7 kg), and contains a proprietary blend of ingredients called Alka-Myte®. All of the ANS ingredients are allowed by both the U.S. and World anti-doping agencies (i.e., WADA), while Alka-Myte® itself has been granted New Dietary Ingredient (NDI) recognition selleck inhibitor by the Food and Drug Administration (FDA). Given the clearance by WADA and the FDA’s NDI recognition, it surprising that there are no published controlled studies to evaluate the efficacy of

the performance-related claims stated earlier. Therefore, the purpose of this study was to investigate the potential influence of this alkalizing nutrition supplement on previously validated correlates of cross-country skiing performance (i.e., upper body power) [6], as well as cardiorespiratory and blood lactate responses in well-trained competitive Nordic skiers both before and after a 7-day loading period. Methods Subjects and study design Competitive Nordic skiers from the surrounding area were recruited

to visit the Movement Science/Human Performance Lab on the Montana State University campus on three separate occasions. Competitive skiers familiar with the test protocols used for this study were recruited to help minimize Combretastatin A4 price changes expected with athletes performing lab-based performance tests for the first time. All subjects were assigned into a treatment or placebo group, but neither the subjects nor the investigators were aware of the either group’s identity until after all data collection was complete (i.e., double-blind placebo-controlled design). Procedures The first visit familiarized

subjects C59 in vitro with the testing protocol to be used for subsequent visits. Dependent measures recorded during the second visit (i.e., pre-testing) served to establish a baseline for both placebo and treatment groups. Following a 7-day supplement loading phase, the same tests were administered and dependent measures collected during the third visit (i.e., post-testing) and then compared directly to the pre-test measures. Dependent measures of interest included measures of upper body power (UBP), as well as cardiorespiratory and blood lactate responses to the UBP tests. During the first visit, subjects read and signed an informed consent document approved by the Montana State University Internal Review Board (IRB). Subjects then practiced with the testing protocols to be used during their second (pre-testing) and third (post-testing) visits to the lab. During the latter two visits, subjects completed a submaximal double poling test (i.e., Constant-Power Test), followed by three trials of a maximal intensity 10-sec upper body power test (UBP10), and then finished with a high intensity 60-sec UBP test (UBP60). An outline of the test protocol administered for both pre- and post-testing is outlined in Figure 1. The third lab visit (i.e., post-testing) occurred within 24 hrs of completing the supplement loading phase and repeated all test measures performed during the second lab visit (Figure 1).

Most professional bodies

and private companies linked to genetics now have LinkedIn groups, e.g. American Society Human Genetics, Illumina, National Society of Genetic Counselors.     Social media and traditional media are often directly linked. For example, television news outlets usually have an online presence as well as a Twitter feed. Each individual online news story can also typically be linked directly to personal social media feeds. Thus, it is possible to affect the momentum of social media by linking into traditional media sources such as TV and radio; in a cyclical motion, each feeds the other. The following Methods section summarises the processes that were followed for recruitment, and the Results section provides details about the sample obtained. Material and methods Overview of methods The overall study adopted find more a mixed methods approach, utilising both quantitative and qualitative techniques. For the quantitative

arm, non-parametric data were gathered via 32 closed questions and explored using descriptive statistics. A web-link to the online survey was made available via the Wellcome Trust Sanger Institute in Cambridge, UK; this could also be accessed through a web-page that described buy EX 527 the background to the study (www.​genomethics.​org). Participants The study aim was to recruit participants who were genomic researchers, genetic health professionals (e.g. clinical geneticists, genetic counsellors, etc.), non-genetic health

professionals (e.g. surgeons, GPs, nurses, etc.) and members of the public. Survey design An extensive discussion on the survey design process can be found in a separate publication (Middleton et al. 2014). Here details are provided about the background work which was done to iteratively create a robust questionnaire; this involved a Focus Group, five pilot studies, readability tests, test-retest reliability measures and numerous stages of face validity testing. The resultant survey includes 32-closed questions gathering mainly categorical, quantitative data. Recruitment strategy A three-phase interlinked recruitment strategy was utilised (Fig. 1). Fig. 1 Three-phase interlinked recruitment strategy 1 Traditional media Together with the media department at the Wellcome Trust Sanger Institute, a press release Non-specific serine/threonine protein kinase was written that advertised the study and invited participation. Following on from this, Channel 4 news and BBC local news created and delivered news stories on the research for the TV, and BBC Radio Cambridgeshire, BBC Radio 4 ‘Material World’ and the BBC World Service aired news stories for the radio. In each media article an interview with AM was conducted, and a link to the survey website was advertised. Each of these media also had an equivalent online news forum where a link to the survey was placed in an article summarising the project.   2.

