Biotechnol Lett 2006,28(4):207–213.PubMedCrossRef 9. Curley JM, Lenz RW, Fuller C: Sequential production of two different polyesters in the inclusion bodies of Pseudomonas oleovorans . Int J Biol Macromol 1996, 19:29–34.PubMedCrossRef 10.

Huisman GW, Wonink E, De Koning GJM, Preusting H, Witholt B: AZD1208 clinical trial Synthesis of poly (3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains. Appl Microbiol Biotechnol 1992, 38:1–5.CrossRef 11. Stuart ES, Foster LJR, Lenz RW, Fuller RC: Intracellular depolymerase functionality and location in Pseudomonas olevorans inclusions containing polyhydroxyoctanoate. Int J Biol Macromol 1996, 19:171–176.PubMedCrossRef 12. Jurasek L, Marchessault RH: The role of phasins in the morphogenesis of poly(3-hydroxybutyrate) granules. Biomacromolecules 2002,3(2):256–261.PubMedCrossRef 13. Prieto MA, Bühler B, Jung learn more K, Witholt B, Kessler B: PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes. J Bacteriol 1999,181(3):858–868.PubMed 14. Ruth K, de Roo G, Egli T, Ren Q: Identification of two acyl-CoA synthetases from Pseudomonas putida GPo1: One is located at the surface of polyhydroxyalkanoates granules. Biomacromolecules 2008,9(6):1652–1659.PubMedCrossRef

15. Huisman GW, Wonink E, Meima R, Kazemier B, Terpstra P, Witholt B: Metabolism of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans . J Biol Chem 1991, 266:2191–2198.PubMed 16. García B, Olivera ER, Minambres B, Fernández-Valverde M, Canedo LM, Prieto MA, García JL, Martínez M, Luengo JM: Novel biodegradable aromatic plastics from a bacterial source. J Biol Chem 1999,274(41):29228–29241.PubMedCrossRef 17. de Eugenio LI, Garcia P, Luengo JM, Sanz JM, San Roman J, Garcia JL, Prieto MA: Biochemical evidence that phaZ gene encodes a specific intracellular medium-chain-length polyhydroxyalkanoate depolymerase in Pseudomonas putida KT2442 – Characterization of a paradigmatic enzyme. J Biol Chem 2007,282(7):4951–4962.PubMedCrossRef 18. Steinbüchel A, Aerts K, Babel W, Follner C, Liebergesell M, Madkour MH, Mayer F, Pieper-Fürst U, Pries A,

Valentin HE, et al.: Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions. Can J Microbiol 1995, 41:94–105.PubMedCrossRef 19. Ren Q, de Roo G, Ruth K, Witholt 17-DMAG (Alvespimycin) HCl B, Zinn M, Thöny-Meyer L: Simultaneous accumulation and degradation of polyhydroxyalkanoates: Futile cycle or clever regulation? Biomacromolecules 2009,10(4):916–922.PubMedCrossRef 20. Doi Y, Segawa A, Kawaguchi Y, Kunioka M: Cyclic nature of poly(3-hydroxyalkanoate) metabolism in Alcaligenes eutrophus . FEMS microbiol Lett 1990, 67:165–170.CrossRef 21. de Roo G, Ren Q, Witholt B, Kessler B: Development of an improved in vitro activity assay for medium chain length PHA polymerase based on CoenzymeA release measurements. J Microbiol Meth 2000, 41:1–8.CrossRef 22.

2 months in the younger group versus only 4.9 months in the elderly group, the number of treatment cycles was 10.0 and 9.5, respectively, showing that administration of mFOLFOX6 was possible in

elderly patients with a good PS. The response rate was 60.0% in the younger group and 50.0% in the elderly group, while the disease control rate was 100% and 83.3%, respectively, showing no this website significant difference in relation to age. When this study was initiated in San-in, a rural region of Japan with a large elderly population, there was an urgent need to establish effective chemotherapy regimens for colorectal cancer, which has recently become much more common in Japan. Accordingly, the present study was intended to assess the feasibility of mFOLFOX6 in Japanese colorectal cancer patients, including elderly patients, with regard to the incidence and severity of adverse events. In an attempt to rapidly investigate the efficacy and safety of mFOLFOX6, the subjects were enrolled during a 1-year period. The limited duration of enrollment resulted in too small a sample size for the study to be adequately powered. Despite this, our findings suggested that mFOLFOX6 is similarly tolerable and effective for elderly patients as it is for non-elderly patients, because the therapy could be administered at its recommended

