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1995,16(6):399–403.CrossRefPubMed 7-Cl-O-Nec1 18. Nielsen HB, Kharazmi A, Bolbjerg ML, Poulsen HE, Pedersen BK, Secher NH: N-acetylcysteine attenuates oxidative burst by neutrophils in response to ergometer rowing with no effect on pulmonary gas exchange. Int J Sports Med 2001,22(4):256–260.CrossRefPubMed 19. Peake J, Suzuki K: Neutrophil activation, antioxidant supplements and exercise-induced oxidative stress. Exerc Immunol Rev 2004, 10:129–141.PubMed 20. Kawada S, Kobayashi K, Ohtani M, Fukusaki C: Cystine and Theanine Supplementation Restores High-Intensity Resistance Exercise-Induced Attenuation of Natural Killer Cell Activity in Well-Trained Men. J Strength Cond Res 2010,24(3):846–851.CrossRefPubMed 21. Pedersen BK, Rohde T, Ostrowski K: Recovery of the immune system after exercise. Acta Physiol Scand 1998,162(3):325–332.CrossRefPubMed 22. Nagatomi R: The implication of alterations in leukocyte subset counts on immune function. Exerc Immunol Rev 2006, 12:54–71.PubMed

23. Shinkai S, Watanabe S, Asai H, Shek PN: Cortisol response to exercise and post-exercise suppression of blood lymphocyte subset counts. Int J Sports Med 1996,17(8):597–603.CrossRefPubMed 24. Suzuki K, Totsuka Unoprostone M, Nakaji S, Yamada M, Kudoh S, Liu Q, Sugawara K, Yamaya K, Sato K: Endurance exercise selleck screening library causes interaction among stress hormones, cytokines, neutrophil dynamics, and muscle damage. J Appl Physiol 1999,87(4):1360–1367.PubMed

25. Ulich TR, del Castillo J, Guo K, Souza L: The hematologic effects of chronic administration of the monokines tumor necrosis factor, interleukin-1, and granulocyte-colony stimulating factor on bone marrow and circulation. Am J Pathol 1989,134(1):149–159.PubMed 26. Tidball JG: Inflammatory cell response to acute muscle injury. Med Sci Sports Exerc 1995,27(7):1022–1032.CrossRefPubMed 27. Bethin KE, Vogt SK, Muglia LJ: Interleukin-6 is an essential, corticotropin-releasing hormone-independent stimulator of the adrenal axis during immune system activation. Proc Natl Acad Sci USA 2000,97(16):9317–9322.CrossRefPubMed 28. Pedersen BK, Steensberg A: Exercise and hypoxia: effects on leukocytes and interleukin-6-shared mechanisms? Med Sci Sports Exerc 2002,34(12):2004–2013.CrossRefPubMed 29. Pedersen BK, Steensberg A, Fischer C, Keller C, Keller P, Plomgaard P, Wolsk-Petersen E, Febbraio M: The metabolic role of IL-6 produced during exercise: is IL-6 an exercise factor? Proc Nutr Soc 2004,63(2):263–267.CrossRefPubMed Competing interests This study was supported by Ajinomoto Co. Inc. The authors declare that they have no competing interests.

Due to the patchiness of the forests, the subplots could not always be realized next to each other, but were selected as close to each other as possible Stattic solubility dmso in apparently homogeneous remnants of forests. The AM plots were visited six times from August 2003 until October 2005, and preferably in or just after the rainy season. Sampling Macrofungi

in all AR plots were recorded during 6 or 7 visits during a three and a half year-period (January 1998 to July 2001), while the AM plots were explored 5 or 6 times during 3 years (August 2003 to October 2005). Each plot was preferably visited in or just after the rainy season as it is well documented that this strongly benefits sporocarp production (Henkel et al. 2005). The sampling efforts took 2 weeks per visit on average. The following definitions were used: sporocarp is mushroom; collection represents the sporocarps of a species that are collected at a site at a time point, and that supposedly, represented a single ‘AZD1390 price mycelium/individual’; record is the number of sporocarps of a species in a sample at a time point; sample is

the assemblage/community at a site/plot at a time point; productivity (=total abundance) is the total number of sporocarps of a species or of the assemblage/community at a site at a time point. During each visit a representative number of sporocarps of each morphological BLZ945 order species was collected, photographed in situ when possible, packed in waxed paper, and transported in a basket for further processing. They were described and preserved according to protocols given by Largent (1986) and Lodge et al. (2004). Morphological identification of specimens was carried out with the RANTES use of keys and, in some cases, in collaboration

