In Pb339, identities between regions are 89% (1a × 1b), 79% (1a × 1c) and 90% (1b × 1c). In Pb18, the structure and sequence of the PbGP43 5′ flanking region (2,047 bp) are quite similar to those in Pb339. Sequence identities are also high when comparing the same regions between Pb339 and Pb18: 99% (1a), 95% (1b) and 97% (1c). Pb3 lacks one repetitive region: 1a in Pb3 is 96% identical to 1a in Pb339, while 1c/a/b carries nucleotides characteristic of the three regions, however the level of identity is higher with 1c (94%) than with 1b (87%) or 1a (78%). Therefore, when sequence alignments of the repetitive regions from

Pb339, Pb18 and Pb3 were compared in a dendrogram, there were two main clusters, one with 1a learn more sequences and another branching into 1b and 1c (and 1c/a/b) regions (data not shown). Pb3 sequences formed individual Pifithrin-�� purchase branches, in accordance with the phylogenetically distinct nature of this isolate detected with PbGP43 gene and other loci [3, 15]. The 442-bp upstream fragment is highly divergent from the repetitive check details regions, but conserved among isolates (about 99% identity). The highly conserved nature of the connector (Figure 4C) drove our attention to a more detailed analysis of its contents. We observed that some oligonucleotide sequences occur exclusively in the connectors, while others can be found

in other positions of the repetitive regions. In Figure 4C, we boxed six sequences (6- to 8-bp long) Ergoloid that can be found in the positions represented in Figure 4B by color-coded arrowheads or bars. Note that the blue oligonucleotide (TTTTCAAG) was invariably found 44 bp upstream of the last base of all repetitive regions. The purple sequence (ATGAAAT) localized 109 bp downstream of the first base of the connector in the three isolates considered; therefore this sequence is not seen in 1c (or 1c/b/a) region. The gray sequence TTGATA in the connector could also be seen in 1b region at -883 (Pb339) and -1006 (Pb3). The green ATGTTA oligonucleotide was detected at -1756 (Pb339 and Pb18) and -1261

(Pb3) and at -268 in all isolates. The orange TATAGA was found exclusively in Pb18 and Pb339 at distances of 186 and 184 bp from the start base of 1a and 1b regions. The red-coded corresponding mutated sequence in Pb3 (TTATTGAT) was also detected 238 bp upstream of the last base in 1c/b/a region; it is not present in Pb18 or Pb339 connector, but it could be detected at distances varying among 237, 234 and 229 bp upstream of regions 1a, 1b and 1c last bases. The brown CTTATTT initial connector sequence was observed only once in 1a region, 67 bp upstream of the last base in Pb339 and Pb18. Although this exact sequence is not observed in the Pb3 connector, which shows a unique CTTCATT oligonucleotide not found elsewhere, in this isolate CTTATTT has been observed twice in 1a region, at 67 bp upstream of the last base, and at a polymorphic -372 site.

Pellets were resuspended in 500 μl of BSK-II lacking GlcNAc and transferred to 2 ml microcentrifuge tubes. One ml of Bacteria RNAProtect (Qiagen, Inc.) was added and mixed by vortexing. Cells were incubated for 5 min at room temperature, and then centrifuged for 10 min at 5,000 × g. Pellets were stored at -80°C for up to 4 weeks prior to RNA extraction. RNA was extracted using the RNeasy Mini kit (Qiagen, Inc.) according to the manufacturer’s instructions. RNA was DNase-treated with RQ1 RNase-free DNase (Promega Corp.), and RNasin (Promega Corp.) was added according to the manufacturer’s instructions. Protein from the DNase reaction was removed using the RNeasy Mini kit according

to the RNA Cleanup protocol supplied by the manufacturer. RNA concentration (OD260) and purity (OD260/OD280) were determined by UV spectrophotometry. RNA integrity was evaluated by gel electrophoresis.

