Objective To analyse the DNA heterogeneity of 20 clinical isolates of Acinetobacteria using infrequet-restriction-site PCR (IRS-PCR ).Methods Strain-specific electrophoretic patterns from PCR products by amplifying DNA sequences flanking infrequent restriction of 20 strains of Acinetobacter were compared with the results of genotype with RAPD as well as the results of biotyping.Results Among the 20 bacteria, ten can be recognized as Acinetobacter haemolytius with biotyping, seven as Acinetobacter lwoffi, while the other 3 bacteria can not be recognized. Acinetobacter haemolytius isolates were divided into 5 gene types with IRS-PCR, isolates of Acinetobacter lwoffi were divided into 4 gene types, meanwhile, the last three bactria were divided into 2 gene types. With RAPD technique, Acinetobacter haemolytius, Acinetobacter lwoffi and the other 3 bacteria were divided into 6 gene types, 4 types, and 2 types by prime 1, respectively, the amplifying results of primer 2 divided Acinetobacter haemolytius into 9 gene types, the Acinetobacter lwoffi ,5 types and the other 3 bacteria,2 types. Conclusion IRS-PCR can be used for typing Acinetobacter and is more aceurate than biotyping. It has the same recogniton abili ty as RAPD while is of better stability and repeatability, so this method is capable of clinical application.

Objective To investigate the effect of cerebral ischemic preconditioning (IP) on the expressions of angiopoietin-1 (Ang-1) and its receptor Tie-2 mRNA in cerebral ischemia in rats.Methods Ninety-nine Wistar rats were randomly assigned to three groups:sham operation (n =9),non-ischemic preconditioning (NIP) (n =45),and IP (n =45).The latter two groups were redivided into 5 subgroups:ischemia-reperfusion 1,3,7,14,and 21 days (n =9 in each group).A model of transient middle cerebral artery occlusion (MCAO) was induced by the intraluminal suture method for focal IP (ischemia for 10 minutes and restoring perfusion).Infarct volume was determined by 2,3,5-triphenyltetrazolium staining.The expression levels of Ang-1/Tie-2 mRNA were detected by in situ hybridization.Results The infarct volumes in the 1 -,3-,and 7-day subgroups of the IP group were significantly smaller than those in the relative subgroups of the NIP group (all P＜ 0.05).The expression of Ang-1 mRNA in the 3- and 7-day subgroups of the IP group and the expression of Tie-2 mRNA in the 1-,3-,and 7-day subgroups of the NIP group were upregulated significantly (all P ＜ 0.05).The infarct volume in the 3-day subgroup of the IP group was reduced most significantly (P ＜ 0.05).The expression of Ang-1 mRNA in the 7-day subgroup was upregulated significantly,and the peak expression of its receptor Tie-2 mRNA appeared at day 3 after IP and continued to day 7.Pearson correlation analysis showed that the expression levels of Ang-1/Tie-2 mRNA were significantly negatively correlated with infarct volume (P ＜0.01).Conclusions The expression of Ang-1/Tie-2 mRNA in the IP group was upregulated within the time window of ischemic tolerance (1 - 7 days after preconditioning),in which Ang-1 may mainly act on the later stage of the cerebral ischemic tolerance.

Science Spirti play a important role in promoting moral standardization and the comcept development.The implication of science to seek truth,rational,criticize and suspicion,freedomand creating,which possesses important significance to cllture value sense of professional ethics of physicians to save life by loveheart,uphold truth,dedceating ond self to medical cause,and to promote sociatist medical moral progress.

