This work was funded in part by the ANR “RhizocAMP” (ANR-10-BLAN-1719) and the Pôle de Compétitivité “Agrimip Innovation Sud Ouest”. This work is part of the “Laboratoire d’Excellence” (LABEX) entitled TULIP (ANR-10-LABX-41). Electronic supplementary material Additional file 1: SpdA, a putative Class III phosphodiesterase. (A) Phylogenetic tree generated with Phylogeny.fr [1]. The tree shows the phylogenetic check details relationship of the 15 IPR004843-containing proteins of S. meliloti with known phosphodiesterases from M. tuberculosis (Rv0805), H. influenzae (Icc) and E. coli

(CpdA and CpdB). (B) Table showing the distribution of the five class III PDE subdomains among the 15 IPR004843-containing proteins from S. meliloti. (PDF 386 KB) Additional file 2: Plasmids used this website in this study. (PDF 364 KB) Additional file 3: Molecules and conditions tested for expression of spdA ex planta. (PDF 429 KB) Additional file 4: Enzymatic characteristics of purified Selleck MDV3100 SpdA. (A)Lineweaver-Burk representation of SpdA kinetics of hydrolysis of 2′, 3′ cAMP. Purified SpdA was assayed as described in methods. (B)SpdA kinetic values. (PDF 237 KB) Additional file 5: SpdA does not require metal cofactor for 2′, 3′ cAMP hydrolysis. (A) Activity assayed in absence (CT) or presence of ions chelators. (B) SpdA activity in absence (CT) or presence of added bivalent ions.

(PDF 245 KB) Additional file 6: 2′, 3′ cAMP weakens smc02178-lacZ expression. (A) smc02178-lacZ expression was monitored ex planta in S.meliloti 1021 WT and ΔSpdA background strains after addition of 2.5 mM 3′, 5′-cAMP and/or 7.5 mM 2′, 3′-cAMP. ***p < 1.3E-06, http://www.selleck.co.jp/products/CAL-101.html **p < 0.0001, *p < 0.003 with respect to the wild type. (B) hemA-lacZ expression was monitored ex planta in S. meliloti 1021 WT and ΔSpdA background strains after addition of 2.5 mM 3′, 5′-cAMP and/or 7.5 mM 2′, 3′-cAMP. (PDF 547 KB) Additional file 7: Growth characteristics and stress adaptability of the ΔSpdA mutant. (A) Growth curves of 1021 WT and ΔSpdA mutant strains in LBMC or in VGM supplemented or not with 7.5 mM

2′, 3′ cAMP. (B and C) sensitivity of 1021 WT and ΔSpdA strains to SDS (B) and heat shock (C) (see methods for details). (PDF 274 KB) Additional file 8: spdA mutant symbiotic phenotype. (A) Nodulation kinetics on M. sativa following inoculation with S. meliloti 1021 and ΔSpdA mutant. (B) Dry weight of M. sativa shoots 35 dpi (C and D). Expression pattern of the smc02178-lacZ reporter gene fusion in young (7dpi) nodules of M. sativa following inoculation with S. meliloti 1021 (C) and ΔSpdA mutant (D). (PDF 513 KB) Additional file 9: Bacterial strains used in this study. (PDF 373 KB) Additional file 10: Primers and oligonucleotides used in this work. (PDF 326 KB) References 1. Jones KM, Kobayashi H, Davies BW, Taga ME, Walker GC: How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model. Nat Rev Microbiol 2007,5(8):619–633.PubMedCentralPubMedCrossRef 2.

The experimental traces in general represent the averages of three samples each illuminated once. The simulation

and fitting of the experimental polyphasic fluorescence induction curve with its algorithmic representation F FIA(t) was done with dedicated optimization routines. The fit parameters (rate constants, heterogeneity, fraction, etc.) of the simulation curve F FIA(t) were estimated after application of dedicated routines provided by appropriate software (Mathcad 13, MathSoft, Inc. Cambridge, MA, USA) which calculates the parameter values (vector) for which the least mean square function is minimal, where NN is the number of data points (in most experiments NN ≥50). Reduction of data points was in some cases purposely applied selleck chemical for F FIA(t) curves to facilitate better comparison with the experimental curve F exp(t). Analysis with fluorescence induction algorithm It has been shown (Vredenberg and Prásil 2009; Vredenberg 2011) that

