Objective To investigate the inhibition mechanism of rohdea roth on hyphae formation by Candida albicans.Methods MTT assay was used to detect the minimum inhibitory concentration(MIC)and minimum fungicidal concentration(MFC)of C.albicans.The inhibitory effect of fungicidal adherence was detected by MTT assay.Inverted fluorescence microscope was used to observe effect on hyphae formation.The influence on Efg1 and Hwp1gene expression were detected by RT-PCR method.ResultsMIC and MFC of C.albicans were 16 mg/mL and 32 mg/mL, respectively.The inhibitory effects of rohdea roth on C.albicans adherence and hyphae formation were significantly inhibited,and the concentration was dose-dependent.After the concentration of 16 mg/mL acted on C.albicans for 6 h, hyphae disappeared completely.The results of RT-PCR showed that the gene expression of Efg1 and Hwp1 could be inhibited by rohdea roth.Compared with the control group,the expression of Efg1 and Hwp1in the experimental groupwere reduced by 84.18% and 59.57%(P<0.01).Conclusion The inhibitory effect of rohdea roth on the adherence and hyphae formation of C.albicans is mainly through inhibiting the expression of Efg1 and Hwp1genes.

AIM: To investigate the frequency of P459H(CCC→CAC) in exon 10 of CYP21 gene among normal population.METHODS: The exons 3-10 of CYP21 gene were amplified with polymerase chain reaction(PCR).The PCR round products were digested by restriction enzyme Pst I to confirm that CYP21 gene was specifically amplified.PCR-based amplification-created restriction site(PCR-ACRS) was performed using the first round PCR products as template.After the second PCR products were digested by restriction enzyme Fsp I,10% polyacrylamide gel electrophoresis was used to screen the frequency of P459H in exon 10.RESULTS: The codon 459 in exon 10 of CYP21 gene was all CCC among 100 normal cases tested.CONCLUSION: P459H(CCC →CAC) of CYP21 gene might be a novel point mutation causing CAH.Furthermore,PCR-ACRS was a fast and safe method for gene mutation screening.

Objective To investigate the inhibition mechanism of gallnut on biofilm formation by MRSA 41577.Methods TTC assay was used to detect inhibitory effects of biofilms formation and mature biofilms.The of PIA on biofilm formation was studied using Congo red agar method.Micro-Ultraviolet Spectrophotometer was used to detect inhibitory effects of the release of eDNA.The influence for Baicalein on icaA and cidA gene expression were detected by RT-PCR method.Results The inhibitory concentration (MIC) and minimum bactericidal concentration (MIC) of MRSA 41577 BF were 0.5 mg/mL and 1 mg/mL, respectively.The inhibitory effect of galla on MRSA 41577BF formation and mature BF was significantly inhibited.Inhibition of MRSA 41577,the MIC and MBC of mature BF were 4 mg/mL and 16 mg/mL.Congo red test results show that Galla can inhibit the synthesis of MRSA 41577 PIA, and the concentration was dose-dependent.The results showed that gallnut could inhibit the release of MRSA 41577 eDNA, and the release amount of eDNA was 3.61μg/OD595 and 11.91μg/OD595 , respectively, when the concentration of gall was 1/2MIC.The release of eDNA was reduced by 69.7% (P<0.01).The expression of icaA and cidA genes in the control group was 9.7% and 6.67%, respectively.The expression of icaA and cidA in the control group was significantly lower than that in the control group ( icaA and cidA, and cidA gene expression were 100%, the expression of icaA and cidA genes were reduced by 90.3%and 93.3%, respectively (P<0.01).Conclusion The inhibitory effect of gallnut on the biofilm of MRSA 41577 is mainly through inhibiting the expression of icaA and cidA genes, and then affecting the synthesis of PIA and the secretion of eDNA .

Objective To analyze the application of clinicopathological features and LEF-1 in the diagnosis of solid pseudopapillary neoplasm of the pancreas (SPN).Methods Clinical and pathological data of 227 cases who were pathologically diagnosed as pancreatic SPN at Changhai Hospital from Jan 2000 to Dec 2015 were collected and analyzed.Immunochemical assay was used to detect the expression of LEF-1 in 132 cases of SPN, and the results were compared with β-catenin, which is most commonly used for diagnosing SPN.Results 81.9% of patients with SPN were female (186/227).Mean age at the onset was 34 years.Mean tumor size was 5.4 cm.48.5% tumors were localized in the pancreatic tail, and 33% in the head.46.3% tumors were cystic and solid, 42.3% were solid, and 11.4% were cystic.There were 2 cases of lymph node metastasis (0.9%), 15 cases of vascular tumor thrombus (6.6%), 14 cases of nerve invasion (6.2%), and 13 cases of adjacent organs invasion (5.7%) based on microscopic observations.Immunochemical analysis showed that 130 of 132 cases with SPN expressed LEF-1 with strong nuclear positivity, and the positivity rate was 98.5%.But no obvious expression of LEF-1 can be seen in normal pancreatic tissue and other pancreatic tumors.The specificity was 100%.The positivity of β-catenin expression in SPN was 96.6%(144/149), and β-catenin was positively expressed in only one case of acinar cell carcinoma.Tumors were completely removed by surgery in 165 cases, and the median follow-up was 51 months.By Oct 31, 2016, 162 patients (98.2%) survived, 5 had liver metastasis, and 1 had recurrence.Conclusions SPN is predominantly encountered in young female patients, and the clinical manifestations are not specific.LEF-1 can be used as a specific marker for the diagnosis and differentiation of SPN, which is more accurate than β-catenin.

Objective To investigate the phenotypes and percentages of B 10 cells in different tis-sues from wild-type mice and to identify their biological functions .Methods The percentages of B10 cells derived from different tissues of mice and their responses to lipopolysaccharide ( LPS) stimulation were ana-lyzed by flow cytometry .Magnetic-activated cell sorting ( MACS ) and fluorescence-activated cell sorting (FACS) were used to purify B10 cells, CD4+CD25-T cells and Treg cells.CD4+CD25-T cells and Treg cells labeled by CFSE were co-cultured with or without B10 cells, and then their proliferation were evaluated after 72 h.Results (1) A subset of CD19+CD5+CD1dhigh regulatory B cells was identified in spleen , pe-ripheral blood and lymph nodes from wild-type mice , of which the highest frequency was detected in spleen (3.95%±0.79%, P<0.05).The isolated B cells from different tissues were stimulated by LPS , PMA, ionomycin and monensin (L+PIM) in vitro to express IL-10.Among them, splenic CD19+IL-10+B cells showed the highest expression of IL-10 (P<0.05).(2) Prolonged LPS stimulation (48 h) to CD5+CD1dhigh B cells enhanced the expressions of IL-10 (P<0.01).(3) CD19+CD5+CD1dhigh B cells inhibited the prolif-eration of CD4+CD25-T cells in vitro in a dose-dependent manner (P<0.01), but increased the secretion of IL-10 by CD4+T cells (P<0.01) and the proliferation of Treg cells in vitro (P<0.01).Conclusion Com-pared with other tissues , the percentage of B10 cell subset in spleen is the highest in wild-type mouse , and B10 cells subset can be activated through Toll-like receptor ( TLR ) signaling pathway .The responses of CD4+CD25-T cells and Treg cells in co-culture with B10 cells are regulated by B 10 cell subset through an increased IL-10 production .B10 cells might be a useful cell population for the treatment of inflammatory au-toimmune diseases.

Mutations P459H and R483W detected in CYP21A2 gene in two Chinese patients with simple virilizing 21-hydroxylase deficiency were studied.Plasmid vectors containing P459H and R483W were constructed and transfected into COS-7 cells.The converting rate of progesterone to 11-desoxycortisone was calculated.P459H reduce 21-hydroxylase activity to 6.8%,while the residual enzyme activity of R483W was only 2.9%.

[Objects]To isolate and identify the pathogen of the large outbreak of dengue in Guangdong province in 2014. To understand the origin and the phylogenetic characteristics of the isolates ,and provide scientific foundation for the surveillance and prevention of dengue fever.[Methods]Collected the patient serum samples over all the Guangdong province during the 2014 outbreakperiod,isolated and identified the virus from these samples. Amplified complete E gene and complete genome with certain primers and sequenced all the products. Then the Phylogenetic ,Bayesian phylogeography and mutations analysis were carried.[Results]40 DENV-1 strains were isolated and identified. 40 complete E gene sequences and 6 complete genome sequences of DENV-1 were obtained. Phylogenetic analysis with E gene sequences revealed that the 40 isolates were classified into two genotypes including 16 genotypeⅠ(Asia)and 24 genotypeⅤ(America/Africa). 14 genotypeⅠisolates were clustered closest with isolates from Guangdong province(2013)and Sigapore(2013)which share the nucletide identities of 99.6% ~ 99.9%,other two genotypeⅠisolates were clustered with strains from Malaysia (2013) and both share the nucletide identities of 99.7%;24 genotypeⅤisolates were all classified in one clade with striains from Bangladesh(2009),China(2009)and Bhutan(2013)which share nucletide identities of 99.0%-99.9%. Further analysis with six complete genome sequences showed that five isolates were clustered closest with strains isolated from Guangdong province(2013)share the nucletide identities of 99.6%-99.8% while the sixth stains closest with strains isolated from Myanmar(2002)share the nucletide identities of 98.8%. The isolates have five amino acid mutations compared with strains epidemic in Guangdong province in 2013,three mutations(S88V,E203G,T275R)are in the EⅡdomain and one mutation (S305P)is in the EⅢdomain which associated with virulence.[Conclusions]During the outbreak in Guangdong province in 2014, DENV-1 is the predominant causative serotype,and there are at least two different kinds of genotypes of DENV-1 largely epidemiced in the whole province. Evolution analysis reveals the multiple origins of the isolates which may origin from Guangdong province , Sigapore,Malaysia,Myanmar so that we should enhance the study and surveillance of autochthonous and vectors in order to understand the epidemic way of dengue in Guangdong province. The isolates have had four mutations in the domain associated with virulence which remain further study to know their biological effects.

