Objective To evaluate the diagnostic value of fluorescent quantitative polymerase chain reaction(PCR) for Mycoplasma pneumoniae (MP) in children with MP pneumonia(MPP).Methods From Jun.2008 to Jan.2009,153 cases hospitalized with pneumonia were enrolled,and 30 cases without respiratory infection were enrolled as control group.Their respiratory secretion (including nasopharyngeal secretion,sputum,bronchialalveolar lavage fluid or pharyngeal swab) samples were collected for fluorescent quantitative PCR for MP.And their single or paired serums were collected for specific MP antibody detection.Results There were 123 cases confirmed with MPP by serology,among whom 114 cases were MP PCR positive.The quantitation of MP DNA was among 1.20?106-3.66?1010 gene copys/L. There were 30 cases with pneumonia negative with MP by the paired serum serology,among whom 2 cases were MP PCR positive,and the quantitation of MP DNA was (1.08-3.02)?107gene copys/L.All cases of control group were MP PCR negative.During the first and second weeks of the MPP onset,the sensitivity of MP-IgM from the first single blood samples were 66.7% and 83.9%,respectively.While the sensitivity and specificity of MP PCR were 92.7% and 93.3%,respectively.From the third week of the disease onset,the sensitivity of MP-IgM from the first single blood samples increased to 90.9%-100%.The clinical manifestations of MPP were nonspecific.Conclusions PCR is superior to serology for early diagnosis on MP infection.Combination of the 2 methods may be helpful to early and accurate diagnosis on MP infection.

Objective To study the feasibility and safety of hand-assisted laparoscopic hepatectomy for huge left hepatoma.Methods Nine patients with huge left hepatoma underwent hand- assisted laparoscopic hepatectomy including hepatocellular carcinoma(4 cases),intrahepatic cholangioearcinoma(1 case),hepatic metastatic squamous carcinoma(1 case),hepatic cavernous hemangioma(2 cases),and hepatic spindle cell tumor(1 case).The mean age was 45.3 years.AFP was positive in 3 cases and CEA was positive in 1 case.The preoperative liver function was Child-Pugh A in all patients.The procedure included dissection of left hepatic ligaments and portal triad clamping with Pringle's maneuver and hepatectomy.Results The laparoscopic procedures were completed safely in all patients including 6 left lateral segmentectomies and 3 left hemihepatectomies.There was no conversion to laparotomy.Mean surgical time was 111.7 minutes.Mean blood loss was 97.8 ml.Portal triad clamping was used in 8 cases and mean clamping time was 13.4 minutes.Neither formidable bleeding nor gas embolism occurred.There were no serious postoperative complications such as postoperative bleeding or bile leak or liver failure.Liver function recovered within 7 to 10 days.Preoperatively positive AFP and CEA turned negative after operation.The mean postoperative hospital stay was 8.4 days.Four patients with HCC underwent postoperative prophylactic hepatic arterial chemoembolization within the first postoperative month. All patients were tumor-free as evaluated by postoperative follow-up of 4～11 months.Conclusions Hand-assisted laparoscopic hepatectomy for huge left hepatoma is feasible and safe in appropriately selected patients.

Spleen kidney Yang deficiency (SKYD) diarrhea is a common syndrome in tranditional Chinese medicine (TCM). Until now, there is not an ideal SKYD diarrhea rat model for the research. In this study, we compared single factor way (method I, injecting hydrocortisone and gavaging Sennae Folium) with compound factors way(method II, gavaging adenine, improper diet, exhaustion, and gavaging Sennae Folium) on establishing SKYD diarrhea rat model. After modelling, diarrhea index, D-xylose excretory rate, NOS/cGMP signal transduction system, organ index and histopathology examination were used to evaluate the two ways. The results showed that, compared with health group, all the assessment criterias of method I and method II had significant differences (P < 0.01, 0.05). In addition, the index such as diarrhea index, NOS/cGMP signal transduction system, organ index (kidney, testis and thymus) and histopathology examination had significant differences (P < 0.01, 0.05) between method I and method II. In conclusion, the compound factors modelling method better conforms to the symptom of diarrhoea model caused by SKYD. This new modelling method provides a basis for studying on TCM astringents warming and tonifying the spleen and kidney, relieving diarrhea.
Animals ; Diarrhea ; metabolism ; pathology ; physiopathology ; Disease Models, Animal ; Humans ; Kidney ; pathology ; physiopathology ; Male ; Rats ; Rats, Sprague-Dawley ; Spleen ; pathology ; physiopathology ; Xylose ; metabolism ; Yang Deficiency ; metabolism ; pathology ; physiopathology

