Objective To clone and express the mouse acidic mammalian chitinase ( AMCase ) gene and protein, and to analyze the hydrolysis of the cell walls of Trichophyton rubrum and Candida a/ft/cans by this enzyme. Methods Total RNA was isolated from the stomach of BALB/c mouse, and AMCase gene was amplified by RT-PCR. The recombinant fusion expression vector of pET28a ( + ) -AMCase was constructed. AMCase protein was expressed in prokaryotic system and purified. After incubation of AMCase with the cell wall extracts, the level of N- acetylglucosamine was measured. Results AMCase gene was cloned and expressed successfully. Purified AMCase protein can hydrolyse the chitin in the cell walls of T. rubrum and C. albicans. Conclusion The AMCase expressed in prokaryotic system can hydrolyse chitin in the cell walls of T. rubrum and C. albicans, which implies its antifungal potential.

ObjectiveTo explore the risk factors for the prognosis of respiratory failure in neonates with gestational age≥34 weeks. MethodsA total of 143 hospitalized neonates with respiratory failure who had gestational age≥34 weeks were enrolled from Jan. 2011 to Jun. 2013. According to the outcome, the neonates were divided into good prognosis group and poor prognosis group. The risk factors for the prognosis of respiratory failure were screened by univariate analysis and mul-tivariate binary logistic regression model.ResultsAmong the 143 neonates, 105 neonates had good prognosis and 38 neonates had poor prognosis. Univariate analysis showed that the mode of delivery, small for gestational age, 5 minutes Apgar score <7, and with primary disease being pneumonia/sepsis were associated with a poor prognosis. The differences were statistically signif-icant (P<0.05). Furthermore, the multivariate binary logistic regression model showed that, small for gestation age (OR=23.87, 95%CI:3.149-180.9) and pneumonia/sepsis (OR=2.996, 95%CI:1.514-5.928) were the independent factors of poor prognosis. ConclusionsSmall for gestation age and primary diseases being pneumonia/sepsis are the independent factors inlfuencing prog-nosis of respiratory failure in neonates born at a gestational age≥34 weeks.

Objective To study the differences of clinical manifestations,etiology and hospitalized outcomes of purulent meningitis in preterm and term infants.Method All preterm and term infants with purulent meningitis hospitalized in the Hospital from 2006 to 2015 were enrolled in this study.The data of neonate's condition,maternal condition,clinical manifestations,complications,etiology,treatment and outcomes of the preterm and term infants groups were compared.Result During the study period,44 preterm infants and 118 term infants were included.The time of onset for purulent meningitis of preterm infants group was statistically earlier than that of term infants group [11.2 (3.2,19.8) d vs.14.3 (5.6,23.9) d,P ＜ 0.05].The prognosis of preterm infants group was statistically worse than that of term infants group (P ＜ 0.05).Among them,the incidences of clinical manifestations in preterm infants group compared to term infants group were:fever (54.5％ vs.78.8％),seizure (11.4％ vs.26.3％),lethargy and poor response (59.1％ vs.38.1％),slow weight gain (9.1％ vs.0％),apnea (45.5％ vs.0.8％) and cyanosis (15.9％ vs.4.2％);all the differences between two groups were significant (P ＜ 0.05).The time of onset for purulent meningitis with complications was statistically earlier than those without complications [9.5 (4.1,20.5) d vs.13.8 (5.9,22.0) d,P＜0.05].The duration of treatment for purulent meningitis with complications was longer than that without complications [(42.2 ± 8.8) d vs.(28.7 ± 7.1) d,P ＜ 0.05],and the positive rate of pathogens was also statistically higher than those without complications (73.8％ vs.26.7％,P ＜0.05).Coagulase-negative staphylococcus was the main pathogen for both preterm and term infants group.Klebsiella pneumoniae was more common in preterm infants group than in term infants group (40.0％ vs.10.4％,P ＜0.05).Conclusion Preterm infants with purulent meningitis had early onset time,atypical clinical manifestations,and poor prognosis.The treatment course for purulent meningitis with complications is prolonged.The pathogens for neonatal purulent meningitis have already changed.The detection rate of conditional pathogens is increasing yearly,for which the clinicians should take note seriously.