dosage without causing more severe adverse events than in non-elderly patients by employing appropriate criteria for patient selection, treatment suspension, and dose reduction in consideration of factors such as the PS and comorbidities. CH5424802 order However, discontinuation

was necessary in 12 patients (including 3 elderly patients) because of adverse reactions, and 5 patients (including 2 elderly patients) discontinued treatment due to peripheral neuropathy PLEKHM2 (the dose-limiting toxicity of oxaliplatin). Therefore, avoiding or reducing the occurrence of such adverse events is necessary for the establishment of safer standard therapy. Conclusion It was confirmed by the present study that mFOLFOX6 therapy, a standard chemotherapy for unresectable advanced/recurrent colorectal cancer, could be performed safely in elderly Japanese patients. The tolerability and efficacy of mFOLFOX6 therapy can be expected to be similar in the elderly, provided that the PS is good, the major organs are functioning well, and there are no uncontrolled complications. The present findings also suggested that withdrawal of bolus 5-FU to avoid severe neutropenia might allow the continuation of treatment. Because discontinuation due to peripheral neuropathy (the dose-limiting toxicity of this regimen) was common, methods to avoid or alleviate such adverse events without reducing efficacy need to be investigated. Acknowledgements We deeply appreciate the assistance of Dr. Kouji Kodama (Department of Radiology, Shimane Prefectural Central Hospital), Dr.

Sydowia 51:167–175 Crous PW, Slippers B, Wingfield MJ, Rheeder J, Marasas WFO, Philips AJL, Alves A, Burgess TI, Barber PA, Groenewald JZ (2006) Phylogenetic lineages in the Botryosphaeriaceae. Stud Mycol 55:235–253PubMed Damm U, Cannon PF, Woudenberg JHC, Crous PW (2012a) The Colletotrichum acutatum species complex. Stud Mycol 73:37–113 Damm U, Cannon PF, Woudenberg JHC, Johnston PR, Weir BS, Tan YP, Shivas RG, Crous PW (2012b) The Colletotrichum boninense species complex. Stud Mycol 73:1–36 Damm U, Crous PW, Fourie PH (2007a) Botryosphaeriaceae as potential pathogens of Prunus species in South Africa, with descriptions of Diplodia africana and Lasiodiplodia plurivora

sp. nov. Mycologia 99:664–680PubMed Damm U, Fourie PH, Crous PW (2007b) Aplosporella prunicola, a novel species of anamorphic Botryosphaeriaceae. Fungal Divers 27:35–43 Denman PW, Taylor JE, Kang JC, Pascoe I, Michael J (2000) An overview of the taxonomic MLN0128 purchase history of Botryosphaeria, and a re-evaluation of its anamorphs based on beta-catenin inhibitor morphology and ITS rDNA phylogeny. Stud Mycol 45:29–140 Denman S, Crous PW, Groenewald JZE, Slippers B, Wingfield BD, Wingfield MJ (2003) Circumscription of Botryosphaeria species associated with Proteaceae based on morphology and DNA sequence data. Mycologia 95:294–307PubMed Denman S, Crous PW, Wingfield MJ (1999) A taxonomic reassessment of Phyllachora proteae, a leaf pathogen of Proteaceae. Mycologia 91:510–516 Doidge

