pleural Effusion ; microbiology ; pathology ; Polymerase Chain Reaction ; Retrospective Studies ; Sputum ; microbiology ; Tuberculin Test ; Tuberculosis, Pleural ; diagnosis ; microbiology ; pathology

Objective: To construct the mutant strain ATP binding cassette transporter SSU05_0948 of Streptococcus suis type 2 and comprehensively study its pathogenicity, and to provide useful insights for understanding the mechanism that Streptococcus suis avoid host innate immunity. Methods: The mutant strain 05ZYH33Δ0948 was constructed through homologous recombination technology. The differences between the mutant strain and the wild type strain were evaluated through bacterial adhesion, whole blood killing, mice meningitis assay, mice and piglets virulence assay. Chi-square test and t test were used. Results: Successfully constructed the mutant strain 05ZYH33Δ0948. The adhesion results showed that the adhesion rate (0.663±0.047)% of the wild strain to A549 cell was significantly higher than that of the mutant (0.246±0.074)%, the difference was statistically significant (χ²=5.267, P=0.014); the adhesion rate (16.540±2.320)% of the wild strain to Hep2 cell was significantly higher than that of the mutant (1.970±0.320)%, the difference was statistically significant (χ²=0.014, P<0.01); the adhesion rate (5.497±0.174)% of the wild strain to Hep2 cell was significantly higher than that of the mutant (1.950±0.335)%, the difference was statistically significant (χ²=0.016, P<0.01). The killing rate (32.970±3.589)% of the wild strain in whole blood is no difference with the mutant (29.560±3.737)% (χ²=1.200, P=0.133). Piglets competitive infection showed that, the competitive index at 12 h, 24 h and 36 h were 0.046±0.003, 0.107±0.003, 0.064±0.001, respectively. 12 h and 24 h was significant differences(t=15.490, P=0.041), 24 h and 36 h was significant differences(t=5.660, P=0.047), 12 h and 36 h was no differences(t=1.445, P=0.285). Conclusions Streptococcus suis type 2 ABC transporter SSU05_0948 is a new adhesion factor and virulence factor of Streptococcus suistype 2, and also a new meningitis factor, which plays important roles in Streptococcus suis against host innate immunity.

Objective To study the main types and their distribution of molecular typing of carbapenem-resistant Acinetobacter baumannii (CRAB)in Beijing.Methods Seventy-eight non-repeated CRAB strains were isolated and collected from patients aged over 60 years in hospitals in Beijing from 2010 to 2014.The drug susceptibilities of 11 antimicrobial agents were tested by microdilution method.A modified Hodge assay was used to preliminarily screen the carbapenemases.A multiplex PCR assay was used for detection of the carbapenemases genes:OXA-23-like,OXA-24-like,OXA-51-like,OXA-58-like,IMP-1,and VIM-2 of Acinetobacter baumannii.We also detected the insertion sequence IsAbal of OXA-23-like and OXA-51-like,and the preliminary classification of homology of carbapenem-resistant Acinetobacter baumannii (CRAB)was conducted by 3LST technique.According to the drug susceptibility,carbapenemases gene typing,3LST primary screen,and hospital distribution,22 strains were selected to conduct MLST classification.Results The test of OXA enzyme gene in 78 strains of CRAB showed that all the strains(100％)carried oxa-51-like,and 74/78 strains(94.8％)carried oxa-23-like.Additionally,all products of ISAbal-oxa-23 were positive in OXA-23-like positive strains.In the examined five β-lactamase genes,74 strains(94.8％)showed positive AmpC gene;50 strains (64.1％) showed positive TEM-1 gene;5 strains (6.4％) showed positive PER-1 gene;48 strains(61.5％)showed positive genes of TEM-1 and AmpC and 3 strains showed positive genes of TEM-1,Amp C,and PER-1.By using 3LST technique for preliminary classification,we found 74 strains were in type Ⅰ of group types belonging to the European Ⅱ cloning spectrum.Of the six ST types found in MLST classification,the ST195,ST208,ST218 and ST368 belonged to the clone complex 92(CC92);the ST103 and ST500 were two newly discovered types in Chinese population.Conclusions CC92 clone cluster is the major epidemic strain of CRAB in Beijing,which belongs to the classic European Ⅱ cloning spectrum,and its insertion sequence,Abalinduced OXA-23-like carbapenemases is the major molecular of carbapenem resistant Acinetobacter baumannii(CRAB) in Beijing.Both AmpC and TEM-type 1 β-lactamase genes are detected to be positive in most of strains.The newly discovered two types are mainly CC103 clone complex.