Objective To study the occurrence of nosocomial infection ( NI ) situation in the neonatal intensive care unit ( NICU ) of Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine from January 2012 to October 2013,and analyze risk factors of NI. Methods A retrospective study was per-formed in the NICU,of which all neonates hospitalized over 48 hours were included and the occurrence situation and risk factors of NI were analyzed. Results This study included 1 357 neonates,and 175 neonates developed 202 times NI. The overall incidence of NI was 14. 89% (202/1 357),and the incidence density was 10. 44 per 1 000 NICU patient-days (202/19 355). The lower the birth weight is,or the smaller gestational age is,the higher the NI rate is. The NI rate was 75. 00% in neonates whose birth weight <1 000 g,and the NI rate was 52. 63%in neonates whose gestational age <30 weeks. The most common infection type was pneumonia[43. 07% (87/202)]and sepsis[26. 73% (54/202)]. The hospital stay of the NI neonates was longer and the cost of hospitali-zation is higher than non-NI neonates ( Median stay:20 days and 10 days. Median cost:21 045. 32 yuan and 8 108. 23 yuan)(both P<0. 01). Univariate analysis showed there were significant differences between NI neo-nates and non-NI neonates on gestational age,birth weight,5 min Apgar score,asphyxia rescue history,previons antibiotics use,mechanical ventilation,peripherelly inserted central catheter( PICC) ,closed thoracic drainage tube and chest or abdominal surgery before NI. The logistic regression analysis indicated that previous antibiotics use, mechanical ventilation and PICC were important risk factors for NI. Conclusion The NI rate in NICU is still high. Previous antibiotics use,mechanical ventilation and central venous catheter are risk factors of NI.

Objective To detect the B cell epitopes of human MCHR1 recognized by serum autoantibodies in patients with vitiligo. Methods The DNA encoding antigen fragments of MCHR1 from amino acid (aa) 1 to 94 were amplified from pcDNA3/MCHR1 by PCR and inserted into the vector pGEX-4T-1. After identification, the recombinant plasmid pGEX-4T-1/MCHR1 was transfected into E. coli for fusion protein production which was purified by GST agarose affinity chromatography later. The sera from 38 vitiligo patients were examined for MCHR1 antibodies by enzyme-linked immunoabsorbent assay. Results Amino acids (aa) 1 to 94 of MCHR1 which most possibly included in B cell epitopes were selected to express. The recombinant plasmid pGEX-4T /MCHR1 was confirmed by sequence analysis. SDS-PAGE showed that a Mr 37 kD fusion protein was expressed by E.coli. Of the sera from vitiligo patients examined by ELISA, 5 of 38 were considered positive for MCHR1 antibody. Conclusion There are B cell epitopes in amino acids(aa) 1 to 94 of MCHR1 which can be recognized by antibodies from vitiligo patients.

Objective To explore the methods for quality management and continuous improvement of nursing care quality in the orthopedic demonstration ward by taking the hospital accreditation as an opportunity. Methods From July 2012 to June 2013, the continuous care quality improvement in the ward was carried out to find out the problems with PDCA (plan, do, check, action) cycle method, including enhancing the function of orthopedic nursing quality management groups, conducting all-staff training and improving the knowing rate by referring to the standards of hospital assessment standards. Results After the performance of whole-process quality management, the percentage of indexes assessed at level A, B and C was increased from 42.2%to 50.0%, 17.2%to 14.7%and 40.2%to 35.3%, respectively. The score of nurses' responsibility accreditation was increased from 92 to 95. The rates of patient and nursing staff satisfaction were increased from 91.8%to 98.9%and 92.57%to 97.7%, respectively. Conclusion In accordance with the standards for hospital accreditation, the continuous improvement of nursing quality in the orthopedic demonstration wards can improve the specialist care of orthopedic care, improve patients' and nurses' satisfaction, thus making the daily work more scientific and standardized.

