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frequency determining quinolone resistance in Chlamydia trachomatis serovars L2 and D. J Antimicrob Chemother 2008, 61:91–94.CrossRefPubMed 25. Shkarupeta MM, Lazarev VN, Akopian TA, Afrikanova TS, Govorun VM: Analysis of antibiotic resistance markers in Chlamydia trachomatis clinical isolates obtained after ineffective antibiotic therapy. Bull Exp Biol Med 2007, 143:713–717.CrossRefPubMed 26. Gieffers J, Rupp J, Gebert A, Solbach W, Klinger M: First-choice antibiotics at subinhibitory concentrations induce persistence of Chlamydia pneumoniae. Antimicrob Agents Chemother 2004, 48:1402–1405.CrossRefPubMed 27. Reveneau N, Crane DD, Fischer E, Caldwell HD: Bactericidal activity of first-choice antibiotics against gamma interferon-induced persistent infection of human epithelial cells by Chlamydia trachomatis. Antimicrob Agents Chemother 2005, 49:1787–1793.CrossRefPubMed 28. Wyrick PB, Knight ST: Pre-exposure of infected

human endometrial epithelial cells to penicillin in vitro renders Chlamydia trachomatis refractory to azithromycin. J Antimicrob Chemother 2004, 54:79–85.CrossRefPubMed 29. Migliorini Sorafenib research buy L, Canocchi V, Zanelli G, Valassina M, Cellesi C: Outbreak and persistence of Chlamydia pneumoniae infection in an Italian family. Infez Med 2003, 11:157–160.PubMed 30. Mpiga P, Ravaoarinoro M:Chlamydia trachomatis persistence: an update. Microbiol Res 2006, 161:9–19.CrossRefPubMed 31. Davis CH, Raulston JE, Wyrick PB: Protein disulfide Tryptophan synthase isomerase, a component of the estrogen receptor complex, is associated with Chlamydia trachomatis serovar E attached to human endometrial epithelial cells. Infect Immun

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PubMedCrossRef 28. Wong KT, Puthucheary SD, Vadivelu J: The histopathology of human melioidosis. Histopathology 1995,26(1):51–55.PubMedCrossRef 29. Wilson K: Preparation of genomic DNA from bacteria. In Current Protocols in Molecular Biology. Edited by: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. John Wiley & Sons, New York; 1987:2.4.1–2.4.5. 30. DeShazer D, Brett PJ, Carlyon Fulvestrant supplier R, Woods DE: Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: Isolation of motility

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pseudomallei type III secretion system mutants exhibit delayed vacuolar escape phenotypes in RAW 264.7 murine macrophages. Infect Immun 2008,76(7):2991–3000.PubMedCrossRef 33. Brett PJ, DeShazer D, Woods DE: Characterization of Burkholderia pseudomallei and Burkholderia pseudomallei-like strains. Epidemiol Infect 1997,118(2):137–148.PubMedCrossRef 34. Simon R, Priefer U, Puhler A: A broad host range mobilization system for in vivo genetic engineering: tranposon mutagenesis in gram negative bacteria. Bio/Technology 1983, 1:784–791.CrossRef 35. Ferenci T, Zhou Z, Betteridge T, Ren Y, Liu Y, Feng L, Reeves PR, Wang L: Genomic sequencing reveals regulatory Selleck Compound Library mutations and recombinational events in the widely used MC4100 lineage of Escherichia coli K-12. J Bacteriol 2009,191(12):4025–4029.PubMedCrossRef 36. Witkin EM: Inherited differences in sensitivity to radiation in Escherichia coli. Proc Natl Acad Sci U S A 1946,32(3):59–68.PubMedCrossRef 37. Holden MT, Titball RW, Peacock SJ, Cerdeno-Tarraga AM, Atkins T, Crossman LC, Pitt T, Churcher C, Mungall K, Bentley SD, et al.: Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl Acad Sci 2004,

