[Association analysis of genetic polymorphisms of TCF7L2, CDKAL1, SLC30A8, HHEX genes and microvascular complications of type 2 diabetes mellitus].

OBJECTIVE: To study the associations of single nucleotide polymorphisms (SNPs) of TCF7L2, CDKAL1, SLC30A8, HHEX with diabetic retinopathy (DR) and nephropathy (DN) in type 2 diabetes mellitus. METHODS: A total of 479 subjects with DR,248 with DN and 650 without DR or DN were recruited to assess the associations between SNPs of TCF7L2 (rs7903146, rs6585205, rs11196218), CDKAL1 (rs10946398,rs4712527), SLC30A8 (rs13266634, rs3802177, rs11558471) and HHEX (rs1111875, rs7923837) and the development of DR and DN. RESULTS: There were significant differences in genotypic and allele frequencies of rs11558471 (SLC30A8) between DR and control groups (P< 0.05), the odds ratio (OR) values of A and AA were 1.27 and 1.68. The distributions of genotype and allele frequency for rs11196218 (TCF7L2) were significantly different between DN and control group (P=0.0051,OR=1.37). However, the P value after Bonferroni correction showed no significant difference. No significant differences were found in the distributions of rs13266634 and rs3802177 (SLC30A8), rs10946398 (CDKAL1), rs6585205, rs7903146 and rs11196218 (TCF7L2) and rs7923837 (HHEX) between DR and control groups, and nor significant differences were found in distributions of rs6585205 (TCF7L2), rs4712527 (CDKAL1), rs13266634, rs3802177 and rs11558471 (SLC30A8), and 7923837 (HHEX) between DN and control groups, though for all comparison the OR values were greater than 1. CONCLUSION: Polymorphisms of SLC30A8 and TCF7L2 genes may be associated with the development of DR and DN, respectively. Association between the polymorphisms of CKDAL1, TCF7L2 and HHEX genes and DR, and between the polymorphisms of SLC30A8, HHEX and CDKAL1 genes and DN, cannot be excluded.

Contribution of ClpE to virulence of Streptococcus pneumoniae.

The ATP-dependent caseinolytic proteases (Clp) play a fundamental role in stress tolerance and virulence in many pathogenic bacteria. Although ClpE of Streptococcus pneumoniae is required for growth at high temperatures, little is known about the role of ClpE in pathogenesis. In this study, we observed that the virulence of the clpE mutant of S. pneumoniae strain D39 was strongly reduced in a mouse intraperitoneal infection model. The clpE mutant also showed substantially reduced adherence to the human lung epithelial carcinoma A549 cell line and human umbilical-vein-derived endothelial cells. The underlying mechanism of virulence attenuation induced by the mutation of clpE was further investigated with real-time RT-PCR and 2-dimensional protein gel analysis. The results indicate that ClpE affects pneumococcal pathogenesis by modulating the expression of some important virulence determinants and metabolism-related factors in S. pneumoniae.

Microarray analysis of microRNA expression in hepatocellular carcinoma and non-tumorous tissues without viral hepatitis.

BACKGROUND AND AIM: MicroRNAs (miRNAs) are non-coding RNA molecules of 21-24 nt that regulate the expression of target genes in a post-transcriptional manner. Evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation, and pathogenesis of human diseases including cancer. METHODS: We analyzed the miRNA expression profiles in 10 pairs of hepatocellular carcinoma (HCC) and adjacent non-tumorous tissue (NT) from 10 non-viral hepatitis patients, using a mammalian miRNA microarray containing whole human mature and precursor miRNA sequences. RESULTS: A total of 15 miRNAs exhibited higher expression in the HCC samples than that in the NT samples, and one miRNA demonstrated lower expression in the HCC samples than in the NT samples. A total of 18 miRNAs identified valid expression only in HCC samples, with six only in NT samples. The chip results were confirmed by Northern blot analysis. CONCLUSION: Our study may help clarify the molecular mechanisms involved in the pathogenesis of HCC, and miRNAs potentially serve as a novel diagnostic tool of HCC.

[Research on the procedure for recovery and species identification of Legionella from surface environmental water].