Figure 9 XRD spectra of polished Cu foil (400 grit) and Cu film specimens before heating. In addition, surface roughness is believed to have an effect on the growth of FGLNAs. Surface topography of unpolished Cu foil, polished Cu foil, and Cu film

specimens was measured by AFM, and the surface roughness was evaluated using the height of ditches, as shown in Figure 10. To compare with the stress condition, measured initial residual stress on the specimen surface before heating is also shown in Figure 10. It can be found that the 400-grit polishing specimen has a similar roughness as the Cu film specimen (around 1.4 μm). It was suspected that the surface roughness may increase the surface area, thereby promoting the surface oxidation

of the specimen (i.e., enhancing VGS), and there is an optimum value for the selleck screening library growth of FGLNAs. It also can be found that the measured compressive stresses for the specimens of 800 and 1,000 grits polished are greatly larger than that of the 400-grit polished specimen. The reason why high-density GSK2879552 nmr FGLNAs were not observed on these high initial stress specimens is that the relatively low surface roughness may lack enough surface area to further enhance the growth of FGLNAs on the specimens. Therefore, there is a balance between the initial compressive stress and surface roughness for the growth of FGLNAs. Figure 10 AFM topography image, surface ditch height, and residual stress. (a) AFM three-dimensional topography image of the unpolished Cu foil specimen. (b) Surface ditch height and residual stress of unpolished Cu foil, polished Cu foil, and Cu film specimens. Conclusions Cu2O FGLNAs which are 3.5 to 12 μm in size with 50- to 950-nm wide petals were successfully fabricated using the thermal oxidation approach with catalyst under moderate humid atmosphere. The effect of surface conditions, such as surface stress, grain size, and roughness, on the growth of

FGLNAs was analyzed. Larger initial compressive stress, optimum grain size, and surface roughness were beneficial for the formation of FGLNAs. Compared with Beta adrenergic receptor kinase other methods for fabricating Cu2O FGLNAs, the thermal oxidation method featured remarkable simplicity and cheapness. Acknowledgements This work was supported by the Japan Society for the Promotion of Science under a Grant-in-Aid for Scientific Research (A) 23246024. References 1. Xiong YJ, Li ZQ, Zhang R, Xie Y, Yang J, Wu CZ: From complex chains to 1D metal oxides: a novel strategy to Cu 2 O nanowires. J Phys Chem B 2003, 107:3697–3702.CrossRef 2. Caballero-Briones F, Palacios-Padros A, Calzadilla O, Moreira I d PR, Sanz Fausto : Disruption of the chemical environment and electronic structure in p-type Cu 2 O films by alkaline doping. J Phys Chem C 2012, 116:13524–13535.CrossRef 3. Akkari FC, Kanzari M: Optical, structural, and electrical properties of Cu 2 O thin films. Phys Status Solidi A 2010, 207:1647.CrossRef 4.

7% of total) bared high/positive BRCA1. An interesting conclusion was found: opposite to platinum-based

treatment, NSCLC patients bearing high/positive BRCA1 were more likely to respond to toxal-based treatment when compared with those bearing the low/negative (low/negative vs high/positive: 26.0% vs 46.1%, OR = 0.41, 95%CI = 0.27-0.64, I2 = 0.0%, P = 0.61 for heterogeneity) selleck chemicals llc (Figure 5). No publication bias existed (P = 0.84). Table 2 The summary meta-analysis results of association between BRCA1 level with objective response rate (ORR), overall survival (OS) and event-free survival (EFS) in platinum- and toxal-based treatment Comparisons No of studies (patients) Percentage of low/negative BRCA1 (%) ORR: low/negative vs high/postive (%) Overall OR/HR (95% CI) fixed and random Heterogeneity test P for publication bias Platinum-based             ORR overall 16(1330) 44.4 48.9 vs 38.1 1.70 (1.32, 2.18), 1.80(1.26,2.55) I 2 = 44.7%,P = 0.03 0.15 Method        