with specialists. Throughout the studies we used the morphological species concept, which may provide an underestimation of the actual number of species present. Fungal nomenclature followed the 10th edition of the Dictionary of the Fungi (Kirk et al. 2008). All specimens collected are preserved in herbarium HUA (Medellín, Colombia, Suppl. Table 1). In addition, the number of sporocarps, their habitat and substrates were recorded. The macrofungi were found to occur on nine substrates, namely soil, trunk (diameter >2.5 cm), twigs (diameter <2.5 cm), living trees, fallen leaves, fruit shell, trash produced by ants, termite nests, and insects. Data on plant diversity present in the AR and AR-PR sites were taken from Vester (1997; Vester and Cleef 1998) and Londoño and coworkers (1995, Londoño and Alvarez 1997), respectively. Because the above mentioned plant inventories were made some time ago, we performed a new inventory of the tree biodiversity in the Araracuara (except AR-PR), and the Amacayacu plots by listing the presence of trees with a diameter at breast height (DBH) equal or thicker than 2.5 cm (Suppl. Table 2). Plant nomenclature followed Mabberley’s Plant Book (Mabberley 2008).

HupF contributes to HupL stability under elevated oxygen tensions The existence of hupF in hydrogenase systems from bacteria synthesizing this enzyme in the presence of oxygen prompted us to study the potential role of this protein in protection against oxygen. To this aim, we analyzed the possible effect of HupF on the status of hydrogenase large subunit in cultures maintained under different

oxygen tensions (1% and 3%). The higher oxygen tension (3%) still allowed the expression of hydrogenase in R. leguminosarum wild-type strain, although at a reduced level (40% of the level induced under 1% O2, Table  2). The presence

and processing status of the hydrogenase large subunit (HupL) were analyzed in crude cell extracts from microaerobic cultures through immunoblot (Figure  2). In these experiments Selleckchem Milciclib Epigenetics inhibitor we found that the wild-type cells contained a clear band associated to the mature form of HupL, irrespective of whether cells were induced under 1% or 3% oxygen (Figure  2A and 2B, upper panel). This band was absent in a ΔhupL mutant used as negative control (Figure  2A). Analysis of the cell extracts from the ΔhupF Vactosertib strain grown at 1% oxygen revealed the presence of HupL, although in the unprocessed form (Figure  2A, upper panel). Interestingly, HupL was not detected when cultures from the same mutant

strain were incubated under 3% O2 (Figure  2B). In contrast, extracts from a R. leguminosarum mutant lacking HypC, used as a hydrogenase non-processing control, showed a clear band of unprocessed HupL after exposure to both 1% and 3% oxygen tension (Figure  2A and 2B). Similar levels of an immunoreactive band corresponding to HypB were detected in all the extracts (Figure  2, lower panels), indicating that the microaerobic induction of Hup expression was equally effective for all strains in each treatment. These data suggest that, for in the presence of 3% oxygen, HupL is either unstable or not synthesized in the absence of HupF. In order to further evaluate these possibilities, we analyzed the in vivo stability of HupL as a function of the presence/absence of HupF. To address this question, we first induced R. leguminosarum cultures for hydrogenase expression under 1% oxygen, and then the induced cells, carrying either processed HupL (wild-type strain) or unprocessed HupL (ΔhupF and ΔhypC mutants), were exposed to atmospheres containing either 1% O2 or 21% O2 for up to 3 hours. After such treatments, the amount and processing status of HupL was determined through immunoblot assay in cell extracts (Figure  3A).

However, in contrast with the yak library, Methanobrevibacter wolinii was only found in the cattle library. Clones related to Methanimicrococcus blatticola Adriamycin cost and Methanomicrobium mobile were found in both libraries. Bacteria and methanogens has constantly interacted with each other in the rumen microbial communities [25], Sustainable growth of bacteria and methanogen in syntrophic communities depend on transfer of hydrogen and formate

and reverse electron transfer [26]. In the present study, methanogens from the TALC cluster were the dominant sequences in the yak and cattle rumen in the QTP area. However, the metabolic mechanism of this methanogen group is not yet clear; the investigation of fermentive bacteria species in yak and cattle could help understanding these syntrophic microbial communities. Conclusions The current study revealed for the first time the molecular diversity of methanogen community HDAC inhibitor in yaks and cattle in Qinghai-Tibetan Plateau area in China. The differences in methanogen