OBJECTIVE: To establish a set of procedure for recovery and species identification of Legionella from the surface environmental water. METHODS: Forty-four water samples were collected in eight parks of Guangzhou city from August to November in 2006. The bacteriologic examination was performed by cultivation on BCYEalpha plate, and 108 presumptive Legionella colonies were picked and their homogeneous relationship was analyzed by using an amplified fragment length polymorphism (AFLP) method. Species identification was carried out by latex agglutination test, biochemical characterization, analysis of cellular fatty acids composition, 16 S rRNA gene and mip gene sequencing. RESULTS: Legionella was recovered among 27 (61.36%) samples of all eight parks, and 31 different strains were identified from those 108 presumptive Legionella isolates by AFLP method, including 20 strains of L. pneumophila, five strains of L. feeleii, four strains of L. longbeachae, one strain of L. oakridgensis and one strain of L. sainthelensi, and L. pneumophila could be easily differentiated by phenotypic and biochemical characteristics, latex agglutination test or analysis of the cellular fatty acids composition . However, uncertain factors were existing in those phenotypic identification methods as compared to the sequence analysis. CONCLUSION: The taxonomic analysis of the Legionellae family should be dependent on the 16 S rRNA gene or mip gene.

Clinical studies of immunomodulatory activities of Yunzhi-Danshen in patients with nasopharyngeal carcinoma.

OBJECTIVES: Nasopharyngeal carcinoma (NPC) is a prevalent tumor in Hong Kong. The immune system of such patients could be adversely affected during the course of conventional chemotherapy or radiotherapy. We investigated the immunomodulatory effects of Traditional Chinese Medicine (TCM) Yunzhi-Danshen capsules in NPC patients treated with radiotherapy. DESIGN: Randomized, double-blind, placebo-controlled 16-week study. SETTING/LOCATION: The Prince of Wales Hospital, the Chinese University of Hong Kong, Hong Kong. SUBJECTS: Twenty-seven (27) patients with histologically proven NPC, at least 18 years of age. METHODS: Twenty-seven patients with histologically proven NPC were recruited to take Yunzhi (3.6 g daily) and Dangshem (1.4 g daily) in the form of 12 combination capsules (TCM group) or placebo (12 capsules) daily for 16 weeks, respectively. Flow cytometry was used to assess the percentages and absolute counts of human lymphocyte subsets in whole blood. Plasma concentration of soluble interleukin-2 receptor and soluble tumor necrosis factor receptor 2 was measured by enzyme-linked immunosorbent assay (ELISA). Ex vivo production of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-10 in the whole blood assay culture supernatant was measured by ELISA. RESULTS: The decreases in percentage and absolute count of T lymphocytes in the TCM group were less than those in the placebo group after they took the capsules for 16 weeks (both p < 0.05). Furthermore, the decreases in absolute count of T suppressor cells plus cytotoxic T lymphocytes, and T helper cells in the TCM group were significantly lower than those in the placebo group after they took the capsules for 16 weeks (both p < 0.05). CONCLUSION: These results suggest that Yunzhi-Danshen can exert an immunomodulating effect in alleviating lymphopenia during radiotherapy in NPC patients.

[Screen and identification of Streptocococcus pneumoniae genes specifically induced in host].

To identify in vivo-induced genes of S. pneumoniae and search new potential drug targets and vaccine candidates, a selection system was developed based on the in vivo expression technology (IVET). Promoter galU gene which is critical for the capsular polysaccharide biosynthesis and lacZY gene which encodes bea-galactosidase were employed as dual reporter genes. The galU-deficient mutant of S. pneumoniae is incapable of utilizing galactose and synthesizing capsular polysaccharide, therefore can't survival in the host. Firstly, the random pieces of S. pneumoniae chromosomal DNA (200-500 bp), obtained by partial Sau3Al restriction digestion, were subcloned into the Bgl II site of pEVP3-galU. Transformation by this plasmid library yielded promoter-trap library in S. pneumoniae. Then, the library was used to infect animals. Bacteria were harvested from lung tissue. White clones on TSA agar containing X-gal were used to reinfect animals. The sequence of infection and sorting was performed twice, 165 white clones harvested from the final round of infection were analyzed. A total of 15 unique sequences were obtained through in vivo screen. The bioinformatics analysis showed that these ivi genes involved in colonization and adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism and cell wall synthesis. And there were some hypothetical proteins have unknown functions. Part of these genes may be related with virulence and can be used as vaccine candidates and drug targets.

The effect of transformation on the virulence of Streptococcus pneumoniae.

Although pneumococcus is one of the most frequently encountered opportunistic pathogen in the world, the mechanisms responsible for its infectiveness have not yet been fully understood. In this paper, we have attempted to characterize the effects of pneumococcal transformation on the pathogenesis of the organism. We constructed three transformation-deficient pneumococcal strains, which were designated as Nos. 1d, 2d, and 22d. The construction of these altered strains was achieved via the insertion of the inactivated gene, comE, to strains 1, 2 and 22. We then conducted a comparison between the virulence of the transformation-deficient strains and that of the wild-type strains, via an evaluation of the ability of each strain to adhere to endothelial cells, and also assessed psaA mRNA expression, and the survival of hosts after bacterial challenge. Compared to what was observed with the wild-type strains, our results indicated that the ability of all of the transformation-deficient strains to adhere to the ECV304 cells had been significantly reduced (p < 0.05), the expression of psaA mRNA was reduced significantly (p < 0.05) in strains 2d and 22d, and the median survival time of mice infected with strains 1d and 2d was increased significantly after intraperitoneal bacterial challenge (p < 0.05). The results of our study also clearly indicated that transformation exerts significant effects on the virulence characteristics of S. pneumoniae, although the degree to which this effect is noted appears to depend primarily on the genetic background of the bacteria.

