3); South Tarawa, Kiribati (DLF 1995); Alofi, Niue (DLF 1995) The island typology can provide a template (checklist) of potential hazards and the nature of potential impacts, but our review has highlighted the critical importance of local place-based

analysis of the coastal biophysical and social-ecological systems. Understanding shoreline stability BV-6 in vitro on atoll islands and projecting long-term land availability under various climate-change scenarios requires detailed data on coastal morphology, including high-resolution digital elevation models, and on the processes that drive coastal change. In this context, Woodroffe (2008) pointed to a number of specific knowledge requirements. He noted the need to watch for thresholds

that might lead to major transformations in the nature and stability of reef and shore systems. Webb and Kench (2010), reporting an analysis of multi-decadal island shoreline change, concluded that “island nations must SRT2104 cell line place a high priority on resolving the precise styles and rates of change that will occur over the next century and reconsider the implications for adaptation”. In another context, evaluating the stability and size of potential tsunami-generating landslide blocks on heavily forested volcanic island slopes in Dominica, Teeuw et al. (2009) identified mapping with suitable tools as a prime requirement. Other critical data needs have also emerged from this study. It is evident that

measurements of vertical crustal motion are a prerequisite for robust projections of future sea levels at any specific island site (Fig. 11). Niclosamide Long-term water level records from tide gauges are equally important, even when complemented by satellite altimetry (Davis et al. 2012). Yet the network of GNSS stations on islands worldwide is extremely sparse and the number of co-located GNSS and tide gauges is even smaller. Even where data are available, as at many of the 18 sites used for SLR projections in this study (Fig. 1), continuity is a challenge and very few islands are represented in the active network of the International GNSS Service (http://​www.​igs.​org/​network/​netindex.​html). EPZ5676 solubility dmso Conclusions Realistic physical hazard and impact projections are a prerequisite for effective adaptation planning. The hazard mix and severity may vary with island type and regional setting. There is a need for monitoring of evolving physical exposure to provide objective data on island responses and early warning of changing risk. Reef islands may be resilient under rising sea level, at least at rates experienced during the twentieth century, maintaining island area but not necessarily fixed shoreline positions. The latter has implications for land ownership, property boundaries, and shorefront infrastructure. Coastal stability requires maintenance of healthy coastal ecosystems, particularly in tropical regions where organisms produce sand.

90 and 0.95 of the maximum density K R The solid gray line describes the prediction for maximum density K T being a fraction of 0.80 of K R . Also, the experimental results of the long term experiment 3 did not show a decrease in the proportion of T in comparison to T + R (Figure 3). This means that the population of T did not decline more than 10 fold compared to T + R, which would have been visible. Because the experiment did not allow distinction between T alone and R + T together, we

cannot determine if R was replaced or if R and T coexisted with R at low numbers. Discussion Fitness costs resulting in a lower bacterial growth rate or a lower maximum density due to the presence of the plasmid IncI1 selleck chemical carrying the bla CTX-M-1 gene were not observed here. No differences were found between donor D, recipient R and transconjugant T in growth rate ψ, maximum density PD0332991 clinical trial K or lag-phase λ in single population experiments 1a-j. Fitness costs might have arisen in a

competition setting with mixed populations of D and R[19] due to competition for resources or inhibition by the competitor. However, also in the mixed populations of the conjugation experiments 2a-b, we could not find a difference in growth parameters BAY 57-1293 clinical trial between the recipient R and donor D. San Millan et al.[20] neither found a difference in percentage of plasmid free and plasmid carrying bacteria for their pB1000 plasmid in the first 12 hours. However, starting at day 2 they observed a clear decrease in Cytidine deaminase the fraction of plasmid carrying bacteria. Also in our experiments, the fitness costs of the plasmid carrying bacteria were not evident in the early phase. Small fitness costs may not be observable at all in experiments with a short duration, but when the experiments are maintained longer, fitness costs other than costs related to the growth rate can play a role. In

12 or 24 hours experiments, these differences might be too small to measure. This is why we conducted the long term experiment 3 both with intervals of 24 and 48 hours, as the duration of our experiments 1 and 2 (up to 24 hours) may have been too short to observe fitness costs. We showed by simulation (illustrated in Figure 3) that only for large fitness costs resulting in a 20% smaller maximum density K by carrying the IncI1 plasmid, a distinct decrease in population size would have been observed within the time-frame of experiment 3. This was, however, not observed in experiment 3, underlining the conclusion that this plasmid does not infer sufficient fitness costs to its host bacterium to let it go extinct in the absence of antimicrobials. Thus, our results suggest that reduction of the use of antimicrobials might not result in a decrease, let alone extinction, of such a plasmid. This is in accordance with the conclusions of Poole et al.[21].