the variable fluorescence during the OJ phase in the 0.01–1 ms time range is nearly exclusively, if not completely due to the release of primary photochemical quenching q PP and is represented by F PP(t) with $$ F^\textPP selleck chemicals (t) = 1 + nF_\textv \cdot q^\textdsq (t) \cdot [(1 - \beta ) \cdot \frack_\textL k_\textL + k_\textAB + \beta \cdot (1 + (1 - e^ - \phi k_\textL t ) \cdot e^ - k_2\textAB t )] $$ (1)in which nF v (=F m STF −F o)/F o) is the normalized variable fluorescence, \( q^\textdsq (t) = 1 – \texte^ – k_\textL t , \) β is the fraction of QB-nonreducing Methamphetamine RCs, Φ(0 ≤ Φ < 1)is an efficiency factor for energy trapping in semi-closed QB-nonreducing RCs, and k L, k AB, and k 2AB are the rate constants of light excitation and of oxidation of the single- and double-reduced primary quinone acceptor QA of PSII, respectively. Similarly it was shown that the variable fluorescence during the JI phase in the 1–30 ms time range is nearly exclusive due to the release of photoelectrochemical quenching q PE and is in approximation represented by F PE(t) with $$ F^\textPE (t) = 1 + nF_\textv \cdot

\ [1 - f^\textPPsc (t)] \cdot [1 - e^ - k_\textqbf \cdot t ] \cdot \frack_\textqbf k_\textqbf + k_\textHthyl + 1\ \cdot [1 - e^ - k_\textqbf \cdot t ] \cdot \frack_\textqbf k_\textqbf + k_\textHthyl $$ (2)in which f PPsc(t) is the fraction of semi-closed RCs containing QA − (see for definitions and equations Vredenberg 2011), k qbf is the rate constant attributed to that of the change in pH at the QA − QB redox side of PSII (related to the actual rate constant of proton pumping by the trans-thylakoid proton pump), and k Hthyl the actual passive trans-thylakoid proton leak (selleck screening library conductance). For the experiments presented in this article changes in k qbf and k Hthyl will be of prime importance to be considered.

Figure 1 Schematic diagram of the CdS/ZnO/Ti nanostructured solar cell. The photovoltaic performance was characterized under an AM 1.5 G filter at 100 mW/cm2 using a Newport Oriel 94022A Solar Simulator (Model 94022A, Newport, OH, USA), as calibrated using a certified OSI standard silicon photodiode. A

sourcemeter (2400, Selleckchem NCT-501 Keithley Instruments Inc., Cleveland, OH, USA) was used for electrical characterization during the measurements. Results and discussion Morphology and crystal structure of the nanostructured photoanodes The employed weaved titanium wire is flexible and of a diameter of about 85 μm with quite smooth surface. The color of the weaved titanium wire changed from gray to white after the deposition of ZnO nanosheets. Figure 2a shows the typical FESEM images of ZnO nanosheet arrays grown on weaved titanium wires. The surface of the titanium cylinder wires is covered totally and uniformly with ZnO nanosheet arrays, which would provide a large area for the deposition of CdS nanoparticles. Figure 2b Trichostatin A shows the cross-sectional

SEM image of ZnO nanosheets. It is apparent that all products consist of a large number of well-aligned sheet-like nanostructures. The SEM image clearly indicates that the film is constructed by assembling nanosheets in a compact way and the nanosheets are vertically oriented to the surface of titanium wires with different angles to each other. The average film thickness is about 8 to 10 μm. Figure 2c,d shows the top view of the ZnO nanosheets and CdS/ZnO nanostructures at a high magnification, respectively. The space www.selleckchem.com/products/AG-014699.html between nanosheets presents an

easily accessed open structure for the deposition of CdS nanoparticles, which is very important aminophylline for the performance of solar cells. Furthermore, this open structure could provide an easy filling of electrolyte into the space between the nanosheets and is beneficial to hole diffusion from CdS nanoparticles to counter electrode. By comparing Figure 2c,d, it can be clearly seen that the CdS nanoparticles were uniformly deposited onto ZnO nanosheets. The CdS nanoparticles make direct contact with the ZnO nanosheet surface, forming a firm connection on the ZnO nanosheets with a type II heterojunction, which may greatly enhance charge transport, charge separation, and overall photocurrent efficiency of the solar device. Figure 2 Typical FESEM images of ZnO nanosheets on weaved titanium wire substrate. (a) The low-magnification and (c) high-magnification FESEM images of ZnO nanosheets. (b) The cross-sectional view of ZnO nanosheets. (d) ZnO nanosheets deposited with CdS nanoparticles for 20 cycles. XRD patterns of ZnO/Ti and CdS/ZnO/Ti nanostructures are shown in Figure 3.