Calcium signaling events in Streptococcus pneumoniae invasion of human type II pneumocytes.

OBJECTIVE: To study whether Streptococcus pneumoniae (S.pn) can provoke filamentous actin (F-actin) rearrangements in vitro through calcium signaling pathways in type II pneumocytes(A549 cells), resulting in S.pn invasion of the cells. METHOD: After FITC-phalloidin labeling of F-actin, F-actin rearrangements were observed by S.pn adhesion to type pneumocyte A549 cells. S.pn invasion of A549 cells was determined by pretreating A549 cells with cytochalasin D. To investigate whether F-actin rearrangements could be blocked by Ca2+ inhibitors, A549 cells were pretreated with Ca2+ inhibitors dantrolene, and loaded in Fura-2/AM probe to determine the concentration of cytosolic free calcium by S.pn adhesion to A549 cells after 30, 60, and 90 min respectively. RESULTS: Intact S.pn can promote F-actin rearrangements. Cytochalasin D was able to prevent S.pn invasion of A549 cells. No invasion of A549 cell can be determined at 0.25 microg/ml of cytochalasin D. One subset of the inhibitors of Ca2+ signal transduction molecules blocked F-actin rearrangements dose-dependently, and S.pn adhesion of A549 cells for 30, 60, and 90 min increased cytosolic free calcium, reaching 487.5+/-38.1 , 548.2+/-35.6 and 557.2+/-47.5 nmol/L, respectively. They were higher than of the control group. CONCLUSION: S.pn can provoke F-actin rearrangements through Ca2+ signaling pathways, which further leads to S.pn invasion of A549 cells.

[Clinical analysis of 22 cases community-acquired Pseudomonas aeruginosa urinary tract infection].

OBJECTIVE: To study the characteristics of community-acquired urinary tract infections (CAUTIs) in children, analyze the risk factors and the susceptibility of antibiotics, thus to provide references to the diagnosis and medication of Pseudomonas aeruginosa (PA)-CAUTIs. Mothod Totally 22 cases of PA-CAUTIs were selected in one hospital from Jan, 2006 to Jan, 2012, their clinical information, laboratory results and radiological images were collected, and were compared with the CAUTIs cased by E. coli of those randomly selected over the same period. RESULT: In those 22 cases with PA-CAUTIs, the mean value of protein level was (32.25 ± 13.81) mg/ml, 19 of them were hospitalized, 6 had urinary operation history, 7 of them had long-term usage of glucocorticoids or immunosuppressive agents, and 20 had underlying diseases. A total of 22 children with 26 PA-CAUTIs episodes were compared to E. coli-CAUTIs. Compared with E. coli-CAUTIs patients, children with PA-CAUTIs more often presented with a lower albumin (P = 0.017), a history of urinary operation(P = 0.03), more cases had a history of urinary operation (P = 0.03), a long-term usage of glucocorticoids or immunosuppressive medication (P = 0.044). Through multivariate logistic regression of variables that were significant in univariate analysis (with hospitalizations, long-term usage of glucocorticoids or immunosuppressive, albumin, underlying disease and urinary operation histories), and it turned out that underlying diseases (odds ratio 8.500, 95% CI 1.513 - 47.761, P = 0.037) and with urinary operation histories (odds ratio 6.196, 95% CI 1.120 - 34.273, P = 0.037) were proved as the independent risk factors for PA-CAUTIs. Those PA bacterial strains had a 36.36% resistance rate to piperacillin, aztreonam and gentamicin, a 31.82% resistance rate to cefepime and ceftazidime, while the resistance rate (4.55%) to carbapenem antibiotics was relatively low, only to bacillosporin all the strains were sensitive. CONCLUSION: Underlying diseases and the urinary operation histories are the independent risk factors of the occurrence of PA-CAUTIs, carbapenem antibiotics and bacillosporin can be considered as the drugs of choice for its treatment.

In vivo characterization of Streptococcus pneumoniae genes involved in the pathogenesis of meningitis by differential fluorescence induction.