EM (1942) Revised descriptions of South African species of Phyllachora and related genera. Bothalia 4:421–463 Eriksson O (1981) The families of bitunicate Ascomycetes. Opera Botanica 60:1–220 Farr ML (1989) Two new species of tropical Vasopressin Receptor fungi. Memoirs of the New York Botanical Garden 49:70–73 Felsenstein J (2004) Inferring phytogenies. Sinauer Associates, Sunderland, Massachusetts Fries E (1823) Systema Mycolgicum 2(2):423–424 Fuckel L (1870) Symbolae mycologicae: Beiträge zur Kenntniss der rheinischen Pilze. Jahrb Nassauischen Vereins Naturk 23–24:1–459 Ghimire SR, Charlton ND, Bell JD, Krishnamurthy YL, Craven KD (2011) Biodiversity of fungal endophyte communities inhabiting switchgrass (Panicum virgatum L.) growing in the native tallgrass

prairie of northern Oklahoma. Fungal Divers 47:19–27 Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 61:1323PubMed Glienke C, Pereira OL, Stringari D, Fabris J, Kava-Cordeiro V, Galli-Terasawa L, Cunnington J, Shivas RG, Groenewald JZ, Crous PW (2011) Endophytic and pathogenic Phyllosticta species, with reference to those associated with Citrus Black Spot. Persoonia 26:47–56PubMed González V, Tello ML (2011) The endophytic mycota associated with Vitis vinifera in central Spain. Fungal Divers 47:29–42 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In: Nucleic Acids Symposium Series.

ulcerans[24]. The C. ulcerans 809 strain was isolated from a patient with a rapid fatal pulmonary infection. The 809 strain-unique virulence factor (shiga toxin-like ribosome-binding protein, Rbp) is located adjacent to the truncated integrase (CULC809_00176)

and corresponds to the integrase of ΦCULC0102-I. It appears that virulence factors have been acquired as a cassette gene in the ΦCULC0102-I-like prophage. Lenvatinib It is intriguing to note that the 0102 strain does not carry the 809 strain-unique virulence factors (Rbp and the additional venom serine protease, Vsp2), but instead carries the tox gene on ΦCULC0102-I, which resulted in a diphtheria-like illness in a 52-year-old Angiogenesis inhibitor woman. Isolates of C. ulcerans are generally obtained from a diverse range of animals, including humans. Isolation of a human pathogen C. diphtheriae from animals has been reported previously, although it is rare [31]. The tox gene might be frequently transmitted through common prophages with the aid of the highly homologous regions among Corynebacterium spp., including C. diphtheriae and C. ulcerans isolated from animal sources. Conclusions Toxigenic C. ulcerans is an emerging pathogen that can be transmitted from animals to humans [5]. In the host organism, as well as in C. diphtheriae, the tox gene [18] is encoded by prophages. Through genome sequencing, we have identified a novel structure

in a tox-positive C. ulcerans prophage with no significant sequence homology to those in C. diphtheriae. This suggests distinct origins of the prophages and thus may also explain the difference in the primary structures of their tox genes. The tox-positive bacteriophages may increase the dissemination risk of toxigenic C. ulcerans isolates, therefore, C. ulcerans isolates from both G protein-coupled receptor kinase human and animal sources should be investigated further to determine the

level of variation. Methods This research was not carried out on humans. No experimental research on animals was carried out. Bacterial strain The toxigenic C. ulcerans isolate 0102 was obtained in 2001 as a human clinical isolate [22, 23]. Preparation of genomic DNA Genomic DNA was isolated by conventional methods, using phenol extraction and ethanol precipitation from heat-killed bacterial cells propagated in brain-heart infusion liquid medium. Short-read DNA sequencing using an Illumina Genome Analyzer IIx DNA libraries of the ~600 bp insert length of C. ulcerans 0102 were prepared using a genomic DNA Sample Prep Kit (Illumina, San Diego, CA, USA). DNA clusters were generated on a slide using a Cluster Generation Kit (ver. 4) on an Illumina Cluster Station (Illumina), according to the manufacturer’s instructions. Sequencing runs for 80-mer short reads were performed using an Illumina Genome Analyzer IIx (GA IIx) and TruSeq SBS kit v5.