Specifically, buy PLX-4720 2 μg of each sample was separated on a 1% agarose gel and the intensity RGFP966 supplier of the 16S and 23S ribosomal RNA bands was determined. RNA was stored at -80°C for subsequent gene expression analysis. Real-time quantitative reverse transcription-PCR (qRT-PCR) qRT-PCR was performed using the Mx4000 or Mx3005P Multiplex Quantitative PCR System and the Brilliant SYBR Green Single-Step qRT-PCR Master Mix Kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. A standard curve (101 to 107 copies per reaction) was generated using a purified chbC PCR product as the template. The following primers were used for all reactions: forward primer chbC F and reverse primer chbC R. Reactions (25 μl) containing 10 ng of total RNA were run under the following conditions:

1 cycle of 50°C for 30 min and 95°C for 15 min, followed by 40 cycles of 95°C for 30 s and 58°C for 30 s 2. Fluorescence was measured at the end of the 58°C step every cycle. Samples were run in duplicate, and all qRT-PCR experiments included both no-reverse transcriptase (RT) and no-template controls. The copy number of chbC mRNA in each sample was determined using the MxPro (Stratagene) DOK2 data analysis Selleck LGK-974 software based on the chbC standard curve described above. The chbC copy number for each sample was normalized based on the total RNA input (10 ng per reaction), and fold differences in chbC expression from the initial time point (44 h) were calculated based on the normalized copy numbers. Identification of the chbC transcriptional start site and promoter analysis Total RNA was isolated from wild-type B. burgdorferi strain B31-A cultured in complete BSK-II as described above. The transcriptional start site was determined using the 2nd Generation 5′/3′ RACE Kit (Roche Applied Science; Mannheim, Germany) according to the manufacturer’s instructions. Briefly, first-strand cDNA synthesis was carried out in a reverse transcription reaction for 60 min at 55°C using primer BBB04 5′ RACE R1 2 and 1 μg of total RNA.

PubMedCrossRef 32. van Vliet AH, Stoof J, Poppelaars SW, Bereswill S, Homuth G, Kist M, Kuipers EJ, Kusters JG: Differential regulation

of amidase- and formamidase-mediated ammonia production by the Helicobacter pylori fur repressor. J Biol Chem 2003,278(11):9052–9057.PubMedCrossRef 33. Wu CC, Lin CT, Cheng WY, Huang Ilomastat purchase CJ, Wang ZC, Peng HL: Fur-dependent MrkHI PD173074 chemical structure regulation of type 3 fimbriae in Klebsiella pneumoniae CG43. Microbiology 2012,158(Pt 4):1045–1056.PubMedCrossRef 34. Hantke K: Iron and metal regulation in bacteria. Curr Opin Microbiol 2001,4(2):172–177.PubMedCrossRef 35. Masse E, Vanderpool CK, Gottesman S: Effect of RyhB small RNA on global iron use in Escherichia coli. J Bacteriol 2005,187(20):6962–6971.PubMedCrossRef 36. Andrews SC, Harrison PM, Guest JR: Cloning, sequencing, and mapping of the

bacterioferritin gene (bfr) of Escherichia coli K-12. J Bacteriol 1989,171(7):3940–3947.PubMed 37. Gruer MJ, Guest JR: Two genetically-distinct and differentially-regulated aconitases (AcnA and AcnB) in Escherichia coli. Microbiology 1994,140(Pt 10):2531–2541.PubMedCrossRef 38. Niederhoffer EC, Naranjo CM, Bradley KL, Fee JA: Control of Escherichia coli superoxide dismutase (sodA and sodB) genes by the ferric uptake regulation (fur) locus. J Bacteriol 1990,172(4):1930–1938.PubMed 39. Masse E, Gottesman S: A small RNA click here regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci USA 2002,99(7):4620–4625.PubMedCrossRef 40. Masse E, Escorcia FE, Gottesman S: Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev 2003,17(19):2374–2383.PubMedCrossRef 41. Dubrac S, Touati D: Fur positive regulation of iron superoxide dismutase in Escherichia coli: functional analysis of the sodB promoter. J Bacteriol 2000,182(13):3802–3808.PubMedCrossRef 42. Davis BM, Quinones M, Pratt J, Ding Y, Waldor MK: Characterization of the small untranslated RNA RyhB and its regulon in Vibrio cholerae. J Bacteriol