Objective To determine whether coagulase-negative non-epidermal staphylococcus is methicillin-resistant coagulase-negative staphylococcus by mecA gene test,when the minimal inhibitory concentration(MIC)of oxacillin is between 0.5-2.0 mg/L.Methods The mecA gene was detected and analyzed by the cefoxitin disk diffusion,E-test,VITEK-2 Compact and polymerase chain reaction (PCR)purification.Results A total of 300 strains of coagulase-negative staphylococci were screened from 4032 patients(7.4％),of which 45 strains of Staphylococcus saprophyticus and 80 strains of Staphylococcus hemolyticus were identified by Compact VITEK-2.There was a statistically significant difference in the positive rate of mecA gene detection between Staphylococcus saprophyticus and Staphylococcus hemolyticus(P ＜0.05).The results of detection of cefoxitin disk diffusion(inhibitory zone diameter ≥ 25 mm),E-test(MIC of oxacillin between 0.5-2.0 mg/L)and Compact VITEK-2 (MIC of oxacillin between 0.5-2.0 mg/L)showed that there were 81 strains of coagulase-negative non-Staphylococci,of which 10 strains with positive mecA gene were confirmed by PCR.Conclusions When the minimal inhibitory concentration (MIC)of oxacillin against coagulase-negative non-Staphylococci stains is between 0.5-2.0 mg/L,the guidelines of the American clinical laboratory standardization institute(CLSI)should be strictly implemented in clinical microbiology laboratory and the mecA gene must be tested.Based on the wide dissemination of the mecA gene in Staphylococcus aureus population,if the mecA gene test is negative,it is reported as methicillin-susceptible coagulase-negative Staphylococcus(MSCNS),and the reverse result is reported as methicillin-resistant coagulase-negative staphylococcus(MRCNS).

Objective To study the mechanism of carboxypeptidase E ( CPE ) in promoting the migration of lymphocytes and their subsets through vascular endothelial cells. Methods CRISPR/Cas9 technology was used to prepare cpe gene-knockout MS1 (Cpe-/-MS1) cells. Adhesion ability of lymphocytes to MS1 and Cpe-/-MS1 cells was analyzed with adhesion assay. Expression of adhesion molecules on these cells were detected by RT-PCR and flow cytometry. Transwell model was used to compare the difference in the transmigration of lymphocytes and their subsets through MS1 and Cpe-/-MS1 cells. Results Cpe-/-MS1 cells were successfully obtained. Under the stimulation of TNF-α, the adhesion ability of lymphocytes to MS1 cells was much better than that of Cpe-/-MS1 cells. Moreover, adhesion molecules expressed on MS1 cells were significantly more than those on Cpe-/-MS1 cells. The percentages of lymphocytes and their sub-sets that transmigrated through MS1 cells were significantly higher than those through Cpe-/-MS1 cells. Con-clusion CPE involved in the adhesion of lymphocytes to vascular endothelial cells and the transmigration of them through vascular endothelial cells, which was of great significance for understanding the migration of lymphocytes across vascular endothelial cells to peripheral lymph nodes.

Objective To construct a mutant strain of Streptococcus suis type 2 05ZYH33 express-ing ABC transporter SSU05 0946 and to study the pathogenicity of ABC transporter SSU05 0946 for better understanding the immune evasion strategies by Streptococcus suis. Methods Genome of the Streptococcus suis type 2 05ZYH33 strain was extracted and used as a template to amplify SSU05 0946 upstream and down-stream homeodomains. Chloramphenicol-resistance gene was amplified by using pSET1 plasmid as the tem-plate. These three amplified fragments were fused and integrated with the thermo-sensitive plasmid pSET4s by using overlap extension PCR. Homologous recombination method was used to construct the mutant strain 05ZYH33Δ0946. Differences between the mutant and wild type strains were evaluated through bacterial ad-hesion assay,whole blood killing assay and challenge test in mice and piglets. Results The mutant strain 05ZYH33Δ0946 was successfully constructed. Results of bacterial adhesion assay demonstrated that SSU05 0946 was not involved in the adherence of Streptococcus suis to human epithelial cells. SSU05 0946 was an ovel anti-phagocytic factor and virulence factor of Streptococcus suis. Conclusion Streptococcus suis type 2 ABC transporter SSU05 0946 is a newly discovered virulence factor of Streptococcus suis, playing an impor-tant role in the evasion of host innate immunity by Streptococcus suis.