diversity found in the present study, may help to explain, to some extent, the differences associated with the low methane production contributed to the adaptation of the yak to the harsh forage environment in the Qinghai-Tibetan plateau. Yaks have co-evolved with a unique rumen microbial ecosystem that is significantly different from that of cattle, even when feed similar diets. Understanding these particularities will yield development of technology for reducing methane emission intensity by optimizing dietary conditions to exploit the full potential of the yak ruminal ecosystem and function. However, native grazing might be a limited Selleck Pembrolizumab factor for this experiment, since feed intake could significantly influence the rumen microbiota. This study also contributes to the understanding of the buy Fedratinib specific features of the rumen microbial ecosystem of yaks

which have adapted to high altitude ecosystems which may help to explain the differential rates of methanogenesis compared to cattle. Methods Animals and diet Samples of individual rumen contents were obtained from four domestic cattle (BW: 160 ± 5kg, Age: 4 ± 0.4 years) and four domesticated yaks (body weight: 180 ± 5 kg, Age: 4 ± 0.6 years) in the Qinghai Tibetan Plateau (QTP) in China. The animals were maintained outdoors, grazing a Kobresia pasture. Approximately 100 ml of rumen contents were collected using a 1.5 cm diameter stomach tube attached to an electric pump. The animal sampling procedure strictly followed the rules and regulations of experimental field management protocols (file No: 2010–1 and 2010–2) which were approved by the Lanzhou University.

The innermost ring again depicts the core (very light green) regions Tariquidar datasheet present in all three strains and the regions absent from strain Pm70 but present in other sequenced strains using the same color scheme. Twelve proteins were also identified that were present in both strains P1059 and X73 at greater than 90% amino acid similarity, but at less than 90% similarity in strain Pm70 (Table 2). Among the twelve proteins identified were several

membrane-associated proteins, including LspB, PfhB3, Opa, and SprT. The presence of divergent protein sequences that are membrane-associated is suggestive of adaptation of P. multocida strains towards particular hosts. SC79 Table 2 Predicted proteins of interest present in P. multocida strains X73 and P1059 at greater than 90% similarity but present at less than 90% similarity in strain Pm70 Gene locus Length (aa) Predicted function 00056 576 Hemolysin activator protein precursor 00060 1767 Exoprotein involved in heme utilization or adhesion – PfhB3 00219 96 Hypothetical protein 00361 617 Outer membrane iron receptor protein-Fe transport 00444 80 Hypothetical protein 00514 116 Hypothetical protein 00515 91 Hypothetical protein 00522 70 Hypothetical protein 00795 972 Beta-1,3-glucosyltransferase CA4P 01068 197 Opacity family integral membrane protein-Opa protein 01069

169 SprT- protein 01350 424 Nucleoside permease -NupC There were also predicted proteins identified as unique to strains P1059 (148 total) and X73 (127 total) compared to strain 17-DMAG (Alvespimycin) HCl Pm70. Many of these proteins were again of unknown

function and/or associated with prophage-like elements (Additional file 1: Table S1 and Additional file 2: Table S2). However, some systems unique to each strain were noteworthy. In strain P1059, one unique region contained six genes predicted as involved in the transport and modification of citrate, and the conversion of citrate to oxaloacetate via citrate lyase (00080 to 00085). This system was absent in all other sequenced P. multocida genomes. The conversion of citrate to oxaloacetate is linked to citrate fermentation. Also unique to strain P1059, but present in strains 36950, 3480, and HN06, are four genes involved in xylose ABC transport system with a transcriptional repressor (01538 to 01541). Present in strains X73 and 36950 was a putative toxin-antitoxin system similar to the HipAB systems (genes 02005 and 02006). Finally, genes for several novel proteins with similarity to the previously described Pfh-type filamentous hemagglutinins were identified in strains P1059 and X73. Strain P1059 contained a novel predicted filamentous hemagglutinin (designated PfhB4 – gene # 00523) that shares similarity with PfhB1 and PfhB2 from P. multocida. PfhB4 has conserved domains related to hemagglutination activity, two-partner secretion, hemagglutinin repeats, and toxicity. PfhB4 is present only in strains P1059, HN06, and 3480 (Figure 3).

