Lactobacillus casei was selected as an antigen delivery vehicle for the development of oral vaccine to express recombinant LTB and porcine parvovirus (PPV) VP2 protein. The fusion protein gene encoding PPV VP2 protein and LTB, was cloned into the surface expression vector pPG, and then the recombinant expression vector pPG-VP2-LTB was electrotransformed into Lactobacillus casei 393, generating recombinant strain pPG-VP2-LTB/L. casei 393. After induced by 2% Lactose in MRS broth, an about 78 kD protein was detected in the recombinant Lactobacillus casei by SDS-PAGE. The result of Western blot indicated that the protein possessed the antigenic specificity same as the native virus protein. The result of the whole bacteria cell ELISA indicated that the LTB protein was expressed at the same time. The results of indirect immunofluorescence test and immuno-gold electron microscopy showed that the interest protein was expressed on the surface of L. casei 393. The results provide potential for the development of lactic acid bacteria oral vaccine of PPV, which used LTB as mucosal adjuvant.

Objective To discuss the risk factors of invasive fungal infection in preterm infants and conduct corresponding nursing intervention.Methods Retrospective analysis was used to investigate 1 146 preterm infants in Neonatal Intensive Care Unit (NICU) of the risk factors of invasive fungal infection.Results There were 66 preterm infants who had invasive fungal infection,with the rate of 5.76％,and it happened in (15.9 ± 4.8) days during hospitalization.Preterm infants with different gestational age,birth weight,time for intravenous nutrition,hospitalization period and mother having complication,asphyxiation,mechanical ventilation,parenteral nutrition,broad spectrum antibiotics or not had different rates of invasive fungal infection (P ＜ 0.05).Multivariate Logistic regression analysis showed that low birth weight,mechanical ventilation,broad spectrum antibiotics and intravenous nutrition for more than one week were the individual risk factors of invasive fungal infection in preterm infants (P ＜ 0.05).Conclusions Risk factors of invasive fungal infection in preterm infants include low birth weight,mechanical ventilation,broad spectrum antibiotics and intravenous nutrition for more than one week.Corresponding intervention methods should be taken to reduce the rate of invasive fungal infections.

Aim To investigate the molecular mecha-nism of mTORC1/2 inhibitor PP242, which inhibiting cholangiocyte cell preliferation and cystic diliatation via inducing apoptosis and autophagy in the polycystic kid-ney ( PCK ) rats. Methods The expression of p-mTOR and p-Akt in the bile duct epithelial cells was examined by immunohistochemistry. The inhibiting effect of rapamycin and PP242 on cell proliferation ac-tivity on bile duct epithelial cells, the effect of gene si-lence on LC3, Beclin-1 and the effect of the authoph-agy-specific inhibitor 3-methyladenine (3-MA) on cell proliferation were respectively analyzed by WST-1 as-say. The expression of PI3K/Akt signaling pathway re-lated proteins, autophagy-related proteins LC3, Bec-lin-1 and clevead caspase-3, which were treated by PP242 were determined by Western blot. The effect of PP242 on apoptosis was detected by Annexin V/PI double staining and ELISA. The expression of LC3 in cytoplasm was detected by immunofluorescence. The a-bility of rat bile duct epithelial cells spheroid formation was detected by 3D cell culture method, and the cells were treated by single applied with rapamycin and ap- plied rapamycin combined with Rictor gene silencing respectively. Results The protein levels of p-Akt and p-mTOR markedly increased in the bile duct epitheli-um of PCK rats. PP242 inhibited the proliferation of bile duct epithelial cells more effectively than rapamy-cin and showed a dose-and time-dependent manner ( P<0.05 ) . PP242 significantly reduced the levels of PI3K/Akt signaling pathway-related proteins in PCK rat cholangiocytes. PP242 induced apoptosis and auto-phagy, up-regulated the levels of cleaved caspase-3, Beclin-1 and increased the ratio of LC3-II/LC3-I. The combination of Rictor gene silencing and rapamycin was more effective than rapamycin alone in inhibiting cholangiocytes in PCK rats. The inhibitory effect of PP242 on the cell viability was significantly weakened by treatment with 3-MA and knockdown of LC3 and Beclin-1 ( P <0.05 ) . Conclusions PP242 inhibits the proliferation of PCK rat cholangiocytes through PI3K/Akt/mTOR signaling pathway, and the mecha-nism is closely related with autophagy and apoptosis.

