performance enhancing) is favouring functional foods However, ex

performance enhancing) is favouring functional foods. However, exercise physiology literature is brimming with experimental studies using foodstuff, CB-5083 fruits and vegetables alike, to find natural sources of performance enhancing substances. For example, red berries are generally known for their antioxidant properties with recent studies looking into tart cherries to prevent symptoms of muscle damage [69]. Future directions arising from this study relate to testing the effect of direct

experience on implicit and explicit attitudes, as well as investigating the stability of the observed change over time. The GW-572016 solubility dmso current study does not offer insight into behavioural intention or volition. Follow up studies should elucidate how attitude change upon vicarious or direct positive experience with functional food lead to behaviour change; and whether it will happen is a desirable direction. Conclusion Effective PED deterrence campaigns should accept that a desire for constant performance enhancement is natural to athletes. Instead of a solely prohibitive approach, anti-doping campaigns should promote acceptable and healthy alternatives to doping and primarily seek to create a community

HKI272 that takes the Olympic spirit further. Promoting the natural form (as opposed to the purified form of the main active ingredient) is key to the ‘alternative means’ approach. In the unrelenting quest for effective but not prohibited substances, athletes may put their health in great danger. There is a wide range of risks associated with the use of performance enhancing substances that do not apply to naturally occurring functional foods which Meloxicam mainly arise from the omission of the concentration step converting the foodstuff to a supplement or allegedly pure therapeutic agent with dosage ramifications. Improvements in our understanding of nutrigenomics and pharmacogenomics warrant caution regarding use of concentrated substances in supplement form. Owing to variations in genetic make-up the effect of a quantity of a supplement can vary enormously in pharmacodynamic and pharmacokinetic effects

leading to large variations in therapeutic efficacy along with toxicity profiles. One of the criteria for a drug to be included into the list of prohibited substances is that it presents a danger to health. Functional foods, whilst aiding athletic performance, are the opposite: they are healthy. The campaign should include an online community that can offer information about comparable healthy alternatives and spread this approach for benefits to all stakeholders. Also better information should be made available about FFs regarding dosage and administration. As FFs are becoming increasingly available in a variety of products [70], wide dissemination of accurate information would facilitate safe intake and thus prevent overdosing. Acknowledgements Christiana Adesanwo assisted AP conducting the literature review on framing effect in social marketing.

Today’s market leader may be rapidly replaced by another temporar

Today’s market leader may be rapidly replaced by another temporary leader. To be able to cover the necessary investments and improve the efficiency of the services, the chances are that larger reference centres with appropriate diversified technological platforms will be set up responsible for the high throughput analysis of thousands of samples a year. Local clinical this website services would then mainly serve as entry point for the patient and interpretation of his/her testing results. In fact this is somehow what the direct to consumer (DTC) services tried to set up. We should actually be grateful to the DTC companies that we were forced to review this new

approach as well as its potential impact on our services and on the

population. The questions to be answered in this regard will be: what service provision will be optimal in the future? What will EPZ-6438 nmr be the role of the geneticists in this? How can we convince the policy makers to follow our suggestions? Should we plan an orderly introduction of these services or wait and see what happens, let market forces decide? Impressive efforts are underway to identify tissue-, organ- and individual-specific networks CP-868596 nmr of interacting proteins (Barabasi et al 2011). Rather than the symptom-based approach we have today, they will undoubtedly become the basis on which diseases and ‘diseasomes’ will be identified in the future. Moreover, they will allow one to measure the effect of genetic polymorphisms and of epigenetic and environmental influences on the function of these networks and give a solid scientific basis for ‘personalized (stratified groups) medicine’. In addition, they will be the basis on which new

treatments will be designed. The available knowledge about these networks can in most Regorafenib supplier cases not yet be used in medical practice. Also in model organisms the role of the ‘dark genome’—the non-coding part of our genome—is being successfully unraveled and opportunities to do the same for humans are becoming available (Blaxter 2010, Davidson 2010). More information—time and research—is needed before the knowledge will be applicable in the clinic, but will we be able to wait? In this regard, as stated in the report, proven clinical validity and utility of the research findings as well as the ethical, legal and societal aspects will be evaluated before their clinical application can be considered. This will require a fundamental change in the regulations about genetic/medical testing. The IVD directive of the EU is under revision. Even in its new formulation, it may not provide sufficient regulation to guarantee that all tests done in academic or private settings in the EU are done under appropriate quality criteria. Moreover, it will probably not be able to regulate tests offered over the internet.