OBJECTIVE: To further understand the pathogenesis of pneumococcal meningitis, and provide some target candidates for the development of drugs. METHODS: This study was performed at the Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine (Ministry of Education), Chongqing Medical University, Chongqing, China from March 2006 to December 2007. A promoter-trap library of Streptococcus pneumoniae TIGR4, reported by green fluorescent protein was constructed, and used to infect BALB/c mice (n=15) intranasally, to set up a meningitis model. The control group (n=5) were inoculated with sterile phosphate buffered saline. The bacteria containing the promoter fusions induced only in meningitis brain tissue, not in vitro were screened by differential fluorescence induction. The obtained bacteria were prepared to re-infect the mice and re-screened, as above. The sorted bacteria were spread on trypticase soy agar with 5% sheep blood agar plates containing chloramphenicol (2.5 g/mL), and were used for DNA cloning, sequencing, and bioinformatics analysis. RESULTS: A total of 52 genes were obtained. Bioinformatics analysis revealed that these in vivo induced genes were involved in functions such as, adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism, as well as, cell wall synthesis. In addition, there were some genes encoding for some hypothetical proteins with unknown, or putative functions. CONCLUSION: Pneumococcal genes involved in meningitis identified in this study are potential targets to understand the pathogenesis of pneumococcal meningitis.

[Effect of ClpE depletion on the bacterial protein expression profiles of Streptococcus pneumoniae].

OBJECTIVE: To investigate the effect of ClpE on the protein expression profiles of Streptococcus pneumoniae. METHODS: clpE-deficient Streptococcus pneumoniae strain was constructed by long flanking homology-polymerase chain reaction (LFH-PCR) and identified by PCR and sequencing. The total bacterial proteins were analyzed by two-dimensional gel electrophoresis and imaging analysis, and the differentially expressed protein spots were excised by dot-gel digestion with trypsin. Peptide mass fingerprinting (PMF) was obtained by analysis of the fragment length by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The PMF was analyzed using software to identify the proteins. RESULTS: The number of matched protein spots of the two gels was 61%. By sequence database searching, 4 out of the 17 differential protein spots were identified, namely hypoxanthine-guanine, pyrrolidone-carboxylate peptidase1, formate-tetrahydrofolate ligase, and bifunctional protein pyrR. CONCLUSION: clpE gene-deficient Streptococcus pneumoniae expresses fewer kinds of proteins at also lower levels than the wild-type bacteria, suggesting that ClpE allows the bacteria to adapt to different host environments by inducing the expression of special proteins.

Identification of Streptococcus pneumoniae genes specifically induced in mouse lung tissues.

To identify Streptococcus pneumoniae genes expressed specifically during infections, a selection system based on the in vivo expression technology (IVET) was established. galU, which is critical for capsular polysaccharide biosynthesis, and lacZY encoding beta-galactosidase were employed as dual reporter genes to screen in-vivo-induced (ivi) genes of S. pneumoniae. The galU-deficient mutant of S. pneumoniae is incapable of utilizing galactose, thus failing to synthesize capsular polysaccharide, and therefore loses its ability to survive in the host. A promoter-trap library was constructed in S. pneumoniae, which was used to infect BALB/c mice in an intranostril model. Those strains recovered from lung tissue of mice and exhibiting a white colony phenotype on tryptic soy agar containing X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) were collected and identificated. A total of 15 unique sequences were obtained through in vivo screening. The ivi genes of S. pneumoniae are involved in many processes, such as colonization and adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism, and cell wall synthesis. There are some hypothetical proteins whose functions are not clear. This novel IVET is a useful tool for identifying ivi genes in S. pneumoniae.

Immunomodulatory effects of lingzhi and san-miao-san supplementation on patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune joint disease. We evaluated a standard preparation of Lingzhi (Ganoderma lucidum) and San-Miao-San (Rhizoma atractylodis, Cortex phellodendri, Radix achyranthes bidentatae) capsules (TCM group) for its supplementary treatment efficacy for RA. There was no significant difference in the absolute count, percentage, and ratios of CD4(+)/CD8(+)/natural killer/B lymphocytes between the TCM and placebo groups after taking the capsules (all p > 0.05). There was no significant change in concentrations of plasma cytokines of interferon-gamma-induced protein-10 (IP-10), monocyte chemoattractant protein-1, monokine induced by IFN-gamma, regulated upon activation normal T-cell expressed and secreted, interleukin (IL)-8, and IL-18 after taking the capsules for 8 and 24 weeks (all p > 0.05). The percentage change in ex vivo-induced level of inflammatory cytokine IL-18 was significantly lower in the TCM group than in the placebo group after taking the capsules for 24 weeks (p < 0.05). Therefore, Lingzhi and San-Miao-San capsules might exert a beneficial immunomodulatory effect in patients with rheumatoid arthritis.