This has already been observed by Wörle-Knirsch et al. [24]. In their work, they showed that single-walled carbon nanotubes (SWNTs) were found to be non-toxic when using assays

such as LDH, annexin V, and PI staining, mitochondrial membrane potential, as well as other tetrazolium salt-based water-soluble assays such as WST-1, XTT, or INT. However, the MTT assay was the only assay which displayed SWNT cytotoxicity. In addition, real-time bright-field microscopy (Figure  3) did not show any morphological features suggestive of cytotoxicity, such as blebbing, membrane rupture, pyknosis, or fragmentation, for concentrations 1 to 10 selleck inhibitor μg/ml. Also, several cells were observed undergoing mitosis (data not shown). These findings suggest that at these low concentrations,

the sulfonation process affords protection to cells against the cytotoxic effects of graphene, similar to the observed protein corona-mediated mitigation of GO cytotoxicity recently published by Hu et al. [17]. However, there was a drastic change in cell morphology for concentrations of 100 μg/ml which shows evidence of pyknosis and fragmented, spindle-like cell features for the SNU449 cell see more lines. In these regard, we suggest that 10 μg/ml should be the upper concentration limit when using SGSs for full biocompatibility and that more work should be undertaken to understand the exact death mechanism of SGSs at concentrations >10 μg/ml. We initially sought to investigate this through the use of propidium iodide and annexin V FITC staining also with cell flow cytometry, but as mentioned in the ‘Methods’ section, we could only perform one time slot (24 h) with one cell line (SNU449) at two concentrations (10 and 100 μg/ml). Figure 3 Optical images

of SNU449 cells exposed to SGSs for 72 h. Images depict control cells (no SGSs) (A) and 1 (B), 10 (C), and 100 (D) μg/ml concentrations. Propidium iodide is a cell impermeable fluorophore that can bind to the DNA of cells which have lost nuclear and plasma membrane integrity. From our fluorescence-activated cell sorting (FACS) analysis shown in Additional file 1: Figure S5, we found that with an increasing concentration of SGS nanoparticles, the intensity of positive PI-stained cells increased from approximately 1.9% to 10.3%, suggesting slight cell membrane structural damage, while the majority of cells remain healthy and viable at approximately 93% ± 2.4%. Phosphatidylserine (PS) externalization is an early event in the apoptosis cascade. Annexin V binds to PS with high affinity. Our FACS analysis hence also demonstrates that very few cells were annexin V positive 24 h after exposure to SGS which ruled out apoptosis as a significant cell death mechanism, as was similarly reported for GO materials [16, 18]. Cellular internalization of SGSs Figure  4 depicts high-resolution SEM images of both SNU449 and Hep3B cancer cells after exposure to SGS at a concentration of 10 μg/ml for 24 h.

The following antimicrobial agents (disk contents indicated in parentheses) were tested: ampicillin (10 μg), chloramphenicol (30 μg), streptomycin (10 μg), sulfonamides (300 μg), tetracycline (30 μg), trimethoprim (5 μg), nalidixic acid (30 μg), kanamycin (30 μg), ciprofloxacin (5 μg), ceftazidime (30 μg), gentamicin (10 μg) and minocycline (30 μg) (OXOID, Hampshire, United Kingdom). Escherichia coli ATCC 25922 was used as the control. Phage typing Phage typing of S. Typhimurium and S. Enteritidis isolates was performed in accordance with the methods of the Laboratory of Enteric Pathogens, Health Protection Agency, Colindale,

Aloxistatin London, United Kingdom [19, 20]. Pulsed field gel electrophoresis Pulsed field gel electrophoresis (PFGE) using the PulseNet standard protocol [21] was performed on selected isolates. DNA was digested using restriction enzymes XbaI (Roche, Basel, Switzerland) and BlnI (Sigma-Aldrich,

Dorset, England) and DNA fragments were separated using the CHEF Mapper XA (Bio-Rad, California) system. Multi-locus variance analysis Multi-locus variable-number tandem-repeats analysis (MLVA) using the method of Linstedt et al. [22] was performed on selected S. Typhimurium isolates. DNA was extracted Selleckchem MLN0128 using Qiaqen QIAamp mini kit (Qiagen, West Sussex, UK) and PCR was performed with flouresent primers (Sigma-Genosys, Suffolk, UK) using Qiagen Multiplex PCR master mix kit (Qiagen) on a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Chesire, UK). Fragments were separated using a Beckman Coulter CEQ™ 8000 DNA analysis system (Beckmann-Coulter, Fullerton, CA). Review of records The collection of isolates and our records Farnesyltransferase were reviewed to identify possible episodes of laboratory cross contamination and sending laboratories were contacted to request submission of quality control strains (where not previously submitted) and to discuss the possibility of cross contamination. Acknowledgements We wish