Objective To detect the ratio of CD19+-CD25+ and CD19+-CD25-B lymphocytes and content of IgA,IgG,IgM and complement C3 in patients with acute cerebral infarction and study their clinical significance. Methods Disease were diagnosed according to the history and cranlal computer tomography or magnetic resonance imagine.Venous blood of 69 cases with acute cerebral infarction and 115 cases with cerebral hemorrhage, 41 cases in normal control group was extrdcted. The ratio of CDl9+-CD25+and CD19+-CD25-B lymphocytes was determined by flow cytometry and content of IgA,IgG,IgM and C3 was measured with scattering turbidimetry.Changes in humoral immunological function were compared among patients with different courses of disease, imaging scores and neurological function scores. Results Differences in CD19+-CD25+and CD19+-CD25-B lymphocytes, IgA, IgG,IgM and C3 were not significant at the acute stage between cerebral infarction and cerebral hemorrhage (P＞0.05,for all).The ratio of CD19+-CD25+B lymphocytes and content of IgG and C3 at the acute stage of cerebral infarction were all higher than that at the recovery stage and in the control group (P＜0.05, for all). There was no statistical signmcance in humoral immunological indices between that at the recovery stage of cerebral infarction and in the control group (P＞0.05, for all). The ratio of CD19+-CD25+ and CD19+-CD25-B lymphocytes was significantly different among patients with different imaging scores (P＜0.05,for all).Neurological function scores at the acute stage of cerebrdl inflarction were not correlated with humoral immunological indices(P＞0.05,for all). Conclusions Same changes occur to humoral immunological function in patients with cerebral infarction and cerebral hemomlage, which might be related with stress,and location and scope of lesions.The larger the lesion of cerebral infarction is,the more obvious changes of humoral immunological function become; with the disappearing of stress,humoral immunological function gradually recovers.

Adult ; Female ; Hepatitis B, Chronic ; complications ; physiopathology ; Humans ; Liver ; physiopathology ; Liver Function Tests ; Male ; Retrospective Studies ; Severe Acute Respiratory Syndrome ; complications ; physiopathology

AIM:To explore the effects of mammalian target of rapamycin (mTOR) double inhibitor AZD8055 on autophagy and apoptosis of human cholangiocarcinoma cell line HuCCT1. METHODS:The effect of AZD8055 on the viability of HuCCT1 cells was detected by MTT assay. Autophagosome was detected by acridine orange (AO) staining. Af-ter treated with AZD8055, the expression levels of apoptosis-related proteins Bcl-2, Bax and cleaved caspase-3 and auto-phagy marker proteins beclin 1, LC3 and p62 were determined by Western blot. Apoptotic rate was analyzed by flow cyto-metry with Annexin V-FITC/PI double staining. RESULTS:AZD8055 significantly inhibited the viability of HuCCT1 cells (P<0.05). AO staining showed that AZD8055 significantly increased orange granules in the cytoplasm. After treated with AZD8055, compared with the control group, the protein level of beclin 1 and the ratio of LC3-Ⅱ/LC3-Ⅰ were enhanced, while p62 was attenuated (P<0.05). The protein expression level of pro-apoptotic regulator Bax was down-regulated and anti-apoptotic regulator Bcl-2 was increased. The protein level of cleaved caspase-3 was reduced (P<0.05). The results of flow cytometry showed that AZD8055 inhibited cell apoptosis. CONCLUSION:AZD8055 inhibits the viability of cholangiocarcinoma cells, and the mechanism is closely related with autophagy induced by AZD8055.

Objectives To establish the normal value of Sniffin' Sticks test in Chinese population and to explore it's clinical application in China.Methods One hundred and five healthy volunteers were choosen from the department of physical examination of Beijing Tongren Hospital between 2007 and 2013.Another 165 patients complained of abnormal olfactory function were obtained from the outpatient clinic of the department of otorhinolaryngology head and neck surgery in the same period and were divided into two groups:92 in hyposmia and 73 in functional anosmia group.The 270 subjects were divided into 3 subgroups:younger group(＜35 years of age),middle-age group(35-55 years of age) and older group(＞55 years of age).The olfactory functions were examined with Sniffin' Sticks test and T&T test,respectively.All analyses were performed using SPSS 12.0 software.Results For the normal value of Sniffin' Sticks test,TDI score was ＞30.12 for younger group,＞27.37 for middle-age group and ＞20.43 for older group; the mean TDI score was 32.12 ± 3.95 for healthy group,17.52 ± 10.37 for hyposmia and 3.56 ± 3.49 for functional anosmia group; the differences in TDI score,olfactory threshold,discrimination threshold and identification threshold between healthy group and olfactory dysfunction group with different ages had statistical significance (Younger group:FTDI =125.136,P =0.000 ; FT =49.454,P =0.000 ; FD =89.037,P =0.000 ; FI =39.888,P =0.000; Middle-age group:FTDI =190.240,P =0.000;FT =128.374,P =0.000;FD =174.122,P =0.000 ; FI =178.945,P =0.000 ; Older group:FTDI =72.992,P =0.000 ; FT =26.599,P =0.000 ; FD =77.119,P =0.000 ; FI =88.107,P =0.000,respectively).The mean T&T value was-1.00 ± 0.98 for healthy group,2.27 ±2.01 for hyposmia and 5.89 ±0.14 for functional anosmia group.T&T score between healthy group and olfactory dysfunction group with different ages had statistical significance (Fyounger =158.144,P =0.000; Fmiddle-age =247.695,P =0.000; Folder =70.579,P =0.000,respectively).TDI score of the Sniffin' Sticks test result was correlated with T&T value (r =-0.927,P ＜ 0.01) ; T&T threshold was correlated with the olfactory threshold,discrimination threshold and identification threshold of Sniffin' Sticks test (rT =-0.846,P ＜0.01,rD =-0.908 P ＜0.01,rI =-0.864,P ＜0.01,respectively).Conclusions Sniffin' Sticks test and T&T olfactometry are able to differentiate normosmia from hyposmia and anosmia with high reliability and consistency in test results.Sniffin' Sticks test can assess subject's olfactory function status more thoroughly and is suitable for application in Chinese population.

