Carbapenem-resistance is an emerging problem in Chinese neonatal intensive care units.Carbapenem-resistant enterobacteriaceae(CRE)can hydrolyze almost all β-lactam antibiotics including carbapenems by producing carbapenemase.There are three groups of carbapenemases, namely Amber A, B and D groups, which have different hydrolytic activities to specific β-lactam antibiotics.Currently, Chinese NICUs have been facing high colonization and infection rates of CRE, with high fatality rate and rapid transmission.The treatment of neonatal CRE infections is extremely difficult.The limited choice of antibiotics, the lack of pharmacokinetic and pharmacodynamic data and the uncertainty of the optimal dose and interval bring great challenges to the effective therapy of neonatal CRE infections.The main antimicrobial agents for CRE in adults and children include carbapenems, ceftazidime/averbactam, fosfomycin, polymyxin, aztreonam, etc., but there are few studies in neonates.Once infants are colonized or infected by CRE, decolonization and treatment are very difficult.Therefore, strict implementation of infection control and neonatal antimicrobial stewardship programs to reduce CRE production, transmission and infection, are the most important measures to cope with the prevalence of CRE.

Objective To explore related factors affecting bone mineral density (BMD) and osteoporosis (OP) in patients with type 2 diabetes mellitus (T2DM). Methods Dual-energy X-ray absorptiometry was used to measure BMD of the femoral neck and the lumbar vertebrae in 220 patients with T2DM. All the patients were divided into three groups:normal bone mass group, decreased bone mass group and OP group according to the value of BMD. The clinical data and biochemical indicators were compared and analyzed between these groups, and the influencing factors of T2DM and OP were explored. Results Compared with normal bone mass group and decreased bone mass group, there were more older patients, longer course of T2DM, lower BMI and high density lipoprotein cholesterol (HDL-C), and higher glycated hemoglobin (HbA1c) level in OP group. There were significantly higher age, longer course of T2DM, higher level of HbA1c, and lower levels of BMI, HDL-C in decreased bone mass group than those of normal bone mass group (P＜0.05). There were no significant differences in waist-to-hip ratio, systolic blood pressure (SBP), diastolic blood pressure (DBP), postmenopausal women (PMW) ratio, fasting plasma glucose (FPG), 2-h postprandial blood glucose (2hPG), blood phosphorus, calcium, total cholesterol (TC), three acyl glycerin (TG) and low-density lipoprotein cholesterol (LDL-C) between three groups. The risk factors for T2DM combined with OP were older, female, long duration of T2DM, higher levels of FPG, 2hPG and HbA1c. Conclusion Older and female T2DM patients were high-risk group of OP. The treatment plan should be timely adjusted by regularly monitoring indexes of blood glucose and HbA1c.

BACKGROUND: Inhibiting the degradation of extracellular matrix in the intervertebral disc can delay the degenerative process of intervertebral disc. Matrix metalloproteinases-3 (MMP3) is considered as a key enzyme for degradation of extracelular matrix components such as type II collagen and aggrecan.
OBJECTIVE:To construct the short hairpin RNA lentiviral vector targeting human MMP3 gene and to detect its efficiency of gene silence by infecting human degenerative nucleus pulposus cells.
METHODS:According to the human MMP3 mRNA (NM_002422.4) sequence, four groups of the short hairpin RNA gene sequences targeting MMP3 were designed, synthesized and annealed to form double stranded DNA fragments, which were connected with the LV3 vectors digested by BamHI andEcoRI enzymes, and then transfected into the competent cels. The positive clones were identified by PCR, and analyzed by sequencing. The packaging and titer of lentivirus were determined after transfecting 293T cells. Human degenerative nucleus pulposus cels were infected with lentivirus vector, and the transfection efficiency of each group was observed under inverted fluorescence microscope. The interfering efficiency was detected by real time-PCR and western blot at 72 and 96 hours.
RESULTS AND CONCLUSION:The ds-oligo DNA was successfully inserted into the lentiviral vector as confirmed by electrophoresis and sequence analysis. The recombinant lentivirus was harvested from 293T cels with a viral titer of 1-5 ×108 TU/mL. RNA interference targeting the GCC AGG CTT TCC CAA GCA AAT sequences with the highest interfering efficiency in MMP3 gene at 72 and 96 hours resulted in suppression of MMP3 mRNA expression by 98% and 72%, respectively; and at 96 hours, the interfering efficiency of protein expression was 57.2%. The recombinant lentivirus vector containing RNA interference targeting MMP3 gene is successfuly constructed, which lays a foundation for further studies on the MMP3 function and gene therapy.