A creatininase produced from a Arthrobacter sp. was purified 145-fold by a series of steps including heat treatment, ammonium sulfate precipitation, DEAE-Cellulose ion-exchange and hydrophobic chromatography. The specific activity of the pure enzyme was 209u/mg. The subunit molecular mass of creatininase was estimated to be 33 700D by SDS-PAGE. The creatininase was stable in the pH range between 6.0 - 9.0 and below 60 degrees C . Its Km value for creatinine was estimated to be 21.14 mmol/L. The enzyme was markedly inactivated by incubation with 1 mmol/L of Hg2+, Ag2+, Li+, Cu2+ and 20 mmol/L of 1, 11-Phananthroline respectively. Activation was observed when the enzyme was incubated with 1 mmol/L of Co2+ and Mn2+.
Amidohydrolases ; isolation & purification ; metabolism ; Arthrobacter ; enzymology ; Bacterial Proteins ; isolation & purification ; metabolism ; Chromatography, DEAE-Cellulose ; methods

Objective To construct the eukaryotic expression vector of LDHA with Flag label and detect its effects on the growth of human choroidal melanoma (CM) MUM-2B cells.Methods CM cells line MUM-2B subcultured by the Military Academy of Sciences were divided into two groups:experimental group and control group.The experimental group was transiently transfected with Flag-LDHA plasmid,and the control group was transiently transfected with Flag plasmid.Using the Flag-LDHA with GST label as a template,the LDHA gene was amplified by polymerase chain reaction (PCR),which then was inserted into eukaryotic expression vector of Flag,and the recombinant plasmid Flag-LDHA was identified by bacterial liquid PCR,double enzyme digestion and sequencing,both which were transiently transfected into human CM MUM-2B cells after successful identification,and finally,its expression was determined by Western blot.The biology behaviors of melanoma cell line MUM-2B transfected with Flag-LDHA and Flag plasmid were analyzed by counting Kit-8 (CCK8) assays.Results The coding region sequence of LDHA gene of approximately 1000 bp was harvested from PCR amplification,which was successfully cloned into the Flag vector.Compared with the control group,the PCR result of the bacterial liquid in the experimental group was positive.The double enzyme digestion results showed that eukaryotic expression vector of Flag with a length of about 4000 bp Flag vector and a 1000 bp LDHA gene band.And the sequencing results indicated that the inserted sequence was completely in consonance with the coding sequence of the LDHA gene.Western blot results showed the successful expression of recombinant plasmid Flag-LDHA in MUM-2B melanoma cells.CCK8 assays demonstrated that Flag-LDHA recombinant plasmid could promote the growth of melanoma cell line MUM-2B.Conclusion The eukaryotic expression vector of Flag-LDHA was successfully constructed,which can promote the growth of melanoma cell line MUM-2B.This will lay the foundation for studying the function of LDHA in the initiation and progression of human CM.