to acknowledge the contribution of the laboratories that have submitted the isolates described in this report and colleagues in Departments of Public Health and Environmental Health for helpful discussion. Electronic supplementary material Additional file 1: Summary of all Suspected Contamination Incidents investigated by NSRL from 2000–2007. The table provided represents all the suspected contamination incidents investigated by the NSRL from the years 2000–2007, including the isolates concerned, their stated source and their probable cause. (DOC 111 KB) References 1. Millar BC, Xu J, Moore JE: Risk assessment models and contamination management: implications for broad-range ribosomal DNA PCR as a diagnostic tool in medical bacteriology. J Clin Microbiol 2002,40(5):1575–1580.CrossRefPubMed 2. Caplan J: Cleaning up Peter Pan’s Mess. [http://​www.​time.

Under our test conditions, the doubling time of E. coli ΔssrA mutant was twice that of the wild type

strain (Figure 2). Interestingly, wild type growth was restored in the E. coli ΔssrA mutant complemented with plasmid pILL788 that expresses high levels of Hp-SsrA (Figure 2) but not with plasmid PD-0332991 order pILL2318 that expresses low levels of Hp-SsrA. As a control, wild type growth was also observed with strain MG1655 ΔssrA pILL2334 expressing wild type Ec-SsrA. This indicated that Hp-SsrA is functional to rescue the growth defect of E coli ΔssrA but is not able to restore the phage propagation deficiency. We then wanted to understand further the functional basis of the partial functionality of Hp-SsrA in E. coli. Analysis of the functionality of mutated Hp-SsrA versions in E. coli In a previous study, we constructed a series of five H. pylori SsrA mutants and evaluated in H. pylori their impact on trans-translation, check details survival and stress-response [10]. Characteristics of these mutations are summarized in Figure 4. Plasmids pILL793, pILL794 and pILL792 express mutant Hp-SsrA that are unable to be alanylated on the TLD (SsrAwobble), to interact with SmpB (SsrASmpB)

and to restart the translation on the MLD (SsrAresume), respectively. Each of this mutation was found to be essential for growth of H. pylori [10]. When these plasmids were tested for complementation of the E. coli GBA3 ΔssrA mutant, neither phage propagation nor growth defective phenotypes was rescued (Figure 2 and Table 3). Figure 4 Mutations introduced into the H. pylori

SsrA molecule. The model of the H. pylori mature SsrA molecule is after the tmRNA website http://​www.​indiana.​edu/​~tmrna/​. As described in [10], the SsrA wobble , SsrA SmpB , SsrA resume mutations that abolish the trans-translation process are boxed in red. Mutations of the mRNA-like domain that affect the tag are also indicated. The amino acid sequence of the tag (wild type or mutant) appended to trans-translated proteins are listed in the table. In H. pylori, two mutations in the MLD of Hp-SsrA were found to be viable but affected the capacity of the corresponding mutant strains to resist to various stresses [10]. One mutation targets the terminal part of the tag sequence, the corresponding mutant gene Hp-SsrADD is carried by plasmid pILL791. This mutation was chosen because it was described to stabilize the trans-translated proteins in species like E. coli. In another mutant, Hp-SsrASTOP (carried by pILL2328) two stop codons were introduced immediately downstream from the resume codon. As a consequence, Hp-SsrASTOP adds a minimal tag (Ala-Val) to trans-translated proteins (Figure 4). These two mutated Hp-SsrA versions did not restore the phage propagation capacity to the E. coli ΔssrA mutant (Table 3). Interestingly, growth defect of the E.

caviae JL006 EcoRV agcaGATATCttaagattctgtttgat incB C. caviae JL014 EcoRI agcaGAATTCatgacctctgtaagaga incC C. caviae JL013 EcoRV agcaGATATCtaaatgtccggtaggag incC C. caviae DA114 Dasatinib research buy EcoRI agcaGAATTCatggtgagcaagggcga GFP DA115 EcoRV agcaGATATCctacttgtacagctccatg GFP The restriction sites built into oligonucleotides for cloning purposes are shown in capital letters. Antibodies, transfection experiments and immunofluorescence microscopy Monoclonal antibody recognizing chlamydial lipopolysaccharide was a gift from Harlan Caldwell of the Rocky Mountain laboratories, Hamilton, MT.

