coli tat mutants BK designed and coordinated the study, and draf

coli tat mutants. BK designed and coordinated the study, and drafted the manuscript. All the authors read and approved the final manuscript.”
“Background TTSS plays a major role in virulence determination in pathogenic Shigella. The expression of TTSS is regulated in response to environmental stimuli, such as changes in salt concentration [1] and growth temperature [2, 3]. This response to environmental factors is appropriate for the life cycle of Shigella, in which the expression of virulence genes is required for invasion and propagation in the host intestinal tract, but might be a potential burden for survival in the natural environment.

The genes this website that encode the components of TTSS in Shigella are located on the virulence plasmid, and are controlled by two regulator proteins, VirF and InvE (VirB) [4, 5]. VirF, an AraC-type transcriptional regulator, activates the transcription of invE (virB) [4, 6–8]. InvE is a homologue of a plasmid-partitioning factor, ParB [7], and possesses DNA binding activity [9]. InvE activates the transcription of the mxi-spa and ipa genes,

which encode the components of TTSS, through competition with the global repressor H-NS, a histone-like DNA binding protein [10]. Recently, we reported that the temperature-dependent expression of TTSS is controlled at the post-transcriptional level, through the Nepicastat nmr regulation of InvE synthesis [11]. The mRNA of invE is highly stable at 37°C, but stability decreases significantly at 30°C JPH203 supplier where the TTSS synthesis is tightly repressed. Deletion

mutants of hfq, which encodes an RNA-binding protein in Gram-negative bacteria, restores the expression of invE and other TTSS genes at low temperature due to the increased stability of the invE mRNA. To date, a detailed mechanism of osmolarity-dependent Metalloexopeptidase regulation of TTSS expression has yet to be elucidated. In the current study, we examined whether osmotic-dependent changes in TTSS expression involved post-transcriptional regulation. We present several lines of evidence that invE expression is regulated at the post-transcriptional level during TTSS synthesis in Shigella, and that the RNA chaperone Hfq plays a key role in regulating invE mRNA stability. Results Osmolarity and TTSS expression The expression of TTSS in Shigella is markedly reduced in low-salt LB medium [1]. However, it is not clear whether the critical factor for the decreased expression of TTSS in LB medium is low osmolarity or low-salt concentration. We analysed the expression of TTSS in the presence of several different osmolytes, but similar osmotic pressures. There was a difference in the growth rate of S. sonnei in LB medium in the absence (doubling time, 42.1 minutes) and presence (doubling time, 30.6 minutes) of 150 mM NaCl. To control for differences in growth rate in LB medium, we used yeast extract and nutrient broth (YENB) medium [12], since growth rate in YENB in the absence (doubling time, 32.2 minutes) and presence (doubling time, 31.

The IC50 values were the drug concentrations causing a 50% reduct

The IC50 values were the drug concentrations causing a 50% reduction in the optical density. The experiments were performed

in triplicate, and expressed as the mean values of three experiments. The relative resistance was calculated by the following formula: Apoptosis analysis On post-transfection day 3, cells were resuspended in 100 μl binding buffer at a concentration of 1 × 106/ml after washing twice with cold PBS and mixed with 5 μl Annexin V-FITC (PharMingen) and 10 μl of 20 μg/ml propidium iodide (Sigma) at room temperature for 15 min. Samples were diluted with 400 μl binding buffer and analyzed by fluorescence activated cell sorting (FACS) using the protocol provided by the manufacturer (ClonTech, Palo Alto, Calif., USA). The apoptotic rate was calculated as the mean fluorescence intensity. Statistical analysis The data are expressed as the mean Mdm2 antagonist ± SEM. Each experiment was repeated at least three times. Bands from Western blots were quantified by Quantity One software (Bio-Rad). The Seliciclib clinical trial differences among means were examined with ANOVA followed by post-hoc

test using SPSS RG-7388 11.0 software (Chicago, Ill., USA). A p value less than 0.05 was considered as statistical significance. Results RT-PCR and Western blots Both mRNA and protein levels of Fas were significantly lower in H446/CDDP and H446/CDDP/Empty cells compared with those in H446/CDDP/Fas cells (p < 0.01), indicating that Fas was successfully transduced into and expressed in H446/CDDP cells. Over-expression of Fas effectively down-regulated ERCC1 and GST-π in both mRNA and protein levels (p < 0.01) compared with the control cells (Figs. 1 and

2). Figure 1 The expression of Fas, ERCC1, GST-π and GAPDH detected by RT-qPCR. GAPDH was used as an internal control. Upregulation of Fas led to Immune system a significant decrease in ERCC1 and GST-π. * p < 0.01 vs H446/CDDP/Empty and H446/CDDP cells. Figure 2 The expression of Fas, ERCC1, and GST-π detected by Western blots. β-actin was used as an internal control. Upregulation of Fas caused the downregulation of ERCC1, and GST-π. Effect of Fas on cisplatin resistance To explore the roles of Fas in cisplatin resistance of SCLC, MTT assays were performed. 72 h after exposure to CDDP, the 50% inhibitory concentration (IC50) of CDDP in H446/CDDP/Fas was 7.6 ± 0.46 μg/ml, significantly lower than 30.8 ± 0.92 μg/ml and 29.7 ± 0.26 μg/ml in H446/CDDP and H446/CDDP/Empty, respectively (p < 0.01). In other words, H446/CDDP/Fas cells showed a 3.9-fold decrease in resistance to CDDP compared with H446/CDDP/Empty cells, suggesting that up-regulation of Fas could inhibit the cisplatin-resistant phenotype of SCLC. Effect of Fas on cell apoptosis The apoptosis rates in H446/CDDP, H446/CDDP/Empty and H446/CDDP/Fas cells were 6.02 ± 0.70%, 7.19 ± 0.89% and 13.17 ± 0.40%, respectively. Compared to H446/CDDP and H446/CDDP/Empty cells, H446/CDDP/Fas cells showed a significantly lower apoptotic rate (p < 0.

V Nutricia, Zoetermeer, The Netherlands) providing 2 1 MJ (500 k

V. Nutricia, Zoetermeer, The Netherlands) providing 2.1 MJ (500 kcal) and 40 g of protein per 500 ml. Furthermore, the dietician made arrangements to solve any problems, e.g. Pictilisib feeding difficulties, in collaboration with the hospital medical and nursing

staff. At the second visit during hospitalization, 7–8 days after surgery, the dietician evaluated food intake and the consumption of the ONS using a 24-h recall and gave individually tailored advice to optimize dietary intake. Furthermore, the transfer of the patient to the rehabilitation centre or the patient’s home was prepared by evaluating the patient’s physical restrictions with regard to nutritional care, i.e. purchasing food products and the preparation of meals, and by making arrangements to enable adequate food intake, e.g. support of informal caregivers and delivery of information on meal services. After hospital discharge, the dietician visited each patient Selleck MLN8237 three times (1, 2 and 6 weeks after discharge) at the patient’s home or in the rehabilitation centre (whatever was applicable) in order to evaluate dietary intake including the intake of the ONS, to evaluate possible bottlenecks in nutritional care at home (e.g. LY2874455 purchase shopping, cooking) and to give dietary advice as needed. In addition, in-between these home visits, weekly telephone calls were made (3, 4, 5, 8 and 10 weeks after discharge) to evaluate dietary

intake (including the ONS) by 24-h recall. If necessary, a telephone call was replaced by a home visit. Usual care Patients allocated to the control group received usual care as provided in the hospital, rehabilitation clinic or at home, i.e. dietetic

care or nutritional supplements were only provided on demand of the medical doctor in charge. In the control group, ten patients (13%) received ONS and 18 patients (23%) received dietetic counseling. Economic evaluation Effect measures Weight At baseline, self-reported weight was used, because patients were not able to stand on a weighing scale because of hip fracture. At 3 months postoperatively, weight was measured using an electronic weighing scale (Seca 862, Seca Ltd, Birmingham, Methamphetamine UK). The difference in weight in kilograms between baseline and 3 months postoperatively was calculated and used to evaluate the effectiveness of the nutritional intervention. Quality adjusted life years Quality of Life was estimated at baseline and at 3 and 6 months postoperatively using the Dutch version of EuroQoL (EQ-5D-3 L) [27–29]. In the EuroQoL, the patient was asked to make a statement on the degree of problems (no problem, some problems or major problems) he/she experienced on the dimensions of mobility, self-care, usual activities, pain or discomfort and anxiety or depression. The degree of problems on each dimension were combined to a health state.

As DPP IV is occasionally expressed in thyrocytes of benign thyro

As DPP IV is occasionally expressed in thyrocytes of benign thyroid disorders [18] a link to proliferation has been suggested [11]. Increased APN expression is correlated with progression and metastasis in colorectal, pancreatic carcinoma and undifferentiated thyroid carcinoma [12, 19, 20]. Dipeptidyl peptidase II (DPP II, EC, a lysosomal protease ubiquitously expressed in many cells including normal thyrocytes of mammalian origin [21], is thought to play find more a role in the release of hormone from thyroglobulin [22]. Dipeptidyl peptidase IV (DPP IV, CD26, EC

is a trans-membrane type II glycoprotein with multifaceted function. As well as the integral membrane form, a soluble form is found in serum and semen. In contrast to thyroid follicle cells, JAK pathway most other types of human cell express DPP IV [23]. DPP IV is most Trichostatin A cell line up-regulated in papillary thyroid carcinoma [24, 25] and apparently induced by RET/PTC mutations [26]. Aminopeptidase N (APN, aminopeptidase M, alanine aminopeptidase, CD13, EC, is expressed in anaplastic thyroid carcinoma cells but not in normal thyrocytes [12]. In porcine thyrocytes, by contrast, APN is a marker of the apical thyrocyte membrane [27, 28]. To identify species with an identical pattern of protease activity compared to human thyrocytes, here we localized protease activities using synthetic substrates. The activities of DPP II, DPP IV and APN were

studied in animal species used for investigating thyroid function, namely human, porcine,

rat, mouse, bovine and ovine thyrocytes. Methods Tissue samples Cadavers of rats (female, Sprague–Dawley) and mice (female, Balb/c), which had been used for other experiments, were obtained from the Institute of Pharmacology and the Institute of Anatomy, respectively. Porcine, bovine and Mirabegron ovine thyroid glands were obtained from the local slaughterhouse. Samples from human thyroid tissue were obtained from the surgical department of the University after informed consent of the patients. Animal procedures were performed according to the guidelines of the local authorities. All experiments on human subjects were conducted in accordance with the Helsinki Declaration of 1975. For the localization of DPP IV and APN activities unfixed tissues were used. For the demonstration of DPP II 0.5 cm3 cubes of thyroid tissue were fixed in neutral buffered 4% formaldehyde solution with 30% sucrose for 24h at RT. After fixation, samples were rinsed for 24h at RT in distilled water containing 30% sucrose and 1% gum arabicum. Tissue samples were embedded in Tissue Tec (Miles) and deep-frozen in isopentane per-cooled with liquid nitrogen. Detection of protease activity Protease activity in tissues and in cells was detected by cleavage of specific synthetic substrates. The synthetic substrate is bound to a tag, which together with the coupler yields a colored product, when released from the substrate.

Microb Pathog 1999, 27:105–117 PubMedCrossRef 13 Samuel G, Reeve

Microb Pathog 1999, 27:105–117.PubMedCrossRef 13. Samuel G, Reeves P: Biosynthesis of O-antigens: genes and pathways involved in nucleotide sugar precursor synthesis and O-antigen assembly. Carbohydrate research 2003, 338:2503–2519.PubMedCrossRef 14. DebRoy C, Fratamico PM, Roberts E, Davis MA, Liu Y: Development of PCR assays targeting genes in O-antigen gene clusters for detection and identification of Escherichia coli O45 and O55 serogroups. Applied and environmental microbiology 2005, 71:4919–4924.PubMedCrossRef 15. Fitzgerald C, Sherwood R, Gheesling LL, Brenner FW, Fields PI: Molecular analysis of the rfb O antigen gene cluster of Salmonella enterica serogroup O:6,14 and development of a serogroup-specific

PCR assay. Applied and environmental microbiology 2003, 69:6099–6105.PubMedCrossRef

16. Tao J, Feng L, Guo H, Li Y, Wang L: The Selleckchem Epoxomicin O-antigen gene cluster of Shigella boydii O11 and functional identification of its wzy gene. FEMS Microbiol Lett 2004, 234:125–132.PubMedCrossRef 17. Bogdanovich T, Carniel E, Fukushima H, Skurnik M: Use of O-antigen gene cluster-specific PCRs for the identification and O-genotyping of Yersinia pseudotuberculosis and Yersinia pestis. J Clin Microbiol 2003, 41:5103–5112.PubMedCrossRef 18. Majed Z, Bellenger E, Postic D, Pourcel C, Baranton G, Picardeau M: Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 2005, 43:539–545.PubMedCrossRef find more 19. Salaun L, Merien F, Gurianova S, Baranton G, Picardeau M: Application of check details multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. Doramapimod solubility dmso J Clin Microbiol 2006, 44:3954–3962.PubMedCrossRef 20. Zuerner RL, Alt DP: Variable nucleotide tandem-repeat analysis revealing a unique group of Leptospira interrogans serovar Pomona isolates

associated with California sea lions. J Clin Microbiol 2009, 47:1202–1205.PubMedCrossRef 21. Zuerner RL, Alt D, Bolin CA: IS1533-based PCR assay for identification of Leptospira interrogans sensu lato serovars. J Clin Microbiol 1995, 33:3284–3289.PubMed 22. Zuerner RL, Bolin CA: Differentiation of Leptospira interrogans isolates by IS1500 hybridization and PCR assays. J Clin Microbiol 1997, 35:2612–2617.PubMed 23. Herrmann JL, Baril C, Bellenger E, Perolat P, Baranton G, Saint Girons I: Genome conservation in isolates of Leptospira interrogans. Journal of bacteriology 1991, 173:7582–7588.PubMed 24. Herrmann JL, Bellenger E, Perolat P, Baranton G, Saint Girons I: Pulsed-field gel electrophoresis of NotI digests of leptospiral DNA: a new rapid method of serovar identification. J Clin Microbiol 1992, 30:1696–1702.PubMed 25. Perolat P, Lecuyer I, Postic D, Baranton G: Diversity of ribosomal DNA fingerprints of Leptospira serovars provides a database for subtyping and species assignation. Research in microbiology 1993, 144:5–15.PubMedCrossRef 26.

But they may provide imaging characteristics similar to the situa

But they may provide imaging characteristics similar to the situation in humans with a single comparatively well-defined lesion. The multifocal tumour growth in the SPC-raf transgenic animal model examined in this study limits direct application of established radiological imaging techniques in humans. However, it has already been reported that this animal model allows examination of the potential relevance of human protooncogenes or disabled tumour suppressor genes in an immunologically competent environment.

Thus, aspects of human carcinogenesis may be better understood highlighting the clinically translational aspects of the research. In the animals examined in this study tumour growth seemed to occur at a later point of time in male animals as compared to female animals, furthermore females MEK inhibitor cancer showed clinical signs of tumour necessitating to sacrifice the animals ICG-001 in vivo earlier compared to male animals (Table1). This has not been reported for the SPC-raf transgenic animal model, yet. However, due to the genetic mechanism of tumour induction in this model it might represent a relevant finding to understand lung tumour carcinogenesis. Further experiments have to be performed in order to validate the potential finding and present a hypothesis