BACKGROUND: This study aimed to determine the plasma levels of urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), D-dimer, IL-6 and TNF-α, and observe the relations among uPA, uPAR, D-dimer, IL-6 and TNF-α in patients with systemic inflammatory response syndrome (SIRS). METHODS: A prospective, clinical case-control study was conducted in patients with SIRS at age of more than 55 years old treated during 2008-2010 at Wuhan Central Hospital. Venous blood samples were collected by routine venipuncture. Eighty-five patients were divided into two groups according to diagnostic criteria of SIRS: SIRS patients from intensive care units (n=50), and non-SIRS patients from medical wards (n=35). Thirty healthy blood donors who visited the General Health Check-up Division at Wuhan Central Hospital served as controls. Excluded from the study were (1) those patients with pregnancy; (2) those with cancer; (3) those died after admission into the ICU in 7 days; (4) those received cardiopulmonary resuscitation; (5) those who had previous blood system diseases; and (6) those with SIRS before admission into the ICU. The levels of uPA, uPAR, D-D, IL-6 and TNF-α in blood were detected by commercial enzyme-linked immunosorbent assay (ELISA) kit. The data were analyzed using SPSS version 17.0 and expressed as mean ± standard. Student's t test and the Mann-Whitney U test were used in the analysis. The relations of uPA, uPAR and D-dimer, IL-6 TNF-α levels were analyzed using Spearman's rank-order correlation coefficient test. RESULTS: The plasma levels of uPA , uPAR, D-dimer,IL-6 and TNF-α in the patients with SIRS were obviously higher than those in the non-SIRS patients and controls (P<0.001). Correlation analysis showed a positive correlation between uPAR and IL-6 levels (r=0.395, P=0.004) and between uPAR and TNF-α levels (r=0.606, P<0.001), but no correlation between uPAR and D-dimer levels (r=0.069, P=0.632). No correlation was observed between uPA, D-dimer, IL-6 and TNF-α levels (P>0.05). The establishment of ROC curve was based on the levels of uPAR, D-dimer, IL-6 and TNF-αin 24 hours for the diagnosis of multiple organ dysfunction syndrome (MODS), and the ROC areas under the curve were 0.76, 0.58, 0.86 and 0.83, respectively. CONCLUSIONS: uPA and uPAR play a major role in patients with SIRS in the process of coagulation disorder, but the mechanism of SIRS is not the same. uPAR may play a central role in the development of SIRS to MODS.

BACKGROUND:Urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR) are known as important factors, which mediate a variety of functions in terms of vascular homeostasis, inflammation and tissue repair. However, their role in systemic inflammatory response syndrome (SIRS) has been less well studied. This study aimed to test the hypothesis that the abnormalities of fibrinolysis and degradation of extracellular matrix mediated by uPA and uPAR are directly related to the patients with SIRS. We therefore analyzed their role and clinicopathological significance in patients with SIRS.METHODS:A case-control study was conducted with 85 patients who were divided into two groups according to the diagnostic criteria of SIRS:SIRS group (n=50) and non-SIRS group (n=35). The SIRS group was divided into MODS group (n=26) and non-MODS group (n=24) by their severity, and survival group (n=35) and non-survival group (n=15) by their prognosis. Another 30 healthy adults served as normal controls. uPA and uPAR in plasma were detected by commercial enzyme-linked immunosorbent assay (ELISA) kits.RESULTS:The plasma level of uPA was lower in the SIRS group than in the non-SIRS group and controls (P<0.001 andP<0.001). It was lower in sepsis patients and the MODS group than in the non-sepsis patients and the non-MODS patients (allP<0.05). However, there was no difference in uPA level between survivors and non-survivors (P>0.05). The plasma level of uPAR increased in the SIRS group compared with the non-SIRS group and controls (P<0.001 andP<0.001). There was a significant elevation of uPAR in sepsis patients, MODS patients and non-survivors as compared with non-sepsis patients, non-MODS patients and survivors respectively (allP<0.05). Plasma uPAR levels were positively correlated with APACHE II score (r=0.575,P<0.001) and SOFA score (r=0.349,P=0.013). AUCs for the prediction of SIRS mortality were 0.67 and 0.51, respectively, for uPA and uPAR.CONCLUSION:uPAR could be a predictor of poor outcome in patients with SIRS.

AIMTo study the physicochemical properties and the structure of Achyranthes bidentata polysaccharide (AbPS).