Atoms are colored according to their height in Y direction. (e,f,g,h) Cross-sectional views of the substrate after scratching with probe radiuses of 6, 8, 10, and 12 nm, respectively. Atoms are colored according to shear strain ranging from 0 Vistusertib to 1. Figure 7 presents numbers of HCP and defect atoms generated within the substrate after penetration and scratching with the four probe radiuses. For each probe, there are more HCP and defect atoms generated in the scratching stage than that in penetration stage, because of the more complex plastic deformation associated with the multi-axial localized stress states. When the probe radius is not larger than 10 nm, there are more defect

atoms than HCP atoms in both penetration and scratching stages for each probe radius. However, the friction with the probe radius of 12 nm results in

more HCP atoms than defect atoms generated within the material. The formation of HCP atoms is associated with the activity of partial dislocations, while defect atoms are composed of not only dislocation STAT inhibitor cores but also vacancies. Therefore, Figure 7 indicates that the dislocation activity plays more pronounced role in governing incipient plasticity for larger probe. In addition, the incipient plasticity shows strong dependence on probe radius: the larger the probe, the larger both the HCP and defect atoms. Figure 7 Influence of probe radius Isoconazole on numbers of HCP and defect atoms generated within the substrate under friction. Conclusions In summary, we perform MD simulations to investigate the atomic scale origin of the minimum wear depth of single crystalline Cu(111) during single asperity friction. Simulation results show that scratching impression can only be made under a scratching depth at which there are permanent defects formed. It is indicated that the minimum wear depth is equivalent to the critical penetration depth associated with the first force-drop observed

in the force-depth curve. The specific permanent defects governing the wear phenomena are composed of stair-rod dislocations and prismatic dislocation loops as well as vacancies. While the contact pressure for the nucleation of initial dislocation is independent on probe radius, the minimum wear depth increases with probe radius. Further CP-690550 mw analysis of the shear strain distribution implies that a larger probe results in more compliant deformation of the material, which leads to larger volume of wear debris and wider extent of defect structures. Acknowledgements The authors greatly acknowledge financial supports from the NSFC (51005059 and 51222504), China Postdoctoral Science Foundation (20100471047 and 2012 M511463), and Heilongjiang Postdoctoral Foundation of China (LBH-Z11143). JZ also greatly acknowledges Dr. Alexander Hartmaier and Dr.

2004). The relative small size (20 kb) of this biosynthetic cluster of citrinin (Sakai et al. 2008) might also be beneficial for maintaining it in the genome during evolution. Another scenario is that horizontal gene transfer of the citrinin

biosynthetic gene cluster occurred several times during the evolution of the selleckchem series Citrina. The evolution of these biosynthetic genes remains unknown and more research is needed. Besides citrinin and a series of derivates or precursors of citrinin (Clark et al. 2006; Wakana et al. 2006; Lu et al. Selleck KU55933 2008; Zhu et al. 2009), several other metabolites are also claimed to be produced by P. citrinum, including compactins (Endo et al. 1976), agroclavine-1 and epoxyagroclavine-1 (Kozlovskiĭ et al. 2003a, 2005), asterric acid (Turner 1971; Turner and Aldridge 1983), cathestatins (Woo et al. 1995), citrinadin A (Tsuda et al. 2004; Mugishima et al. 2005), quinocitrinines and ergot alkaloids (Kozlovskiĭ et al. 2005), quinolactacins (Kakinuma et al. 2000; Takahashi et al. 2000; Kim et al. 2001), quinolactacide

(Abe et al. 2005), tanzawaic acids (Kuramoto et al. 1997), scalusamides A-C (Tsuda et al. 2005), perinadine A (Sasaki et al. 2005), cyclocitrinols (Kozlovskiĭ et al. 2000a; Amagata et al. 2003), ergosta-4,6,8(14),22-tetraen-3-one (Price and Worth 1974), 2,3,4-trimethyl-5,7-dihydroxybenzofuran (Chen et al. 2002) and gibberellins (Khan et al. 2008). Neuronal Signaling Of these metabolites, we have confirmed the production of citrinin and some of its derivatives, quinolactacins (= quinocitrinins), and citrinadins. Compactins have been incorrectly linked to “P. citrinum” NRRL 8082 and re-examination of this isolate showed it was a P. solitum (Frisvad and Filtenborg 1983). Clavine ergot alkaloids and citrinin have been linked to P. citrinum,

VKM F-1079 (Kozlovskiĭ et al. 2000b), but the strain that was used has been re-identified as P. gorlenkoanum. Penicillium sizovae was claimed to produce agroclavine-I and epoxyagroclavine-I and 1,1-bis(6,8-dimethyl-8,9-epoxy-5a,10e)-ergoline, Bcl-w a dimer of epoxyagroclavine-I (Kozlovskiĭ et al. 1986). The P. citrinum strain VKM FW-800 was isolated from 1.8 to 3 million years old Arctic permafrost sediments. This strain produces quinolactacin (= quinocitrinin) and the ergot alkaloids agroclavine-I and epoxyagroclavine-I, which indicates that this isolate is not P. citrinum, and if it is not a contaminant, then it maybe a ancestor of the group of fungi treated here. Of the investigated group of species, P. citrinum is most commonly occurring. This species has a worldwide distribution and has been isolated from various sources, such as soil, indoor environments and foodstuffs. In our study we found that P.