2005,187(12):4005–4014.PubMedCrossRef 43. Argaman L, Elgrably-Weiss M, Hershko T, Vogel J, Altuvia S: RelA protein stimulates Bcl-w the activity of RyhB small RNA by acting on RNA-binding protein Hfq. Proc Natl Acad Sci USA 2012,109(12):4621–4626.PubMedCrossRef 44. Mey AR, Craig SA, Payne SM: Characterization of Vibrio cholerae RyhB: the RyhB regulon and role of ryhB in biofilm formation. Infect Immun 2005,73(9):5706–5719.PubMedCrossRef 45. Murphy ER, Payne SM: RyhB, an iron-responsive small RNA molecule, regulates Shigella dysenteriae virulence. Infect Immun 2007,75(7):3470–3477.PubMedCrossRef 46. Blumenkrantz N, Asboe-Hansen G: New method for quantitative determination of uronic acids. Anal Biochem 1973,54(2):484–489.PubMedCrossRef 47. Kuhn J, Briegel A, Morschel E, Kahnt J, Leser K, Wick S, Jensen GJ, Thanbichler M: Bactofilins, a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus.

Also, C. jejuni bacteria have been observed in the haemocoel and gut of infected larvae, and have been demonstrated to induce damage to the midgut [36]. In this study,

we demonstrate that G. mellonella is susceptible to infection with H. pylori and may represent a valuable model to identify virulence factors and pathogenic mechanisms of H. pylori. Methods Bacterial strains and growth conditions A total of eleven H. pylori strains were included in this study. In particular, we used: a) the this website wild-type H. pylori strain G27 (VacA+/cagPAI+/urease+) and its isogenic mutants in which click here the cagA (G27ΔcagA) or cagE (G27ΔcagE) gene or the entire cagPAI (G27ΔcagPAI) were disrupted by insertional mutagenesis [3,37]; b) the wild-type H. pylori strain 60190 (ATCC 49503; VacA + s1/i1/m1/cagPAI+/urease+) and its isogenic mutants in which vacA (60190ΔvacA), or cagA (60190ΔcagA), or cagE (60190ΔcagE) were disrupted by insertional mutagenesis [38,39]

as well as its urease-negative spontaneous mutant urease (60190 Urease-negative) [40]; c) the mouse-adapted H. pylori strain M5 and its GGT-defective isogenic mutant (M5ggt::aph) in which ggt was disrupted by insertional mutagenesis [8]. Bacteria were cultured on Columbia agar supplemented with 10% defibrinated horse blood, 1% Vitox and Skirrow’s supplement under microaerophilic conditions in anaerobic jars https://www.selleckchem.com/products/SB-202190.html with microaerobic System CampyGen (all from Oxoid, Milan, Italy) at 37°C for 3 days. Preparation of broth culture filtrates (BCFs) BCFs were prepared as previously described [41,42]. Briefly, bacteria were grown in Brucella broth medium supplemented with 1% Vitox and Skirrow as well as 5% heat-inactivated fetal calf serum (FCS; Sigma-Aldrich, Milan, Italy) in anaerobic jars with microaerobic System CampyGen with gentle shaking (150 oscillations/min) for 24–48 h at 37°C. When bacterial suspensions reached 1.0 optical density units at 450 nm (corresponding to a bacterial concentration of 5 × 108 colony-forming units (CFUs/ml), bacteria were removed by centrifugation