Monoclonal antibody A57B9 (anti-HSP60) recognizes a genus common epitope on chlamydial HSP60 protein [25]. Monoclonal antibodies used in the analysis of CT223p localization in C. trachomatis-infected HeLa or McCoy cells were produced and used as previously described [25]. Rabbit polyclonal anti-CT223p antisera was generated against the peptide sequence NH3-NGINDLSPAPEAKKTGSGL and were produced commercially (Proteintech, Chicago, IL). For these experiments, cells were infected with chlamydiae and incubated for time periods indicated in the figure legends. Cells were then fixed with 100% methanol and used for immunofluorescence. Transfection of plasmids into HeLa or McCoy cells grown on sterile glass coverslips was conducted using Lipofectamine 2000 (Gibco) according to the manufacturer’s

instructions. Transfected cells were Staurosporine incubated for 36 hours and then fixed with methanol. The efficiency of transfection acetylcholine was determined by labeling with monoclonal anti-6-His antibody (Clontech) and secondary FITC or TRITC fluorescent antibodies (Southern Biotechnology Associates) to detect the product of the transgene. Monoclonal anti-γ-tubulin antibodies (Sigma)

were used to detect centrosomes. Cells expressing gfp were analyzed without labeling. Coverslips were examined under 1000× magnification using a Leica fluorescence microscope and images were collected using the SPOT digital camera system (Diagnostic Instruments Inc., Sterling Heights, MI). The rates of cells with a polynuclear phenotype were determined by counting transfected cells with two or more nuclei among the total population of transfected cells. Statistical analysis The number of transfected cells having a polynuclear phenotype was evaluated in at least three independent experiments for each plasmid construct tested. A total of at least 500 individual transfected cells were counted for each tested plasmid construct. Standard deviations were calculated for each individual plasmid construct examined and the significance of differences between means was evaluated using both the Student’s t-test and the Kruskall-Wallis test, as calculated using the Instat software program (GraphPad Software, San Diego, CA).

0 or PB pH 7.5 to a 30 μl volume that was poured on NGM agarized media (peptone, 2.5 g/L; NaCl, 3 g/L; MgSO4,

1 mM; CaCl2, 1 mM; agar 17 g/L) supplemented with 25 mM PB pH 6.0 or pH 7.5, respectively. PAO1 lawns were grown during 24 hrs at 37°C buy SRT1720 following overnight incubation at room temperature, and then were used for feeding C. elegans. As a control of phosphate limitation, P. aeruginosa PAO1 lawns were prepared on NGM containing 0.1 mM PB, pH6.0. Pre-fasted worms were transferred onto lawns and mortality followed for up to 60 hrs. Genome-wide transcriptional analysis All samples for gene expression analysis were prepared in triplicate. P. aeruginosa MPAO1 cells collected from lawns grown on NGM/[Pi]25 mM, pH 6.0 or NGM/[Pi]25, pH 7.5 were used for RNA isolation as previously described. Microarray analysis was performed using Affymetrix P. aeruginosa GeneChips (Affymetrix, Santa Clara, CA) at the University of Chicago Functional Genomics Facility and data were analyzed as previously described [9]. Microarray data were deposited in GEO database, accession number GSE29789. QRT-PCR analysis Multiplex qRT-PCR was performed to simultaneously analyze the expression of selected genes in P. aeruginosa