p. 253–307. 9. Nachman PH, Jennette C, Falk RJ. Primary glomerular disease. In: Taal MW, Chertow GM, Marsden PA, Skorecki K, Yu AL, Brenner BM, editors. Brenner & Rector’s The Kidney. 9th ed. Elsevier Saunders: Philadelphia; 2012. p. 1100–91. 10. Rennke HG. Secondary membranoproliferative glomerulonephritis. Kidney

Int. 1995;47(2):643–56.PubMedCrossRef www.selleckchem.com/products/anlotinib-al3818.html 11. Ferri C, Sebastiani M, Giuggioli D, Cazzato M, Longombardo G, Antonelli A, Puccini R, Michelassi C, Zignego AL. Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients. Semin Arthritis Rheum. 2004;33(6):355–74.PubMedCrossRef 12. Yamabe H, Johnson RJ, Gretch DR, Fukushi K, Osawa H, Miyata M, Inuma H, Sasaki T, Kaizuka M, Tamura N, et al. Hepatitis C virus infection and membranoproliferative glomerulonephritis in Japan. J Am Soc Nephrol. 1995;6(2):220–3.PubMed NCT-501 13. Nasr SH, Satoskar A, Markowitz GS, Valeri AM, Appel GB, Stokes MB, Nadasdy T, D’Agati VD. Proliferative glomerulonephritis with monoclonal IgG deposits. J Am Soc Nephrol. 2009;20(9):2055–64.PubMedCrossRef 14. Sethi S, Nester CM, Smith RJ. Membranoproliferative glomerulonephritis and C3 glomerulopathy: resolving the confusion. Kidney Int. 2012;81(5):434–41.PubMedCrossRef 15. Bomback AS, Appel GB. Pathogenesis of the C3 glomerulopathies and reclassification of MPGN. Nat Rev Nephrol. 2012;8(11):634–42.PubMedCrossRef”
“Introduction

Adrenomedullin (AM) is comprised of 52 amino acids and was originally isolated in pheochromocytoma tissue by its ability to elevate cAMP in rat platelets. It is now recognized as a potent circulating vasodilatory peptide which is secreted by ubiquitous cells and organs [1]. Because the cytoprotective effect of AM is mediated by the cAMP signaling pathway, it is expected that AM is involved in various cellular processes [2]. Circulating AM is mainly secreted from vascular endothelial and smooth muscle cells. AM is processed from its

precursor as the intermediate form. Subsequently, the intermediate form is converted by enzymatic amidation [3] to the biologically active next mature form of AM (mAM). Since AM is biologically active only after C-terminal amidation of immature AM, it is necessary to determine the level of mAM in order to investigate the pathological role of AM [4]. It has also been reported that hyperglycemia enhances AM expression in the vessels, indicating that AM is involved in the regulation of glycemic PF-01367338 molecular weight control [5]. Plasma AM concentration in diabetic patients is closely associated with diabetic vascular complications [6]. However, only limited information on mAM level or amidation activity is available. Generally, the dialysate used in peritoneal dialysis (PD) has a high glucose concentration of 1.5–2.5 %; this high glucose concentration leads to deterioration of the peritoneum.

Scripta Mater 2009, 60:240. 10.1016/j.scriptamat.2008.10.019CrossRef 21. Li W, Liu P, Zhao YS, Ma FC, Liu XK, Chen XH, He DH: Structure, mechanical properties and thermal stability of CrAlN/ZrO 2 nanomultilayers deposited by magnetron sputtering. J Alloys Compd 2013, 562:5–10.CrossRef 22. Li W, Liu P, Zhao YS, Zhang K, Ma FC, Liu XK, Chen XH, He DH: SiN x thickness dependent morphology and MI-503 solubility dmso mechanical properties of CrAlN/SiN x nanomultilayers. Thin Solid Films 2013, 534:367–372.CrossRef 23. Kato M, Mori T, Schwartz LH: Hardening by spinodal modulated structure.