    IHC 13(1066) 44.5 50.7 vs 39.0 1.54(1.17,2.00), 1.59(1.07,2.36) I 2 = 44.8%,P = 0.03 0.41 RT-PCR 4(264) 44.3 43.7 vs 25 2.91 (1.55, 3.83), 2.91(1.55,5.47) I 2 = 0.0%, P = 0.52 0.76 Origin             East-Asian 14(1133) 45.4 51.0 vs 36.0 1.68(1.30,2.19), 1.79(1.24,2.60) I 2 = 39.9%,P = 0.04 0.10 Caucasian 3(197) 38.6 39.8 vs 33.4 1.79 (0.84, 3.83), 1.77(0.50,6.28) I 2 = 63.6%,P = 0.06 0.90 OS 8(733) – - 1.58(1.27,1.97), 1.65(1.19,2.89) I 2 = 48.4%,P = 0.03 0.13 EFS 6(599) – - 1.62(1.28,2.05), 1.60(1.07,2.39) I 2 = 54.5%,P = 0.02 0.88 Toxal-based             ORR overall 4(376) GSK1120212 cost 41.3 26.0 vs 46.1 0.41(0.26,0.64), 0.41(0.27,0.64) I 2 = 0.0%, P = 0.61 0.84 Discussion Although the relationship between

BRCA1 expression and chemotherapy outcomes of NSCLC has been investigated by previous studies, the results were inconsistent and some were even conflicting. So a systematic review and meta-analysis based on the published literature was necessary to give further insights on this conflicting issue. Our meta-analysis showed that for platinum-based chemotherapy, low/negative BRCA1 expression were associated with not only better ORR, but also longer OS and EFS, but for toxal-based chemotherapy, high/positive BRCA1 was associated FER with better ORR. Platinum agents can bind to DNA and form complexes thus inducing intra- and inter-strand DNA, as well as DNA-protein cross-links and results in cell growth inhibition and apoptosis. As one of ant-tubulin agents, taxol inhibits cell division by enhancing formation and stabilization of microtubules and disrupts the mitotic spindle assembly, and a surveillance mechanism known as the spindle checkpoint at the metaphase-anaphase transition have been activated.

For the determination of steroids binding activity, the medium was discarded and the cells were washed twice with ice-cooled HBSS (0.14 M NaCl, 5.4 mM KCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 5.6 mM glucose, 1 mM CaCl2, 6 mM Compound C nmr HEPES, 4 mM NaHCO3 pH 7.4). Cells were then harvested using a cell scraper and pelleted by centrifugation. Steroid binding activity was determined in homogenised COS-7 cell extracts prepared by re-suspending cell pellets in 10 mM Tris, 250 mM sucrose pH 7.4 buffer and disruption using a Turrax homogenisor. The homogenate was then centrifuged at 13,000 g for 5 minutes at 4°C. The supernatant was retained and assayed for protein concentration using the method

of Lowry and binding activity using 100 nM [3H]dexamethasone with or without excess unlabelled dexamethasone. After overnight incubation on ice, free ligand was removed by charcoal dextran adsorption and bound ligand determined in supernatants by liquid scintillation essentially, as previously described [9–11]. Westerns Western Blotting was performed after SDS-PAGE under reducing conditions using a MiniP2 Biorad electrophoresis apparatus. Protein was transferred onto nitrocellulose and blocked overnight with 3% (w/v) milk protein/0.3%

(w/v) Tween 20. Antibody raised against the C-termini of CYP3A1/3A23 (IITGS) was used, as described previously Small molecule library price [11]. The anti-α-smooth muscle actin and anti-β-actin (cross reacts with all actin isoforms) antibodies were purchased from the Sigma Chemical Co (Poole, UK) and Chemicon (Chandlers Ford, UK), respectively. The anti-CYP2E1 and anti-LAGS (IZ-Ab) Montelukast Sodium antibodies were obtained from Prof. M. Ingelman-Sundberg, Karolinska Institutet, Stockholm, Sweden, and Prof. Gavin Vinson, Queen Mary College, London, UK. After incubation with primary antibodies, blots were incubated with the appropriate horseradish peroxidase conjugated anti-IgG antibody. Detection was accomplished using chemiluminescence with the ECL kit (Amersham).