METHODSAbPS was isolated from the roots of Achyranthes bidentata Bl., and purified by gel filtration chromatography. The distribution of the molecular weight of AbPS was determined by ESI-MS. The structure of AbPS was deduced by methylation analysis, reductive-cleavage and 13CNMR spectroscopy.

RESULTSAbPS was shown to compose of fructose residues and glucose residues and the molar ratio was 8:1. AbPS contain 2,1-linked fructose residue, 2,1-linked fructose residue, 1,2,6-linked fructose residue, terminal fructose residue and terminal glucose residue.

CONCLUSIONAbPS is a fructan and belong to graminan.

Achyranthes ; chemistry ; Fructans ; chemistry ; isolation & purification ; Methylation ; Molecular Structure ; Molecular Weight ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry

OBJECTIVETo study the epidemiological and clinical characteristics and risk factors of cirrhosis-related hepatocellular carcinomas (HCC) in patients with hepatitis C virus (HCV) infection.

METHODSEighty-nine compensated and decompensated HCV cirrhosis patients were analyzed and followed-up. The main clinical and laboratory variables were analyzed as incidence factors of HCC with univariate analysis and multivariate analysis regression models.

RESULTSThe patients were followed-up for 86 months. Thirty-five of the 89 patients had HCC during the 86 months follow-up. Their five and ten-year cumulative incidences were 16.9% and 40.4% respectively. Of the 35 HCC patients, 4 had a family history of hepatitis C, 12 had a familial history of HCC, and 7 had a history of alcohol ingestion. Five and ten-year cumulative incidences of HCC in patients with hepatic steatosis were 24.6% and 51.0% respectively. Five-year and ten-year cumulative incidences of HCC in patients with non-hepatic steatosis were 8.7% and 26.2% respectively, and the difference in the cumulative incidences between them was significant (P < 0.05). Hepatic steatosis severity was associated with the severity of the cirrhosis. ALT and TBil levels were higher in the HCC group than in the non-HCC group, ALB was lower in the HCC group than in the non-HCC group, and the differences between them were significant (P < 0.05). Child-Pugh score and the severity of the hepatic steatosis during follow-up were independently correlated with HCC.

CONCLUSIONHCC is the most important and frequent outcome of chronic hepatitis C cirrhosis. Child-Pugh score and the severity of the hepatic steatosis are related to the risk factors. History of alcohol ingestion and family history of hepatitis C are also related to liver cancer.

Aged ; Carcinoma, Hepatocellular ; etiology ; Female ; Follow-Up Studies ; Hepatitis C, Chronic ; complications ; Humans ; Liver Cirrhosis ; complications ; Liver Neoplasms ; etiology ; Male ; Middle Aged ; Risk Factors

BACKGROUNDAccurate identification of bacterial isolates is an essential task in clinical microbiology. This study compared culturing to analyzing 16S rRNA gene sequences as methods to identify bacteria in clinical samples. We developed a key technique to directly identify bacteria in clinical samples via nucleic acid sequences, thus improving the ability to confirm pathogens.

METHODSWe obtained 225 samples from Beijing Tongren Hospital and examined them by conventional culture and 16S rDNA sequencing to identify pathogens. This study made use of a modified sample pre-treatment technique which came from our laboratory to extract DNA. 16S rDNA was amplified by PCR. The amplified product was sequenced on a CEQ8000 capillary sequencer. Sequences were uploaded to the GenBank BLAST database for comparison.

RESULTSAmong the positively cultivated bacterial strains, seven strains were identified differently by Vitek32 and by 16S rDNA sequencing. Twelve samples that were negative by standard culturing were determined to have pathogens by sequence analysis.

CONCLUSIONThe use of 16S rRNA gene sequencing can improve clinical microbiology by providing better identification of unidentified bacteria or providing reference identification of unusual strains.

Bacteria ; isolation & purification ; DNA, Ribosomal ; chemistry ; Humans ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; genetics ; Sequence Analysis, DNA ; methods