Objective To explore the effects of NF-E2-related factor 2 (Nrf2) overexpression on mitochondrial biosynthesis and function in melanocytes.Methods An immortalized human vitiligo melanocyte cell line PIG3V was used in this study.An overexpression plasmid Nrf2-pEX-1 containing the full-length Nrf2 gene was constructed.PIG3V cells were divided into 3 groups:blank group receiving no treatment,control group transfected with the pEX-1 plasmid,overexpression group transfected with the Nrf2-pEX-1 plasmid.After transfection,real-time quantitative reverse transcription-PCR (RT-PCR) and Western blot were performed to determine the mRNA and protein levels of mitochondrial biosynthesis-related factors (including Nrf2,nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM)) respectively;RT-PCR was also conducted to measure the copy number of mitochondrial DNA (mtDNA),and flow cytometry to estimate mitochondial membrane potential (MMP);luciferase reporter system was used to estimate the intracellular adenosine triphosphate (ATP) level.Statistical analysis was carried out by using a two-sample t-test.Results After transfection,a significant increase was observed in the mRNA expression levels of Nrf2 and NRF1 at 24 hours (both P ＜ 0.001) and in those of Nrf2 and TFAM at 48 hours (both P ＜ 0.05),but no significant change was noted in the mRNA expression level of TFAM at 24 hours (P ＞ 0.05) or in that of NRF1 at 48 hours (P ＞0.05) in the overexpression group compared with the control group.In the case of Nrf2,NRF1 and TFAM protein levels,the overexpression group showed significant increases compared with the control group at 48 hours after transfection (all P ＜ 0.05),while no significant difference was noted between the two groups at 24 hours.Compared with the control group,MMP in the overexpression group increased by 2.313％ at 24 hours (t =5.546,P =0.005) and by 14.872％ at 48 hours (t =8.537,P =0.001) after transfection.Both the relative copy number of mtDNA and ATP level were similar between the overexpression group and control group at 24 hours after transfection (both P ＞ 0.05),but significantly higher in the overexpression group than in the control group at 48 hours (t =5.760,P =0.005;t =22.040,P =0.008).Conclusion Up-regulation of Nrf2 pathway can improve mitochondrial function and biosynthesis in PIG3V cells likely by promoting the expressions of mitochondrial biosynthesis-related genes and proteins.

Objective To discuss the clinical application of percutaneous radiologic gastrostomy (PRG) in treating dysphagia associated with amyotrophic lateral sclerosis (ALS),and to evaluate its safety and improvement effect on patient's nutritional status in ALS patients with pulmonary insufficiency.Methods The clinical data of 51 ALS patients who received PRG were retrospectively analyzed.The success rate of surgery and postoperative complications were recorded.All patients were regularly followed up,and the longterm complications as well as the one-,3-and 6-month mortality rates after the surgery were documented.The improvement of patient's nutritional status was evaluated.Results PRG was successfully accomplished in all 51 patients,the technical success rate was 100％.Mild postoperative complications occurred in 7 patients (13.73％) and severe massive hemorrhage in one patient (2.0％).After PRG,no signs or symptoms of impaired respiratory function were observed.No death occurred in one month and in 3 months after PRG.Six months after PRG,three patients died(6.8 ％,3/44).One month after PRG,31 patients had an increase in body weight of more than 1 kg,and the mean BMI was increased from preoperative t8.60±2.14 to postoperative 19.27±1.81 (one month after PRG),19.17±1.93 (3 month after PRG) and 18.89±2.33 (6 month after PRG).Conclusion For the performance of PRG no gastroscopy or anesthesia is needed,thus,the risk of aspiration asphyxia can be reduced in ALS patients complicated by pulmonary insufficiency and the success rate as well as the safety can be improved.Therefore,this technique is an effective means to ensure that the ALS patients with pulmonary insufficiency can get adequate energy intake to improve their nutritional status.