Objective:To investigate the effects of ovarian cancer ascites-derived exosomes on the stem cell properties and invasion ability of ovarian cancer stem-like cell (OCS-LC).Methods:(1) A2780 cells were induced into OCS-LC in serum-free medium, and authenticating their stem-like properties by sphere-forming test, differentiation test and CD 133 marker detection. (2) Exosomes from ascites and A2780 cell were extracted by ultracentrifugation, then authenticating them. The morphology of exosomes was observed by the transmission electron microscope, exosome particle size was measured by nanoparticle tracking analysis (NTA). The expressions of heat shock protein 70 (HSP-70), CD 63 and CD 9 were detected by western blot. (3) The exosomes from ovarian cancer ascites and tumor cell supernate were co-cultured with OCS-LC. The groups were divided into control group, ascites-derived exosomes (ADE) group (ADE+OCS-LC group), and cells-derived exosomes (CDE) group (CDE+OCS-LC group). The sphere-forming ability was evaluated by sphere-forming cycle, maximum sphere diameter and sphere-forming rate of each group; the expression of CD 133 was detected by immunofluorescence staining under microscope; quantitative real-time (qRT)-PCR was used to detected the expression levels of octamer-4 (Oct-4), Nanog mRNA of the signature genes in the stem cells of each group; the metastasis ability of each group was measured by transwell assay. Results:(1) Identification of OCS-LC: sphere-forming experiment showed that the suspension of OCSC single cells was grown in serum-free medium in secondary sphere-forming. Differentiation function experiment showed that OCS-LC were differentiated into adherent A2780 cells by changing the growth mode in serum containing medium. Flow cytometry showed that the proportion of CD 133 positive ( CD133+) cells in OCS-LC group was (18.9±0.9)%, significantly higher than that of control group (0.6±0.5)% ( t=38.570, P<0.01). (2) Under transmission electron microscope, clear lipid bilayer structure was observed in ADE and CDE, and one side presented a concave hemispheric or cup like structure. NTA showed that the diameter of exosomes mainly ranged from 30 to 100 nm, with an average diameter of 67.2 nm. Western blot analysis showed that the specific protein molecules HSP-70, CD 63 and CD 9 were positive. (3) Three groups′ OCS-LC could continue to grow into spheres, and the group of ADE+OCS-LC showed two growth modes, most of the cells continued to grow into spheres, while a small part of cells grew in adherent differentiation. The sphere-forming rate of OCS-LC in the control group, ADE+OCS-LC group, and CDE+OCS-LC group were (1.05±0.20)%, (4.15±0.10)%, and (10.45±0.25)%, the sphere-forming cycle of OCS-LC in the three groups were (15.3±1.5), (10.3±0.6), and (6.7±0.6) days, and the maximum diameters of OCS-LC were (100.3±3.2), (145.2±5.1) and (170.0±2.1) μm, respectively. And the differences were statistically significant (all P<0.05). After co-culture of exosomes with OCS-LC, the sphere-forming ability of cells in the group of CDE+OCS-LC was prior to ADE+OCS-LC group (all P<0.05). Immunofluorescence staining showed that the number of CD 133 green fluorescent chromophore cells in OCS-LC groups [(46.2±2.1)%, (58.4±2.2)%] was significantly higher than that in the control group [(26.6±1.5)%] after the addition of exosomes in co-culture, the positive rate of CD 133 was higher than that in the control group( F=187.588, P<0.05). The qRT-PCR results showed that the expression levels of Oct-4 mRNA in ADE+OCS-LC and CDE+OCS-LC groups were 3.46±0.24, 4.03±0.31, compared with that in control group (1.04±0.12), the differences were statistically significant ( F=134.932, P<0.05). The mRNA expression levels of Nanog were 1.57±0.32, 2.66±0.15, which were significantly higher than that in the control group (1.00±0.07), and the differences were statistically significant ( F=49.329, P<0.05). And the expression of both in CDE+OCS-LC group increased more significantly than ADE+OCS-LC group (all P<0.05). The number of invasive cells in the three groups of OCS-LC were: control group 30±5, ADE+OCS-LC group 102±4, CDE+OCS-LC group 210±7, and there were statistically significant differences among three groups ( F=820.800, P<0.05). Compared with the control group, the number of invaded cells in the co-culture group were significantly increased ( P<0.05), and the CDE+OCS-LC group had the higher cell invasion ability then the ADE+OCS-LC group ( t=23.202, P<0.05). Conclusions:Exosomes derived from ovarian cancer ascites could enhance and maintain the stemness of OSC-LC, and promote the invasion of tumor cells. Moreover, CDE is superior to ADE.