(12,000 g Tolmetin for 15 min), and the supernatants were sterilized by filtering through a 0.22-μm-pore-size cellulose acetate filter (Sartorius Minisart SM 16534, Sigma-Aldrich) to obtain BCFs. Purification and use of VacA toxin VacA (s1/m1 genotype) was purified by ammonium sulphate precipitation and gel filtration chromatography from wild-type H. pylori 60190 strain grown in Brucella broth in which foetal calf serum was replaced by 0.2% β-cyclodextrins (Sigma-Aldrich) [43,44]. Purified VacA was stored in melting ice and, immediately before use on G. mellonella larvae, was activated or not by dropwise acidification to pH 3.0 with 0.2 N HCl. Vacuolating activity of purified VacA was determined by means of neutral red uptake as previously described [45].

However, the present interpretation system for CT has not kept up with

the modality’s technological development, AMN-107 and real-time interpretation by radiologists is not available in many institutions in Japan because of a nationwide shortage of radiologists. Many EPs, therefore, must make decisions regarding trauma treatment plans without radiological support. Hunter et al. reported that only wet reading was available in the majority of medical institutions surveyed and that emergency CT was usually supported only by radiology residents even in university hospitals [15]. Torreggiani et al. reported that real-time interpretation by radiologists was not available in many institutions and that, in some, radiologist interpretation took more than 48 hours to prepare [16]. They also reported that EPs and radiologists felt very differently about whether the interpretation system was adequate. Many EPs complained of a deficiency in the current interpretation system. Such problems are likely to continue into the long term unless effective

measures are taken. Our hope is that this study may provide an effective CT interpretation system for EPs to use in blunt trauma cases. In this study, EPs misinterpreted 40 of 1606 cases (2.5%) in the first period. Seven of the 365 total C646 mw patients (1.9%) were most likely placed at a disadvantage by a major misinterpretation; these patients were categorized as gravity level 2 or 3, and they required additional treatments (such as emergency surgery). Chung et al. studied the accuracy of 4768 selleck chemical interpretation reports of torso CT performed by a radiology resident [9]. In this study, serious misdiagnosis occurred in 2.0% of the cases, and changes in treatment were required in 0.3%. Petinaux et al. reported major discrepancies between the interpretations

from EPs and radiologists in 3% of cases (for plain chest and abdominal X-rays) [17]. Most of the discrepancies were considered misdiagnoses, and changes Methane monooxygenase in treatment were required in 0.05% of the cases. Gray comprehensively surveyed the occurrence of diagnostic mistakes in the ED [18] and found that 79.7% of mistakes were associated with bone trauma and that most misdiagnoses could likely be avoided by careful interpretation. There were no large differences in the number and level of diagnostic mistakes between these studies and our study. However, even a small misinterpretation by the EP may lead to irrelevant treatment or a potentially fatal delay in appropriate treatment. This must be avoided wherever possible, but is difficult to achieve in actuality. One solution is to further train EPs to improve their interpretations of CT results. However, a high level of skill is required to interpret CT results, and we believe that it would be almost impossible to improve interpretation ability with unsystematic short-term training. Keijzers et al.

(b) Segmentation of the QDs in the tomogram, showing that the stacking of QDs follows a straight line that deviates 10° from the growth direction. (c) Slice through the upper QD of the reconstructed tomogram where we have superimposed a circle to evidence the elongation in the direction of the optical axis of the microscope. The upper and lower QDs of the Figure 2b have been included with a white and black dotted line respectively. It is worth mentioning that often the 3D information obtained from tomography analyses suffers from the missing Pexidartinib wedge artifact due to a lack

of information for high rotation angles. This causes an elongation of the features in the sample along the microscope optical axis (in our