Objective To investigate the respiratory burst function of neutrophils in very low birth weight infants (VLBWI).Methods Twenty two VLBWI was divided into two groups:neonatal respiratory distress syndrome (NRDS) and non NRDS (11 in each).The respiratory burst function of neutrophils in the peripheral blood of VLBWI within 48 hours after birth was determined using the flow cytometrydihydrorhodamine 1,2,3 method before and after the chemical stimulation of phorbol-12-myrismte 14 acetate (PMA),and the gp91Phox was also measured in resting neutrophils by flow cytometry.Twenty healthy term neonates served as controls.Mann-Whitney U test was used for statistical analysis.Results Before the stimulation of PMA,the percentage of activated neutrophils of VLBWI [(49.10±20.19) ％] producing a respiratory burst was higher than that of term neonates [(18.73 ±6.81) ％] (Z--4.911,P=0.000),however,after the stimulation of PMA,the percentage of activated neutrophils of VLBWI [(96.58 ± 3.44) ％] was lower than that of term neonates [(99.20±0.62) ％] (Z--3.186,P=0.001),and the stimulation index (SI) of VLBWI (171.40 ± 103.35) was lower than that of term neonates (306.30 ± 138.47),with significant difference (Z=-3.413,P=0.001).The geometric mean of gp91Phox in VLBWI (21.66± 19.87) was higher compared with term neonates (19.60±8.03),however,the difference was not significant (P=0.350).The percentage of neutrophils that expressed gp91Phox [(56.11 ± 29.40) ％] was lower in VLBWI than that in term neonates [(80.14± 14.87) ％],with significant difference (Z=-2.374,P=0.018).Before the stimulation of PMA,the percentage of activated neutrophils of VLBWI with NRDS (63.40± 16.45) ％] was higher than that of VLBWI without NRDS [(34.80± 11.65) ％],with significant difference (Z=-3.382,P=0.001),the SI of VLBWI with NRDS (129.46 ± 75.36) was significantly lower than that of VLBWI without NRDS (213.35 ± 113.49) (Z=-2.331,P=0.020).Conclusions Neutrophils producing a respiratory burst in both VLBWI and term neonates are active without stimulation of PMA,while the phenomenon is more obvious in VLBWI.Neutrophils in VLBWI and term infants can be activated by the stimulation of PMA,and express gp91Phox.The activation and gp91Phox expression of neutrophils in VLBWI with NRDS tend to be lower than those in VLBWI without NRDS.

Objective To establish a rabbit VX2 liver tumor model by transplanting tumor tissue mass into the rabbit's liver,to analyze and observe the growing features of the liver tumor.Methods The tumor tissue mass(about 106-108 VX2 liver tumor cells)was inoculated into the left hepatic labe in 20 rabbits to establish rabbit VX-2 hepatic carcinoma model.The observation included the following two respects.(1)The tumor's volume at 7,10,14,17 and 21 days after the procedure was measured by ultrasonography and the growth rate of tumor was calculated.(2)The morphological feature of the tumor was inspected both macroscopically and microscopically.Results The growing pattern of the tumor was compatible with the exponential curve.Seventeen days after transplantation the increase rate of the tumor volume was much higher than that of the tumor diameter. Histopathologjcally,the growing pattern of the tumor took the form of infiltrative way,with its appearance being quite similar to the VX2 squamous cell carcinoma.Conclusion Transplantation of tumor tissue mass is the technique of first choice to establish the VX2 liver carcinoma model in rabbits.This experimental model is a very ideal animal form for both clinical and fundamental studies of liver carcinoma.

Oligonucleotide microarray technology is a powerful data-mining platform and has been widely applied in biosciences. To improve the performance of assays on the oligonucleotide microarray, the factors that influence the hybridization effects such as surface chemistry, probe size, spacer length, hybridization conditions etc were intensely studied and optimized. However, it is a key problem with DNA microarrays how to generate higher fluorescent signals to improve the detection sensitivity. Two types of multiply labeled primers, termed multiply labeled linear primer and multiply labeled branched primer, were used to enhance the fluorescent signal obtained from two-dimensional DNA microarrays.The signal was intensified by increasing the number of fluorophores labeled on the target DNA segment. It was indicated that the detection limit (minimum template amoumt for detection) of the multiply labeled primers is about 1% of that of the singly labeled primer. Multiple labeling is an effective signal amplification method to increase the detection sensitivity of the probes in a miniaturized array format.