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The reaction between PbMLSr and the antibody anti-PbMLSr was used as a positive control (Fig. KPT-330 manufacturer 4A, lane 7). The binding between PbMLS and ECM compounds was also evaluated by ELISA assay. The results reinforced that PbMLSr binds to fibronectin, type I and IV collagen (Fig. 4B). Negative controls were performed using PbMLSr (Fig. 4B) or ECM only (data not shown). The positive control was performed using anti-PbMLSr, anti-laminin, anti-fibronectin, anti-colagen I or anti-colagen IV antibody (data not shown). Figure 4 (A) Binding of Pb MLSr to ECM by Far-Western blot. PbMLSr (0.5 μg) was subjected to SDS-PAGE and electroblotted. Membranes were reacted with fibronectin (lane 1), type I collagen

(lane 2), type IV collagen (lane 3) and laminin (lane 4), and subsequently incubated with rabbit IgG anti-fibronectin, mouse anti-type I and anti-type IV collagen antibodies,

and anti-laminin, respectively. Peroxidase-conjugated anti-rabbit and anti-mouse IgG revealed the reactions. Negative control was obtained by incubating PbMLSr with peroxidase-conjugated anti-rabbit IgG (lane 5), and PbMLSr with ECM (lane 6). Positive control was obtained by incubating PbMLSr with polyclonal anti-PbMLSr antibody (lane 7). (B) Binding of PbMLSr to ECM fibronectin, types I and IV collagen (10 μg/mL). The interaction was revealed by ELISA with peroxidase-conjugated streptavidin. The results were expressed in absorbance units. The negative controls were performed using PbMLSr only. (C) Reactivity of PbMLSr to PCM patient sera. 1.0 μg of purified PbMLSr was electrophoresed and reacted

to the sera of patients with PCM, diluted 1:100 (lanes 1 to 3) and to IWR1 control sera, diluted 1:100 (lane 4). The positive control was obtained by incubating PbMLSr with its polyclonal antibody (lane 5). After reaction to the see more anti-human IgG alkaline phosphatase-coupled antibody (diluted 1:2000), the reaction was developed with BCIP-NBT. (D) Biotinylation assay by Western blot. Lysed A549 cells incubated with biotinylated PbMLSr (lane 1); Lysed A549 cells (lane 2) as negative control. PbMLSr was reacted with three sera of patients with PCM and one serum from a healthy individual in immunoblot assays (Fig. 4C). Strong reactivity was observed with the PCM-patient sera (Fig. 4C, lanes 1 to 3). No cross-reactivity was observed with control serum (Figure 4C, lane 4). Reaction between PbMLSr and anti-PbMLSr was used as positive control (Fig. 4C, lane 5). Binding of PbMLSr to pneumocytes The ability of PbMLSr to bind to A549 cells was evaluated. PbMLSr was biotinylated and incubated with A549 cells. After lyses, proteins from A549 cells were electrophoresed by SDS-PAGE and blotted onto a membrane to perform Western blot with anti-PbMLSr antibody. A positive signal was detected from lysed pulmonary A549 cells treated with biotinylated PbMLSr (Fig. 4D, lane 1). The negative control was obtained using supernatant of A549 cells untreated with biotinylated protein (Fig. 4D, lane 2).

The A20.IIA-GFP cell culture was also supplemented with 0.5 mg/mL neomycin (G418; Gibco-Invitrogen). To obtain the A20.IIA-luc2 cell line, A20.IIA cells were transfected with pGL4.50[luc2/CMV/hygro] (Promega), in the AMAXA Nucleofector II device (Lonza, Switzerland) and were cultured in 0.75 mg/mL hygromycin B (Gibco-Invitrogen) medium.

Proliferation assay A20.IIA cells at a concentration of 105cells/mL were incubated with serial dilutions of CpG 1826 or control 1826 ODNs at concentrations ranging from 0.0003 to 60 μg/mL or with complete RPMI medium alone. After 3 days, [3H] thymidine BIBW2992 order (GE Healthcare) was added for the last 4 h. Cells were harvested onto fiber filters and [3H] thymidine incorporation was measured in a scintillation counter (Microbeta, Perkin Elmer). Apoptosis assay A20.IIA cells (104) were cultured in complete RPMI medium in 96-well plates in the presence or absence of Ensartinib supplier 3 μg/mL or 30 μg/mL of CpG or control ODNs. Staining with Annexin V/allophycocyanin (APC) and propidium iodide (PI)