for the origin. The primary focus of this study was to demonstrate the use of micro-CT for assessment of tumour load and growth. The issue demonstrates the potential additional benefit of the method for assessment of cofactors affecting carcinogenesis applying intraindividual time-course assessment. Micro-CT imaging applying the setup described above did not result in adverse events, even though

animals had advanced tumour stages at the later time points. In synopsis with other studies performed we attribute this to the use of isoflurane inhalation anaesthesia, respiratory monitoring and the use of a heating blanket. We performed prospective respiratory gating as it has been reported to significantly increase image quality. However, more sophisticated gating techniques such as retrospective or intrinsic gating or high-speed single-breath hold techniques could further optimize imaging [19–22]. MRI has been reported to allow high spatial resolution imaging of the lung. Martiniova et al. even second reported better detection of small lesions with MRI as compared to micro-CT [7]. Optical imaging techniques or micro-PET enable assessment of functional parameters but have limitations in high resolution imaging of morphology [8, 23]. Various post-processing strategies have been reported allowing precise evaluation of specific aspects of the image data. Dose measurements for the applied micro-CT protocol were performed in a phantom and ex-vivo in previous studies. The effective dose calculated from these measurements was 154 mGy for the selected protocol.

While ProLIFT can be used to fill the PS pores prior to the appli

While ProLIFT can be used to fill the PS pores prior to the application of photoresist in step I, it is not UV sensitive but can be removed by standard alkaline developer during the photoresist development step. This allows ProLIFT to be patterned in the same wet process that defines the photoresist but requires accurate timing of the development time. If the developing time is too short, exposed photoresist will be removed but ProLIFT residue will remain in the PS film slowing the RIE removal of PS, as shown in Figure 6a. Furthermore, any residual ProLIFT in the PS film once released is expected

to introduce stress in released microbeams, resulting in beam breakage (poor yield). On the other hand, if the developing time is too AZD1152 price long, the photoresist will be over developed, ICG-001 in vivo causing a large side wall angle of the photoresist pattern, resulting in poorly defined PS structures as shown in Figure 6b. Worse, over developing can result in lift off of the patterned photoresist if it is not well attached to the PS film. Repeated experiments have shown the development time when using ProLIFT becomes a significant issue when patterning PS films above 1-μm thick, as they require a much longer developing time (>60 s) to remove all the ProLIFT in the PS films than typically required for photoresist development (approximately 30 s). Figure 6 Comparison of pore

fill techniques utilizing ProLIFT and SOG. Different techniques: (a) ProLIFT pore filling technique with short developing time, (b) ProLIFT pore filling technique with long developing time and (c) SOG pore filling technique. At three steps: (I) UV light exposure with photoresist patterning, (II) developing to remove exposed positive photoresist and (III) RIE and photoresist/pore filling material removal. On the contrary, SOG can be used to form a layer of SiO2

to fill the pores of PS at step I of Figure 6, which is not removed during the developing process at step II. This guarantees the accurate Teicoplanin control of developing time for the photoresist layer, resulting in well-patterned PS structures at step III, as shown in Figure 6c. Our tests showed a 10-s dip in 10% HF/DI is sufficient to remove all SOG in an exposed PS film (where there was no photoresist) up to 2.45-μm thick. The short dip resulted in an optical thickness change of less than 4.4%, suggesting the short dip had very little Mdm2 antagonist effect on the PS layer. In this work which used PS layers of 2.45-μm thickness, SOG as a pore filling layer was more advantageous than ProLIFT and was used as described. These results show a complete MEMS fabrication process using a single material system can be achieved using combination of anodization and electropolishing. No sacrificial layer was required to achieve release of the beams.

