Objective To investigate the distribution of 16S rRNA methylase genes in Klebsiella pneumoniae strains producing Klebsiella pneumoniae ealbapenenase type 2(KPC-2).Methods A total of 37 Klebsiella pneumoniae isolates producing KPC-2 were collected.The minimal inhibitory concentrations (MICs)of these strains to amikacin,gentamyein and netilmicin were determinated by agal dilution method.Six 16S rRNA methylase genes(armA,rmtA,rmtB,rmtC,rmtD and npmA)were detected by PCR.Results The resistant rates to amikacin,gentamycin and netilmicin were 97.3%(MIC50≥1024μg/mL).Among those resistant strains,8 were armr/A positive,25 were rmtB positive,4 were both armA and rmtB positive.and no other 16S rRNA methylase genes were found.The total positive rate of 16S rRNA methylase genes was 78.4%(29/37).Conclusion 16S rRNA methylase genes armA and rmtB ale prevalent in Klebsiella pneumoniae strains producing KPC-2.

Objective:Human adipose-derived stem cells (hASCs)are a highly attractive source in bone tissue engineering.To generate a luciferase reporter system that could be used to quantitatively and rapidly examine osteogenic differentiation potential of human adipose-derived stem cells (hASCs ) in vitro,and eventually make it possible to monitor the osteogenic differentiation of transplanted cells in vi-vo.Methods:The genomic DNA harboring promotor regions of osteocalcin and DNA sequences encoding luciferase genes were amplified by PCR and cloned into the pLVX-pTRE-puro vector to generate the OCpro-Luc-Puro construct.Then,the OCpro-Luc-Puro construct together with three assistant vectors:pM-DLg/pRRE,pRSV-REV,and pVSVG,were transiently transfected into HEK293T cells followed by viral supernatants collection,filtration and concentration.Next,the hASCs stably expressing luciferase repor-ter gene driven by osteocalcin promotor were created with the lentivirus carrying OCpro-Luc-Puro cassette under puromycin selection.The OCpro-Luc-hASCs were then cultured in the absence or presence of osteo-genic differentiation medium.On the 7th and 1 4th days,after osteogenic induction,cellular extracts were collected and analyzed by luciferase reporter assay.Meanwhile,alizarin red staining and quantification as well as quantitative reverse transcription PCR (qRT-PCR)analysis of osteogenic associated genes osteo-calcin (OC),runt-related transcription factor 2 (Runx2)and alkaline phosphatase (ALP)were used to assess the osteogenic differentiation ability of OCpro-Luc-hASCs.Results:OCpro-Luc-Puro plasmid and OCpro-Luc-hASCs were successfully generated.On the 7th and 1 4th days after osteogenic induction,the luciferase activity of the cellular extracts from OCpro-Luc-hASCs was dramatically increased.Consistently, the extracellular matrix mineralization,as shown by Alizarin red S (ARS)staining and quantification was also markedly intensified and a marked increase of the mRNA expression levels of OC,Runx2 and ALP, although to variable extent,was observed upon osteogenic differentiation.These results indicated that the observations from traditional experiments examining hASCs osteogenic differentiation were largely in agreement with that of our luciferase reporter assay in OCpro-Luc-hASCs.Conclusion:We established a luciferase reporter system that could be used to rapidly,quantitatively and specifically determine osteo-genic differentiation ability of hASCs.Comparing with the traditional time-consuming methods,the system we generated here was highly effective.This system not only can be used to examine ostogenic differentia-tion of hASCs in a high throughput manner,but also provides a way to monitor ostogenic differentiation of cells in vivo.

Purpose To study the cytopathologic features of CT-guided percutaneous lung biopsy samples and to evaluate the role of cytopathology in the diagnosis and staging of lung carcinomas, as compared to histopathology. Methods Four-hundred twenty-five specimens were collected by CT-guided percutaneous lung biopsy which were also confirmed by histological diagnosis. Direct smears were performed for each case. Cytological and histological examination was carried out. Results The sensitivity, specificity, false positive rate, false negative rate and accuracy of cytopathology in diagnosing lung carcinomas by CT-guided percutaneous lung biopsy was 86. 6% (264/305), 100% (120/120), 0 (0/120), 13. 4% (41/305), 90. 4% (384/425), respectively. Overall 51. 1%(135/264) of the cases were precisely typed, including 77. 6% (83/107) of adenocarcinoma, 76. 9% (40/52) of squamous cell car-cinoma and 75. 0% (9/12) of small cell carcinoma. Conclusions Cytopathology of CT-guided percutaneous lung biopsy specimens is sensitive and accurate for diagnosing pulmonary carcinomas. In some cases, the lung carcinoma can be precisely typed. Therefore, it is useful for diagnosing and staging lung carcinomas.

Objective:To construct and evaluate a novel tissue-engineered bone composed of murine stromal cell-derived factor 1(mSDF-1), simvastatin (SIM) and collagen scaffold (Bio-Oss?), serving as a cell-homing approach for bone formation .Methods: In the study , 32 ICR mice were randomly divided into 4 groups,each group including 8 mice.The drug-loaded collagen scaffolds were implanted subcutaneously onto the cranium of each mouse according to the groups: ( 1 ) 1 ∶50 ( volume ratio ) dimethyl sulfoxide ( DMSO ) /phosphate-buffered saline ( PBS ) solution +collagen scaffold ( blank control group ); ( 2 ) 10 -3 mol/L SIM solution +collagen scaffold ( SIM group ); ( 3 ) 200 mg/L mSDF-1solution +collagen scaffold (mSDF-1 group); and (4) 10 -3mol/L SIM +200 mg/L mSDF-1 solution +collagen scaffold ( SIM +mSDF-1 group) .One week after implantation , the mice were trea-ted by injecting the same drug solution mentioned above around the scaffold once a day for two days .The specimens were harvested 6 weeks after implantation and the bone formation was evaluated by soft X-ray analysis , HE staining and immunohistochemical staining .Angiogenesis of each group was checked by calculation of vessels in each tissue section .Results:Six weeks after implantation , the collagen scaffolds were retrieved.The value of gray scale for the SIM +mSDF-1 group[(421 836.5 ±65 425.7) pixels] was significantly higher than that of the blank control group [(153 345.6 ±45 222.2)pixels, P<0.01], the SIM group [(158 119.2 ±100 284.2) pixels, P<0.01], and the mSDF-1 group[(255 529.5 ± 152 142.4) pixels, P <0.05 ]; HE staining analysis revealed that significant bone formation was achieved in the SIM +mSDF-1 group; The immunohistochemical staining showed the existence of os-teopontin and osteocalcin in the SIM +mSDF-1 group; There were more vessels in the SIM +mSDF-1 group[(46 ±8)vessels/mm2] than in the blank control group [(23 ±7) vessels/mm2, P<0.01], and the SIM group[(24 ±6) vessels/mm2 , P<0.01].Conclusion:The novel tissue-engineered bone com-posed of mSDF-1, SIM and collagen scaffolds has the potential to form bone subcutaneously in vivo.It re-presents a novel method of in vivo bone re-generation without seed cell delivery .

Objective To analyze the curative effect of traditional Chinese medicine(TCM)combined with early enteral nutrition(EN)for treatment of patients with severe acute pancreatitis(SAP). Methods 70 SAP patients were randomly divided into TCM plus EN group(36 cases)and conventional therapy group(34 cases). Both groups received routine treatment. Additionally,TCM+EN group received early EN and TCM decoction treatment〔the ingredients of decoction including radix bupleuri,radix paeoniae alba,radix scutellariae,fructus aurantii immaturus, magnolia bark,raw rhubarb(rhubarb was added at last during cooking the decoction)and natrii sulfas exsiccatus (dissolved in water to be administered)each 10 g,the decoction was concentrated to 150 mL and then administered via a stomach tube to the patient,afterwards the tube was clipped for 2.5-3 hours,twice a day,4-7 days constituting a therapeutic course〕. After treatment,the time for patients' symptoms improvement,the situation of intestinal recovery, the length of stay in hospital,the total medical cost,serum C-reactive protein(CRP),aspartate aminotransferase (AST), lactate dehydrogenase (LDH), amylase (AMY), acute physiology and chronic health evaluationⅡ(APACHEⅡ)score and complications,intensive care unit(ICU)transfer rate and case fatality rate in two groups were observed. Results The time for symptoms improvement of abdominal tenderness(day:1.68±1.01 vs. 3.89±1.07), abdominal distension(day:2.17±1.48 vs. 4.24±3.23),abdominal pain(day:3.12±1.14 vs. 4.94±3.21)and the intestinal recovery of exhaust defecation time(day:3.48±0.92 vs. 5.32±3.30)of SAP patients after treatment in the TCM+EN group were faster significantly than those in the conventional therapy group(all P<0.05). The length of stay in hospital(day:15.50±1.75 vs. 19.35±1.69)and total cost(wan yuan:1.812±0.424 vs. 3.292±1.081) of TCM+EN group were less than those of conventional therapy group(P<0.05 or P<0.01). After treatment,the levels of serum CRP,AST,LDH,AMY,APACHEⅡscore in TCM+EN group and conventional therapy group were all lower than those before treatment,and on day 10,the degree of descent was more prominent in TCM+EN group〔CRP(mg/L):98.972±43.384 vs. 122.392±71.621,AST(U/L):75.952±55.668 vs. 126.391±47.431, LDH (μmol?s-1?L-1):1.48±0.21 vs. 2.61±1.46,AMY(U/L):146.362±58.792 vs. 226.392±37.692,APACHE Ⅱscores:6.978±3.352 vs. 13.652±7.621,P<0.05 or P<0.01〕. There was no death in TCM+EN group,while in the conventional therapy group,there was 1 case dead. ICU transfer rate in TCM+EN group was less than that in the conventional therapy group(2.78% vs. 11.76%),but there was no statistical significant difference between the two groups(χ2=0.99,P>0.05). Among the 70 patients with SAP,the cause of the disease due to gallstone accounted for 55.72%,hyperlipidemia for 37.14%,alcoholic for 4.28%and other 2.86%. Conclusion The use of TCM combined with early EN for treatment of patients with SAP can enhance the curative effect of SAP,reduce the hospitalization time and the total cost of patients,and decrease complications and mortality,that is conducive to the faster recovery of patients.