Objective To analyze the incidence,bacterial spectrum and clinical features of peripherally inserted central catheter-associated bloodstream infection in neonates, and to investigate the effect of infection control strategies on the occurrence of infection,by which it is helpful in choosing effective strategies for clinical practice. Methods Clinical data, results of blood culture and culture of the catheter tip were collected from 72 infants (66 of them were very low birth weight) admitted to NICU from July, 2007 to August, 2008. The incidence of PICC-associated bloodstream infection (PICC-BSI) in the old hospital was compared with that of the new hospital. Results Bacterial colonization was not found. PICC-BSI was found in 15 cases (18. 1%), including 11 cases (13.2%) with definite sepsis and 4 cases (4. 8 %) with clinical sepsis.The incidence of PICC-BSI was 10. 2/1 000 PICC-days as a whole,with 16. 1/1 000 PICC-days in the old hospital and,7. 7/1 000 PICC-days in the new hospital respectively. Eleven organisms were isolated including Coagulase negative staphylococcus (CoNS) (4 strains), Acinetobacter baumannii (3 strains), Klebsiella pneumonia (2 strains), Enterococcus(1 strain), and Candida parapsilosis (1 strain). The incidence of PICC-BSI in the new hospital was lower compared with that of the old hospital. Conclusion Most of the isolated organisms of PICC-BSI are opportunistic and multi-drug resistant pathogen. PICC-BSI can be reduced by the effective infection control strategies in NICU.

Objective To identify the clinical and imaging characteristics in neonatal refractory purulent meningitis. Methods Clinical data of 70 cases of neonatal purulent meningitis admitted to the neonatal intensive care unit at Children's Hospital of Fudan University from January, 2009 to December, 2014 were reviewed retrospectively. The patients were divided into refractory group (n=28) and non-refractory group (n=42) according to the course of antimicrobial therapy.The clinical and brain MRI characteristics of neonatal refractory purulent meningitis were analyzed. Parameters were compared between the two groups using Chi-square or Fisher's exact tests, and Wilcoxon tests where appropriate. Risk factors of neonatal refractory purulent meningitis were investigated by univariate and multivariate Logistic regression analysis. Results Among the 70 cases, 31(44.3%) were positive for cerebrospinal fluid (CSF)/blood culture. The positive rate was higher in the refractory group than in the non-refractory group [75.0%(21/28) vs 23.8%(10/42),χ2=17.843, P<0.01]. The most common pathogenic bacteria isolated in the refractory group were Escherichia coli [8 cases (38.1%)] and group B streptococci [5 cases (23.8%)]. Compared to the non-refractory group, patients in the refractory group were more likely to have seizure, higher CSF white blood cell count, higher CSF protein concentration and lower CSF glucose concentration [53.6%(15/28) vs 7.1% (3/42), 965.0 (463.0-2 200.0)×106/L vs 116.5 (61.0-327.5)×106/L, 3 221.1(2 354.3-4 633.5) mg/L vs 1 487.6(988.2-1 924.1) mg/L, and 0.2 (0.1-0.8) mmol/L vs 1.5 (1.2-1.8) mmol/L; all P<0.01]. Multivariate Logistic regression analysis showed that seizure, low CSF glucose concentration on admission, and a positive CSF/blood culture result neonatal refractory purulent meningitis (OR=9.6, 95%CI: 1.2-76.0; OR=15.0, 95%CI: 5.6-63.3; and OR=7.3, 95%CI: 1.5-36.0, respectively). Abnormal brain MRI findings, including intracranial extracerebral space abnormality, ventricular dilatation and periventricular white matter injury, were more common in the refractory group [100.0%(28/28) vs 61.9%(26/42), χ2=13.827 totally; 64.3%(18/28) vs 21.4%(9/42), χ2=13.023 for intracranial extracerebral space abnormality; 60.7%(17/28) vs 19.0%(8/42), χ2=12.704 for ventricular dilation and 28.6%(8/28) vs 2.4%(1/42) for periventricular white matter injury; all P <0.01]. Compared with the non-refractory group, the refractory group had a longer hospital stay [(48.0±17.4) d vs (26.0±10.2) d, t=6.016, P<0.01] and more adverse events [67.9%(19/28) vs 31.0%(13/42), χ2=9.220, P=0.002], including hearing impairment and requirement of neurosurgical intervention [14/18 ears vs 10/46 ears (21.7%), χ2=4.292, P=0.038]. There was no death in both groups during hospitalization. Conclusions Neonates with seizure, low CSF glucose concentration and positive CSF/blood culture results are more likely to have refractory purulent meningitis. Brain MRI abnormalities are more common in neonatal refractory purulent meningitis.