case, parallel to the learn more wetting layers). Figure 2c shows an axial slice through the reconstructed needle, where this elongation is observed. We have superimposed a circle along the surface of the needle to evidence this elongation more clearly. From this figure, we have calculated an elongation percentage due to the missing wedge of 1.14%. We have measured the vertical alignment of the dots using the location of the center of each dot and because of the calculated elongation, this position will be displaced from its real selleck products location. The maximum error in the location of the QDs would occur for dots placed close to the surface of the needle, and where the QDs alignment has a component parallel to the optical axis of the microscope. In this case, the error in the angle between the QDs vertical alignment and the growth direction would be of 3.5°. This error could be minimized using needle-shaped specimens in combination with last generation tomography holders that allow a full tilting range. On the other hand, for QDs stacking included in a plane perpendicular to the microscope optical axis

located in the center of the needle (as shown in Figure 2c), there would be no error in the measurement of the angle. In our case, the vertical alignment of the dots is closer to this second case. In Figure 2c we have included the position of the upper QD in the stacking with a white dotted line, and of the lower QD with a black dotted line. As it can be observed, both dots are very close to the center of the needle, and the vertical alignment forms an angle close to else 90° with the optical axis; therefore, the error in the measurement of the QDs vertical alignment is near to 1°. The observed deviation from the growth direction of the stacking of QDs is caused by the elastic interactions with the buried dots and by chemical composition fluctuations [16, 30]. However, other parameters such as the specific shape of the QDs [4, 5, 31], elastic anisotropy of the material [4, 5, 30, 31], or the spacer layer thickness [4, 5, 30] need to be considered as well to predict the vertical distribution of the QDs.

J Antimicrob Chemother 2009,64(6):1175–1180.PubMedCrossRef 38. Chamot D, Owttrim GW: Regulation of cold shock-induced RNA helicase gene expression in the Cyanobacterium anabaena sp. strain PCC 7120. J Bacteriol 2000,182(5):1251–1256.PubMedCrossRef 39. Chinni SV, Raabe CA, Zakaria R, Randau G, Hoe CH, Zemann A, Brosius J, Tang TH, Rozhdestvensky TS: Experimental identification and characterization of 97 novel npcRNA candidates in Salmonella enterica

serovar Typhi. Nucleic Acids Res 2010,38(17):5893–5908.PubMedCrossRef 40. Rosenblum R, Khan E, Gonzalez G, Hasan R, Schneiders T: Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae. Int J Antimicrob Agents 2011,38(1):39–45.PubMedCrossRef 41. Horiyama T, Nikaido E, Yamaguchi A, Nishino K: Roles of Salmonella OSI-906 in vitro multidrug efflux pumps in tigecycline

resistance. J Antimicrob Chemother 2011,66(1):105–110.PubMedCrossRef 42. Tjaden B: TargetRNA: a tool for predicting targets of small RNA action in bacteria. Nucleic Acids Res 2008,36(Web Server issue):W109–113.PubMedCrossRef 43. Darwin AJ: The phage-shock-protein response. Mol Microbiol 2005,57(3):621–628.PubMedCrossRef FK228 price 44. Santiviago CA, Reynolds MM, Porwollik S, Choi SH, Long F, Andrews-Polymenis HL, McClelland M: Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice. PLoS Pathog 2009,5(7):e1000477.PubMedCrossRef 45. Papenfort K, Pfeiffer V, Lucchini S, Sonawane A, Hinton JC, Vogel J: Systematic deletion of Salmonella small RNA genes identifies CyaR, a conserved CRP-dependent riboregulator of OmpX synthesis. Mol Microbiol 2008,68(4):890–906.PubMedCrossRef

46. Ontiveros-Palacios N, Smith AM, Grundy FJ, Soberon M, Henkin TM, Miranda-Rios J: Molecular basis of gene regulation by the THI-box riboswitch. Mol Microbiol 2008,67(4):793–803.PubMedCrossRef 47. Valentin-Hansen P, Eriksen M, Udesen C: The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Microbiol 2004,51(6):1525–1533.PubMedCrossRef 48. Hoiseth SK, Stocker BA: Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 1981,291(5812):238–239.PubMedCrossRef 49. Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, see more Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, click here Mayhew GF, et al.: The complete genome sequence of Escherichia coli K-12. Science New York, NY 1997,277(5331):1453–1462.CrossRef 50. George AM, Hall RM, Stokes HW: Multidrug resistance in Klebsiella pneumoniae: a novel gene, ramA, confers a multidrug resistance phenotype in Escherichia coli. Microbiology (Reading, England) 1995,141(Pt 8):1909–1920.CrossRef 51. Andrews JM: BSAC standardized disc susceptibility testing method (version 8). J Antimicrob Chemother 2009,64(3):454–489.PubMedCrossRef 52.

