Objective To construct markless gene deletion mutant at the clpP loci on the chromosome of Streptococcus mutans(S.mutans).Methods ASp resistance gene was amplified by PCR,to construct the Sp resistance cassette where the Sp resistance gene was flanked with two loxP site.After the clpP gene was cloned into the pGEM-T-Easy TA cloning vector,it was digested and linked with the Sp resistance cassette,yielding homologous recombination vector pIB △ clpP-Sp.The vector was linearized and used for the transformation of S.mutans UA159,with transformants selected on TPY plates containing Sp.The selected strain was transformed with the thermosensitive plasmid pCrePA to excise the Sp resistance gene.The pCre-PA was then easily eliminated at nonpermissive temperature,resulting in a markless mutant strain carrying a deletion at the clpP loci,which was verified by PCR and DNA sequencing.Results The result of the PCR analysis and DNA sequencing indicated that a part of the clpP gene was deleted.There was a loxP at this loci without the Sp resistance gene.Conclusion The markless clpP-deletion mutant of S.mutans was constructed successfully,which laid a foundation for further study of its biological function and its influence on the cariogenicity of S.mutans.

Sepsis is a frequent condition with high mortality,and its early diagnosis in the pediatrics department is one of the keys to improve survival. C-reactive protein and procalcitonin have been used as bio-markers in the diagnosis of sepsis,but they have limited susceptibility and specificity,and can be elevated in non inflammatory condition. Presepsin is the free fragment of a glycoprotein expressed on monocytes/macro-phages. Preliminiary reports suggest that levels of presepsin are significantly higher in septic patients com-pared to healthy individuals, and presepsin is useful for evaluating the severity of sepsis.

Objective To investigate the technical protocols and feasibility of subtraction computed tomography perfusion imaging (sCTP) on observation of ischemic necrosis of femoral head (ANFH) on dog. Methods Sixteen laboratory canines underwent CT perfusion imaging (CTP) of femoral head before and after selective femoral circumflex artery embolization, and ANFH were observed. Then new sequence imaging data, created by sources imaging of CT perfusion scan using subtraction software, were analyzed at workstation (AW 4.2) with CT perfusion 3 analysis program, and data of sCTP were obtained. The parametric maps and indexes of capillary-level hemodynamics including blood volume (BV), blood flow (BF) and mean transit time (MTT) of CTP and sCTP were compared. Results ①The technical success rate of sCTP post-processing created from CTP sources imaging data was 100%. The values and mappings of BF, BV and MTT of region of interest (ROI) were all obtained from subtraction sequence images data. The post-processing time of sCTP was about 1-5 h. ② sCTP depicted ANFH well, though the values and mappings of BF, BV and MTT were different from those obtained with conventional bone CTP. Conclusion sCTP can be generated with subtraction and perfusion analysis program and techniques. It can be applied to bone CT perfusion imaging and early quantitative diagnosis of necrosis.

BACKGROUND:At present, col agen as a material in periodontal tissue engineering has some disadvantages such as poor mechanical strength and rapid degradation speed. Col agen combined with chitosan can improve above-mentioned problems.
OBJECTIVE:To evaluate the biocompatibility of a novel chitosan-col agen scaffold in vitro.
METHODS:Cytotoxicity of the extract of chitosan-col agen scaffold in different concentrations (100%, 75%, 50%, 25%) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Human periodontal ligament cel s at 4-6 passages were cocultured with the chitosan-col agen scaffold. Cel growth on the scaffold was observed. Changes in alkaline phosphatase activity were detected in periodontal ligament cel s before and after coculture.
RESULTS AND CONCLUSION:The novel chitosan-col agen scaffold was made up of double layers with one dense layer and another loose layer. The grade of the cytotoxicity of the scaffold was from 0 to 1. Scanning electron microscope and histological observation demonstrated that cel s grew wel on the chitosan-col agen scaffold;the dense layer could prevent cel s to migrate into the scaffold. There were no significant differences in alkaline phosphatase activity in human periodontal ligament cel s before and 24 hours after combined culture (P>0.05). Alkaline phosphatase activity in human periodontal ligament cel s was greatly higher at 48 and 72 hours after combined culture compared with that before culture (P<0.05). Above results indicated that the novel chitosan-col agen scaffold has a good biocompatibility and barrier function, and potential as a scaffold for periodontal tissue engineering.

Objective To develop a new quasi-zero stiffness isolator for the vibration isolation of the sick and wounded on the ambulance stretcher.Methods The isolator was designed and verified on the basis of the quasi-zero stiffness of slotted disk spring, vibration isolation design method with high static while low dynamic stiffness and co-laminated structure of the spring.Results The theoretical analyses and tests proved the force-displacement curves of the new quasi-zero stiffness isolator fit well. The frequency-weighted acceleration at the buttock and head under different speeds on the asphalt roads was less than 0.315 m/s2.Conclusion The new quasi-zero stiffness isolator developed behaves well in vibration isolation and comfortability for the ambulance stretcher sick and wounded.

Objective To explore ear reconstruction using autologous rib cartilage ear framework by multi-layer spliced sculpture in microtia patients.Methods From Feb 2010 to May 2011,29 microtia patients were subjected to ear reconstruction using autologous rib cartilage ear framwork by four-layer spliced sculpture.Results In one operation 29 patients achieved 2 cm transverse height of reconstructed ears which were basically coincidence with the normal side.Patents and their families were all satisfied with the results.Follow-up of 3-12 months showed that only 1 case reconstructed-ear height was significantly lower transverse process.Owing to sleeping position,the patient did not protect the reconstructed ear,leading to frequent reconstructed-ear pressure.Conclusions The method of multi-layer spliced sculpture autologous rib cartilage ear reconstruction has good clinical effect.It can make reconstructed ear reach nomal transverse height and avoid the third rib cartilage transplant operation to continue increasing the transvers height.