After the determination of gfp and 16S rRNA gene copies of Gfp-ta

After the determination of gfp and 16S rRNA gene copies of Gfp-tagged Asaia, total Asaia, and bacteria, the following ratios were calculated: Gfp-labelled Asaia to total Asaia ratio,

Gfp-labelled Asaia to bacteria ratio (GfpABR), and Asaia to bacteria 16S rRNA gene copy ratio (ABR), the latter according to Favia et al. [6]. These ratios were used to estimate the relative abundances of the introduced strain within total Asaia population in S. titanus individuals and of Gfp-labelled Asaia and Asaia sp. in the bacterial community associated with the insect samples. Statistical analyses To compare the Gfp Asaia density detected in co-feeding or venereal transmission experiments for every tested period, q-PCR data relative to the gfp gene VRT752271 ic50 concentration were log-transformed, after YH25448 mw adding the constant 10, and PX-478 analyzed by one-way analysis of variance (ANOVA). In addition, means were separated by Tukey test (P<0.05) when variance homogeneity was satisfied (Levene test, P<0.05). Fluorescent in situ hybridization Fluorescent in situ hybridization analysis was carried out on organs dissected in a sterile saline solution from donor and recipient S. titanus individuals that were not used for Real time PCR experiments. The dissected organs were fixed for 2 min at 4°C in 4% paraformaldehyde and washed in

PBS. All hybridization experiment steps were performed as previously described [4] using specific and universal fluorescent probes. For detection of Gfp-labelled Asaia, probes gfp540 (5’-CCTTCGGGCATGGCACTCTT-3’) and gfp875 (5’-GGTAAAAGGACAGGGCCATCGCC-3’) were labelled with Cy5.5 (indodicarbocyanine, absorption/emission at 675-694 nm). Probes Asaia1 and Asaia2, labelled with Cy3 (indocarbocyanine, absorption/emission at 550/570 nm), were used to observe the total

Asaia population hosted by S. titanus individuals [6]. As a positive control for the hybridization experiment, a universal bacterial probe EUB388 labelled with fluorescein isothiocyanate (FITC, absorption/emission at 494/520 nm) was also used [32]. After hybridization, the samples were mounted in antifading medium and then observed in a laser scanning confocal until microscope SP2- AOBS (Leica). Authors’ contributions EG designed and performed most of the experiments, analyzed data and wrote the manuscript. EC and AR provided the Asaia strain SF2.1(cGfp) and designed the experiments, MM designed FISH experiments and performed confocal microscopy observations. GF gave suggestions and contributed to data analysis. AA and DD designed and supervised all the experiments. All authors have read and approved the final manuscript. Acknowledgements We are grateful to Greg Hurst for English editing of the manuscript.

Besides the absolute dependence of EBPs and ATP hydrolysis for th

Besides the absolute dependence of EBPs and ATP hydrolysis for the formation of the RNA polymerase open complex on the promoters, another unique feature of σ54 is the recognition of

-24/-12-type promoters with consensus sequence TGGCACG-N4-TTGC [17, 18]. The σ54 regulon was Sepantronium datasheet estimated in several organisms, such as E. coli [19], Pseudomonas putida [20] and several species of Rhizobiaceae [21] by use of powerful computational methods that took advantage of the high conservation of σ54 promoter sequences throughout diverse bacterial groups. Alternative sigma factors provide effective mechanisms check details for regulating a large numbers of genes in response to several environmental stresses. In the genome of X. fastidiosa there are genes encoding each of the sigma factors RpoD, RpoH, RpoE and RpoN [22]. Large-scale this website studies using microarrays and in silico analyses have permitted to determine the RpoH and RpoE regulons and their contribution to the heat shock response [23, 24]. Recently, we have established that RpoN controls cell-cell aggregation and biofilm formation in X. fastidiosa by means of differential regulation of genes involved in type I and type IV fimbrial biogenesis. We have also characterized the first σ54-dependent promoter in X. fastidiosa, controlling expression of the pilA1 gene [25].

