Finally, a lift-off process was performed to get the final Al/Cu/GeO x /W (device S1) memory device, i.e., called Cu/GeO x /W structure hereafter. Similarly, an Al/GeO x /W (device S2) memory device without a Cu layer was also prepared for comparison. Table  2 shows the structures of the fabricated memory devices. A schematic illustration of the fabricated GeO x -based click here cross-point memory device is shown in Figure  1a. The GeO x solid electrolyte

is sandwiched between Cu or Al TE and W BE. An optical micrograph (OM) of 4 × 5 cross-points is shown clearly in Figure  1b. All cross-points are clearly observed. Table 1 Deposition parameters of different materials Materials Target/granules Methods Vacuum (Torr) Ar gas (SCCM) Power (Watt) Deposition rate W W target RF sputtering 1 × 10-5 25 150 12 nm/min GeO x Ge target RF sputtering 2 × 10-5 25 50 5.3 nm/min Cu Cu granules Thermal evaporator 8 × 10-6 – - 2-3 Å/s GW-572016 nmr Al Al granules Thermal evaporator 8 × 10-6 – - 2-3 Å/s Table 2 Structures of the cross-point resistive switching memory devices Devices BE ~ 200 nm

Switching layer (10 nm) TE       Cu ~ 40 nm Al ~ 160 nm S1 W GeO x √ √ S2 W GeO x × √ Figure 1 Schematic illustration and optical image of the Cu/GeO x /W cross-point memories. (a) Schematic illustration and (b) optical image of our fabricated cross-point memory devices. Active area of the cross-point memory is approximately 1 × 1 μm2. The thickness of the GeO x solid electrolyte film is approximately 10 nm. 2-hydroxyphytanoyl-CoA lyase The cross-point structure and thicknesses of all materials were evaluated from a HRTEM image. HRTEM was carried out using a FEI Tecnai (Hillsboro, OR, USA) G2 F-20 field emission system. Memory characteristics were measured using an HP4156C semiconductor parameter analyzer (Agilent Technologies, Santa Clara, CA, USA). For electrical measurements,

the bias was applied to the TE while the W BE was grounded. Results and discussion Figure  2 shows the TEM image of the Cu/GeO x /W structure (device S1). The area of the cross-point is approximately 1.2 × 1.2 μm2 (Figure  2a). Films deposited layer by layer are clearly observed in the HRTEM image, as shown in Figure  2b. The thickness of the SiO2 layer is approximately 200 nm. The thicknesses of W, Cu, and Al metals are approximately 180, 38, and 160 nm, respectively. The thickness of the GeO x solid electrolyte is approximately 8 nm, as shown in Figure  2c. The formation of a thin (2 to 3 nm) WO x layer is observed at the GeO x /W interface. The HRTEM image of the Al/GeO x /W cross-point memory devices is also shown in Figure  3a. It is interesting to note that the AlO x layer with a thickness of approximately 5 nm at the Al/GeO x interface is observed (Figure  3b). The Gibbs free energies of the Al2O3, GeO2, CuO, and Cu2O films are -1,582, -518.8, -129.7, and -149 kJ/mol at 300 K, respectively [43]. Therefore, the formation of AlO x at the Al/GeO x interface will be the easiest as compared to those of other materials.

canaliculatus or H. geminus, the beetle S. halensis makes up an assemblage of argillophilous

beetles preferring waters with poor vegetation and a higher content of minerals, usually shallow selleck chemicals llc ones, which warm up very quickly. This explains why argillophiles comprise species typical of Mediterranean countries (e.g. N. canaliculatus), which—by inhabiting man-made ponds—expand the borders of their distribution north- and eastwards. The presence of thermophilous species in anthropogenic ponds has also been observed in studies on other taxonomic groups, for example dragonflies (Donath 1980; Ohnesorge 1988; Buczyński 1999; Buczyński and Pakulnicka 2000). Another distinct group of beetles combined species such as H. lineolatus, H. flavicollis, H. fluviatilis, H. fulvus, H. versicolor and H. hamulatus. These beetles are correlated with water conductivity and concentration of SO 4 2 ions, as well as water saturation and dissolved oxygen. These species prefer well-oxygenated waters and are frequent Mocetinostat in vivo in clean lakes, in habitats with sandy substrate,

overgrown with scattered Phragmites australis, or in quiet sites located in slowly flowing rivers. Other species demonstrating a strong relationship with NH4-N, organic P, total P and CO3 2− create a community of eurytopic species, primarily associated with small eutrophic water bodies, abundantly overgrown with aquatic plants. Summary Anthropogenic ponds located in the Masurian Lake District, owing to their environmental characteristics, including the type of substrate, development of macrophytes, age of the pond as well as the physical and chemical parameters of the water it holds, are inhabited by a rich and diverse

fauna of beetles. The physical and chemical parameters of water in the analyzed ponds correspond to the ones assigned to oligotrophic lakes in very good ecological condition. This is the reason why they have been colonized by several species whose natural habitats are disappearing, especially the ones which have been ascribed different statuses of threatened species in Europe, including H. aterrimus (VU), Anacetrapib in Poland under total protection, H. fulvicollis (VU) and G. caspius (EN). At the same time, such ponds create suitable conditions for many rare species of the Polish fauna, which helps to sustain biodiversity, both locally and on a scale surpassing a single region. Thus, anthropogenic ponds are a valuable component of the ecological landscape and deserve to be subjected to a special nature conservation program. Acknowledgments The authors would like to thank Prof. Eugeniusz Biesiadka for his suggestions concerning the research materials as well as his valuable comments during this study. Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

This methodology is probably not restricted to pyrosequencing datasets, and could be, after some modifications, applied to datasets obtained with any kind of sequencing techniques. Acknowledgements This research was financed by

the Swiss National Science Foundation, Grants No. 120536, 138148 and 120627. We recognize the excellent assistance selleck compound of Yoan Rappaz in molecular biology analyses. We acknowledge Scot E. Dowd, Yan Sun, Lars Koenig and at Research and Testing Laboratory (Lubbock, Texas, USA), Timothy M. Vogel, Sébastien Cecillon and the Environmental Microbial Genomics Group at Ecole Centrale de Lyon (France), and GATC Biotech (Konstanz, Germany) for pyrosequencing analyses and advice. We are grateful to Ioannis Xenarios for support and access to the Vital-IT HPCC of the Swiss Institute of Bioinformatics (Lausanne, Switzerland). Electronic supplementary

material Additional file 1: Quality plots generated for samples pyrosequenced with LowRA (>3′000 reads) and HighRA methods (>10′000 reads). Sequence quality PHRED scores over all bases (A): PHRED scores are defined as the logarithm of the base-calling error probability Perror = 10-PHRED/10 and PHRED = −10 log Perror. Box plots represent the distribution of reads quality at each sequence length. The black curve represents the mean sequence quality in function of the sequence length. Distribution of the mean sequence quality PHRED score over the pyrosequencing reads (B). Distribution of sequence lengths over EGFR inhibitors list all pyrosequencing reads (C). Only sequences between 300 and 500 bp were kept for dT-RFLP analysis. (PDF 163 KB) Additional

file 2: Assessment of mapping performances with pyrosequencing datasets denoised without (0–500 bp) and with (300–500 bp) minimal read length cutoff. Examples are given for the groundwater sample GRW01, the flocculent activated sludge sample FLS01 and the aerobic granular sludge sample AGS01. After denoising with the one or the other method, each dataset was mapped against a reference database with MG-RAST [66]. No cutoff was set for e-value, minimum identity and minimum Parvulin alignment length. After having observed that between 35-45% of the sequences were unassigned with Greengenes, RDP – the Ribosomal Database Project [67] was used as reference database for this assessment (only 4% unassigned sequences). Correlations between bacterial community profiles obtained with both denoising methods and both reference databases were analyzed with STAMP [68]. (PDF 375 KB) Additional file 3: Comparison of the distributions of the SW mapping score and of the traditional identity score used by microbial ecologists in the field of environmental sciences for phylogenetic affiliation of sequences.

Solid State Ion 2003, 165:139.CrossRef 10. Guillén C, Herrero J: Transparent conductive

ITO/Ag/ITO multilayer electrodes deposited by sputtering at room temperature. Opt Commun 2009, 282:574.CrossRef 11. Sun X, Huang H, Kwok H: On the initial growth of indium tin oxide on glass. Appl Phys Lett 1996, 68:2663.CrossRef 12. Kim DH, Park MR, Lee HJ, Lee GH: Thickness dependence of electrical properties of ITO film deposited on a plastic substrate by RF magnetron sputtering. Appl Surf Sci 2006, 253:409.CrossRef 13. Jeong JA, KiKim H: Low resistance and highly transparent ITO–Ag–ITO multilayer electrode using surface plasmon resonance of Ag layer for bulk-heterojunction organic solar cells. J Sol Energ Mat Sol C 1801, 2009:93. Competing interests The authors declare that they have no competing interests. Authors’ contributions ZQS and QPX prepared the films and tested the surface topography. X-ray Protein Tyrosine Kinase inhibitor diffraction was investigated by XPS and MCZ. The optical properties were measured by GH. The calculations were

carried out by ZQS who also wrote the manuscript. Besides, MCZ helped draft the manuscript. All authors read and approved the final manuscript.”
“Background The use of nanosized colloids offers exciting new opportunities for biomedical HM781-36B concentration applications as they have the potential to overcome significant limitations associated with therapeutic drugs (e.g., physical, chemical, or biochemical instability). In addition, encapsulation of pharmacologically active agents into such nanocarriers allows for spatial and temporal control of drug release, which can significantly improve clinical effects (e.g., controlled and targeted delivery) [1, 2]. Superparamagnetic Fe3O4 nanoparticles (SPIONs) are explored as novel drug delivery systems as their orientation within a magnetic field offers new opportunities Carbohydrate to manipulate accumulation and/or drug release in desired target tissues by an externally applied magnetic field

[3]. Similar to other biomedical applications of SPIONs, including magnetic resonance imaging, biosensing, and cell separation, clinical development critically depends on efficient magnetization and favorable pharmacokinetic properties that minimize clearance by the reticuloendothelial system. It is generally accepted that nanoparticles with hydrophilic surfaces and those less than 200 nm in diameter are compliant with these desired specifications [4, 5]. The large surface-to-volume ratio of small magnetic nanoparticles increases surface energy and, thus, enhancing particle aggregation. As a consequence, chemical reactivity decreases, magnetic properties deteriorate, and clearance within a biological system increases [6–9]. Particle stability in an aqueous vehicle can be augmented by electrostatic repulsion using charged surface coatings and/or surface-associated ions, including OH-, H3O+, or buffer ions [10].

A large central necrotic/fibrotic area could be observed surrounded by peripherally arranged vital tumor cells (Figure 3C). Figure 3 Analysis of contrast agent induced interior structuring of tumours. (A): Transaxial

NMR images of a mouse (face-down position) bearing two s.c. xenografts; left: HT29 colon carcinoma, right HCT8 colon carcinoma. Images were taken to the indicated time points after i.v. application of higher dosed Gd-BOPTA (0.1 mmol/kg). A time dependent alteration of contrast enhancement with initial enhancement of the tumor rim followed by a centripetal progression of the signal is observed in the HT29 tumor. The HCT8 tumor was too small for detailed analyses although a time dependent alteration Tariquidar cost of the signal could also be observed. (upper panel – grayscale, lower panel – pseudocolor) (B): Transaxial NMR images of a mouse (face-down position) bearing two s.c. HT29 xenografts 15 min and 30 min after i.v. application of Gd-BOPTA. One tumor showed strong contrast enhancement and an interior structuring click here could be observed (white arrow). (C): HE staining of the well structured left HT29 xenograft shown in (A). Depicted is a section at the side of the tumor to represent the whole structure composed of a large central necrotic/fibrotic area (white star) surrounded by peripherally arranged vital tumor cells (white arrow). Monitoring of xenograft tumor growth Apart from tumor detection the quantification of tumor burden

is one important aspect of non-invasive in vivo imaging techniques. To test whether buy Hydroxychloroquine the BT-MRI system is suitable for following s.c. xenograft growth the tumor burden was examined in 2 groups of 3 mice each bearing 2 different tumors: one group with 1411HP germ cell tumor and DLD-1 colon carcinoma, one group with HT29 colon carcinoma and DLD-1 colon carcinoma. Growth of tumors was followed using (a) calliper measurement and volume calculation and (b) BT-MRI and measurement of pixel extensions of tumor sections based on NMR images. For both methods comparable progression profiles could be observed, which was independent of Gd-BOPTA injection. A representative example

of one individual is presented in Figure 4A and 4B. In addition, all values calculated by pixel extension analyses were plotted dependent on respective values calculated by calliper measurement. This demonstrates the correlation of both applications (Figure 4C). Figure 4 Monitoring of xenograft tumor growth. (A): Transaxial NMR images of a mouse (face-down position) bearing two s.c. xenografts (left: 1411HP germ cell tumor, right: DLD-1 colon carcinoma) analysed over 5 weeks (d13, d20, d27, d34 post cell injection). Depicted images were taken 10 min after i.v. application of Gd-BOPTA. White arrows point at tumors. (B): Following tumor growth of example shown in Figure 4A as analysed by calliper measurements and volume calculation compared to analyses by pixel extension of tumor sections based on NMR images (with or without Gd-BOPTA (CA)).

: from the strain to gene study. Environ Microbiol 2008, 10:228–237.

18. Kashyap DR, Botero LM, Franck WL, Hassett DJ, McDermott TR: Complex regulation of arsenite oxidation by Agrobacterium tumefaciens . J Bacteriol 2006, 188:1081–1088.PubMedCrossRef 19. Hamamura N, Macur RE, Korf S, Ackerman G, Taylor WP, Kozubal M, Reysenbach A-L, Inskeep WP: Linking microbial oxidation of arsenic with detection and phylogenetic analysis of arsenite oxidase genes in diverse geothermal environments. Environ Microbiol 2009, 11:421–431.PubMedCrossRef 20. Magurran AE: Ecological click here diversity and its measurement. London: Chapman; 1996. 21. Cullen WR, Polishchuk E, Reimer KJ, Sun YM, Wang L, Lai VWM: Arsenic in Yellowknife, North West Territories, Canada. In Arsenic exposure and health effects V. Edited by: Chappell WR, Abernathy CO, Calderson RL, Thomas this website DJ. USA: Elsevier; 2003:79–88. 22. Walker SW, Jamieson HE, Lanzirotti A, Andrade CF: Determining arsenic speciation in iron oxides derived from a gold-roasting operation: application of synchrotron micro-XRD and micro-XANES at the grain scale. Can Mineral 2005, 43:1205–1224.CrossRef 23. Meng SG, Bang SB, Korfiatis GP: Effects of silicate, sulphate, and carbonate on arsenic removal by ferric chloride. Water Res 2000, 34:1255–1261.CrossRef 24. Meng XG, Korfiatis GP, Jing CY, Christodoulatos C: Redox transformations of arsenic

in water treatment sludge during aging and TCLP extraction. Environ Sci Technol 2001, 35:3476–3481.PubMedCrossRef 25. Tu S, Ma LQ, MacDonald GE, Bondada B: Effects of arsenic species and phosphorus on arsenic absorption, arsenate reduction and thiol find more formation in excised parts of Pteris vittata L. Environ Exp Bot 2004, 51:121–131.CrossRef 26. Lane DJ: 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics. Edited by: Stackebrandt E, Goodfellow M. UK: John Wiley & Sons; 1991:115–163. 27. Alschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local

alignment search tool. J Mol Biol 1990, 215:403–10. 28. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 1997, 25:4876–4882.PubMedCrossRef 29. Felsenstein J: PHYLIP – Phylogeny Inference Package (Version 3.2). Cladistics 1989, 5:164–166. 30. Page RDM: TREEVIEW: An application to display phylogenetic trees on personal computers. Comput Appl Biosci 1996, 12:357–358.PubMed 31. Hurlbert SH: The nonconcept of species diversity: a critique and alternative parameters. Ecology 1971, 52:577–586.CrossRef 32. Tipper JC: Rarefaction and rarefiction – the use and abuse of a method in paleontology. Paleobiol 1979, 5:423–434. Authors’ contributions THO performed the majority of the experiments (clone libraries, 16S rRNA gene sequencing, phylogenetic analyses, GM1 growth experiments and enzyme assays).

Intensity of each protein was quantified by calculation of spot volume after H 89 normalization of the image using the total spot volume normalization method multiplied by the total area of all the spots. The calculation of the theoretical molecular weight and pI values of the identified protein spots is based on algorithms included in the ImageMaster 2D Elite 4.01 analysis software package. Statistical analysis was carried out with SPSS for Windows 10.0 and Excel. MALDI-TOF-MS Differential protein spots were excised from preparative gels using biopsy

punches and transferred to a 1.5 ml siliconized Eppendorf tube. Proteins in-gel was digested as previously described [6]. The gel-spots were destained in the destaining solution consisted of 100 mmol/L Na2S2O3 and 30 mmol/L K3Fe(CN)6 (1:1). The proteins-contained gel-spots were reduced in the reduction buffer consisted of 100 mmol/L NH4HCO3, 10 mmol/L DTT for

1 h at 57°C, and alkylated in the alkylation buffer consisted of 100 mmol/L NH4HCO3and 55 mmol/L iodocetamide in the dark for 30 min at room temperature. The gel pieces were dried in a vacuum centrifuge. The dried gel-pieces were incubated in the digestion solution Doramapimod nmr consisted of 40 mmol/L NH4HCO3, 9%ACN and 20 μg/mL however trypsin(Sigma, St. Louis, USA) for 16 h at 37°C. The tryptic peptide mixture was extracted and purified with Millipore ZIPTIP™C18 column. The purified tryptic peptide mixture was mixed with α-cyano-4-hydroxycinnamic acid (CCA) matrix solution, and vortexed lightly. A volume (1 μl) of the mixture containing CCA matrix was loaded on a stainless steel plate, and dried in the air. The samples were analyzed with Applied Biosystems Voyager System 4307 MALDI-TOF Mass Spectrometer (ABI). The parameters were set up

as following: positive ion-reflector mode, accelerating voltage 20 kV, grid voltage 64.5%, mirror voltage ratio 1.12, N2 laser wavelength 337 nm, pulse width 3 ns, the number of laser shots 50, acquisition mass range 1000–3000 Da, and delay 100 nsec, and vacuum degree 4×10-7Torr. A trypsin-fragment peak was served as internal standard for mass calibration. A list of the corrected mass peaks was the peptide mass fingerprinting (PMF). Database analysis Proteins were identified with peptide mass fingerprinting data by searching software PeptIdent http://​www.​expasy.​org/​ and Mascot http://​www.​matrixscience.​com. Mascot Distiller was used to detect peaks by attempting to fit an ideal isotopic distribution to the experimental data. The searching parameters were set up as following[6, 7]: the mass tolerance was ± 0.

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

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

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

to fill the pores of PS at step I of Figure 6, which is not removed during the developing process at step II. This guarantees the accurate Orotidine 5′-phosphate decarboxylase control of developing time for the photoresist layer, resulting in well-patterned PS structures at step III, as shown in Figure 6c. Our tests showed a 10-s dip in 10% HF/DI is sufficient to remove all SOG in an exposed PS film (where there was no photoresist) up to 2.45-μm thick. The short dip resulted in an optical thickness change of less than 4.4%, suggesting the short dip had very little effect on the PS layer. In this work which used PS layers of 2.45-μm thickness, SOG as a pore filling layer was more advantageous than ProLIFT and was used as described. These results show a complete MEMS fabrication process using a single material system can be achieved using combination of anodization and electropolishing. No sacrificial layer was required to achieve release of the beams.

There is evidence to suggest that dietary supplements such as omega-3 containing fish-oil, specifically the polyunsaturated fatty acid 20:5n3 component (also commonly known as eicosapentaenoic acid or EPA), may be efficient at reducing the pro-inflammatory cytokines associated with inflammation [18, 19]. Magee et al. [18] demonstrated in vitro that EPA inhibited the effects of TNF-α by reducing its apoptotic effects and enabling myogenesis, thus allowing optimal skeletal muscle cell differentiation from myoblasts into myotubes, a process which is key in the regeneration Fosbretabulin clinical trial of muscle following damage. Complimentary evidence was provided in vivo by Matsuyama

et al. [19] who worked with patients suffering

from chronic obstructive pulmonary disease (COPD). COPD is characterised by chronic inflammation and pain in the throat and chest when breathing. Matsuyama et al. [19] treated patients for 24 months with EPA supplementation. With treatment, participants exhibited lower TNF-α levels and reported a reduction pain in comparison with baseline values. The findings from these two studies suggest a link between elevated levels of pro-inflammatory cytokines and pain [6] and also that EPA may be beneficial in reducing the symptoms of DOMS and the level of inflammation associated SCH772984 datasheet with it. In this potential therapeutic context, several studies have already queried whether Enzalutamide clinical trial omega-3/EPA doses between 300 mg/day to 2224 mg/day can affect the acute inflammation response and symptoms associated with DOMS after a single bout of exercise [3, 20, 21]. Lenn et al. [3], using 1800 mg/day of omega-3, reported that EPA had no effect on range of motion, pain, IL-6, TNF-α and creatine kinase levels. However, Phillips