Microsomal receptor-ligand binding assay Rat liver microsomes were prepared and incubated with [3H] dexamethasone to determine LAGS activity, as previously outlined [9–11]. In brief, rats were anaesthetized with pentobarbital and a 16G cannula inserted into the hepatic portal vein and secured. The blood was cleared from the liver by pumping ice-cooled perfusion buffer (0.14 M NaCl, 5.4 mM KCl, 0.34 mM Na2HPO4, O.44 mM KH2PO4, 15.7 mM NaHCO3 and 5.6 mM glucose, pH 7.4) through the liver at 50 mls per minute. The liver was then excised and chopped roughly with ice-cooled TS buffer (10 mM Tris/HCl pH 7.4 containing 250 mM sucrose) and disrupted using a Potter-Elvehjem homogenisor. The resultant homogenate was then centrifuged at 12,000 g for 20 minutes at 4°C and the supernatant retained and centrifuged at 100,000 g for 60 minutes at 4°C.

As we can see from the SEM images with low magnification, the cell concentration with N 8.67% (Figure 5b,e) is significantly less than that with N 9.28% (Figure 5c,f), which is consistent with the results given by Figure 4 and Figure 5a,d. And, the adhered cells all spread flat with richer pseudopod and microvilli, as shown at a high magnification. These results add to growing evidence that the increase of nitrogen content

promoted cell adherence and growth. The ability of substrates to promote adhesion of cells depends on how well they adsorb proteins from the culture medium that interact with receptors on the cell surface [31]. Adsorption of proteins in an active conformation, in turn, is likely to be affected by the functional groups of the substrate. All proteins have NH2 and COOH groups at the ��-Nicotinamide solubility dmso ends, where the NH tends to be positively charged and the COOH negatively charged [32]. Thus, a surface with an organized arrangement of functional groups can act as a site for cell growth.

The formation of functional sp 2 C-N and sp 3 C-N bonds on the N+-bombarded MWCNTs by N ion beam bombardment induces polarization at Cediranib the surface due to the difference in electronegativity between carbon and nitrogen [33]. In addition, from the XPS results (Figure 1d,e,f), it is clear that with the increase of nitrogen concentration, the ratio of the sp 2 C-N bond decreases and the sp 3 C-N bond increases while the unsaturated degree of the N bond increases. Therefore, the number of protein attached on the material’s surface increases with increasing unsaturated degree of the N bond, and adhesion of cells are promoted. Blood platelets are anucleated cells that originate from bone marrow megakaryocytes and circulate in the blood as

sentinels for vascular integrity [34]. Platelets play a vital role in hemostasis; however, derangement of their functions can lead to thrombosis, which is a leading cause of death and disability in the developed world [35]. Figure 6 displays the statistical results of the platelets adhered on the surfaces of three N+-bombarded MWCNTs with different nitrogen content and the glass with and without methylsilicone oil. Each value represents the mean ± SD for five measurements. And, each experiment is performed three times. From the Isotretinoin average platelet adhesion rates, it is observed that the number of adherent platelets decreases with increasing nitrogen concentration. In addition, as shown in Figure 7c,d, the platelets show less pseudopodium as demonstrated by the isolated and nearly round state when the nitrogen concentration is higher. The morphology of the red blood cell (RBC) on N+-bombarded MWCNTs is perfect round. It is demonstrated that higher nitrogen concentration is contributive to the improvement of hemocompatibility. Figure 6 Platelet adhesion rates on the different materials. Figure 7 SEM images of platelet adhesion testing for N + -bombarded MWCNTs. Nitrogen contents are (a, b) 8.67% and (c, d) 9.28%.

Federal crop insurance programs The additional support programs available for all farmers are important for the continuing success of non-program crops. These programs provide assistance for the development, commercialization, and continuation of farms and provide incentives for environmentally sound farming practices. The largest of these programs, in which all farmers (including those of aquaculture and livestock) can participate, is the selleck inhibitor crop insurance program. The original crop insurance program began in 1938 and only covered major crops (Agricultural Adjustment Act of 1938, 1938), but the passing of the Federal

Crop Insurance Act of 1980 expanded the program to be universal (Federal Crop Insurance Act of 1980, 1980). Crop insurance is run by the USDA Risk Management Agency (RMA) and paid for by the separate Federal Crop Insurance Corporation (FCIC). Over 100 crops are currently eligible for the Federal Crop Insurance (FCI) program, in which farmers pay a subsidized premium for insurance delivered by private companies. While program crops are eligible for revenue-based CCI-779 cost loss insurance, specialty