Acta Metall 1980, 28:285–290. 10.1016/0001-6160(80)90163-7CrossRef 24. Mirkarimi PB, Barnett SA, Hubbard KM, Jervis TR, Hultman L: Structure and mechanical properties of epitaxial TiN/V 0.3 Nb 0.7  N(100) superlattices. J Mater Res 1994, 9:1456–1467. 10.1557/JMR.1994.1456CrossRef 25. Shinn M, Barnett SA: Effect of superlattice layer elastic moduli on hardness. Appl Phys Selleckchem Nutlin 3 Lett 1994, 64:61–63. 10.1063/1.110922CrossRef 26. Hsu TY, Chang HB: On calculation of M S and driving force for martensitic transformation in Fe-C. Acta Metall 1984, 32:343–348. 10.1016/0001-6160(84)90107-XCrossRef

27. Hsu TY: An approach for the calculation of M S in iron-base alloys. J Mater Sci 1985, 20:23–31. 10.1007/BF00555894CrossRef 28. Chang HB, Hsu TY: Thermodynamic prediction of M S and driving force for martensitic transformation in Fe-Mn-C alloys. Acta Metall 1986, 34:333–338. 10.1016/0001-6160(86)90204-XCrossRef 29. Hsu TY, Chang HB, Luo SF: On thermodynamic calculation of M S and on driving force for martensitic transformations in Fe-C. J Mater Sci 1983, 18:3206–3212. 10.1007/BF00544144CrossRef 30. Gautier E, Simon A, Collette G, Beck G: Effect of stress and strain on martensitic transformation in a MTMR9 Fe-Ni-Mo-C alloy with a high M S temperature. J de Phys 1982, 43:473–477. RG-7388 Competing interests The authors declare that they have no competing interests. Authors’ contributions WL designed the experiment and

wrote the article. PL, KZ, and FM carried out the synthesis of the monolithic FeNi film and FeNi/V nanomultilayered films. XL, XC, and DH assisted in the technical support for measurements (XRD and HRTEM) as well as the data analysis. All authors read and approved the final manuscript.”
“Background One of the important applications of nanomaterials metallic nanoparticles (NPs) is to manufacture fine-pitch electrical line patterns for organic transistors, radio frequency identification (RFID) antennas, or ultra-large-scale integration (ULSI) interconnections not only because of the high electrical conductivity and flexibility in handling, but also the low processing temperature [1, 2]. The reduced processing temperature is due to the large surface-to-volume ratio of the particles leading to a dramatic lowering of the melting point and sintering transition.

Antimicrob Agents Chemother 2003,47(2):665–669.PubMedCrossRef 24. Ruzin A, Keeney D, Bradford PA: AcrAB efflux pump plays a role in decreased susceptibility to tigecycline in Morganella morganii. Antimicrob Agents Chemother 2005,49(2):791–793.PubMedCrossRef 25. Ruzin A, Visalli MA, Keeney D, Bradford PA: Influence of transcriptional activator RamA on expression SB202190 purchase of multidrug efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneumoniae. Antimicrob Agents Chemother 2005,49(3):1017–1022.PubMedCrossRef 26. Keeney D,

Ruzin A, Bradford PA: RamA, a transcriptional regulator, and AcrAB, an RND-type efflux pump, are associated with decreased susceptibility to tigecycline buy AZD1152 in Enterobacter cloacae. Microbial drug resistance (Larchmont,

NY 2007,13(1):1–6.CrossRef 27. Keeney D, Ruzin A, McAleese F, Murphy E, Bradford PA: MarA-mediated overexpression of the AcrAB efflux pump results in decreased susceptibility to tigecycline in Escherichia coli. J Antimicrob Chemother 2008,61(1):46–53.PubMedCrossRef 28. Hentschke M, Christner M, Sobottka I, Aepfelbacher M, Rohde H: Combined ramR mutation and presence of a Tn1721-associated tet(A) variant in a clinical isolate of Salmonella enterica serovar Hadar resistant to tigecycline. Antimicrob Agents Chemother 2010,54(3):1319–1322.PubMedCrossRef 29. Horiyama T, Nikaido E, Yamaguchi A, Nishino K: Roles of Salmonella multidrug efflux pumps in tigecycline resistance. J Antimicrob Chemother 2010,66(1):105–110.PubMedCrossRef 30. Vogel J: A rough guide to the non-coding RNA world of Salmonella. Mol Microbiol 2009,71(1):1–11.PubMedCrossRef 31. Brown DG, Swanson JK, Allen C: Two host-induced Ralstonia solanacearum genes, acrA and dinF, encode multidrug efflux pumps and contribute to bacterial wilt virulence. Appl Environ Microbiol 2007,73(9):2777–2786.PubMedCrossRef 32. Zhang XS, Garcia-Contreras R, Wood TK: YcfR (BhsA) influences Escherichia coli