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England (2013c) Higher Level Stewardship, 4th Edition. http://​publications.​naturalengland.​org.​uk/​file/​2819648 Nix J (2010) Farm management pocketbook, 41st edn. The Andersons Centre, Melton Tolmetin Mowbery Ollerton J, Winfree R, Tarrant S (2011) How many flowering plants are pollinated by animals? Oikos 120(3):321–326CrossRef Potts SG, Woodcock BA, Roberts SPM, Tscheulin T, Pilgrim ES, Brown VK, Tallowin JR (2009) Enhancing pollinator biodiversity in intensive grasslands. J Appl Ecol 46(2):369–379CrossRef Potts SG, Roberts SPM, Dean R, Marris G, Brown MA, Jones R, Neumann P, Settele J (2010) Declines of managed honeybees and beekeepers in Europe. J Apic Res 49:15–22CrossRef Pywell RF, Meek WR, Loxton RG, Nowakowski M, Carvell C, Woodcock BA (2011) Ecological restoration on farmland can drive beneficial functional responses in plant and invertebrate communities. Agric Ecosyst Environ 140:62–67CrossRef Ricketts T, Lonsdorf E (2013) Mapping the margin: comparing marginal values of tropical forest remnants for pollination services. Ecol Appl 25:1113–1123 SAFFIE (2007) Cost:benefit analysis of the best practices for increased biodiversity, Chapter 8, HGCA. http://​www.​hgca.​com/​publications/​documents/​cropresearch/​PR416_​SAFFIE_​8_​Cost_​benefit_​analysis.

Data processing The microarray data obtained was analysed by using

the EMMA 2.8.2 software [74]. The mean signal intensity (A i) was calculated for each spot using the formula A i = log2(R i G i)0.5[75]. R i = I ch1(i) − Bg ch1(i) and G i = I ch2(i) − Bg ch2(i), where I ch1(i) or I ch2(i) is the intensity of a spot in channel 1 or channel 2, and Bg ch1(i) or Bg ch2(i) is the background intensity of a spot in channel1 or channel 2, respectively. The log2 value of the ratio of signal intensities (Mi) was calculated for each spot using the formula Mi = log2(Ri/Gi). Spots were flagged as “empty” if R ≤ 0.5 in both channels, where R = (signal mean–background mean)/background standard deviation [76]. The raw data were normalized selleck compound by the method of LOWESS (locally

weighted scattered plot smoothing). A significance test was performed by the method of false discovery rate (FDR) control and the adjusted p-value defined by FDR was called q-value [77, 78]. An arbitrary cutoff, fold change (FCH) greater than 1.5, was applied to the genes with a q-value of ≤0.01. Only those genes which meet both filter conditions (q ≤ 0.01 & FCH ≥ 1.5) were regarded to be significantly differentially expressed. Real-time PCR The first-strand cDNA was obtained by reverse transcription with RevertAidTM Premium Reverse Transcriptase (Fermentas, St. Leon-Rot, Germany), using random hexamers as primers. Oligonucleotide GSK3326595 cell line primers were designed by the software PrimerExpress and VX-809 in vivo listed in supplemental materials (Additional files 1: Table S4). Real-time PCR was performed with SYBR® Green PCR Master 5-Fluoracil order Mix kit (Carlsbad, California, USA) using 7500 Fast Real-Time PCR System (Carlsbad, California, USA) according to the manufacturers’ instructions. As an internal control, the housekeeping gene gyrA was used as its expression was not significantly altered in all microarray experiments. Three