Influenza A/H1N1 virus-encoded nonstructural, or NS1, protein inhibits the 3'-end processing of cellular pre-mRNAs by binding the cellular protein: the 30-kDa subunit of CPSF (cleavage and polyadenylation specificity factor, CPSF30). CPSF30 binding site of the NS1 protein is a potential target for the development of drugs against influenza A/H1N1 virus. A yeast two-hybrid screening system was constructed and used for screening Chinese medicines that inhibit the interaction of the A/H1N1 flu NS1 protein and human CPSF30 protein. The NS1 gene of A/H1N1 virus was amplified by consecutive polymerase chain reaction (PCR), and the human CPSF30 gene of HeLa cell cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). Then the two gene fragments confirmed by sequencing were subcloned into the yeast expression vectors pGBKT7 and pGADT7, respectively. The two constructs, bait vector pGBKNS1 and prey vector pGADCPSF, were co-transformed into yeast AH109. The eight individual yeast colonies were picked and subjected to verification by PCR/gel electrophoresis. The inhibition of the NS1-CPSF30 interaction was allowed the identification of selective inhibitors. The four of more than thirty identified Chinese medicines, including 'Shuanghuanglian oral liquid', showed the strong inhibition of the NS1-CPSF30 interaction.
Base Sequence ; Binding Sites ; Cleavage And Polyadenylation Specificity Factor ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Gene Amplification ; HeLa Cells ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; Peptide Fragments ; genetics ; Plasmids ; Protein Binding ; drug effects ; Transformation, Genetic ; Two-Hybrid System Techniques ; Viral Nonstructural Proteins ; genetics ; metabolism

Animals ; Cell Proliferation ; Male ; Mice ; Mice, Inbred C57BL ; Spermatogonia ; Stem Cells ; Testis

OBJECTIVEDuring 2003-2005, an outbreak of meningitis due to Neisseria meningitidis serogroup C occurred in China. With the aim to find strain clues result in the final epidemics, the ancestral strain 053442, a clinical isolate, and a carrier strain 053426 with different gene type were analyzed.

METHODSClinical strain 053442 and carrier strain 053426 were cultured on GC agar plates under the same condition. Two-dimensional electrophoresis was performed using the pH 3-10 nonlinear IPG strips of 24 cm length, and all the protein spots were identified by matrix-assisted laser desorption/ionization time of flight spectrometry.

RESULTS502 and 380 protein spots were identified in 053426 and 053442 respectively, relating to 266 and 202 different genes covering a wide range of cellular functions. The express volume and number of proteins involved in energy metabolism, protein synthesis and amino acid biosynthesis in 053426 were higher than in 053442. Virulence factor Opa, Opc and a series of proteins involved in pilus assembly and retraction were identified in 053442, which appear to be of primary importance in colonization and invasion of human cells. Compared to 053442, virulence protein species were less in 053426, with lower express volumes too. No Opa and Opc were detected in 053426.

CONCLUSIONSThe different protein expression profiles of the clinical strain 053442 and carrier strain 053426 in the present study provide some clues of the different pathogenicity of the two strains, which may account for result in the final epidemics.

Bacterial Proteins ; analysis ; Bacterial Typing Techniques ; China ; epidemiology ; Disease Outbreaks ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Meningitis, Meningococcal ; cerebrospinal fluid ; epidemiology ; microbiology ; Neisseria meningitidis, Serogroup C ; classification ; isolation & purification ; Proteome ; analysis ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization