Acknowledgments The research was supported

by the Wroclaw

Acknowledgments The research was supported

by the Wroclaw Research Center EIT+ under the Project “Biotechnologies and advanced medical technologies – BioMed” (POIG 01.01.02-02-003/08-00) financed from the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2). The cytotoxic investigations were carried out with the equipment purchased, thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (Contract No. POIG.02.01.00-12-023/08). Conflict of interest The authors declare that they have no conflict of interest. Open AccessThis article is distributed under the terms of the Creative CUDC-907 price Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary material 1 (DOCX 120 kb) References Balenci D, Bonechi G, D’Amelio N, Gaggelli E, Gaggelli N, Molteni E, Valensin G, Szczepanik W, Dziuba M, Święcicki G, Jeżowska-Bojczuk M (2009) Structural features and oxidative stress towards plasmid DNA of apramycin Selleck GDC 0068 copper complex. Dalton Trans 7:1123–1130PubMedCrossRef Baron ESG,

DeMeio RH, Klemperer F (1936) Studies on biological Nintedanib (BIBF 1120) oxidations: V. Copper and hemochromogens as catalysts for the oxidation of ascorbic acid. The mechanism of the oxidation. J Biol Chem 112:625–640 Bertini I, Pierattelli R (2004) Copper(II) proteins are amenable for NMR investigations. Pure Appl Chem 76:321–333CrossRef Chibber S, Hassan I, Farhan M, Naseem I (2012) Light-mediated interaction of methotrexate with this website transition metal Cu(II). Med Chem Res 21:2379–2387CrossRef de Hoog P, Boldron C, Gamez P, Sliedregt-Bol K, Roland I, Pitie M, Kiss R, Meunier B, Reedijk J (2007) New approach for the preparation of efficient DNA-cleaving agents: ditopic copper–platinum complexes based on 3-clip-phen and cisplatin. J Med Chem 50:3148–3152PubMedCrossRef Devereux M,

Shea DO, Kellett A, McCann M, Walsh M, Egan D, Deegan C, Kedziora K, Rosair G, Muller-Bunz H (2007) Synthesis, X-ray crystal structures and biomimetic and anticancer activities of novel copper(II) benzoate complexes incorporating 2-(4′-thiazolyl)benzimidazole (thiabendazole), 2-(2-pyridyl)benzimidazole and 1,10-phenanthroline as chelating nitrogen donor ligands. J Inorg Biochem 101:881–892PubMedCrossRef Dunger A, Limbach HH, Weisz K (1998) NMR studies on the self-association of uridine and uridine analogues. Chem Eur J 4:621–628CrossRef Franco R, Panayiotidis MI, Cidlowski JA (2007) Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation.

After cultivation, the optical density at 600 nm of the cell cult

After cultivation, the optical density at 600 nm of the cell cultures was adjusted to 0.5 with each respective medium. The cells were collected by centrifugation (10,000 g for 15 min), and the resulting click here supernatants were filtered (low protein binding Durapore membrane, 0.45 mm polyvinylidene fluoride,

Millipore, Bedford, Mass.). The filtrates were centrifuged (40,000 g, 2 h at 4°C), washed with PBS and re-centrifuged (40,000 g, 2 h at 4°C). The pellets were next resuspended in PBS supplemented with 0.2 M NaCl. The media without the bacteria were used as controls. The OMV of strain TK1402 in Brucella broth supplemented with 0.2% β-cyclodextrin buy Trichostatin A or 7% horse serum were also isolated in a similar manner. Sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques The fractionated OMV (OMV-fraction) were treated with sodium dodecyl sulfate (SDS) loading buffer including 5% 2-mercaptoethanol at 100°C for 5 min and separated by polyacrylamide gel electrophoresis (PAGE). The separated OMV proteins were stained with Coomassie brilliant blue. For Western blotting assays, the OMV-fractions were loaded onto gels and transferred to polyvinylidene difluoride membranes (Atto, Tokyo, Japan). After transfer, the membranes were blocked with 3% bovine serum albumin in PBS for 60 min and incubated with H. pylori strain NCTC 11638 whole-cell antiserum (1:2,000) [36] for 60 min. After washing with PBS containing 0.05% Tween 20 (PBST), peroxidase-labeled goat anti-rabbit Selonsertib price immunogloblins (Dako A/S, Glostrup, Denmark) were used at 1:2,000 dilution as secondary antibodies. After washing with PBST, the blots were developed. Complementation of biofilm forming ability using the OMV The OMV-fraction from Brucella broth supplemented with 7% FCS (OMV-fraction) and the medium fraction (control-fraction) in PBS were adjusted to an optical density of 2.0, or 1.0 at 280 nm. The OMV-fractions from Brucella broth supplemented with 0.2% β-cyclodextrin were also adjusted to optical densities

of 1.0. After filtration, 100 μl of the fractionated OMV were added Interleukin-2 receptor to Brucella broth with 0.2% β-cyclodextrin for TK1402 biofilm formation assays (described above). Statistical analysis Statistical analysis was performed using the Mann-Whitney U test. P values of 0.05 or less were considered to indicate statistical significance. Acknowledgements This work was supported by Grants for Scientific Research 18590437 from the Ministry of Education, Culture, Sport, Science and Technology and a grant from the Dental Research Center, Nihon University School of Dentistry. References 1. Marshall BJ, Warren JR: Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984, 16:1311–1315.CrossRef 2. Blaser MJ:Helicobacter pylori : its role in disease. Clin Infect Dis 1992, 15:386–391.PubMed 3.

For comparison, we prepared TiO2 nanoparticles with an average di

For comparison, we prepared TiO2 nanoparticles with an average diameter of 50 nm through a sol–gel method (Figure  1f). Figure 1 XRD patterns and SEM, TEM, and HRTEM images of the hybrid [email protected] 2 . XRD patterns (a) and SEM image (b) of the [email protected] hybrids, SEM image (c) of a single [email protected] hybrid, TEM (d) and HRTEM (e) images of the tip of a [email protected] hybrid with red arrows indicating TiO2 nanoparticles, CP673451 cell line and SEM image (f) of TiO2 nanoparticles prepared through a sol–gel method. The present [email protected] feature a favorable porous structure and improved electrical conductivity, which are attractive for addressing the existing issues for

TiO2 as anodes of LIBs; therefore, we systematically investigated the electrochemical performance of the [email protected] as anode of LIBs. We first applied the techniques of galvanostatic charge/discharge and CV to compare and study the electrochemical properties of lithium insertion/deinsertion in half-cells based on CNT,

TiO2, and [email protected] materials. Figure  2a,b,c and Figure  2d,e,f display the SBE-��-CD chemical structure initial two charge–discharge profiles and CV curves for the CNT, TiO2, and [email protected] electrodes, respectively. learn more The initial two charge–discharge profiles are generally consistent with the corresponding CV results. For CNTs, there is no pronounced peak in the range of 1.0 to 3.0 V with a remarkable discharge capacity loss from 55 mAh g-1 in the first cycle to 20 mAh g-1 in the second cycle. In contrast, both TiO2 and [email protected] electrodes show a discharge plateau at around 1.70 V and a charge plateau at about 1.90 V in the first cycle, which is basically consistent with those reported previously [20, 21]. In particular, the TiO2 electrode exhibits a pronounced capacity loss of 20.0% in the second discharge process, while the [email protected] electrode only shows a capacity loss of less than 10.0% in the initial two cycles. As expected, there is a pair of peaks in the CV curves of the TiO2 and

[email protected] electrodes, namely, the cathodic peak at 1.69 V and the anodic peak at 2.08 V, corresponding with the reversible biphasic transition between the tetragonal anatase and orthorhombic Li x TiO2, respectively (Equation 1). (1) Oxalosuccinic acid Figure 2 The first two charge/discharge profiles and CV curves. CNTs (a), TiO2 nanoparticles (b), and [email protected] (c) LIB anodes at a current density of 100 mA g-1. The initial two cyclic voltammograms of CNTs (d), TiO2 (e), and [email protected] (f). There is an observable decrease of cathodic current in the second CV compared with the first CV for the TiO2 electrode, which agrees with the previous report on TiO2 anode materials and can be attributed to the irreversible lithium insertion-deinsertion reaction, indicating a large capacity loss during the first two cycles. The [email protected], however, only display a small change during the initial two CVs, suggesting a small capacity loss in the initial two cycles.

With a $50 annual difference,

With a $50 annual difference, check details every 20,000 users of alendronate or risedronate instead of etidronate costs the public system $1 million. The difference in costs between agents may be justifiable if one agent is more effective at reducing fracture risk. However, little comparative effectiveness data are available to support the superiority of any of the oral bisphosphonates in reducing fracture risk. To our knowledge, only a single study has directly compared the effects of etidronate to alendronate or risedronate in reducing fracture risk

[10]. Authors found little difference in hip fracture rates within 2 years between female fracture patients receiving etidronate compared to alendronate or risedronate (HR = 1.0, 95%; CI = 0.6–1.6) Vadimezan solubility dmso [10]. More data are needed to clarify the comparative effectiveness of oral bisphosphonates in reducing fracture risk. Many provinces in Canada continue to restrict access to alendronate and

risedronate through public drug plans. In the absence of clear evidence of superiority compared with etidronate—despite differences between agents based on placebo-controlled trials—it may be difficult for policy makers to justify the additional costs to the public healthcare system by covering second-generation bisphosphonates without restriction. Our study is subject to some limitations. First, we were limited to publicly funded drug claims in Ontario, restricting us from assessing drugs dispensed yet processed through private insurance or out-of-pocket. Thus, we are limited in ability to assess the use of medications that are not listed on the Ontario formulary such as calcitonin, teriparatide, and zoledronic acid, as well as alendronate and risedronate dispensing outside the public plan. Second, we are limited to pharmacy claims data and do not have a record of medications prescribed yet not dispensed in community pharmacies. Despite these limitations, our study Niclosamide has significant strength. We were

able to generate temporal trends in drug dispensing patterns and identify significant differences in osteoporosis pharmacotherapy between provinces in Canada related to drug coverage policies. BC recently broadened coverage for alendronate (November 2009) and risedronate (January 2011) to remove the need for a prior trial of etidronate. However, access to these second-generation bisphosphonates through BC PharmaCare still requires clinical or radiographically confirmed fracture or long-term glucocorticoid use. Our results identify that physicians prefer to prescribe following evidence-based guidelines that rank treatment as first-line (e.g., alendronate, risedroante) or second-line (e.g., etidronate) based on placebo-controlled efficacy in reducing fracture risk, with a shift toward alendronate and risedronate when available. Better evidence regarding the comparative effectiveness of oral bisphosphonates is needed to inform drug policy decision making in Canada.

Electroanalysis 2007, 19:1023–1031 CrossRef 8 Wang Y, Yuan H, Lu

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properties. J Solid State Chem 2011, 184:1333–1338.CrossRef 11. Vick D, Friedrich L, Dew S, Brett M, Robbie K, Seto M, Smy T: Self-shadowing and surface diffusion effects in obliquely deposited thin films. Thin Solid Films 1999, 339:88–94.CrossRef 12. Dolatshahi-Pirouz A, Hovgaard MB, Rechendorff K, Chevallier J, Foss M, Besenbacher F: Scaling behavior of the surface roughness of platinum films grown by

oblique angle deposition. Phys Rev B 2008, 77:115427.CrossRef 13. Poziotinib in vitro Wolcott A, Smith WA, Kuykendall TR, Zhao Y, Zhang JZ: Photoelectrochemical water splitting using dense and aligned TiO2 nanorod arrays. Small 2009, 5:104–111.CrossRef 14. Xie Z, Zhang Y, Liu X, Wang W, Zhan P, Li Z, Zhang Z: Visible light photoelectrochemical properties of N-Doped TiO 2 nanorod arrays from TiN. J Nanomater 2013., 2013: 15. Dohnalek Z, Kimmel GA, Ayotte P, Smith RS, Kay BD: The deposition angle-dependent density of amorphous solid water films. J Chem Phys 2003, 118:364.CrossRef 16. Zhao J, Wang X, Chen Z, Yang S, Shi T, Liu X: Overall energy model for preferred growth of TiN films MLN4924 in vivo during filtered arc deposition. J Phys D Appl Phys 1997, 30:5.CrossRef 17. Ni J, Zhu Y, Wang S, Li Z, Zhang Z, Wei B: Fenbendazole Nanostructuring HfO2 thin films as antireflection coatings. J Am Ceram Soc 2009, 92:3077–3080.CrossRef 18. Ho PK, Stephen D, Friend RH, Tessler N: All-polymer optoelectronic devices. Science 1999, 285:233–236.CrossRef 19. Qian L, Yang X: Composite film of carbon nanotubes and

chitosan for preparation of amperometric hydrogen peroxide biosensor. Talanta 2006, 68:721–727.CrossRef 20. Miao Y, Tan SN: Amperometric hydrogen peroxide biosensor based on immobilization of peroxidase in chitosan matrix crosslinked with glutaraldehyde. Analyst 2000, 125:1591–1594.CrossRef 21. Wang G, Xu J-J, Chen H-Y, Lu Z-H: Amperometric hydrogen peroxide biosensor with sol–gel/chitosan network-like film as immobilization matrix. Biosens Bioelectron 2003, 18:335–343.CrossRef 22. Liu Y, Chu Z, Jin W: A sensitivity-controlled hydrogen peroxide sensor based on self-assembled prussian blue modified electrode. Electrochem Commun 2009, 11:484–487.CrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions ZX carried out the fabrication and characterization of the study and drafted the manuscript. XL participated in the design and coordination of the study.

J Biol Chem 286:35683–35688PubMedCrossRef Jordan DB, Ogren WL (19

J Biol Chem 286:35683–35688PubMedCrossRef Jordan DB, Ogren WL (1981) A sensitive assay procedure for simultaneous determination of ribulose-1,5-bisphosphate carboxylase and oxygenase activities. Plant Physiol 67:237–245PubMedCentralPubMedCrossRef

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Lan Y, Woodrow IE, Mott KA (1992) Light-dependent changes in Ribulose bisphosphate carboxylase activase activity in leaves. Plant Physiol 99:304–309PubMedCentralPubMedCrossRef Larson EM, O’Brien CM, Zhu G, Spreitzer RJ, Portis AR Jr (1997) Specificity for activase is changed by a Pro-89 to Arg Amino acid transporter substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. J Biol Chem 272:17033–17037PubMedCrossRef Li C, Salvucci ME, Portis AR Jr (2005) Two residues of Rubisco VRT752271 clinical trial activase involved in recognition of the Rubisco substrate. J Biol Chem 280:24864–24869PubMedCrossRef Lorimer GH, Badger MR, Andrews TJ (1977) D-ribulose-1,5-bisphosphate carboxylase-oxygenase. Improved methods for the activation and assay of catalytic activity. Anal Biochem 78:66–75PubMedCrossRef Mueller-Cajar O, Stotz M, Wendler P, Hartl FU, Bracher A, Hayer-Hartl M (2011) Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase. Nature 479:194–199PubMedCrossRef Ott CM, Smith BD, Portis AR Jr, Spreitzer RJ (2000) Activase region on chloroplast Ribulose-1, 5-bisphosphate carboxylase/oxygenase:

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Nanoparticles behave differently from their respective bulk mater

Nanoparticles behave differently from their respective bulk materials and thus the unique properties of the nanoparticles might also cause adverse health effects on human, animal and environment. The speedy commercialization of nanotechnology requires thoughtful and careful environmental, animal and human health safety assessment [18,19]. The detection and quantification of viable bacteria plays a critical role in quality control programs of the food, cosmetics and drug industry to prevent Selleckchem Pevonedistat illness and in clinical diagnosis and therapeutics. Currently there are many methods used for the detection and quantification of bacteria,

ncluding conventional and molecular approaches PD0332991 price [20-24]. Conventionally identification of bacteria is usually performed by three methods including culture-based counting for colony-forming units Selleckchem Tariquidar (CFU) [22,25], spectrophotometer method of optical density (OD) measurement

[23,24], and flow cytometry (FCM) [26,27]. In spite of the sensitivity and reliability, counting CFU is time-consuming and labor-intensive [28,29]. CFU determination is the conventional method to quantify bacteria, but only detects those that are able to grow on specific solid media, which excludes the detection of unculturable live, inactive or damaged bacterial cells [30,31]. Therefore, CFU counting tends to undercount the actual number of the bacteria. For example, anaerobic bacteria are not able to grow on the media and cultural conditions suitable for growth of aerobic bacteria. Optical density method measures turbidity associated directly with bacterial growth which is rapid, low cost and non-destructive,

however, it measures live as well as dead bacterial cell debris. Flow cytometry is a relatively newly developed technique and enables a fast and reliable detection of all bacteria including the non-cultivable microorganisms. It enables researchers to reliably distinguish and quantitate live and dead Isotretinoin bacteria with the aid of a flow cytometer in a mixed population containing various bacterial types [32]. Besides, Flow cytometry method is able to provide morphometric and functional properties of the detected bacteria [33,34]. Currently all these three methods are employed to quantify bacterial contents in the presence of nanoparticles [35-39]. So far there has not been any research performed concerning potential interference by nanoparticles on the bacterial counting methods. The aim of this study was to compare three commonly used conventional methods for bacterial detection and quantification in the presence of nanoparticles.