(BD Biosciences, France) was performed 72 h later and then analyzed by flow cytometry. Apoptotic cells were defined as those positive for Annexin V and PI. Mice Female BALB/c mice (H-2d) were obtained from Charles River Laboratories (L’Arbresle, France) and used between 6 and 8 weeks of age. They were provided with sterile food and water ad libitum and kept on a 12-hour light–dark cycle. All procedures involving mice conformed with European Union guidelines, French regulations for animal experimentation (Ministry of Agriculture Act No. 2001–464, May 2001), and the guidelines of the Institut Fludarabine National de la Santé et de la Recherche Médicale Committee on Animal Research, and were approved by the relevant local committees (Charles Darwin Ethics Committee for Animal Experiments, Paris, France; Permit Number: p3/2009/004). Tumor implantation Mice

were first anesthetized by intraperitoneal injection of a mixture containing 120 mg/kg of ketamine (Virbac, France) and 6 mg/kg of xylazine (Rompun 2%; Bayer Healthcare). To obtain a subcutaneous lymphoma (SCL) murine model, BALB/c mice were inoculated subcutaneously with 5 × 106 A20.IIA-GFP tumor cells in a final volume of 50 μL of RPMI, at 2 different sites: the right and left abdomen. For the intracerebral tumor implantation, anesthetized mice were immobilized on a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). Tumor cells (5 × 104 in a final volume of 2 μL RPMI) were injected into the specific cerebral location (right striatum), located 2 mm to the right of the medial suture and 0.

Discussion Trehalose in rhizobia is a key compound for signaling plant growth, yield and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plant. In this work we reconstructed trehalose metabolism in R. etli, and investigated the role of trehalose in the response to high temperature and desiccation stress, as well as symbiotic performance. By using13C-NMR, we showed that besides trehalose as the major compatible solute, R. etli CE3 also amasses glutamate. In addition, it can accumulate

mannitol if present in the external medium. The same compatible solute profile was recently reported for the strain R. etli 12a3, isolated from P. vulgaris nodules BGB324 purchase in Tunisian fields [6]. Two successive genome-based metabolic reconstructions of R. etli have been reported, covering in total 405 reactions and 450 (but not trehalose-related) genes [57, 58]. In this study, we reconstructed the metabolism of trehalose in R. etli, including trehalose uptake, degradation, and synthesis (see Figure 2). Our data suggest that uptake and catabolism of trehalose in R. etli uses the same pathways as in S. meliloti, since

orthologs to the S. meliloti AglEFGK/ThuEFGK ABC trehalose/maltose/sucrose transporters [22, 23], as well as the ThuAB catabolic route [21], were found in R. etli. In addition, R. etli genome accounts for up to 3 putative copies of the trehalose-6-phosphate hydrolase (TreC). Only TreC3 was in the same group as the characterized TreC protein from E. coli, suggesting that the other copies might have a slightly different function. Interestingly, treC2 (annotated as aglA) was located mTOR inhibitor upstream of the aglEFGK genes encoding the alpha-glucoside ABC transporter. In S. meliloti, aglA, encoding an alpha-glucosidase with homology to family 13 of glycosyl hydrolases, forms part of the aglEFGAK operon, suggesting a possible function in sucrose, maltose and/or trehalose catabolism. Further work is necessary to elucidate the role of the different systems involved in trehalose transport and degradation in R. etli. Regarding trehalose synthesis, Suarez

et al. [10] already suggested the presence in R. etli of the three trehalose biosynthetic pathways so far known in rhizobia (OtsAB, TreS, and TreYZ). In this work, we precisely located the DNA ligase corresponding genes, and proposed the most plausible route of glucose synthesis from mannitol, and subsequent OtsAB-mediated trehalose synthesis (see Figure 2). We found that genes for trehalose metabolism were scattered in the genome, and sometimes present in more than one copy (i.e., otsA, treZ, treS, treC). This high enzyme redundancy seems to be a general characteristic of R. etli CFN 42, and was proposed to correlate with the different degrees of metabolic responses and alternative regulation necessary to cope with a challenging environment without compromising the integrity of the pathways [30].