A target molecule affinity-ultrafiltration liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MSn) method was established for rapid screening acetylcholinesterase (AChE) inhibitors from total alkaloids of fibraurea recisa Pierre.A total of 12 potential inhibitors were screened from Fibraurea recisa Pierre.and 6 compounds were identified including palmatine,berberine,jatrorrhizine,palmatrubine,7,8-dihydro-8-hydroxyberberine and groenlandicine.The AChE inhibitory activity of these 6 compounds was validated in vitro.Palmatine showed the strongest inhibitory activity for AChE,which was stronger than that of donepezil hydrochloride,demonstrating the potential of palmatine as anti-Alzheimer's drug.This method is simple,rapid,and accurate for directly screening active ingredients which can inhibit AChE from complex extract of traditional Chinese medicines.

The metabolism of thyroid hormone changes during pregnancy. Subclinical hypothyroidism during pregnancy may adversely affect the pregnancy outcomes and neuropsychological development of child, controversially. The diagnostic criteria of subclinical hypothyroidism during pregnancy need to be specific. It is important to figure out when should pregnancy with subclinical hypothyroidism be treated and what is the specific goal of the treatment.

Objective We established a rapid detection method of Pasteurella spp.and provided a reference for microbiological quality control of laboratory animal .Methods According to the β subunit of bacterial RNA polymerase ( rpoB) protein multiple alignments of 13 different Pasteurella spp.published in NCBI .The degenerate primers were designed by CODEHOP designer online .CODEHOP PCR method was applied to detecting Pasteurella spp.after the specificity and sensitivity of the method had been evaluated by 21 reference strains .Results Standard strain amplified fragment were about 200 bp by degenerate primers PastF6/PastR5.The primers are able to distinguish between Pasteurella spp.and the other pathognic organisms of laboratory animal respiratory tracts .Sensitivity of this method were 0.2 pg/μL~2 pg/μL to different Pasteurella.The Pasteurella positive rate was 19.1% in 609 animal ' s respiratory samples .The accuracy of positive results was 100%through verifying by sequenced and blast .Conclusions The established method has good specificity and sensitivity .It can be used to detect Pasteurella spp.in animal samples .

Objective To prepare a kind of superparamagnetic iron oxide (SPIO) and doxorubicin loaded multifunctional polymer microbubbles (MPMBs),and to explore its potential application as an ultrasound(US)/magnetic resonance (MR) imaging contrast agent in vitro.Methods The MPMBs and normal polymer microbubbles (PMBs) were made by double emulsion and freeze-drying methods.The physical property,drug encapsulation efficiency and the drug-loading efficiency of MPMBs were determined,and the release property and US/MR imaging enhancement of MPMBs were observed.Results The MPMBs had a regular shape and narrow size distribution.The drug encapsulation efficiency was (60.20±2.69) ％,and the drug-loading efficiency was (6.02 ± 0.27) ％.The in vitro release experiment showed that ultrasound can promote the release of doxorubicin in MPMBs.US imaging in vitro showed that the enhancement of MPMBs was better than PMBs,and MR imaging in vitro conformed that MPMBs could well enhance MR imaging.Conclusions The MPMBs is a multifunctional contrast agent with the treatment function as well as US/MR dual-mode imaging enhancement effect.