biofilm CHIR98014 supplier formation through stress response and surface hydrophobicity. J Bacteriol 2007,189(8):3051–3062.PubMedCrossRef 33. Vanderpool CK, Gottesman S: The novel transcription factor SgrR coordinates the response to glucose-phosphate Atezolizumab stress. J Bacteriol 2007,189(6):2238–2248.PubMedCrossRef 34. Kroger C, Dillon SC, Cameron AD, Papenfort K, Sivasankaran SK, Hokamp K, Chao Y, Sittka A, Hebrard M, Handler K, et al.: The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium. Proc Natl Acad Sci U S A 2012,109((20):E1277-E1286.PubMedCrossRef 35. Waters LS, Storz G: Regulatory RNAs in bacteria. Cell 2009,136(4):615–628.PubMedCrossRef 36. Biedenbach DJ, Rhomberg PR, Mendes RE, Jones RN: Spectrum of activity, mutation rates, synergistic interactions, and the effects of pH and serum proteins for fusidic acid (CEM-102). Diagn Microbiol Infect Dis 2010,66(3):301–307.PubMedCrossRef 37.

2012). Previous genetic comparisons involving several

marine species have shown that most Baltic populations contain lower levels of variation than conspecific Atlantic ones (reviewed in Laikre et al. 2005a; selleck kinase inhibitor Johannesson and André 2006; Johannesson et al. 2011). In addition, several species show large genetic differences at the entrance of the Baltic Sea (Johannesson and André 2006). Further, a genetic barrier near to the Islands of Åland has been identified DNA-PK inhibitor in both herring (Clupea harengus; Jørgensen et al. 2005) and perch (Perca fluviatilis; Olsson et al. 2011), separating northern populations from southern ones. An important question is whether this and other barriers are consistent across taxa. Testing the hypothesis of shared overall genetic structures is of high relevance to management. The present study is based on population genetic data from seven species of key socio-economic and/or ecological importance sampled from each of seven geographic regions throughout the Baltic Sea. The key question is whether

genetic divergence patterns of these different species are similar over the Baltic Sea. Despite the adaptive relevance of such ecological variables as temperature and salinity, our data sets are not designed to address levels or types of selection affecting specific loci, noting the ambiguity of interpreting such effects on outlier loci even from extensive genomic scans (Bierne et al. p38 MAPK cancer 2011, 2013). Rather, we assume an overall signal of neutrality as a first approximation of reality (Ihssen et al. 1981) as balanced by divergent, convergent, and nonselective forces. O-methylated flavonoid This interpretation has been widely validated for diverse organisms and is particularly applicable to initial comparisons among heterogeneous data sets such as those used in this study (Utter and Seeb 2010). Each species diverges uniquely from the null hypothesis of panmixia,

reflecting factors including barriers to effective migration, isolation by distance, and repeated colonizations. Genetic data of Baltic species Genetic data were compiled or generated for each of the following seven species selected for this study: (1) Atlantic herring (C. harengus), one of the most economically important species fished in the Baltic Sea, (2) Northern pike (Esox lucius), and (3) European whitefish (Coregonus lavaretus), two ecologically important predators and popular targets for commercial and recreational fishing, (4) three-spined stickleback (Gasterosteus aculeatus), and (5) nine-spined stickleback (Pungitius pungitius), abundant mesopredators; and two important habitat forming species, (6) the blue mussel (Mytilus trossulus) including collections from populations putatively hybridized with M. edulis at the Baltic/Atlantic interface (Väinölä and Strelkov 2011; Zbawicka et al.