J Dairy Res 2006, 73:417–422.CrossRef 15. Fallingborg J: Intraluminal pH of the human gastrointestinal tract. Danish Med Bull 1999, 46:183–196.PubMed 16. Fallingborg J, Christensen LA, Jacobsen BA, Ingeman-Nielsen M, Rasmussen HH, Abildgaard K, et al.: Effect of olsalazine and mesalazine on intraluminal pH of the duodenum and proximal jejunum in healthy humans. Scan J Gastroenterol 1994, 29:498–500.CrossRef 17.

Fallingborg J, Pedersen P, Jacobsen BA: Small intestinal transit selleck chemical time and intraluminal pH in ileocecal resected patients with Crohn’s disease. Digestive Dis Sci 1998, 43:702–705.CrossRef 18. Andres MR Jr, Bingham JR: Tubeless gastric analysis with a radiotelemetering pill (Heidelberg capsule). Can Med Assoc J 1970, 102:1087–1089.PubMed 19. Fallingborg J, Christensen LA, Ingeman-Nielsen M, Jacobsen BA, Abildgaard K, Rasmussen HH: pH-profile and regional transit times learn more of the normal gut measured by a radiotelemetry device. Aliment Pharmacol Ther 1989, 3:605–613.CrossRefPubMed 20. Fallingborg J, Christensen LA,

Ingeman-Nielsen M, Jacobsen BA, Abildgaard K, Rasmussen HH, et al.: Measurement of gastrointestinal pH and regional transit times in normal children. J Ped Gastroenterol Nutr 1990, 11:211–214.CrossRef 21. Huang Y, Adams MC: In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria. Int J Food Microbiol 2004, 91:253–260.CrossRefPubMed 22. Mojaverian P: Evaluation of Gastointestinal pH and Gastric Residence Time via the Heidelberg Radiotelemetry Capsule: Pharmaceutical Application. Drug Devel Res 1996, 38:73–85.CrossRef 23. Thews G, Mutscheler E, Vaupel E: Anatomie, Physiologie, Pathophysiologie des Menschen (4. Auflage) 1991. 24. Driessche M, Van Malderen N, Geypens

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This was caused by severe scoliosis (n = 17), Torin 1 chemical structure inability to lay supine because of clinical condition (n = 23), severe adiposity (n = 5), and miscellaneous reasons (n = 31). In the remaining 2,424 patients, VFA was considered reliable. Image quality was subjectively scored as “good” in 2097 (87%), “moderate”

in 294 (12%), and “poor” in 33 patients (1%), and was based on assessment of the whole image. Despite “poor” or “moderate” VFA image quality results in those patients were considered sufficiently reliable to allow analysis. The levels that were adequately visualized by VFA were from vertebra L4 up through vertebra T4 in 1,991 (82%) patients,

from L4 through T5 in 2,247 (93%), and from L4 through T6 in 2,402 (99%). In total, around 30,000 vertebral bodies were analyzed. Vertebral Fracture Assessment results 17-AAG supplier VFA demonstrated a vertebral fracture in 541 (22%) of the patients. An example is presented in Fig. 1. These 541 patients together had 954 vertebral fractures, which amounts to a mean of 1.8 selleck kinase inhibitor fractures per patient with a fracture. In 375 patients (69% of those with a fracture, or 16% of the whole cohort), these fractures were not demonstrated earlier and were unknown according to the patient. Fig. 1 Example of a VFA study result with left the image after placing marker points, upper right the Genant classification and lower a table with the percentages of deformity. In this patient, one moderate vertebral fracture was detected: wedge shaped in L1 The distribution of the fractures over the individual vertebral levels showed the well-known dual-peak distribution with a peak

at T7 (119 fractures, 13% of total) and at T12 (169 fractures, 18% of total) (Fig. 2). The severity of the fractures was “mild” in 458 (48% of all fractures), “moderate” in 295 (31%), and “severe” in 201 (21%). Vertebral fractures were wedge shaped in 79% (n = 759), biconcave in 19% (n = 178) see more and “crush” in 2% (n = 17). Mild fractures were often accompanied by moderate or severe fractures, and on a per patient analysis 219 patients (9% of all patients) had mild fractures only. Fig. 2 Frequency distribution of vertebral fractures assessed with VFA As there has been controversy in the definition of mild fractures we also analyzed the data for moderate and severe fractures only, after excluding mild fractures. The prevalence of moderate or severe vertebral fractures was 322 (13%) in this cohort, 180 (56%) were unknown.