Each graph represents the mean of three independent experiments ± standard deviation. Proteome analysis of B. suis after six weeks of nutrient Tipifarnib cell line starvation Figures 2 and 3 each show one representative gel out of three featuring the proteomes of B. suis under long-term starvation conditions (left panels) versus late log/early stationary phase in rich medium (right panels). On the 2D-DIGE

reference gels, a total of 2553 and 2284 different protein spots were detected in the pI ranges 4–7 and 6–11, respectively. Figure 2 Up- regulated proteins of Brucella under starvation conditions. Protein profiles of B. suis 1330 after six weeks under starvation conditions in a salt solution (left panels), or during early stationary phase in TS broth (right panels). Proteins with a pI 4–7 are shown in (A), those with a pI 6–11 in (B). Proteins up-regulated during starvation are encircled.

Figure 3 Down- regulated proteins of Brucella under starvation conditions. Protein profiles of B. suis 1330 after six weeks under starvation conditions in a salt solution (left panel), or during early stationary phase in TS broth (right panel). Proteins down-regulated during starvation are encircled. Only proteins with pI 4–7 are shown, as no down-regulated proteins with pI 6–11 were detected. Up- and down-regulated Fer-1 ic50 proteins during starvation are separately marked in Figures 2 and 3, respectively. Details of these gels together with the tags identifying the spots of interest are available in the Additional files 1 and 2. The proteins with either increasing or decreasing concentrations under long-term starvation are presented in Table 1 and have been classified according to their potential

functions. Table 1 Up- or down-regulated Brucella suis proteins under nutrient starvation conditions Spot IDa ORFb Protein functionc Theoret.Mr/pId Fold changee t-Testf   Adaptation to atypical conditions   2146 BR2149 Dps family protein (DNA-binding proteins from selleck compound starved cells) 18.2/5.3 2.63 0.00019 429 BR0685 organic solvent tolerance, putative Edoxaban 88.7/5.4 1.53 0.024 2122 BR2149 Dps family protein 18.2/5.3 1.52 0.006 438 BR0685 organic solvent tolerance, putative 88.7/5.3 1.49 0.0004   Stress proteins/chaperones, protein folding   1624 BR0171 heat shock protein GrpE 25.2/4.7 −1.42 0.039 662 BR2125 chaperone protein DnaK 68.2/4.9 1.65 0.0056   Cell envelope   1653 BRA0423 31 kDa outer-membrane immunogenic protein (“Omp31-2”) 23.2/5.2 1.45 0.00034 1874 BRA0423 31 kDa outer-membrane immunogenic protein (“Omp31-2”) 23.2/5.2 1.34 0.026   Transport and binding proteins   1415 BR0639 porin Omp2a (omp2b) 40.5/4.6 1.41 0.03 1410 BR0639 porin Omp2a (omp2b) 40.5/4.6 1.4 0.028 2176 BRA0565 bacterioferritin 18.7/4.6 1.38 0.00065 1229 BRA0655 glycerol-3-phosphate ABC transporter, periplasmic 47.2/5.4 1.33 0.

It has also been shown that A. hydrophila produces an array of virulence factors that induce strong inflammatory responses [34–36]. The induction kinetics of some of the zebrafish intestinal immune system https://www.selleckchem.com/products/cb-839.html genes revealed an Acute Phase Response (APR), that is

the immediate host inflammatory reaction which counteract challenges such as tissue injury and infection [37]. In the current study A. hydrophila infection resulted in a clear GDC-0973 chemical structure increase in expression of the genes encoding the pro-inflammatory cytokines TNF α, IL-1β and IL-8. These cytokines are important inducers of APR resulting in increased production of Acute Phase Proteins (APPs) [38], such as C3. C3 is central in elimination of bacterial threats [39]. A systematic study of APR in zebrafish has shown striking similarities with mammals in function and induction of involved genes [25]. The fact that 1 IL-1β and IL-8 are highly induced while C3 remains moderately expressed is consistent with the expected expression profile at the early stages of infection (3 days in our case). The composition of the zebrafish intestinal bacterial microbiota and its interaction with the host and the environment has previously been studied by cultivation and culture-independent methods [28, 40]. In the present study this microflora and the experimentally introduced pRAS1 harboring A.