coli as soluble in the cell lysate following IPTG induction. For preparation of immunogen, the soluble NS1 was purified by Amylose Resin according to pMAL™ Protein Fusion and Purification System, Version 5.01 (New England Biolabs, Inc., USA). Purified NS1 was used for immunization. Hybridoma cells secreting anti-NS1 antibodies were generated according to standard procedures [45]. Briefly, six-week-old female BALB/c mice were immunized subcutaneously with purified NS1 emulsified C646 manufacturer with an equal volume of Freund’s complete adjuvant (Sigma, St. Louis, MO, USA). Two booster injections containing purified NS1 with equal volume of Freund’s incomplete

adjuvant were given at 2-week intervals. The final immunization, purified NS1 without adjuvant was given intraperitoneally. Three days after the

final dose, mice were euthanized and spleen cells were URMC-099 research buy harvested and fused with SP2/0 myeloma cells at 5-10:1 ratio using polyethylene glycol (PEG 4000, Sigma). Hybridoma cells were seeded into 96-well plates and selected in HAT medium (DMEM containing 20% fetal bovine serum, 100 ug ml-1 streptomycin, 100 IU ml-1 penicillin, 100 mM hypoxanthine, 16 mM thymidine and 400 mM aminopterin), and after 5 days, the medium was removed and replaced with fresh HT-DMEM medium. After HAT/HT selection, culture supernatants of surviving clones were screened for reactivity and specificity by indirect ELISA, WB and IFA. The ELISA was described previously [46]. Briefly, microplates were sensitized Thymidine kinase at 4°C overnight with affinity-purified WNV-NS1 antigen at 100 ng ml-1. The sensitized plates were incubated with culture supernatants from hybridoma cells at 37°C for 1 h, with HRP-conjugated goat anti-mouse Selleck GSK458 secondary antibodies (LICOR Biosciences) at a 1:4,000 dilution at 37°C for 1 h, followed

by color development with substrate solution containing o-phenylenediamine (OPD). WB was performed as described above, but the primary antibodies were the mAbs supernatant and HRP-conjugated goat anti-mouse secondary antibodies were used. The IFA results were supplied by Beijing Institute of Microbiology and Epidemiology. WNV, JEV, DENV1-4, YFV and TBEV antigen slides were prepared on porous slides using WNV, JEV, DENV1-4, YFV and TBEV infected and uninfected C6/36 cells. Cell suspensions were dripped onto slides, fixed using acetone, air dried and stored at -20°C. Next, anti-NS1 mAbs supernatant and WNV-, JEV-, DENV1-4-, YFV- and TBEV-positive/negative mouse sera (working dilution was 1:100) (positive/negative control) were incubated on acetone-fixed antigen slides for 2 h. A FITC-conjugated goat anti-mouse IgG (Sigma, USA) was used as a secondary antibody at a 1:50 dilution, and slides were viewed at a magnification of ×40 on a fluorescence microscope (Leica, Germany) [47]. The positive cell clones were subcloned three times by limiting dilution method.

In Handbook of methods in aquatic microbial ecology. Edited by: Kemp PF, Sherr BF, Sherr EB, Cole JJ. Boca Raton: Lewis; 1993:509–512. 62. Simon M, Azam F: Protein content and protein synthesis rates of planktonic bacteria. Mar Ecol Prog Ser 1989, 51:201–213.CrossRef 63. Wilhelm Nutlin-3 mw SW, Brigden SM, Suttle CA: A dilution technique for the direct measurement of viral production: A comparison in stratified and tidally mixed coastal waters. Microb Ecol 2002, 43:168–173.PubMedCrossRef 64. Hewson I, Fuhrman JA: Covariation of viral parameters with bacterial assemblage richness and diversity in the water column and sediments. Deep-Sea Res I 2007, 54:811–830.CrossRef 65. Sime-Ngando

T, Colombet J, Personnic S, Domaizon I, Dorigo U, Perney P, Hustache JC, Viollier E, Jacquet S: Short-term variations in abundances and potential activities of viruses, bacteria and nanoprotists in Lake Bourget. Ecol Res 2008, 23:851–861.CrossRef 66. Weinbauer MG, Rowe JM, Wilhelm SW: Determining rates of virus production in

aquatic systems by virus reduction approach. MAVE 2010, 1:1–8. 67. Del Giorgio PA, Gasol JM, Vaqué D, Mura P, Agusti S, Duarte CM: Bacterioplankton community structure: protists control net production and the proportion of active bacteria in a coastal marine community. Limnol Oceanogr 1996, 41:1169–1179.CrossRef 68. Dorigo U, Fontvieille D, Humbert JF: Spatial variability in the abundance and composition of the free-living bacterioplankton community in the pelagic zone of Lake Bourget (France). Cell Cycle inhibitor FEMS Microbiol Ecol 2006, 58:109–119.PubMedCrossRef 69. Schauer M, Balagué V, Pedrós-Alió C, Massana R: Seasonal changes in the taxonomic composition of bacterioplankton in coastal oligotrophic system. Aquat Microb Ecol 2003, 31:163–174.CrossRef 70. Nicholas KB, Nicholas HBJ:

Genedoc: a tool for editing and annoting multiple sequence alignments. 1997. 71. Huber T, not Faulkner G, Hugenholtz P: Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics Applications Note 2004., 20: 72. Cole JR, Chai B, Farris RJ, Wang Q, Kulam SA, McGarrell DM, Garrity GM, Tiedje JM: The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucl AC Res 2005, (33 Database):D294–296. Authors’ contributions All authors read and approved the final manuscript. SJ was the responsible of this study and participated in the experimental design. LB realised all analyses except for the flagellate counting and phylotype analysis. TP made the cloning-sequencing analysis of the selected DGGE bands. ID participated to the experimental design and realised the flagellate counting. Writing was mainly done by LB, helped and corrected by ID and SJ. Authors’ MK5108 cell line information LB and TP have been PhD students, working in the BioFEEL group between 2007 and early 2011. ID and SJ have obtained permanent positions since 2000, as research scientists.

The structural analysis revealed a close proximity of T. denticola and P. gingivalis in the top layer of the biofilms, which might indicate a high pathogenic potential of these in vitro formed subgingival model biofilms. V. dispar appeared in the top layer as well, forming tight microcolonies. Figure 9 Schematic structure of the 10-species in vitro biofilms after 64 h of incubation in iHS medium. Distribution of the 10 species and EPS as observed by CLSM. The scale is not representative The use of 50% heat-inactivated

human serum in the growth medium improved the stability of the biofilms, resulting in significantly thicker biofilms. Under these conditions the fastidious T. denticola was able to establish in significantly higher densities compared to the media with 10% or no human serum. Surprisingly, neither P. gingivalis nor T. Selleck eFT-508 forsythia were affected by the concentration of human serum, and neither by the addition SGLT inhibitor of saliva. Methods Biofilm generation and fixation The biofilms used in this study are produced using

a similar protocol as described before [11]. However, there are some key changes in the growth media and the strain composition that are described below. In the present study, Streptococcus oralis SK248 (OMZ 607), Streptococcus anginosus click here ATCC 9895 (OMZ 871), Actinomyces oris (OMZ 745; formerly Actinomyces naeslundii), Fusobacterium nucleatum subsp. nucleatum OMZ 598, Veillonella dispar ATCC 17748T (OMZ 493), Campylobacter rectus OMZ 698, Prevotella intermedia ATCC 25611T (OMZ 278), Porphyromonas gingivalis ATCC 33277T (OMZ 925), Tannerella forsythia OMZ 1047, and Treponema denticola ATCC 35405T (OMZ 661) were used. All strains, except for T. forsythia and C. rectus, were maintained on Columbia blood agar (CBA). T. forsythia and T. denticola were maintained in liquid culture using the media outlined in Table 1. Prior to the onset of

biofilm experiments, all strains were transferred into adequate liquid media (Table 1) for two cycles of precultures. The slow growing T. forsythia, C. rectus and T. denticola were precultured for 64 h (first cycle), then diluted 1:2 in fresh media and incubated these for another 24 h (second cycle). All other strains were incubated over night (first cycle), diluted 1:10 in fresh media and incubated again for 8 h (second cycle). Prior to biofilm inoculation, all strains were adjusted to a defined optical density (OD550 = 1.0 except for C. rectus, T. denticola with OD550 = 0.5) and mixed in equal volumes. Sintered circular HA discs with a diameter of 10.6 mm (Clarkson Chromatography Products, South Williams-port, USA) were coated with 1:2 diluted saliva for pellicle formation. Discs were placed in 24-well polystyrene cell culture plates and covered with 1.5 ml of growth medium. In this study three different growth media, all based on mFUM [12], were used (Table 1).