Am J Pathol 2003, 162:1139–1149.PubMed 52. Korkolopoulou P, PLX4032 cell line Goudopoulou

A, Voutsinas G, Thomas-Tsagli E, Kapralos P, Patsouris E, Saetta AA: c-FLIP expression in bladder urothelial carcinomas: its role in resistance to Fas-mediated apoptosis and clinicopathologic correlations. Urology 2004, 63:1198–1204.PubMed 53. Ohta T, Elnemr A, Kitagawa H, Kayahara M, Takamura H, Fujimura T, Nishimura G, Shimizu K, Yi SQ, Miwa K: Fas Trametinib ligand expression in human pancreatic cancer. Oncol Rep 2004, 12:749–754.PubMed 54. Ho SY, Guo HR, Chen HH, Hsiao JR, Jin YT, Tsai ST: Prognostic implications of Fas-ligand expression in nasopharyngeal carcinoma. Head Neck 2004, 26:977–983.PubMed 55. Osaki M, Kase S, Kodani I, Watanabe M, Adachi H, Ito H: Expression of Fas and Fas ligand in human gastric adenomas and intestinal-type carcinomas: correlation with proliferation and apoptosis. Gastric Cancer 2001, 4:198–205.PubMed 56. Kase H, Aoki Y, Tanaka K: Fas ligand expression in cervical adenocarcinoma: find more relevance to lymph node metastasis and tumor progression. Gynecol Oncol 2003, 90:70–74.PubMed 57. Younes M, Schwartz MR, Ertan A, Finnie D, Younes

A: Fas ligand expression in esophageal carcinomas and their lymph node metastases. Cancer 2000, 88:524–528.PubMed 58. Bennett MW, O’Connell J, O’Sullivan GC, Roche D, Brady C, Kelly J, Collins JK, Shanahan F: Expression of Fas ligand by human gastric adenocarcinomas: Montelukast Sodium a potential mechanism of immune escape in stomach cancer. Gut 1999, 44:156–162.PubMed 59. Bernstorff WV, Glickman JN, Odze RD, Farraye FA, Joo HG, Goedegebuure PS, Eberlein TJ: Fas (CD95/APO-1)

and Fas ligand expression in normal pancreas and pancreatic tumors. Implications for immune privilege and immune escape. Cancer 2002, 94:2552–2560.PubMed 60. Ibrahim R, Frederickson H, Parr A, Ward Y, Moncur J, Khleif SN: Expression of FasL in squamous cell carcinomas of the cervix and cervical intraepithelial neoplasia and its role in tumor escape mechanism. Cancer 2006, 106:1065–1077.PubMed 61. O’Connell J, Bennett MW, O’Sullivan GC, Roche D, Kelly J, Collins JK, Shanahan F: Fas ligand expression in primary colon adenocarcinomas: evidence that the Fas counterattack is a prevalent mechanism of immune evasion in human colon cancer. J Pathol 1998, 186:240–246.PubMed 62. Gastman BR, Atarshi Y, Reichert TE, Saito T, Balkir L, Rabinowich H, Whiteside TL: Fas ligand is expressed on human squamous cell carcinomas of the head and neck, and it promotes apoptosis of T lymphocytes. Cancer Res 1999, 59:5356–5364.PubMed 63. Niehans GA, Brunner T, Frizelle SP, Liston JC, Salerno CT, Knapp DJ, Green DR, Kratzke RA: Human lung carcinomas express Fas ligand. Cancer Res 1997, 57:1007–1012.PubMed 64.

CHIR98014 surface smooth, with rare remnants of short, collapsed, brownish hyphae. Cortical layer (14–)16–26(–33) μm (n = 30) wide, a distinct, yellow t. angularis of isodiametric to oblong, thick-walled, angular cells (4–)6–11(–13) × (3–)4–8(–10) μm (n = 60) in face view and in vertical section. Cortex turning bright orange in KOH.