hydrophila were impacted by various antibiotic treatments. Recent studies have shown that Real-Time PCR with species-specific Idasanutlin or universal probes is an accurate and sensitive method Cell press for quantification of total bacterial populations as well as individual species from the intestinal contents

[41–45]. In our study a broad spectrum of 16S rDNA primers were used since bacteria can have different genome sizes and different rrn operon copy numbers. There are different concepts for considering the rrn operon numbers in quantitative 16S rDNA-based experimental systems [43, 44, 46]. Ott et al. [47], have provided accurate and stable figures of similar bacterial concentrations in clinical samples with application of universal primers and specific probes. In the present study, 16S rDNA gene copy numbers were significantly decreased after effective flumequine treatment, whereas sub-lethal flumequine or the clinically relevant ineffective tetracycline, trimethoprim and sulphonamide treatments caused minimal change. The reduction in 16S rDNA gene copy number following treatment with flumequine might be the result of killing of pathogenic A. hydrophila and a disturbed and reduced commensal flora. In mammals and humans, it is well known that antibiotics can change the composition of the bacterial populations in the intestines [48–50]. Studies concerning the distribution of antibiotic resistant bacterial isolates in zebrafish facilities are, however, limited. Previous studies performed in our laboratory Cantas et al.

Pyocyanin was added to Congo red plates at a final concentration of 50 μM. HHQ (a gift from M. whitelely, University of Texas) and HNQ (a gift from P. Williams, University of Nottingham) were added to MOPS-buffered Congo red plates at a final concentration of 50 μM or directly to the bacterial inoculum at final concentrations of 20, BI 10773 solubility dmso 100 and 500 μM. The respective solvents ethyl acetate, dimethyl sulfoxide (DMSO), and methanol were used as controls. Pel’-lacZ-reporter construction and β-galactosidase measurements A 555 bp promoter region of the pel operon was amplified from the ZK strain using

the primers listed in Additional file 1: Table S1 and cloned upstream of the lacZ gene in the integration vector mini-CTX-lacZ [44]. The Inhibitor Library research buy resulting plasmid pRG11 was then inserted into the chromosome of the wild-type and the lasR mutant as described [44]. As a control, the mini-CTX-lacZ parent vector was also integrated into the genome. The colonies of the ZK wild-type and the lasR mutant grown on Congo red plates at 37°C were used to measure β-galactosidase levels. A colony was cut

out on the 3rd, 4th, and the 5th day and suspended in 2 ml of 50 mM phosphate buffer, pH 7.4, in a 15 ml conical tube. Cells were lysed by sonication. The total protein was estimated by Bradford assay [49]. The sonicated sample was centrifuged at 4°C for 30 min. The resulting supernatant was check details used to measure β-galactosidase activity as described previously [50]. Pellicle biofilm assay Cultures were inoculated in tryptone broth at an OD600 of 0.0025 and incubated at 22°C and 37°C without shaking [11]. After 24, 48 and 72 h, pellicle formation was observed at the air-liquid interface. Microtiter plate biofilm assay Biofilm formation in a microtiter format was assayed as described [11]. Overnight cultures of the wild-type and the lasR mutant grown in LB broth at 37°C were diluted 1:100 in tryptone Temsirolimus price broth. One hundred and fifty μl of the diluted culture was added to 96-well polystyrene microtiter plates (Cellstar-Greiner Bio-one) and incubated at 22°C and 37°C without shaking for 48

and 72 h. Microtiter plates were rinsed in running hot water. Adherent cells were then stained with 1% crystal violet for 20 min. The microtiter plate was again rinsed in running hot water. Ethanol was added to each dry well and the samples were allowed to stand for 20 min. Absorbance was measured at 590 nm. Flow-cell biofilm assay Biofilms were grown at 37°C in flow chambers. The system was assembled as described [33, 51]. The cultures for inoculation were prepared from mid-exponential phase (OD600 of 0.4-0.8) TSB cultures grown at 37°C. The cultures were diluted to an OD600 of 0.05 in 1:100 diluted TSB medium and injected into the flow cell. Flow was initiated after 1 h. The diluted TSB was supplied at a flow rate of 180 μl/min using a peristaltic pump (Watson Marlow 205S).