Any volume of

Any volume of output INCB28060 chemical structure for greater than 14 days would indicate failure of conservative therapy and the need for surgical intervention as well [10]. Conservative treatment of chylothorax begins with prompt drainage via

tube thoracostomy and dietary manipulation. The goal of dietary manipulation is to dramatically decrease lymph production since sixty percent of the dietary fat is absorbed by the lymphatic system, and approximately 1500 to 2000 ml of lymph is produced daily. This can be accomplished by completely bypassing the lymphatic circulation with Total Parenteral Nutrition (TPN), or by strict dietary manipulation based on a very low fat diet. We chose the latter to avoid some of the known infectious complications associated SCH727965 with TPN, especially in this high risk patient with multi-system trauma. With either approach, volume status, electrolyte abnormalities and nutritional parameters of the patient should be followed closely and aggressive replacement of nutritional losses of fat soluble vitamins and proteins should be carried out [10, 14, 15]. A diet strategy to limit chyle production involves avoidance of long chain triglycerides (LCTs).

Medium chain triglycerides (MCTs), however, are absorbed directly into the portal circulation without stimulating lymphatic flow, so inclusion of these MCTs in the diet can help to increase caloric intake for healing [11]. These dietary changes 4��8C as in our case were accomplished by a strict low fat diet supplemented with extra protein powder and MCT oil. Elemental peptide-based enteric formulas with less than 3% LCTs and MCTs added are also ideal supplements, as they have been shown to reduce the MG-132 in vivo quantity and duration of chyle output. Though there is no consensus on the definitive duration of this nutritional management, the literature supports that these dietary measures be continued for approximately two weeks [10–12]. In general, once the chyle leak is resolved, then a regular diet may be resumed. In complicated cases, however, it may be advisable to ensure leak resolution

with a provocative high-fat meal before removing a drainage tube. Finally, there are anecdotal data supporting the use of octreotide to promote decreased lymphatic production. The mechanism is based on the reduction of gastrointestinal secretions and sphlancnic blood flow associated with octreotide, thus decreasing lymphatic production. The drug can be given as a continuous infusion or bolus injections [16, 17] Conclusions Although rare, traumatic chylothorax can be a difficult entity to manage especially in a patient with additional traumatic injuries. This case reflects a successful approach to the management of a traumatic chyle leak, using drainage and strict dietary changes, which precluded the need for surgical intervention. Consent Written informed consent was obtained from the patient for publication of this case report and accompanying images.

Arch Microbiol 2003, 180:498–502 CrossRefPubMed 27 Jiang H, Lin

Arch Microbiol 2003, 180:498–502.Napabucasin clinical trial CrossRefPubMed 27. Jiang H, Lin JJ, Su ZZ, Goldstein NI, Fisher PB: Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma

differentiation, growth and progression. Oncogene 1995, 11:2477–2486.PubMed 28. Gueta-Dahan Y, Yaniv Z, Zilinskas A, Ben-hayyinm G: Salt and oxidative stress: similar I-BET-762 molecular weight and specific responses and their relation to salt tolerance in Citrus. Planta 1997, 203:460–469.CrossRefPubMed 29. Kurtzman CP, Robnett CJ: Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek 1998, 73:331–371.CrossRefPubMed 30. Tekaia F, Blandin G, Malpertuy A, Llorente CFTRinh-172 nmr B, Durrens P, Toffano-Nioche C, Ozier-Kalogeropoulos O, Bon E, Gaillardin C, Aigle M, Bolotin-Fukuhara