crops typically only participate in physical crop-loss insurance. If a crop is ineligible for the program, then it can still be insured through the Non-insured Crop Disasters Assistance program, established in the 1996 farm bill and run by the Farm Service Agency (FSA), which functions similarly to FCI (Federal Agriculture Improvement Methocarbamol & Reform Act of 1996, 1996). Sea grass, a similar crop to algae that requires a blend of agriculture and aquaculture, is eligible for Non-Insured Crop Disasters Assistance (FSA 2011). Additional insurance support is available for all farmers to cover losses from natural disasters under the Supplemental Revenue Assurance Program. This program provides additional assistance beyond crop insurance to farmers who experience a decrease in revenue due to natural disasters and is only available for crops that are enrolled in one of the crop insurance

programs. The expansion of crop insurance programs to specialty crops, aquaculture, and livestock was important for the development and protection of these industries. Farms of these commodities are all affected by the same environmental factors as those of program crops, such as lower-than-expected production due to droughts, natural disasters, soil quality, water availability, etc. The farming of algae is equally susceptible to different but similar factors that affect biomass and crop yields. Farm loan programs Farm loans are essential in successful agriculture as up-front capital is needed to make purchases of inputs such as fertilizer, equipment, land, etc. Most farm loans are authorized by the Consolidated Farm and Rural Development Act (1961) and can be in the form of direct loans, guaranteed loans or emergency loans.

2001). The summer or southwest monsoon brings heavy rain from the warm Indian Ocean PLX-4720 mw from June through August. In contrast, the typically drier northeast monsoon winds blow in the reverse direction from January through March. Between the two monsoons, or following the summer monsoon if there is only one, there is a hot dry season of 1–7 months duration (December through May is typical). Plant distribution and phenology is associated with rainfall seasonality and variability, and animals in turn tend to track plant productivity (see Brockelman

2010 for a recent discussion of the implications of seasonality at one site). This annual monsoonal pattern has been disrupted by ENSO events every 4–6 years (during in the 20th century) that are associated with drought and increased fire frequency (e.g., 1997–8, 2006–7) (Berger 2009; Taylor 2010). There are also super-droughts, some associated with ~40 year global drought cycles and others with 10–15 years concordance of ENSO and Indian Ocean dipole cycles. It is in this setting that Wallace first recognized the four zoogeographic subregions and the major zoogeographic transition between Oriental and Australian regions. That transition, which lies between the Sundaic and Wallacean subregions, is associated with Makassar Strait, which

serves as a marine barrier to the dispersal of land animals between Borneo and Sulawesi. This Strait is better known as the Liothyronine Sodium location of Wallace’s Line and is discussed at great length elsewhere (Whitmore 1987; Hall and Holloway 1998; Metcalfe ARN-509 in vitro et al. 2001; Hall et al.

2010; Gower et al. 2010). Plants show a different pattern with a significant transition between Continental Asiatic and Malesian floral regions occurring, not at Wallace’s Line, but at a line drawn between Kangar (Malaysia) and Pattani (Thailand) on the peninsula near the Thai-Malay border (van Steenis 1950) (Fig. 1). The Malesian floral region encompasses the peninsula south of the Kangar-Pattani Line and all of the islands of Southeast Asia from Sumatra to the Philippines and New Guinea (Morley 2000; Wikramanayake et al. 2002). The Malesian forests differ from the Indochinese in having far more species and series of ecologically sympatric congeneric species (especially dipterocarps), and the tendency to exhibit synchronous mass [mast] fruiting. To locate the Malesian-Asian transition van Steenis used distribution maps for 1,200 genera of plants; he found that 375 genera of Sundaic plants reach their northern limits, and 200 genera of Indochinese plants reach their southern limits, at the Kangar-Pattani Line at 6–7°N. This transition is twice the magnitude to that occurring in plants at Wallace’s Line.

This difference in the distribution of environmental/animal and human clinical strains was statistically significant (P value = 5.10-4) for the 3 main clades and for the A. veronii (P value = 0.02) and A. caviae (P value = 0.05)

clades. Finally, a non-random eFT508 in vivo distribution of strains was observed among the different CCs according to their site of isolation and/or colonizing/pathogenic status. CC “C” grouped 3 out of the 5 non-pathogenic, colonizing A. caviae strains in the dataset, and this rate was significantly different from that of the non-pathogenic A. caviae strains found outside of the CC (P value = 0.04) (Table 1, Figure 1). In contrast, some other clusters included strains involved only in infectious processes (Table 1, Figure 1). Finally, the A. veronii ST13 cluster appeared to be associated with a particular type of disease, i.e., wound infection. Indeed, 5 out of the 12 A. veronii strains in the dataset involved in wound infection were grouped into this cluster, representing a frequency that was significantly different from the rest of the A. veronii population (P value = 0.0001). Recombination events in the aeromonad population The sIA value was 0.30 at the genus level, ranged from 0.15 to 0.42 at the clade level and was significantly different from 0, indicating Protein Tyrosine Kinase inhibitor the existence of significant linkage disequilibrium, showing