Subcortical tissue a pale yellowish t. angularis of thin-walled cells (4–)5–11(–16) × (3–)3.5–6(–7) μm (n = 30), mixed with scant, subhyaline to yellowish hyphae (2.5–)3–5(–6) μm (n = 30) wide. Subperithecial tissue a hyaline to yellowish t. epidermoidea of thin-walled cells (6–)10–28(–42) × (4–)7–15(–19) μm (n = 30), extending into the substrate. Asci (50–)60–75(–85) × (3.3–)3.8–4.7(–5.5) μm, stipe (1–)5–15(–25) μm ACY-1215 long (n = 80); fasciculate on long ascogenous hyphae. Ascospores hyaline,

often yellow or orange after ejection, SAHA HDAC nearly smooth to minutely verruculose, cells dimorphic; distal cell (2.5–)2.8–3.2(–3.5) × (2.3–)2.5–3.0(–3.2) μm, l/w (0.9–)1.0–1.2(–1.4), (sub-)globose or oblong; proximal cell (2.8–)3.3–4.2(–5.0) × (1.8–)2.2–2.5(–2.8) μm, oblong or wedge-shaped (or subglobose), l/w (1.2–)1.4–1.8(–2.3) (n = 100). Anamorph on natural substrate observed as a white, thin, loose, crumbly layer in association with stromata; dense conidial heads on small regular conidiophores with 1–3(–4) terminal phialides. Phialides (6–)8–15(–17) × (2.5–)3–4(–4.1) μm, l/w (2–)2.5–4.3(–5.4), (1.9–)2.2–2.8(–3.1) μm (n = 20) wide at the base, lageniform, pointed, straight to sinuous, often collapsed. Conidia (2.8–)3.0–4.5(–5.6) × (2.3–)2.4–3.0(–3.6)

μm, l/w 1.2–1.6(–2.4) (n = 30), hyaline, mostly subglobose to pyriform, less commonly broadly ellipsoidal or oblong, smooth, scar sometimes distinct. Cultures PRKACG and anamorph: optimal growth at 25°C on all media, at 30°C hyphae soon dying after onset of growth; no growth at 35°C. On CMD after 72 h 5–8 mm at 15°C, 7–10 mm at 25°C, 0–3 mm at 30°C; mycelium covering the plate after ca 2 weeks at 25°C. Colony hyaline, thin, smooth, homogeneous, not zonate. Mycelium loose, little on the surface; hyphae generally narrow, curly, without specific orientation. Margin ill-defined, diffuse, of solitary strands. Aerial hyphae infrequent, loose, thick, becoming fertile. Surface becoming indistinctly downy by conidiation mainly on the distal and lateral margins. Autolytic activity moderate to strong, coilings abundant. Sometimes fine whitish granules 0.5–0.7 mm diam of aggregated conidiophores with dry conidiation appearing in distal and lateral areas of the plates. No chlamydospores seen, but globose or irregularly thickened cells appearing in surface hyphae in aged cultures. Conidia swelling on the agar surface forming clumps, probably wrapped in an excreted substance. Agar hyaline, sometimes becoming faintly yellowish, 2AB3.

The KU-60019 datasheet sensitization effect of saikosaponin was mainly through enhancing the cisplatin-induced apoptosis, which was accompanied by enhanced activation of caspase 3 and the cleavage of caspase 3 substrate PARP, and was blocked by the caspase inhibitor z-VAD. It is noteworthy that Siha cell, which is a well known cervical cancer cell line resistant

to cisplatin, was significantly sensitized to cisplatin-induced cell death, suggesting that saikosaponins are potent adjuvant that are able to override primary cisplatin resistance in cancer. Thus, results from this study reveal a novel function of saikosaponins that adds up the anticancer value of these naturally occurring compounds. Many naturally occurring compounds have been reported to exert anti-cancer effect through BAY 63-2521 in vivo ROS induction. For example, d-Limonene, a bioactive food component from citrus, was found to augments the cytotoxic effects of docetaxel through induction of cellular H2O2 [25]. Our finding in this study also showed that both SSa and SSd induced significant cellular ROS accumulation in cancer cells, which substantially contribute to synergistic cytotoxicity in saikosaponin and cisplatin cotreated cell. It was previously found that saikosaponins exhibit antioxidant activity in normal hepatocytes [24]. The reason of discrepancy is currently unclear, but could be explained by differences in cellular contents. Indeed, redox regulating compounds such

as flavonoid luteolin can function as an antioxidant in normal cells while as a pro-oxidant