Infect Immun 1997, 65:2707–2716.PubMed 21. Ward TJ, Gorski L, Borucki MK, Mandrell RE, Hutchins J, Pupedis Selleckchem 5-Fluoracil K: Intraspecific phylogeny and

lineage group identification based on the prfA virulence gene cluster of Listeria monocytogenes. J Bacteriol 2004, 186:4994–5002.PubMedCrossRef 22. Orsi RH, Bakker HC, Wiedmann M: Listeria monocytogenes lineages: Genomics, evolution, ecology, and phenotypic characteristics. Int J Med Microbiol 2010, 301:79–96.PubMedCrossRef 23. Ragon M, Wirth T, Hollandt F, Lavenir R, Lecuit M, Le Monnier A, Brisse S: A new perspective on Listeria monocytogenes evolution. PLoS Pathog 2008, 4:e1000146.PubMedCrossRef 24. Yan H, Neogi SB, Mo Z, Guan W, Shen Z, Zhang S, Li L, Yamasaki S, Shi L, Zhong N: Prevalence and characterization of antimicrobial resistance of foodborne Listeria monocytogenes isolates in Hebei province of Northern China, 2005–2007.

Int J Food Microbiol 2010, 144:310–316.PubMedCrossRef 25. Zhou X, Jiao X, Wiedmann M: Listeria monocytogenes in the Chinese food system: strain characterization through partial actA sequencing and tissue-culture pathogenicity assays. J Med Microbiol 2005, 54:217–224.PubMedCrossRef 26. Chao G, Zhou X, Jiao X, Qian X, Xu L: Prevalence and antimicrobial resistance of foodborne pathogens isolated from food products in China. Foodborne Pathog Dis 2007, 4:277–284.PubMedCrossRef 27. Chen J, Zhang X, Mei L, Jiang L, Fang W: Prevalence of Listeria in Chinese food products from 13 provinces Phenylethanolamine N-methyltransferase between 2000 and 2007 and virulence characterization of www.selleckchem.com/products/NVP-AUY922.html Listeria monocytogenes isolates. Foodborne Pathog Dis 2009, 6:7–14.PubMedCrossRef 28. Jiang L, Chen J, Xu J, Zhang X, Wang S, Zhao H, Vongxay K, Fang W: Virulence characterization and genotypic analyses of Listeria monocytogenes isolates from food and processing environments in eastern China. Int J Food Microbiol 2008, 121:53–59.PubMedCrossRef

29. Sauders BD, Fortes ED, Morse DL, Dumas N, Kiehlbauch JA, Schukken Y, Hibbs JR, Wiedmann M: Molecular subtyping to detect human listeriosis clusters. Emerg Infect Dis 2003, 9:672–680.PubMedCrossRef 30. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 24:1596–1599.PubMedCrossRef 31. Zhang W, Jayarao BM, Knabel SJ: Multi-virulence-locus sequence typing of Listeria monocytogenes. Appl Environ Microbiol 2004, 70:913–920.PubMedCrossRef 32. Chenal-Francisque V, Lopez J, Cantinelli T, Caro V, Tran C, Leclercq A, Lecuit M, Brisse S: Worldwide distribution of major clones of Listeria monocytogenes. Emerg Infect Dis 2011, 17:1110–1112.PubMedCrossRef 33. Rasmussen OF, Skouboe P, Dons L, Rossen L, Olsen JE: Listeria monocytogenes exists in at least three evolutionary lines: evidence from flagellin, invasive associated protein and listeriolysin O genes. Microbiology 1995,141(Pt 9):2053–2061.PubMedCrossRef 34.