M, Casarégola S, de Montigny J, Lépingle A, Neuvéglise C, Potier S, Souciet J, Wésolowski-Louvel M, Dujon B: Genomic exploration of the hemiascomycetous yeasts: 3. Methods and strategies used for sequence analysis and annotation. FEBS Lett 2000, 487:17–30.CrossRefPubMed 31. Rouhier N, Jacquot JP: Plant peroxiredoxins: alternative hydroperoxide scavenging enzymes. Photosynth Res 2002, 74:259–268.CrossRefPubMed 32. Jeong JS, Kwon SJ, Kang SW, Rhee SG, Kim K: Purification and characterization of a second type thioredoxin peroxidase (type II TPx) from Saccharomyces cerevisiae. Biochem 1999, 38:776–783.CrossRef 33. Christman MF, Morgan RW, Jacobson FS, Ames BN: Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium. Cell 1985, 41:753–762.CrossRefPubMed 34. Armstrong-Buisseret L, Cole MB, Stewart GS: A homologue to the Escherichia coli alkyl hydroperoxide reductase AhpC is induced by osmotic

upshock in Staphylococcus aureus. Microbiol 1995, 141:1655–1661.CrossRef 35. Leblanc L, Leboeuf C, Leroi F, Hartke A, Auffray Y: Comparison between NaCl tolerance response and acclimation to cold temperature in Shewanella putrefaciens. Curr Microbiol 2003, 46:157–162.CrossRefPubMed 36. Chauhan R, Mande SC: Characterization of the Mycobacterium tuberculosis H37Rv alkyl hydroperoxidase AhpC points to the importance of ionic interactions in oligomerization and activity. Biochem J 2001, 354:209–215.CrossRefPubMed Methocarbamol 37. Rhee HJ, Kim GY, Huh JW, Kim SW, Na DS: Annexin I is a stress protein induced by heat, oxidative stress and a sulfhydryl-reactive agent. Eur J Biochem 2000, 267:3220–3225.CrossRefPubMed 38. Irani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon ER, Sundaresan M, Finkel T, Goldschmidt-Clermont PJ: Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science 1997, 275:1649–1652.CrossRefPubMed 39. Yuan L, Hillman JD, Progulske-Fox A: Microarray analysis of quorum-sensing-regulated genes in Porphyromonas gingivalis. Infect Immun 2005, 73:4146–4154.CrossRefPubMed 40.

pertussis DNA using primers with the restriction sites BamHI (Prn

pertussis DNA using primers with the restriction sites BamHI (PrnProF-BamHI) and NdeI (PRNProR-NdeI). The plasmid pSKPD25FpPRN3 was cut with BamHI and NdeI to generate a fragment which had lost the FHA promoter. The PRN promoter Bcl-2 inhibitor was ligated in its place. After transformation into E. coli and verification by restriction analysis, the resulting plasmid was designated as pSKPD25PRN3 (Figure 5C). The plasmid was

cut with NotI and inserted into pSS4245 cut with the same enzyme. The resulting construct, pSSPD2prn was transferred into E. coli SM10 to conduct the allelic exchange. The resulting B. pertussis strain was designated as Bp-WWE. Integration of the prn gene at its designated position was confirmed by PCR with the primers that specifically bind only to the upstream 5′ (5′FPD2-int and PRNProR-NdeI primers), 3′ (PRNF-int and 3′RPD2-int primers) downstream flanking regions, and inside the prn gene. PT, FHA and PRN expression in shake flask culture The Bp-WWC, Bp-WWD and Bp-WWE strains were grown in shake flasks with 100 mL MSS medium supplemented with methylated β-cyclodextrin (1 g/l) at 35°C with shaking speed of 200 rpm. After 32-48 h of growth, the culture supernatants were collected and assayed by ELISA to quantify the PT and FHA expression level. As PRN releasing from its membrane-bound precursor is the result of an imprecise cleavage by unidentified proteases

[34], PRN expression was determined by Western blot with densitometric analysis to evaluate the integrity of the antigen. Selleckchem GW786034 Org 27569 This assay was conducted both on the clarified culture supernatant and the cell extract obtained by heating cell suspension in isotonic buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.002% NaN3, and 1 mM PMSF) at 60°C for 30 min and the supernatant was collected after centrifugation

at 10,000 × g, 4°C for 30 min. ELISA assay for PT and FHA Purified rabbit polyclonal antibodies against PT or FHA (NLAC, Thailand) with the dilution of 1:1000 in carbonate/bicarbonate buffer (pH 9.6) were coated in 96-well plates (NUNC Maxisorp, Denmark) for 100 μL per well and incubated overnight at 4°C. After 3 time-washing with phosphate-buffered selleckchem saline pH 7.4 containing 0.1% Tween 20 (PBST), blocking was performed using 100 μL per well of 3% bovine serum albumin (BSA)-PBST then incubated at 37°C for 1 h. After discarding the blocking buffer and washing, dilutions of the standard PT, FHA or samples were loaded and incubated at 37°C for 1 h. Then, anti-PT mouse monoclonal antibody (Abcam, USA) at 1:30,000 dilution or anti-FHA mouse monoclonal antibody (NIBSC, UK) at 1:10,000 dilution in blocking buffer was added and incubated under the same conditions. After washing the wells for three times with PBST, 100 μL of rabbit anti-mouse (H + L) IgG-HRP conjugate (Abcam, USA) in blocking buffer at 1:10,000 dilution was used as secondary antibody and incubated for 37°C for 1 h.