in cancer cells [26]. It remains to be determined that how distinct redox modulating functions are executed in normal and cancerous condition. Conclusion Our results suggest that saikosaponin-a and -d are potent in sensitizing cancer cells to cisplatin-induced apoptosis through ROS accumulation. Thus, the combination of saikosaponins with cisplatin could increase the therapeutic effect of cisplatin against solid tumors. Acknowledgements This study was supported in part by grants 30772539 and 30973403 from National Natural Science R406 Foundation of China and by a grant from the Scientific Research Foundation for the Returned Overseas Chinese Scholar, State Education Ministry of China. Electronic supplementary material Additional file 1: Figure S1. Saikosaponins Forskolin order induce intracellular ROS accumulation in Siha cells, A549 cells, and SKOV3 cells. Siha cells, A549 cells, and SKOV3 cells were treated with saikosaponin-a (10 μM) or saikosaponin-d (2 μM) for 30 min respectively and stained with 5 μM of CM-H2DCFDA. The fluorescent intensities were detected by flow cytometry. (JPEG 46 KB) References 1. Bermejo Benito P, Abad Martinez MJ, Silvan Sen AM, et al.: vivo and in vitro antiinflammatory activity of saikosaponins. Life sciences 1998, 63 (13) : 1147–56.PubMedCrossRef 2. Dang SS, Wang BF, Cheng YA, Song P, Liu ZG, Li ZF: Inhibitory effects of saikosaponin-d on CCl4-induced hepatic fibrogenesis in rats. World J Gastroenterol 2007, 13 (4) : 557–63.PubMed 3.

All these observations indicate a rearrangement of the Si buy MK-4827 nitride network toward that of

the stoichiometric structure with a lower structural disorder. This can be due to a phase separation between Si and Si nitride. Figure 6 Effect of the annealing temperature on the FTIR spectra of SiN x . The FTIR spectra were recorded under normal incidence (a) and with an angle of 65° (b). Raman spectroscopy Figure 7 shows LDN-193189 research buy the evolution of the Raman spectra of SiN x thin layers deposited on fused silica with various Si contents and with various annealing temperatures. Again, it is seen that the evolution of the Raman spectra does not depend on the deposition methods but only on the composition that is set by n. Upon annealing at 900°C, the two broad vibration bands of the transverse acoustic (TA) phonon and of the TO

phonon of a-Si at 150 and 480 cm−1, respectively, became clearly narrower and more pronounced (Figure 7). This evolution can be explained by the formation of small amorphous Si-np [45]. Unlike this deduction, the appearance of new sharp peaks slightly shifted towards lower wavenumbers compared to bulk crystalline Si (c-Si) at approximately 520 cm−1 upon annealing at 1100°C as shown in Figure 7b, which unequivocally demonstrates the formation of small crystalline Si-np. Besides, the formation of a c-Si phase is also consistent with the appearance of a weak peak at 300 cm−1 that is attributed to the second order of the transverse acoustic (2TA) phonon mode in the thin films containing a high Si content (n = 2.89 and 2.98). It is seen PCI-32765 in vivo GBA3 that the condensation of the excess of Si in small crystalline Si-np during the annealing at 1100°C occurs but only in thin films having a refractive index higher than 2.5 (Figure 7b) or maybe equal to 2.5 as indicated

by the presence of a weak shoulder (see the arrow) in Figure 7a. Nevertheless, thin films with a low Si content (SiN x > 0.8, see Figure 3) could also contain small Si-np upon annealing at 1100°C but having an amorphous structure. Figure 7 Evolution of the Raman spectra of SiN x with the refractive index and the annealing temperature. Effect of the annealing temperature on the Raman spectra of SiN x thin layers deposited on fused silica with a refractive index below 2.5 (a) and above (b). It independently concerns films produced by the N2-reactive (full symbols) and the co-sputtering (empty symbols) methods. The excitation power density was 0.46 MW/cm2. Figure 8 shows the Raman spectra of the thin films with n > 2.5 (Figure 7b) after annealing at 1100°C. A low excitation energy density of 0.14 MW/cm2 was used to record these spectra in order to avoid any heating and induced stress of the films that may affect the Raman spectra of crystalline Si-np [46]. One can observe that the c-Si peaks progressively shift to higher wavenumbers toward the peak position of bulk c-Si with increasing n.