1 to 100 μg/ml) of the tested agents. The compounds were dissolved in 10% DMSO to concentration of 1 mg/ml, and subsequently diluted in culture medium to reach the required

concentrations. DMSO, which was used as a solvent did not exert any inhibitory effect on cell proliferation. The cells attached to the plastic were fixed by gently layering cold 50% TCA (trichloroacetic acid, Aldrich-Chemie, Germany) on the top of the culture medium in each well. The plates were incubated PD-0332991 molecular weight at 4°C for 1 h and then washed five times with tap water. The background optical density was measured in the wells filled with culture medium, without the cells. The cellular material fixed with TCA was stained with 0.4% sulforhodamine B (SRB, Sigma, Germany) dissolved in 1% acetic acid (POCh, Gliwice, Poland) for 30 min. Unbound dye was removed by rinsing (4×) with 1% acetic acid. The protein-bound dye was extracted with 10 mM unbuffered tris base (POCh, Gliwice, Poland)

for determination of optical density (at 540 nm) in a computer-interfaced, 96-well microtiter plate reader Multiskan RC photometer (Labsystems, Helsinki, Finland). Each compound LBH589 in vitro in given concentration was tested in triplicates in each experiment, which was repeated 3–5 times. MTT assay This technique was applied for the cytotoxicity screening against mouse leukemia cells growing in suspension culture. An assay was performed after 72-h exposure to varying concentrations (from 0.1 to 100 μg/ml) of the tested agents. The compounds were dissolved in 10% DMSO to concentration of 1 mg/ml, and subsequently diluted in culture medium to reach

the required concentrations. DMSO, which was used as a solvent did not exert any inhibitory effect on cell proliferation. For the last 3–4 h of incubation 20 μl of MTT solution were added to each Interleukin-2 receptor well (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; stock solution: 5 mg/ml). The mitochondria of viable cells reduce the pale yellow MTT to a navy blue formazan: the more viable cells are present in well, the more MTT will be reduced to formazan. When incubation time was completed, 80 μl of the lysing mixture was added to each well (lysing mixture: 225 ml dimethylformamide, 67.5 g sodium dodecyl sulfate, and 275 ml of distilled water). After 24 h, when formazan crystals had been dissolved, the optical densities of the samples were read on an Multiskan RC photometer at 570 nm wavelength. Each compound in given concentration was tested in triplicates in each experiment, which was repeated 3–5 times. The results of cytotoxic activity in vitro were expressed as an ID50—the dose of compound (in μg/ml) that inhibits proliferation rate of the tumor cells by 50% as compared to the control untreated cells. Acknowledgments This work is supported by Polish Ministry of Science and Higher Education, Grant No. N405 036 31/2655 and the Medical University of Silesia, Grant No. KNW-1-029/09.

References Antal TK, Krendeleva TE, Laurinavichene TV, Makarova VV, Ghirardi ML, Rubin AB, Tsygankov AA, Seibert M (2003) The dependence of algal H2-production on photosystem II and O2 consumption activities in sulphur-deprived Chlamydomonas reinhardtii cells. Y-27632 chemical structure Biochim Biophys Acta 1607:153–160. doi:10.​1016/​j.​bbabio.​2003.​09.​008 CrossRefPubMed Arnon D (1949) Copper enzymes in isolated chloroplasts and polyphenol

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The DSSC cell was sealed using the polymer resin to act as a spacer. The electrolyte was injected into the space between the electrodes from these two holes, and

then these two holes were sealed completely by Surlyn (DuPont, Taipei, Taiwan). Results and discussion In this study, high-density long-branched tree-like ZnO structures and NRs were grown on AZO/FTO substrates of photoanodes to increase the optical absorption of the dye. Figure 2 shows the XRD GSK1120212 research buy patterns for the AZO thin film, ZnO nanorods, and tree-like ZnO nanostructures, respectively. The crystalline structure was analyzed using XRD measurements according to a θ/2θ configuration. According to the XRD database, all of the diffraction peaks can be indexed to the hexagonal

wurtzite phase of ZnO. In principle, the XRD spectra show that the ZnO films developed without the presence of secondary phases and groups. No Al2O3 phase was found. Moreover, the much higher relative intensity of the (002) diffraction peak provides evidence that the nanorods are preferentially oriented in the c-axis direction perpendicular to the substrate. No other ZnO phase was found. Regarding tree-like ZnO nanostructures, the presence of secondary phases and groups was observed. These secondary phases and groups result from the thin AZO film coating on the ZnO NRs, which served as a seed layer for the tree-like nanostructures. Figure 2 XRD patterns. The XRD patterns of different ZnO nanostructures. ZnO NRs and tree-like ZnO structures were obtained on JAK inhibition an FTO substrate, and DSSCs were constructed, as shown in Figure 3. Figure 3a,b,c,d shows the FE-SEM images of the ZnO ‘NRs’ and ‘tree-like structures’ on the FTO substrate, respectively, indicating that the ZnO NRs