Here, we analyzed the global transcriptional profile of X. fastidiosa under nitrogen starvation conditions using DNA microarrays. A more complete description of the X. fastidiosa σ54 regulon was achieved using microarray data from an rpoN mutant integrated with an in silico analysis of RpoN-binding sites. The regulatory below region of the glnA gene that encodes the enzyme glutamine synthetase was

further characterized, and confirmed to have a σ54-dependent promoter, suggesting an important role of ammonium assimilation mediated by σ54 in X. fastidiosa. Methods Bacterial strains and growth conditions The citrus strain J1a12 of Xylella fastidiosa [26] was cultivated in PW medium [27] without bovine serum albumin and phenol red and supplemented with 0.5% glucose (w/v) (PWG) at 25°C with no agitation. Cultures were also grown in defined XDM2 medium [28] or XDM2 medium lacking all nitrogen sources (XDM0) at the same conditions. For the rpoN mutant strain [25], 10 μg ampicillin ml-1 was supplemented to the PWG medium. Growth of Xylella cells in nitrogen starvation For time course studies, late-exponential phase cells in PWG medium were used to inoculate a culture in 100 ml XDM2 medium to an optical density at 600 nm (OD600 nm) of 0.1. Cells were grown during 12 days in the XDM2 medium (mid-log phase) and harvested by centrifugation.

The intracellular location for these bacteria appears to be a com

The intracellular location for these bacteria appears to be a comfortable niche for growth, allowing them to be

more aggressive and more protected against immune response and antibiotics. Although U. diversum is a little studied species, its intracellular location adds this important feature to the understanding of mollicutes and explains their importance in bovine diseases. Methods Ureaplasma diversum and click here cell lines Four isolates of U. diversum and two type-strains, ATCC 49782 and 49783, were studied. Isolates 77 and A203 were recovered from the vaginal mucus of a bovine vulvovaginitis (high passage), and the isolates 10T and S8 recovered from frozen bovine semen previously mixed with antibiotics in an artificial insemination center in Brazil (low passage). The isolates were initially identified with culturing characteristics and specie-specific PCR [26]. The Hep-2 (ATCC-USA CCL-23) cell lines were hosts to ureaplasmas in the present study and were previously certified to be free of mycoplasma by culture and PCR [27]. The cells were cultured in 5% of CO2 at 37°C in Minimum Essential Medium (MEM) containing 2 mM L-glutamine and Earl’s balanced salts, supplemented with 10% fetal calf serum Cult Lab, São Paulo, Brazil). Twenty-four hours prior to mycoplasma infection, Hep-2 cell monolayers were established for 10-20% confluence on 13 mm glass slides, in 24-well micro plates (TPP -

Switzerland), with one ml of MEM medium (Cult Lab, São CB-5083 chemical structure Paulo, Brazil) for analysis by confocal microscopy. The Hep-2 cells used in the present study were analyzed for presence of BAY 1895344 order mycoplasmas by culture and PCR. Labeling Mycoplasma cells The methodology was based on Basemam et al. [28]. The ureaplasmas were first cultured in 2 ml of ureaplasma medium (UB) at 37°C and expanded to 50 ml in the same broth. In a logarithmic growth phase (based in colorimetric changes), the culture

was centrifuged at 20,600 g for 30 minutes at 25°C. The pellets were homogenized by washing twice with PBS and incubated with carbocyanine dye solution (Vybrant™ Dil cell-labeling solution-Dil, V-22885, Molecular Probe, Eugene, Oregon, USA). Two-hundred microliters of Vibrant Dil (diluted at 1:200) were added to 105 – 107 mycoplasma cells in one ml of PBS and incubated at 37°C, for 40 minutes. The number of ureaplasma cells was determined by 10-fold dilution in UB medium and expressed as Changing Color Units/ml (CCU/ml). The labeled bacteria were centrifuged for 10 minutes at 20,600 g, at 25°C, washed twice with PBS, gently homogenized and transferred to the monolayer of Hep-2 cells. Inoculation of ureaplasma on Hep-2 cells [28] The Hep-2 cells at 60 to 70% confluence corresponding to approximately 106 cells/glass slide were selected for ureaplasmal infection. These cells were initially washed with PBS and inoculated with 105 to 107 of labeled mycoplasmas contained in one ml of MEM with 2% bovine fetal serum.