Objective To analyze the methods and effects for treating unstable posterior pelvic ring fracture combined with sacral nerve injury and further identify the relationships among the diagnostic methods,surgical approaches and clinical outcomes.Methods A total of 38 patients with posterior pelvic ring fracture combined with sacral plexus injury treated from January 2000 to January 2010 were enrolled in the study.There were 20 males and 18 females at an average age of 35 years (range,10 to 59 years).The causes of fractures included traffic injury in 20 patients,fall injury in 12,weighty object impingement injury in five,and stabbing injury in one.Classification of posterior pelvic ring fractures included fracture and dislocation of sacroiliac joints in eight patients,fracture of ilium wing in two and sacrum fracture in 28.According to the Denis typing of sacrum fractures,there was one patient with type Ⅰ fracture,14 with type Ⅱ fracture and 13 with type Ⅲ fracture.All 38 patients presented the decrease or loss of skin sensation around the lower extremities,perineal region and crissum.Simultaneously,30 patients suffered motor dysfunction of the lower extremities,while 20 patients had bladder and anus sphincter dysfunction or sexual disorder.Thirteen patients were suspected of sacral plexus avulsion and four of them were confirmed by myelography or MRI examination.All patients had at least one associated injury.The average ISS was 21.9 points ( range,9 to 47 points).Therapeutic methods were fracture reduction and fixation in the absence of nerve decompression for eight patients and nerve decompression for 30 patients including 26 patients being also managed by fracture reduction and fixation.Operation time ranged from 6 days to 6 months.The clinical outcomes were evaluated according to the British Medical Research Council (BMRC) evaluation criteria of sensation and movement function.Results Thirty-four patients were followed up for average 4.9 years ( range,1 to 10 years),during which their pelvis obtained stable recovery.The neurological outcome was excellent in two patients,good in four and unchanged in two in the nondecompression group and was excellent in 16 patients,good in nine and unchanged in one in the decompression group,with the decompression group superior to the non-decompression group ( P ＜ O.05 ).Conclusions For unstable posterior pelvic ring fracture combined with sacral nerve injury,nerve decompression and release combined with internal fixation can better improve the sacral nerve function and obtain good pelvic ring stability and is worth of clinical application.

Objective:To establish a melanoma cell line derived from a Chinese female patient of Han nationality with metastatic melanoma, and to study its basic biological characteristics.Methods:Metastatic melanoma cells were isolated from axillary lymph nodes of a 17-year-old female patient with malignant melanoma, and subjected to culture in vitro to establish a cell line. Short tandem repeat (STR) genotyping was performed to compare the information about the cell line and its derived tissue, and to detect gene mutations; cell counting kit-8 (CCK8) assay was conducted to assess the cellular proliferative activity, and soft agar cloning assay to assess the anchorage-independent proliferation; karyotype analysis was performed to determine the number and structure of chromosomes; with the highly aggressive melanoma cell line A2058 and keratinocyte line HaCaT serving as controls, Transwell assay was conducted to detect differences in cell migratory and invasive abilities, cell immunofluorescence assay and Western blot analysis were performed to determine the protein expression of melanoma-specific markers HMB45, S100 and Melan-A; the tumorigenicity was evaluated in vivo in a tumor-bearing mouse model. Results:A melanoma cell line was successfully established and named ZJMM-45, which was cultured for more than 70 passages over a 1-year period, and showed a stable shape and proliferative activity. The cells were spindle-shaped or polygonal, and could produce melanin. STR matching analysis showed that the ZJMM-45 sample was 96% matched with the cryopreserved lymph node tissue of the patient, suggesting that they were from the same source. A tumor-related gene BRAF V600E mutation (c.1799T>A) was identified in the ZJMM-45 cell line; karyotype analysis revealed that ZJMM-45 cells had triploid chromosomes and abnormal structures. ZJMM-45 cells grew exponentially in vitro and reached a plateau-phase in growth on day 5; the cells grew clonally and formed colonies in agar, showing anchorage-independent and malignant proliferative activity. Cell immunofluorescence assay and Western blot analysis showed that both ZJMM-45 and A2058 cells expressed HMB45, S100 and Melan-A; Transwell assay revealed that the numbers of invasive and migratory ZJMM-45 cells (300 ± 14, 260 ± 14, respectively) were significantly higher than those of invasive and migratory A2058 cells (150 ± 6, 160 ± 19, t = 13.25, 11.76, respectively, both P < 0.001) . The tumor-bearing mouse experiment showed that all 5 nude mice developed tumors with an inner diameter of 1.0 cm after 4 weeks, and the tumors were histopathologically characterized by proliferating hyperchromatic melanoma cell nuclei and formation of small nests, which were similar to primary solid tumors. Conclusion:The metastatic melanoma cell line ZJMM-45 derived from a Chinese patient with melanoma was successfully constructed, which carried the BRAF V600E mutation and expressed melanoma-specific markers, and was characterized by rapid proliferation, invasion and metastasis in both in vivo and in vitro culture, as well as obvious tumorigenicity in the in vivo experiment.