(C) upper panel depicts detection of gp340 in parotid saliva alon

(C) upper panel depicts detection of gp340 in parotid saliva alone and after incubation with five different L. gasseri isolates and the L. gasseri type strain; (D) upper panel depicts detection of gp340 and lower panel detection of MUC7 in submandibular/sublingual saliva alone and after incubation with five different L. gasseri isolates and the type strain. Numbers below lanes in panels C and D refer to the following contents: (1) Saliva alone (+ve control), (2) Saliva after L. gasseri CCUG31451T incubation, (3) Saliva after L. gasseri isolate A241 incubation, (4) Saliva after L. gasseri

isolate A274 incubation, (5) Saliva after L. gasseri isolate B1 incubation, (6) Saliva after L. gasseri isolate learn more B16 incubation, (7) Saliva after L. gasseri isolate L10 incubation. MUC7 (mw ≈150 kDa) was detected using Western blot analysis with mAb LUM7-2 antibodies in submandibular saliva (Figure 4, lower panels A and B, lane 6, lower panel D lane 1) but not in parotid saliva (data not shown). MUC7 selleck products levels were reduced in submandibular saliva after incubation with L. gasseri (Figure 4, click here lower

panel A, lane 7) and S. mutans (Figure 4, lower panels B, lane 7). MUC7 was detected bound to L. gasseri (Figure 4, lower panel A, lane 8) and S. mutans (Figure 4, lower panel B, lane 8) after incubation with submandibular saliva. SDS treatment

released the MUC7 bound to L. gasseri (Figure 4, lower panel A, lane 9) and to S. mutans (Figure 4, lower panels B, lane 9). Similar results were observed for MUC7 binding to six additional isolates of L. gasseri (Figure 4D, lower panel). L. gasseri binds to human epithelial cells Adherence of FITC-tagged L. gasseri strains was detected by fluorescence microscopy as illustrated for strain A274 (Figure 5). All L gasseri strains were observed only adjacent to epithelial cells. Figure 5 Adhesion PRKACG of L. gasseri to human epithelial cells. Field of view containing differentiated human gingival epithelial cells (HGEP.05) and fluorescently stained L. gasseri A274 (in green). Bacteria were detected only in association with gingival epithelial cells. Images were captured using a Zeiss imager Z1 upright microscope. Bars in panels equal 20 μm. Discussion In this study lactobacilli were detected more frequently in breastfed than formula-fed 4 month-old infants in saliva and mucosal swab samples as we previously observed in a different population of infants [13]. L. gasseri was the dominant Lactobacillus species detected, which was identified from 16S RNA gene sequences of isolates. Probiotic potential of L. gasseri was found to include growth inhibition of F. nucleatum, A. naeslundii, A. oris, S. sobrinus and C.

A pathologist scored protein

expression as the percentage

A pathologist scored protein

expression as the percentage of positive tumor cells (scale 0–100%) 17-AAG with a staining intensity from 0–3+. Positive IHC expression was defined as >25% staining with an intensity of 2–3 +. Cell culture and RNA interference (RNAi) Human GC cell lines SGC7901 and MGC803 (CBTCCCAS, Shanghai, China) were cultured in RPMI-1640 (Life Technologies, Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen), penicillin/streptomycin (1:100 dilution; Sigma, St. Louis, MO), and 4 mM glutamine (Life Technologies, Gibco BRL) at 37°C/5% CO2. RNAi assays were conducted according to previous methods [18]. Western blotting assays Western blotting was used to detect expression levels of proteins as described previously [18, 23]. We used antibodies against AQP3 (Santa Cruz Biotechnology, Santa Cruz, CA), vimentin, E-cadherin, Snail, AKT, phospho-AKT(Ser473) (Cell Signaling Technology, Beverly, MA), fibronectin (R&D systems, Minneapolis, MN), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime Institute of Biotechnology,