Objective To study the expressions of osteopontin (OPN),caspase-3 and mt-P53 proteins, and their relationship in gliomas. Methods Seventy gliomas specimens of patients (glioma group) were selected, and 10 samples of non-glioma brain tissue were used as control group. The SP method was used to detect the positive rates of protein expressions of OPN, caspase-3 and mt-P53 between two groups. The relationship between protein expressions of OPN, caspase-3 and mt-P53 in gliomas and grade of gliomas were detected by Western blot assay. Spearman rank correlation was compared between the positive expression of OPN, caspase-3 and rate mt-P53. Results The positive expression rates of OPN and mt-P53 were significantly higher in glioma group (64.29%and 60%) than those of control group (no positive expression), but the positive expression rate of caspase-3 was significantly lower than that of control group (47.14%vs. 90%, P<0.05). There were no significant differences in OPN, caspase-3 and mt-P53 expressions between different gender, age, tumor size and tumor position (P>0.05). The higher the WHO classification, the higher the positive expression rates of OPN and mt-P53 (P<0.001), and the lower the positive expression rate of caspase-3 (P<0.001). With the increased level of glioma grade, OPN and mt-P53 protein levels were increased, but caspase-3 protein expression level was decreased. There was a negatively correlation between OPN and the positive expression of caspase-3, but there was a positive correlation between OPN and the expression of mt-P53 (rs=-0.720 and 0.722, P<0.05). There was a negative correlation between caspase-3 and mt-P53 expressions (rs=-0.556, P<0.05). Conclusion The higher the WHO classification, the higher the positive expression rates of OPN and mt-P53, while the lower the positive expression rate of caspase-3. The study reveals that OPN, caspase-3 and mt-P53 expressions are associated with the occurrence and the progress of gliomas. The combined detection of them can contribute to the judgment of biological behavior of gliomas.

Objective To investigate the clinical features of neonatal invasive fungal infection(IFI) so as to guide diagnosis,prevention and treatment of IFI.Methods Seventy-six neonates with IFI admitted to the Neonatal Intensive Care Unit (NICU) at Children's Hospital of Fudan University from 2004 to 2014 were included in the study.Pathogens,clinical manifestation,risk factor exposure,laboratory findings,complications,and clinical outcome of neonatal IFI were analyzed.Results Seventy-six cases were diagnosed as IFI between 2004 and 2014,with an yearly increasing trend.Sixty-eight patients were premature infants (89.5％).Of the 76 cases,except one with unknown birth weight,11(14.7％),34(45.3％),20(26.7％)and 10 (13.3％) cases had birth weight ＜ 1 000 g,(≥ 1 000-＜1 500) g,(≥ 1 500-＜2 500) g and ≥ 2 500 g,respectively.The pathogens were mainly Candida (74/76,97.4％),including 26 cases of Candida albicans (34.2％).However,the incidence of non-Candida albicans infection was increasing.Candida guilliermondii was the most common in nonCandida albicans,accounting for 29.2％ (14/48).All Candida albicans were sensitive to fluconazole.One strain of Candida glabrata was resistant to fluconazole.The most common risk factors included use of broad-spectrum antibiotics(93.3％,56/60),parenteral nutrition(70.0％,42/60),central vein catheterization(53.3％,32/60),invasive ventilation(40.0％,24/60) and history of abdominal surgery(21.7％,13/60).Clinical manifestations of IFI included temperature instability,frequent apnea,increased requirement of respiratory support and feeding intolerance.Among all cases,six were diagnosed as central nervous system infection.Of the patients who received cranial MRI,46.8％(22/47) showed multiple abnormal signals in cerebral parenchyma.Fiftytwo patients were cured and seven patients died before discharge,including one death due to fungal infection.Conclusions There is an increasing trend of IFI cases in NICU,especially in premature infants.Non-Candida albicans has become the main pathogenic fungus.There are no specific clinical manifestations in neonatal IFI.Use of broad-spectrum antibiotics,parenteral nutrition and central venous catheterization are common risk factors,and preventive measures should be taken in high-risk infants.In addition,IFI in neonates may affect important organs such as central nervous system,thus early treatment is necessary in suspected patients.