are well-grown on the substrates with a distinctive, clear morphology. Both the lengths of the NRs and tree-like structures are in the range of 2 to 3 μm, as shown in Figure 3a,c. Figure 3a,b,c,d shows that the pillar-shaped tree-like structures form upright against the FTO substrate, whereas Figure 3a,c indicates that the NRs grow randomly on the FTO substrate. The eventual growth of tree-like ZnO structures or NRs was highly dependent on the preexisting textured seed layers on the FTO substrate. NADPH-cytochrome-c2 reductase According to Greene et al., the factor causing the upright growth of ZnO NRs is the temperature during growth. In the present case, the growing temperature for the FTO substrate was set to be 90°C. Accordingly, the ZnO NRs grow on the FTO substrate, as shown in Figure 3c. To synthesize the branched structures of tree-like ZnO, a second set of AZO seeds containing the previously grown ZnO NRs were sputtered. The growth procedures at the same growth conditions were repeated. Figure 3a,b shows the tree-like ZnO with a branched structure. The dye loading at an approximate wavelength of 370 and 530 nm corresponds to the absorption edge of D-719 dye. Figure 4 shows the absorptions of solutions containing 0.

The selective PKA activator phorbol myristate acetate (PMA) was purchased from Promega (Madison, WI, USA). Immunohistochemical staining and assessment find more of COX-2, VEGF, and MVD Immunohistochemical staining was carried out using the streptavidin-peroxidase method. Briefly, each tissue section was deparaffinized, rehydrated, and then incubated with fresh 3% hydrogen peroxide in methanol for 15 min. After rinsing with phosphate-buffered saline (PBS), antigen retrieval was carried out by microwave treatment in 0.01 M sodium citrate buffer (pH 6.0) at 100°C for 15 min. Next, non-specific binding

was blocked with normal goat serum for 15 min at room temperature, followed by incubation at 4°C overnight with different primary antibodies. Antibodies, clones, dilutions, pretreatment conditions, selleck kinase inhibitor and sources are listed in Table 2. After rinsing with PBS, slides were incubated with biotin-conjugated secondary antibodies for 10 min at room temperature, followed by incubation with streptavidin-conjugated peroxidase working solution for 10 min. Subsequently, sections were stained for 3-5 min with 3,39-diaminobenzidine

tetrahydrochloride (DAB), counterstained with Mayer’s hematoxylin, dehydrated, and mounted. Negative controls were prepared by substituting PBS for primary antibody. For this study, the intensity of VEGF and COX-2 staining were scored on a scale of 0-3: 0, negative; 1, light staining; 2, moderate Vitamin B12 staining; and 3, intense staining. The percentages of positive tumor cells of different intensities (percentage of the surface area covered) were calculated as the number of cells with each intensity score divided by the total number of tumor cells (x 100). Areas that were negative were given a value of 0. A total of 10-12 discrete foci in every section were analyzed to determine average staining intensity and the percentage of the surface area covered. The final histoscore was calculated using the formula: [(1× percentage of weakly positive

tumor cells) + (2× percentage of moderately positive tumor cells) + (3× percentage of intensely positive tumor cells)]. The histoscore was estimated independently by two investigators by microscopic examination at 400× magnification. If the histoscores determined by the two investigators differed by more than 15%, a recount was taken to reach agreement. The results of COX-2 and VEGF immunostaining were classified into high and low expression using cut-off values based on the median values of their respective histoscores. Table 2 Multivariate analysis of VEGF and MVD expression in NSCLC specimens     VEGF expression     MVD expression     β HR (95% CI) P β HR (95% CI) P COX-2 expression                 High 2.286 9.836 (3.387 – 28.564) 0.000 1.146 3.147 (1.152 – 8.598) 0.025     Low 1.000     1.000     TNM stage                 III + IV 0.061 1.063 (0.493 – 2.289) 0.877 0.025 1.025 (0.493 – 2.132) 0.947     I + II 1.000     1.