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transmission. Infect Control Hosp Epidemiol 2003,24(Suppl 5):S6–52.CrossRefPubMed 7. Aronoff SC: Outer membrane permeability in Pseudomonas cepacia : diminished porin content in a beta-lactam-resistant mutant and in resistant cystic fibrosis isolates. Antimicrob Agents KU55933 chemical structure Chemother 1988,32(11):1636–1639.PubMed 8. Moore RA, Hancock RE: Involvement of outer membrane of Pseudomonas cepacia in aminoglycoside and polymyxin resistance. Antimicrob Agents Chemother 1986,30(6):923–926.PubMed 9. Parr TR Jr, Moore RA, Moore LV, Hancock RE: Role of porins in intrinsic antibiotic resistance of Pseudomonas cepacia. Antimicrob Agents Chemother 1987,31(1):121–123.PubMed 10. Trépanier S, Prince A, Huletsky A: Characterization of

the penA and penR genes of Burkholderia cepacia 249 which encode the chromosomal class A penicillinase and its LysR-type transcriptional regulator. Antimicrob Agents Chemother 1997,41(11):2399–2405.PubMed 11. Burns JL, Lien DM, Hedin Microbiology inhibitor LA: Isolation and characterization of dihydrofolate reductase from trimethoprim-susceptible and trimethoprim-resistant Pseudomonas cepacia. Antimicrob Agents Chemother 1989,33(8):1247–1251.PubMed 12. Burns JL, Wadsworth CD, Barry JJ, Goodall CP: Nucleotide sequence analysis of a gene from Burkholderia ( Pseudomonas ) cepacia encoding an outer membrane lipoprotein involved in multiple antibiotic resistance. Antimicrob Agents Chemother 1996,40(2):307–313.PubMed 13. Fehlner-Gardiner CC, Valvano MA: Cloning and characterization of the Burkholderia vietnamiensis norM gene encoding a multi-drug efflux protein. FEMS Microbiol Lett 2002,215(2):279–283.CrossRefPubMed 14. Wigfield SM, Rigg GP, Kavari M, Webb AK, Matthews RC, Burnie JP: Identification of an immunodominant drug efflux pump in Burkholderia cepacia. J Antimicrob Chemother 2002,49(4):619–624.CrossRefPubMed 15. Poole K, Srikumar R: Multidrug efflux in Pseudomonas aeruginosa : components, mechanisms and clinical significance.

Conclusions We established an important role of SspA in the regulation of LEE- and non-LEE-encoded virulence factors of a T3SS, which is important for A/E lesion formation by EHEC. SspA downregulates H-NS levels allowing the expression of EHEC virulence genes, which are part of the H-NS/Ler regulon. Virulence genes in many bacteria are horizontally acquired genetic elements and subject to repression by H-NS.

Thus, our study indicates that SspA potentially plays an important role in the pathogenicity of many bacterial pathogens in general. Methods Standard procedures Standard DNA selleck inhibitor techniques, agar plates and liquid media were used as described [60]. Restriction check details endonucleases, T4 DNA polynucleotide kinase- and ligase (New England Biolabs) and the Expand High Fidelity PCR System (Roche Applied Sciences) were used according to manufacturer’s instructions. DNA sequencing

was performed by the National Cancer Institute DNA Sequencing MiniCore facility. Bacteria were grown at 37°C in LB or DMEM (Invitrogen #11885) media supplemented with ampicillin (100μg/ml), chloramphenicol (25 μg/ml) or kanamycin (25 μg/ml) as needed. HEp-2 cells (ATTC # CCL-23) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO2. Strain and plasmid constructions Oligonucleotides used in this study Rigosertib mw are listed in Table  1. Gene deletions were constructed in EHEC O157:H7 EDL933 strain ATCC 700927 (Perna et al. 2001) by Lambda Red-mediated recombination using linear DNA fragments as described [61]. An in-frame deletion of sspA was created as previously described [44] resulting in strain DJ6010 (ATCC 700927 ΔsspA). The DNA fragment used for making the sspA deletion was amplified by PCR from pKD13 with primers PKD13sspAUS2 and PKD13sspADS. An hns deletion mutant derivative of strain ATCC 700927 was made by inserting a chloramphenicol

resistance-encoding cat cassette, which was PCR amplified from pKD3 however [61] using primers Δhns92-1 and Δhns92-2, 276 nt from the hns translation initiation codon (strain DJ6011). An sspA hns double mutant (DJ6012) was constructed by introducing the Δhns::cat deletion into strain DJ6010. All gene deletion constructs were verified by PCR amplification using primer sets sspABUS/sspABDS and hnsUS2/hnsDS2. In addition, Western blot analysis using polyclonal antibodies specific to the respective proteins confirmed the sspA and hns mutant strains. Plasmid pACYCler (pDJ610) contains a ~ 800 bp DNA fragment encoding ler expressed from its two native promoters cloned into the HindIII/BamHI sites of pACYC184. The DNA fragment was PCR amplified from EDL933 genomic DNA using oligos lerUS2/lerDS2.