et al. [20] (using a daily cocktail of 300 mg of tocopherols plus 800 mg of docosahexaenoate plus 300 mg of flavonoids) and Bloomer et al. [21] (using 2224 mg/day of EPA) both reported a reduction in IL-6, CRP and TNF-α respectively, following a single bout of exercise. These studies in conjunction with the in vivo and in vitro work mentioned earlier [18, 19] exemplify the confusion as to whether EPA may be beneficial in reducing pro-inflammatory cytokines linked with the inflammatory response and the symptoms associated with DOMS. To date the impact of fish oils on the acute and chronic response to a single bout of exercise remains unclear. Moreover, the conventional dose of 1000-2000 mg per day (of total fish oil or 180-360 of EPA) has mainly been far exceeded in the research to date. Aims and Objectives The aims of the present study were therefore to investigate the effects of a dose of EPA supplementation just above standard recommendations, on basal inflammation, as well on both the acute and the chronic resistance exercise responses.

3 Kb Pst 1 fragment in fur:kanP mutant but not in the wild type. These results confirm that a single copy

of Kmr was correctly inserted in the Fur box Trichostatin A order located in the promoter region of NE0616 gene of the N. europaea genome (Figure 4A). A fur transcript was not detected in the fur:kanP mutant by either RT-PCR or qRT-PCR analysis (up to 28 cycles) indicating the inactivation of fur gene due to Kmr insertion in its promoter region. Transcripts of ammonia monooxygenase C (amoC) component used as positive control both for the efficiency of the RT-PCR procedure and for RNA and cDNA recovery showed no significant difference in expression in wild type and the fur:kanP mutant (data not shown). Figure 4 In vitro transposon mutagenesis scheme and mutant confirmation.

(A) The physical structure of a 5,810-bp fragment Selleck Ku-0059436 of the N. europaea chromosome is shown in the center (heavy black line), with positions of NE0616 (fur) gene shown as grey arrow, the fur box (fb) located in NE0616 promoter region shown as white rectangle. The regions covered by the plasmids pFur616, pFur616-kanP, pFur616-kanC whose DNA sequences were determined are shown as thin black lines with the names of the respective plasmids shown below each line. The position and relative orientation of each in vitro-constructed Tn5-Kan2 cassette insertion mutation are indicated by a flag on the lines. The restriction endonuclease sites P (Pst 1) and E (Eco R1) used for Southern blot confirmation are indicated. (B) Verification of mutagenesis of fur:kanP in N. europaea by Southern hybridization. Genomic DNA from the

wild type (WT), fur:kanP mutant (MT) were digested with E (Eco RI) and Phospholipase D1 P (Pst 1), and probed with (left) fur ORF sequence and (right) kan sequence. Effect of fur:kanP mutation on growth of N. europaea Growth of the N. europaea fur:kanP strain was compared to that of the wild-type strain in both Fe-replete (10 μM Fe) and Fe-limited (0.2 μM Fe) media. Surprisingly, there was no significant difference in growth of fur:kanP in both Fe-replete and Fe-limited media compared to the wild-type strain (Figure 5A). The fur:kanP mutant did not exhibit a growth advantage over the wild type when iron was limiting or show increased sensitivity to iron-induced redox stress when grown in the presence of Fe (up to 250 μM Fe; data not shown). However, growth of fur:kanP mutant was affected when grown in medium containing 500 μM Fe (Figure 5B). The mutant was unable to grow in media containing more than 500 μM Fe (data not shown). Growth of wild type was inhibited only when concentrations of Fe exceeded 1 mM [14]. Figure 5 Growth curves of the N. europaea wild type (solid lines, filled symbols) and fur:kanP mutant (dotted lines, open symbols) as measured by OD. (A) Fe-replete (squares) and Fe-limited (triangles) medium. (B) 500 μM Fe medium (circles) and in Fe-limited medium with 10 μM ferrioxamine (diamonds).