that the studied Aeromonas population was not panmictic but clonal. Events of recombination among the clonal population were then analyzed via RDP, decomposition analysis and phylogenetic incongruence. Considering the recombination events detected using at least 4 methods of the RDP software, 14 types of recombination events leading to 166 recombinant sequences were detected among the population and are detailed in an additional table (Additional file 2: Table S2). All but two loci (radA and rpoB) were affected by recombination events that occurred in 89

STs (50.9%). dnaK and gyrB were the most affected loci (4 events each, 75 and 13 recombinant sequences, respectively), Cytidine deaminase followed by tsf and zipA (3 events each, 73 and 5 recombinant sequences, respectively) and gltA (1 event and 3 recombinant sequences) (data not shown). One to four types of significant recombination events occurred in most clades, except for the A. hydrophila, A. piscicola and A. tecta clades and the A. fluvialis type strain and strain CCM 1271. Five events could not be significantly linked to parental sequences, suggesting the occurrence of transfer from strains that are not represented in our collection. Recombination was also investigated for the 3 main clades via split decomposition in the concatenated sequences (Additional file 3: Figure S3 a-c). Most of the STs were not affected by recombination, and the trees showed a limited parallelogram formation, notably including A. hydrophila STs (Additional file 3: Figure S3 b).

The isthmal epithelium of the oviduct was washed extensively with HBSS containing 200 U/ml penicillin and 200 mg/ml streptomycin and treated with 20 ml of HBSS containing 1 mg/ml collagenase (Sigma) for 30 min at 37°C. Following collagenase treatment, the supernatant was discarded and the tissue fragments were digested three

times with 0.25% trypsin and 3 mM EDTA in 20 ml of HBSS for 10 min at 37°C. The cells suspension was supplemented with 10% of heat-inactivated fetal bovine serum (FBS) to stop the activity of trypsin. I-BET151 datasheet To remove undigested tissue clumps, the cell suspension was passed through cell strainers (100-micro pores). To separate epithelial cells, which quickly formed

cell aggregates, from erythrocytes, platelets, and other immune cells, the cell suspension was centrifuged at 50 × g for 5 min. Following centrifugation, supernatant containing fibroblasts, erythrocytes, and immune cells, was discarded and the loose pellet containing epithelial cells and cell sheets was resuspended in 20 ml of HBSS. After three low-speed centrifugations, the cell pellet was resuspended in minimal essential FHPI in vivo medium (MEM, ATCC) supplemented with 10% FBS, 2% heat-inactivated chicken serum (CS), insulin (0.12 U/ml), and estradiol (50 nM). The COEC cells were incubated in Petri dishes for 2 h at 39°C in 5% CO2 to allow fibroblast cells to attach. Following incubation, epithelial cells were collected by http://www.selleck.co.jp/products/Abiraterone.html gentle pipetting and subsequent centrifugation at 125 × g for 10 min. The pelleted epithelial cells were resuspended in fresh MEM medium and seeded into 48-well tissue culture plates at a density of approximately 8 × 104 cells per well and incubated for 24 h to 48 h at 39°C in 5% CO2 until infection took place. Immunohistochemistry COEC cultures were incubated with monoclonal anti-pan cytokeratin

mouse Ab (epithelial cell marker) for 2 h at 37°C, washed three times, then incubated with fluorescein isothiocyanate (FITC) anti-mouse IgG for 1 h at 37°C. Staining of cytoskeleton of COEC was viewed with an Olympus IX81 FA scope. Cultures with more than 80% of cytokeratin-positive cells were used in subsequent infections. Thus, the COEC preparations consisted of more than 80% epithelial cells, less than 20% fibroblast, and possibly residual amount of immune cells. Infection of cell culture Infections were conducted using the gentamicin protection method as described previously [25]. Prior to inoculation, cell cultures were washed 3 times with pre-warmed Hanks’ Balanced Salt Solution (HBSS) without antibiotics. For each bacterial strain/time point combination, 500 μl of bacterial suspension containing approximately 16 to 24 × 105 CFU was added into each of the six wells to reach a multiplicity of infection (MOI) of 20:1 to 30:1 (bacteria:cells).