OBJECTIVE To observe the neurotoxicity of 1-bromopropane(BP) and investigate the protective effects of edaravone(Edv) against BP-induced deficits of spatial learning and memory ability in rats by its anti-inflammatory mechanism. METHODS Adult male Wistar rats were ig given BP 800 mg·kg-1 to develop the model, followed by Edv 1, 3 and 5 mg·kg-1 ip treatment respectively 4 h later for consecutive 12 d. From the 7th day (d 7), all rats were subjected to the five-day place navigation in Morris water maze (MWM) to measure the escape latency and the total swimming distance. On d 6 of MWM, spatial probe test was performed and the crossing times of rats were recorded to evaluate the spatial memory ability. At the end of the behavioral experiment, four rats in each group were randomly selected and the frozen section of the whole brain was sliced for thionin staining and immunohisto?chemistry. The other eight sacrifced rat brains from each group were harvested for the determination of the tumor necrosis factor-α (TNF-α) and nitric oxide (NO) by ELISA and nitrate reductase method, respectively. RESULTS The results of MWM test showed that compared with control rats the escape latencies of rats in BP group were increased by 60.8%, 81.9%,124.0% and 323.3%, respectively, during the d 2-d 5 of MWM, and the total swimming distance increased by 47.0%, 66.4%, 106.0% and 277.6%, respectirely. All the differences between BP group and control group were significant (P<0.05, P<0.01). In the spatial probe trial, the crossing times of rats in BP group were significantly decreased, compared with the control rats (P<0.01). Morphologically, thionin staining and immunohistochemistry revealed significant microglia activation and neuron loss in the rat forebrains, accompanied by a 147.6% and 18.7% increase in NO and TNF-α levels in rats treated with BP respectively compared with control values (P<0.05, P<0.01). After co-treatment at different dosages of Edv with BP, the escape latencies of rats in BP+Edv 5 mg·kg-1 group were decreased by 38.4%and 44.3%(P<0.01), and the total swimming distance decreased 34.5%and 43.3%(P<0.05, P<0.01), respectively, compared with the BP treated rats on the d 4 and d 5 of MWM test. The microglia activation and neuron damage in the brain of rats induced by BP treatment were significantly alleviated in BP+Edv groups. In addition, the contents of NO and TNF-α were decreased in BP+Edv 1, 3 and 5 mg · kg-1 groups, with a decrease of 53.8%, 55.4% and 59.8% in NO, and 12.2%, 15.8% and 22.2% in TNF-α(P<0.05, P<0.01), respectively. CONCLUSION Edv could effectively protect against central neurotoxicity induced by BP via anti-neuro?inflammation.

AIM:To investigate the effect of quercetin on the biological functions of rat bone marrow-derived endothelial progenitor cells (EPCs) and its potential mechanisms.METHODS:The bone marrow-derived mononuclear cells of Sprague-Dawley rats were isolated by density gradient centrifugation.The differentiated EPCs were cultured specially and stained with DiI-Ac-LDL and FITC-UEA-1.CD133+ and FLK-1+ were detected on the cell surfaces.After 14 d, the EPCs were incubated with a PI3K inhibitor BYL719 (3 μmol/L) and an ERK inhibitor FR180204 (15 μmol/L).After incubation of the inhibitors for 2 h, the cells were treated with quercetin at different concentrations (0, 10, 20, 40, 80 and 100 μmol/L).MTT assay and Transwell assay were used to detect cell viability and the number of migratory cells.The protein levels of AKT, eNOS, ERK and their phosphorylated status were determined by Western blot.RESULTS:Quercetin enhanced the viability and migration of the EPCs at a dose-dependent manner.However, the PI3K inhibitor BYL719 suppressed the QUE-induced cell viability and migration.Moreover, ERK inhibitor FR180204 exerted the similar inhibitory effect on the cell viability but had no effect on cell migration.Quercetin activated the phosphorylation of AKT, eNOS and ERK.On the other hand, BYL719 was observed to inhibit the phosphorylation of AKT and ERK.FR180204, however, was showed to inhibit the phosphorylation of ERK only.On the contrast, the stimulatory effects that quercetin exerted on the expression of eNOS and its phosphorylation were suppressed by BYL719 and FR180204.CONCLUSION:Quercetin stimulates the viability and migration of EPCs via PI3K/AKT/eNOS and ERK/eNOS signaling pathway, which would be beneficial for cardiovascular health.

Mechanical ventilation (MV) is a common way of respiratory support during general anesthesia and in critically ill patients. Recently people gradually realize that MV is a double-edged sword, which itself can also induce or aggravate lung injury as a damage factor, that is, ventilator induced lung injury (VILI). Current researches on the mechanisms of VILI have changed from traditional barotrauma/volutrauma/atelectasis to biotrauma. In particular, it should be noted that MV can not only cause damage to the lung, but also to the distant organs. This article reviews current researches on the molecular mechanisms of VILI, updates our understanding of them, and provides a theoretical basis for the prevention and treatment of VILI and distant organ damage.