technical replicates were carried out for each target gene. Quantification was analysed based on the threshold cycle (Ct) values as described by Pfaffl [79]. The raw data of the Micro-array experiments, described here, are available in the ArrayExpress database under the accession numbers: E-MEXP-3421, E-MEXP-3550, E-MEXP-3551, E-MEXP-3553, E-MEXP-3554, respectively (see also Additional file 3: Table S6). Acknowledgements The financial support for FB by the Priority Academic Development Program of Jiangsu Higher Education Institutions and the National Natural Science Foundation of China (No. 31100081) and the German Academic Exchange Service (DAAD) is gratefully acknowledged, as well as, the financial support given to RB in-frame of the competence network Genome Research on Bacteria (GenoMikPlus, GenoMikTransfer) and of the Chinese-German collaboration program by the German Ministry for Education and Research (BMBF).

HeLa cells were grown in 24-well tissue culture plates Selleck GSK126 until they formed semi-confluent monolayers. The culture medium used was RPMI1640 supplemented with 10% fetal calf serum (FCS), and 1% penicillin-streptomycin; and cultures were incubated at 37°C/5% CO2. Cells were washed three times with phosphate-buffered

saline (PBS), and bacteria added to the semi-confluent HeLa cultures at a multiplicity of infection (MOI) of 100. After incubating at 37°C for 90 min, growth medium containing 5% (w/v) agar and 20 μg/mL gentamicin was poured into the 24-well plates, then incubated at 37°C/5% CO2 for 72 h. HeLa cells were inoculated with SF301 as a positive control, and with E. coli ATCC 25922 as a negative control. Sequence and analysis of virulence genes on PAI-1 of SF51 SF51 genomic DNA was extracted using a QIAamp DNA Mini Kit (Qiagen). PCR primers for amplification of pic, sigA, int and

orf30 from PAI-1 of the SF51 clinical isolate were designed according to the SF301 sequence. Amplicons were cloned into a pCR-XL-TOPO vector using a TOPO® XL PCR Cloning Kit (Invitrogen), and the inserts were sequenced by Sangon CB-839 cell line Biotech (Shanghai, China) Co. Ltd, then selleck chemicals llc identified using the standard nucleotide basic local alignment search tool (BLASTn; NCBI). Construction of SF301-∆ pic The upstream and downstream portions of pic were amplified by PCR. Primers uppic-F-NotI and uppic-R-XbaI (Table 1) were used to amplify the upstream fragment of pic, with primers

downpic-F-XbaI and downpic-R-BamHI TCL (Table 1) used to amplify the downstream fragment. The amplified downstream fragment of pic was digested with XbaI and BamHI and ligated into pSB890 which had been cut with the same restriction endonucleases [27]. We designated the resulting plasmid pSB890-pic downstream. The amplified upstream pic fragment was digested with NotI and XbaI and ligated into pSB890-pic downstream that had been digested with NotI and XbaI. The resulting vector was designated pSB890-∆ pic and transformed into E. coli SM10 λpir cells, then introduced into SF301 through a bacterial conjugation test. After culturing on a sucrose LB agar plate at 22°C, sucrose-tolerant colonies were screened using Shigella-specific minimal medium [7] and a PCR employing primers Upuppic-F and Downdownpic-R (Table 1). The mutant strain with the pic deletion was identified by sequencing and named SF301-∆ pic. Construction of complementation strains SF301-∆ pic/pPic and SF51/pPic A plasmid containing pic was constructed using pSC modified from pREP4. The pic gene was amplified from SF301 genomic DNA using PCR. The PCR primers used were pic-pSC-F-PfMlI and pic-pSC-R-AclI (Table 1). Amplicons were inserted into pSC, creating pSC-pic, which was verified by restriction enzyme digestion and nucleic acid sequencing.