Objective:To investigate the effect of compound 48/80(C48/80) activated mast cell on dendritic cell homing to draining lymph nodes(DLN) in order to explore the potential mechanism of C48/80 promoting adaptive immune response.Methods: C57BL/6 mice bone marrow derived dendritic cells(BM-DC) and MC/9 mast cell line were propagated in vitro.Chemotaxis of BM-DC supplemented with supernatant of C48/80 treated MC/9 to CCL21 was measured by transwell chemotaxis assay.C48/80 was injected into murine scapular site,local skin mast cell degranulation was detected by toluidine blue staining method.The number of CD11c+ DC in draining lymph nodes(DLN) was detected by Flow cytometry.Exogenous green microbeads labeled BM-DC homing to DLN was detected by fluromicroscope.The mice pretreated with or without C48/80 were vaccinated by ovalbumin pulsed DC(OVA-DC),DLN lymphocyte proliferation was detected by WST-8 reagent.Results: A large amount of typical BM-DC were harvested from media supplemented with murine recombinant IL-4 and GM-CSF.Product of activated MC/9 enhanced chemotaxis of BM-DC to CCL21(P<0.01).Intradermal injection of C48/80 induced local mast cell obvious degranulation and local inflammation.Mice pretreated with C48/80 demonstrated high number of total cells and DC cells in DLN.The number of fluorescent positive BM-DC in DLN also increased in C48/80 pretreated mice.Antigen specific lymphocyte proliferation enhanced in OVA-DC inoculated C48/80 pretreated mice.Conclusion: Mast cell activation induced by C48/80 can enhance DC homing to DLN and increase specific lymphocyte proliferation,which may be one of a mechanism of C48/80 in promoting adaptive immune response.

Objective To investigate the incidence and the risk factors for hospital-acquired pneumonia(HAP)in the elderly in Shanghai.Methods This was a muhicenter prospective clinical cohort study.A total of 5299 patients more than 65 years old.admitted into 31 secondary or tertiary hospitals in Shanghai,were enrolled.Measurements of the demographic and potential risk factors reflecting illness severity,nutrition,drug exposure,surgery and ventilation were performed.Pneumonia was classified by the definition of Chinese Medical Association.Risk factors were analyzed by univariate Pearson Chi-squared test and multivariable logistic regression analysis with backward(Likelihood ratio).Resuits Of the enrolled patients,2805 male and 2494 female,255(4.81%)developed hospital-acquired pneumonia.The incidence was 46.75/1000 hospitalizations.Among them 38 died:and the rough mortality was 14.90%.The incidence of HAP was higher in ICU(21.43%),hematology(12.17%),chest surgery(11.41%),and respiratory medicine(7.92%)departments.The mean of acute physiology and chronic health evaluation (APACHE Ⅱ)score was 8.3±3.4(5-31).Multivariable logistic regression analysis with backward (Wald)method found that admission into secondary hospitals.admission into ICU,history of chronic obstructive pulmonary disease≥10 years,immunosuppression,administration of antibiotics,insertion of nasogastric tube,mechanical ventilation,administration of H-2 antagonists or antacid and≤7 d,central nervous system diseases,depressed level of consciousness,supine position,albumin＜35g/L were independent risk factors of HAP in the elderly.Conclusion Hospital-acquired pneumonia in the elderly was the usual type of nosocomial infections.The risk factors identified from this study may prove useful to target future clinical interventions to prevent HAP in the elderly.

Aim To investigate the anti-proliferating effect of tetrandrine ( Tet ) on colon cancer cells and its possible molecular mechanism. Methods We intro-duced crystal violet staining and flow cytometry to ana-lyze the effect of Tet on proliferation in LoVo cells. Flow cytometry was used to detect the effect of Tet on apoptosis in LoVo cells. Western blot assay was taken to analyze the effect of Tet on the expression of insulin-like growth factor binding protein 5 ( IGFBP5 ) . Final-ly, luciferase reporter assay, recombinant adenovirus mediated over-expression or silence of IGFBP5 were used to analyze the possible role of IGFBP5 in the anti-proliferating effect of Tet on colon cancer cells. Re-sults Crystal violet staining and flow cytometery anal-ysis results showed that Tet could exert an anti-prolifer-ating effect and induce apoptosis in LoVo cells. Tet de-creased the expression of IGFBP5 in a concentration-dependent manner. Tet inhibited the transcriptional ac-tivity of pTOP-luc reporter, which could be reversed by exogenous expression of IGFBP5 mostly. Similar results were found in the expression of c-Myc, but IGFPB5 knockdown couldn’ t reverse this effect. Conclusion Tet can inhibit the proliferation of colon cancer cells, and this effect may be mediated by down-regulating the expression of IGFBP5 to inhibit Wnt/β-catenin signa-ling transduction partly.

Objective To evaluate the therapeutic effect of pulmonary carcinoma by bronchical arterial infusion (BAI) and bronchical arterial infusion with the addition of pulmonary arterial infusion (PAI).Methods Totally 69 cases with pulmonany cancinoma were treated by intervention therapy .Of them ,38 cases were undergone the bronchical arteriography and arterial infusion and 31 cases were undergone the brochical pulmonany arterial dual infusion.Results The bronchical arteryies taken part in the blood supply of neoplasmin all cases ,while the pulmonany artery only in 77% of cases .The effective ratio of BAI +PAI was 87%, and BAI was 65.7%,there were markly different (?