Objective To evaluate the predictive accuracy of several risk-assessment strategies to predict the risk of significant neonatal hyperbilirubinemia, and to establish the best prediction model.Methods The transcutancous bilirubin (TcB) levels of 4907 term and near-team infants were measured.Trace blood bilirubin levels of the infants whose TcB levels ≥250 μmol/L were detected. Clinical data of newborns and their mothers were collected and were analyzed with Logistic regression model to investigate its correlation with signifrcant hyperbilirubinemia. Clinical high risk factors of significant neonatal hyperbilirubinemia were determined. Accuracy of three prediction methods for significant hyperbilirubinemia was compared by receiver operating characteristic (ROC) curve. The three methods included: whether predischarge bilirubin level (within 72 hours after birth) expressed in risk zone on an hour-specific bilirubin nomogram; clinical risk factors other than predischarge bilirubin level; and combination of the predischarge bilirubin risk zone and other clinical risk factors. Results Two hundred and eighty-six newborns (5.8%) were found with significant hyperbilirubinemia. The risk factors of significant neonatal hyperbilirubinemia were divided into three groups according to OR: (1) Major risk factors:predischarge (within 72 hours after birth) bilirubin level in the high risk-zone (OR=96. 39, 95% CI:53.32-174.27, P = 0. 000), large cephalohematoma (OR = 36.45, 95% CI: 10. 02-132.56,P=0. 0076), gestational age 35-36+6 weeks (OR= 30. 72, 95% CI 14.47-65.23, P=0. 0001) and exclusive breast feeding and weight loss was ＞9% of birth-weight (OR=22.44, 95% CI: 4.42-114. 03, P=0. 0016). (2) Minor risk factors: gestational age 37-37+6 weeks (OR=3.26, 95% CI:1.92-5. 55, P=0. 0232), predischarge bilirubin level in P76-P95(OR=13. 64, 95% CI: 8. 10-22.97,P=0. 0001) and bruising (OR = 2.32, 95% CI: 1.14-4.71, P = 0. 0497). (3)Protective factors (those factors associated with decreased risk of hyperbilirubinemia): predischarge bilirubin level in low-risk zone (≤P40) (OR=0. 00), gestational age ≥40 weeks (OR=0.21, 95% CI: 0.09-0.44,P=0. 0402) and mixed breeding (OR=0. 75, 95% CI: 0. 58-0.95, P=0.0059). The area under the ROC curve of predischarge bilirubin level was 0. 8687 and 0. 7375 for clinical risk factors other than predischarge bilirubin level. The area under the ROC curve of a combination of the predischarge bilirubin risk zone and additional clinical risk factors was 0. 9367. Conclusions The risk of significant neonatal hyperbilirubinemia could be simply and accurately predicted by infant's predischarge bilirubin level and the combination of predischarge bilirubin level, and clinical risk factors might improve the accuracy of prediction significantly.

Background and purpose:Pathological lymph node metastasis (LNM) is not rare in clinical lymph node negative (cN0) papillary thyroid carcinoma (PTC). The aim of this study was to investigate the risk factors of LNM, especially of high volume LNM (more than 5 metastatic lymph nodes) and contralateral central compartment LNM, in cN0 PTC.Methods:Medical records of 350 PTC patients (265 female, 85 male, 212 patients with solitary lesion in unilateral lobe) were reviewed. All operations of these patients were performed by one surgical team. The clinical pathological data were collected, and univariate and multivariate analysis was performed.Results:LNM was conifrmed in 138 patients (39.4%) and 20 patients had high volume LNM. In 169 patients with solitary lesion in unilateral lobe with total thyroidectomy and bilateral central neck dissection, 24 patients had contralateral metastasis (14.2%). In univariate analysis, tumor size (58.5% in >1 cmvs 33.6% in≤1 cm) and tumor with calcification in preoperational ultrasonography (43.7% withvs 31.7% without) showed signiifcant difference in prevelance of LNM. In multivariate analysis, tumor size >1 cm (OR=2.792) was the independent risk factor of LNM. Gender (3.8% in male vs 11.8% in female), age (10.7% <40 yearsvs 3.4%≥40 years ), tumor size(13.4% in >1 cmvs 3.4% in≤1 cm) and tumor with low echo in preoperational ultrasonography (13.9% withvs 4.8% without) showed signiifcant difference in univariate analysis of high volume LNM. Male (OR=5.152), tumor size >1 cm (OR=5.712) and age <40 years (OR=3.959) were conifrmed as independent risk factors of high volume LNM. Male (OR=3.105) and tumor size >1 cm (OR=3.863) were also demonstrated as independent risk factors of contralateral LNM in patients with solitary lesion in unilateral lobe, the prevalence of LNM were 26.5% in male and 26.1% in tumor size >1 cm, respectively.Conclusion:LNM was not “rare” in cN0 PTC patients. Prophylactic central neck dissection should be performed in cN0 patients with tumor size >1 cm. For cN0 microcarcinoma, more active surgical treatment may be considered in male and young patients.