Objective To compare anesthetic effect and safety of general anesthesia and combined spinal and epidural anesthesia used in autonomic nervous hyperresponsive surgery for patients with paraplegia.Methods 26 paraplegic patients were randomly divided into two groups-control group and treatment group from February 2011 to November,2015,each with 13 cases.The control group used general anesthesia,while the treatment group used combined spinal and epidural anesthesia,to observe onset time,duration,intraoperative hemodynamic changes and complications,Complications,length of stay and cost,Days and costs of hospitalization,satisfaction of patients and their families,of anesthesia in two groups.Results The dosage of narcotics and the onset time of the treatment group were better than that of the control group.The difference between the two groups was significant,and had statistical significance (P＜0.05) Two groups of patients after surgery,diastolic blood pressure,systolic blood pressure and heart rate were lower in the treatment group than in the control group,and had statistically significant difference (P＜0.05);The postoperative complications of the treatment group were significantly better than those of the control group,and had statistically significant difference (P＜0.05);There were statistically significant differences in postoperative pain degree between the two groups (P＜0.05);Two groups of patients in hospital days,hospital costs,satisfaction rate had statistically significant difference,Have statistical significance (P＜0.05).Conclusion In autonomic nervous hyperresponsive surgery for patients with paraplegia,anesthetic effect and safety of combined spinal and epidural anesthesia is significantly better than that of general anesthesia,featured by the rapid onset of action,long duration,fewer complications,strong safety and patients' great satisfaction.It is worth generalizing and applying clinically.

Objective:To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2),and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo.Methods:The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study.Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces.The release kinetics was measured to evaluate the slow-release characteristics in vitro.BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM),respectively.The supernatants were collected and used to culture hASCs in vitro.Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation.After 7 and 14 days,alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP.After 14 and 21 days,the calcification deposition was determined by alizarin red S (ARS) staining and quantification.The expressions of the osteoblast-related genes were tested on day 4 and day 14.In the in vivo study,6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups:(1) BioCaP scaffold only,(2) BioCaP scaffold + hASCs,(3) BMP-2-BioCaP scaffold,(4) BMP-2-BioCaP scaffold + hASCs (test group).After 4 weeks of implantation,hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs.Results:SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight,plate-like and sharp-edged crystal units,and the length of the crystal units varied between 5 and 10 μm.Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days,and the accumulative protein release could reach 20％.CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2BioCaP.ALP activity was higher by the induction of OM + BMP-2-BioCaP than of the other groups (P ＜0.01).More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2),ALP,osteopontin (OPN) and osteocalcin (OC) were determined in the OM + BMP-2-BioCaP group at different time points (P ＜0.01).HE staining showed that,in the test group and BMP-2-BioCaP scaffold group,the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs,and newly-formed bone-like tissues could be found in ECM around the scaffold materials.Moreover,compared with the BMP-2-BioCaP scaffold group,more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups.No obvious positive results were found in the other groups.Conclusion:BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo.The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.