MPAO1 grown under pH 6.0 and pH 7.5 in NGM-Pi 25 mM. Gene clusters for the analysis were chosen as representatives of phosphate signaling and acquisition, quorum sensing, and iron acquisition. Overnight P. aeruginosa MPAO1 culture was diluted 1:50 in triplicate Ferroptosis inhibitor drugs Oxalosuccinic acid in 25 mM phosphate NGM media at pH 6.0 and 7.5, and grown for 9 hrs at 37°C. RNA was isolated and reversed to cDNA as previously described [7]. QRT-PCR analysis was performed as previously described [9]. Briefly, gene specific primers (Tm = 60°C) to amplify 100 bp fragments of target

mRNA were designed based on in silica analysis for amplification specificity by BLAST search against the database of P. aeruginosa PAO1 genome. Gene expression was normalized to tpiA (PA4748) whose expression was not influenced by pH in microarray analysis, and which was used in our previous QRT-PCR analyses [9]. Fold changes of expression levels were determined by normalization to expression at pH 6.0. Pyoverdin assay Pyoverdin production was measured by fluorescence at 400 ± 10/460 ± 10 excitation/emission, and measurements of relative fluorescence units (RFU) were normalized to cell density units as absorbance at 600 nm in bacterial cultures growing in black, clear bottom 96-well plates (Corning Incorporated, Corning, NY, Costar 3603) using a 96-well Microplate Fluorimeter Plate Reader (Synergy HT, Biotek Inc., Winooski, VT). In the experiments with iron supplementation, pyoverdin was measured in supernatants by absorbance at 405 nm as previously described [17], and normalized to initial cell density.

The diffraction peaks obtained with the addition of both PD332991 KOH and EDA into the reaction system correspond to the phase of Fe3O4, JCPDS card no. 19-0629, which is a face-centered cubic structure with space group . The characteristic reflections in the Fe3O4 phase and the γ-Fe2O3 phase are about the same [38]. Here diffraction of the (221), (210), and (213) planes for the γ-Fe2O3 phase does not exist. To further clarify the phase of polyhedral particles, the Raman spectra of α-Fe2O3 hexagonal plates and Fe3O4 polyhedral particles are shown in Figure 2. α-Fe2O3 here can be characterized by four strong peaks at around 225, 299, 412,

and 613 cm-1 and two weak peaks around 247 and 497 cm-1. The peaks at 538 and 668 cm-1 were selleck chemical attributed to Fe3O4, while the peaks at 350, 500, and 700 cm-1 belonging to γ-Fe2O3 were not observed

[39, 40]. The appearance of the Fe3O4 phase during reaction is a clear evidence that the valence change from Fe3+ to Fe2+ must occur due to the fact that Fe2+ ions occupy the octahedral sites of Fe3O4. Figure 1 SEM images and corresponding XRD patterns of iron oxide particles. SEM images of iron oxide particles prepared with the addition of (a) 5 ml of 10.67 M KOH, (b) 1 ml of EDA, and (c) both 5 ml of 10.67 M KOH and 1 ml of EDA into the ferric solutions. (d) The corresponding XRD patterns of the iron oxide particles obtained for the cases of (a), (b), and (c). Figure 2 Raman spectra of α-Fe 2 O 3 hexagonal aminophylline plates and Fe 3 O 4 polyhedral particles. The α-Fe2O3 hexagonal plates have an average size of about 10 μm in edge length and about 500 nm in thickness. The average lateral size of the α-Fe2O3 particles with the shape of a hexagonal bipyramid is about 120 nm. The Fe3O4 polyhedral particles with mainly octahedral shape have an average lateral size in the range of 5 to 25 μm. The particles obtained from the reaction system with the addition of KOH and EDA alone have the same phase but different shapes. One would assume that the reaction system with the addition of both KOH and EDA would produce particles with maybe different shapes but still maintain the

phase of α-Fe2O3. However, the results show that the particles that we obtained have a different phase, Fe3O4, and, surely, a different shape. The transmission electron microscopy images and the corresponding selected area electron diffraction (SAED) patterns of iron oxide particles are shown in Figure 3. The diffraction patterns of the particles confirmed the results of the XRD diffractions. In Figure 3b, the zone axis of the hexagonal plate is [0001] and the six directions normal to the edge are and its other five equivalent directions. In Figure 3d, the hexagonal bipyramid shows that the pyramid is pointed in the direction of <0001>. According to the literatures, the bipyramidal structure was enclosed by crystal planes [41].