Henan, China). Densitometric analysis of proteins was conducted and normalized against GAPDH. The PI3 kinase inhibitor LY294002, was obtained from Cell Signaling Technology (Beverly, MA). Real-time quantitative polymerase chain reaction (qPCR) assays We conducted qPCR assays using previously NU7441 solubility dmso described protocols [18, 23] and the manufacturer’s instructions. We used GAPDH as the reference gene for analysis, with observed expression levels normalized to the expression level of GAPDH. Specific primer sequences check details were used to amplify targets for AQP3 (5′-CTC GTG AGC CCT GGA TCA AGC-3′ and 5′-AAA GCT GGT TGT CGG CGA AGT-3′), vimentin (5′-ATC TGG ATT CAC TCC CTC TGG TTG-3′ and 5′-CAA GGT CAT CGT GAT GCT GAG AAG-3′), fibronectin (5′-TGT TAT GGA GGA AGC CGA GGT T-3′ and 5′-AGA TCA TGG AGT CTT TAG GAC GCT C-3′), E-cadherin (5′-AAT CCA AAG CCT CAG GTC ATA AAC A-3′ and 5′-GGT TGG GTC

GTT GTA CTG AAT GGT), and GAPDH (5′-CGC TGA GTA CGT CGT GGA GTC-3′ and 5′-GCT GAT GAT CTT GAG GCT GTT GTC-3′). All qPCR assays were performed in triplicate. Cell proliferation assays Cells (3 × 104) were seeded in triplicate in 96-well plates and allowed to incubate for 48 h at 37°C/5% CO2. An EdU Fludarabine cell line incorporation assay was used to determine cell proliferation according to the manufacturer’s protocol (RiboBio, Guangzhou, China). We used a fluorescence microscope (Olympus Corporation, Tokyo, Japan) to visualize our results. All experiments were performed in triplicate and repeated three times. Transwell migration and invasion assays According to a previous protocol [5], cells (3 × 105 cells/well) were seeded in the upper chambers of 24-well transwell inserts (8.

Lens, Pseudomonas fluorescens SBW25, Saccharophagus degradans Feb

Lens, Pseudomonas fluorescens SBW25, Saccharophagus degradans Feb-40 and Xanthomonas campestris pv. vesicatoria str. 85–1). CusC was the second most abundant protein of the ensemble and its presence clearly correlated with CusA and CusB (124 out of 206 genomes); however the three genes are contiguous in only 44 Enterobacterial genomes. CopA, the most abundant protein of the sample with a physiological role as an internal membrane ATPase, was identified in the chromosomes of 70 genera with few exceptions:

Baumania, Buchnera, Coxiella, Dichelobacter, one Escherichia, Francisella, two Haemophilus, Wigglesworthia, seven Xanthomonas and Xylella. CueP CueP was found in 35 organisms from 6 genera click here (Citrobacter, Salmonella, Pectobacterium, Yersinia, Ferrimonas and Shewanella) belonging to only three families (Enterobacteriaceae, Cell Cycle inhibitor Ferrimonadaceae and Shewanellaceae). The presence correlation of CueP was the lowest of the experiment, coexisting with PcoC-CutF-YebZ-CueO and CopA-CusC in Enterobacteriaceae (ten Yersinia, one Citrobacter and sixteen Salmonella); with PcoC-CueO-YebZ-CutF, CopA-CusA-CusB-CusC and CusF in one Yersinia and one Citrobacter; with CopA-CusA-CusB-CusC and CusF or CutF in Ferrimonas and Pectobacterium; and with PcoA-PcoB, PcoC, PcoE, CopA-CusA-CusB-CusC and CusF in Shewanella. From this analysis, an apparent phylogenetic

consistency in the distribution of the clusters at the family level was evident. Double optimization and repertoire identification With the aim to identify particular combinations of the 14 seed proteins without the restrain imposed by a phylogenetic classification, we decided to perform the double optimization of the presence/absence profile (Figure 4). This analysis allowed the identification of nine clearly defined clades which represent the existing repertoires of periplasmic copper homeostasis proteins in gamma proteobacteria. In the

first one (clade 0) we identified 13 organisms from seven genera that lack all seed proteins: Baumannia, Carseonella, Riesia, Buchnera, Hamiltonella, Blochmannia and Wigglesworthia. All these organisms are endosymbionts with reduced genomes suggesting the loss of copper homeostasis genes in response to the negligible role of copper homeostasis in their biological not functions and environment. Figure 4 Two-dimensional optimization of the phylogenetic profile of periplasmic copper homeostasis proteins. Clustering optimization was rearranged for taxonomic categories preserving the previously optimized arrangement of protein presence. Eight proteins repertoires were identified (marked with dots). Shade scale represents the fractional abundance of a seed protein within a genus. The second repertoire (clade 1) is depicted in Figure 5a and comprises two organisms from the same genus, Thioalkalovibrio.