Objective To investigate the resistance of carbapenems,the production of metallobeta-lactamases(MBLs)and homology analysis of Psendomonas aeruginosa in China.Methods A total of 654 Pseudomonas aeruginosa isolates were collected from 28 hospitals in 16 difierent regions during the period from July 2006 to July 2007 as part of the Nosocomial Pathogen Resistance Surveillance(NPRS).MICs of imipenem and meropenem were determined by agar dilutiom PFGE was used to analyze the molecular epidemiology of carbapenem-resistant strains.MBLs were detected by Etest and PCR among carbapenem-non-sensitive strains.The transcription levels of MBLs were determined by real-time reverse transcriptase(RT)-PCR.Results Of the 645 Pseudomonas aeruginosa strains,245(37.5%)were resistant to imipenem and 183(28.0%)were resistant to meropenem.A total of 275 carbapenemnonsusceptible strains and 259 carbapenem-resistant strains were chosen for further study. Among the carbapenem-nonsusecptible strains,8.73%(24/275)of the strains carrried MBLs genes hy PCR,but only 6.55%(18/275)of the strains were detected as MBls-producers bv MBL Etest. The MBLs relative expression levels of the 6 MBL genotype-positive but phenotype-negative strains were significantly lower than those of the other 18 MBL genotype-and phenotype-positive strains(P＜0.05).The 259 carbapenemresistant Pseudomonas aeruginosa strains were divided into 89 clones by PFGE.The 24 MBL genotypepositive strains collected from 6 cities were divided into 9 clones by PFGE.Conclusions The carbapenems resistance of Pseudomonas aeruginosa is severe in China.Clonal dissemination of Pseudomonas aeruginosa has not been detected throughout China.MBLs are not the major carbapenems resistance mechanism in Pseudomonas aeruginosa.

Objective To investigate the security and result of operation of one side polycystic kidney removal and homonymy kidney transplantation simultaneously for giga-polycystic kidney di-sease of terminal stage. Methods Forty-five patients with polycystic kidney of transplantation were retrospectively analyzed. The patients were divided into 2 groups. Patients of group A (n=23) under-went resection of the cystic kidney by extraperitoneum and the other 22 patients(group B) didn't re-move the cystic kidney. The data including average length of hospital stay, variance of blood pressure, lessen of abdominal circumference, lung capacity, total lung capacity, FEV1.0/FVC, incidence rate of delayed graft function (DGF) and 1 year patient/kidney survival rate of the 2 groups were compared. Results The average length of post-operative hospital stay of group A was (14.5±2.6)d,lessen of blood pressure was (30.0±0.7/13.34±8.4)mm Hg, lessen of abdominal circumference was (11.0+ 6. 3) cm, lung capacity increased (1.4±0.3)L, total lung capacity increased (2.0±1.0)L, FEV1.0/ FVC increased (5.3±1.0) %, the incidence rate of DGF was 8.7% (2/23), 1 year patient/ kidney survival rate was 100.0%/95.7%. The average length of post-operative hospital stay of group B was (28.45±7.9)d,lessen of blood pressure was (3.9±11.2/2.9±12. 0)ram Hg, lessen of abdominal circumference was (3.3±2.2)cm, lung capacity increased (0.44±0.3)L, total lung capacity increased (0.8±0.2) L, FEV1.0/FVC increased (2.0±0.9)%, the incidence rate of DGF was 9.1%(2/22), 1 year patient/kidney survival rate was 100.0%/95.5%. There were no significant differences of the incidence rate of DGF and 1 year patient/kidney survival rate between the 2 groups. While there were significant differences of the other data between the 2 groups(P＜0.05). Conclusions It is safe and convenient for one side polycystic kidney removal and homonyrny kidney transplantation simultaneous-ly for giga-polycystic kidney disease of terminal stage. The procedure could be applied to the patients of graveness complication or giga-polycystie kidney hampering operation of transplantation.

The diagnosis and treatment of invasive fungal infections has been attracting more and more attention along with the increase incidence in the current clinical practice.We hereby work out this paper to elaborate some key issues,coveting the history and methodology of anti-fungal susceptibility testing, the MIC recognition of yeast fungus and mycelial fungus,breakpoint interpretation,and finally the guidance for clinical practice.To better undemtand susceptibility test results and improve the guality of susceptibility tests in this review,we chosed several most-often used anti-fungal drugs and explained their effect activity.