Two of the most

Two of the most Evofosfamide price frequently used general bacterial PCR primers, targeting the 16S rRNA gene around E. coli positions 8-27 and 338-355, contain mismatches against planctomycete sequences [27, 28]. This may have caused planctomycete abundances to be underestimated in many

habitats, leading investigators to turn their attention towards bacterial groups that appear more abundant. Despite awareness of this problem, the literature and the sequence databases probably reflect a tradition of neglect towards the planctomycetes. In the light of this, it is difficult to say whether the dominance of planctomycetes on Laminaria hyperborea surface biofilms represents a unique feature of this habitat, or if other planctomycete-dominated bacterial communities selleckchem have been overlooked until now. For example, Staufenberger and co-workers

[29] did not detect planctomycetes in surface biofilms of another species of kelp (Saccharina latissima) using general bacterial primers for cloning and DGGE analysis. Yet, use of different primers has let to the detection of planctomycetes on both the kelps S. latissima and Laminaria digitata (Bengtsson, unpublished results). A possible explanation for the suitability of kelp as a habitat for planctomycetes is its content of sulfated polysaccharides, a class of molecules that some marine planctomycetes are known for being able to degrade [10]. For example, Laminaria hyperborea contains fucoidan, a class of complex brown algal sulfated polysaccharides. SB-3CT These see more substances are secreted to the surface of L. hyperborea via mucilage channels [30]. It is reasonable to assume that planctomycetes living on kelp surfaces utilize substances produced by the kelp, for example fucoidan, as carbon sources. However, the presence of suitable carbon sources appears insufficient to explain the observed dominance of planctomycetes, as they must not only be able to grow and divide, but also outcompete other bacteria to be successful. Another contributing factor to the success of planctomycetes on kelp

surfaces may be resistance to chemical antimicrobial defense compounds produced by the kelp. Antibacterial activity has been detected in extracts from many species of kelp, yet the substances responsible for the activity have often not been identified [31]. The lack of peptidoglycan in planctomycete cell walls makes them resistant to conventional cell wall targeting antibiotics like ampicillin. Resistance to other antibiotics, targeting for example protein synthesis (streptomycin) has also been reported in some marine planctomycetes [32, 33]. In many cases the reference sequences that are the most closely related to kelp surface planctomycetes are obtained from other marine eukaryotes such as for example red and green seaweeds, corals, crustaceans and sponges (Figure 4). The frequent association of planctomycetes to eukaryotes has previously been noted [34].

These cultures were either the same as (Cyanidioschyzon and Synec

These cultures were either the same as (Cyanidioschyzon and Synechococcus)

or only slightly lower in biomass (Chlamydomonas) over the 48 h growth period by comparison to the metal-free controls. Although cadmium stress has been shown to induce sulfur limiting conditions [7, 19], this was not entirely alleviated by the simultaneous provision of sulfate in any of the studied species, thus indicating that established metabolic reserves of sulfur other than sulfate itself, may be involved in cellular protection. Furthermore, it has been find more demonstrated that Cd exposure triggers a decline of photosynthetic apparatus thereby liberating sulfur as well as nitrogen and iron, which can be subsequently used for the synthesis of Cd detoxification enzymes [12]. Assimilated sulfate appears ISRIB ic50 to create an organic sulfur pool that can be readily employed to biotransform Cd(II) as it enters the cell in a similar

manner to that proposed for Hg(II) where chemical modification of thiols severely lessened HgS production [14, 15]. Why this cannot be provided by simultaneous sulfate provision is likely to be a product of the high energy demand (732 kJ mol-1) required to reduce sulfate to sulfide for thiol production, energy required for sulfate uptake, and the decline in sulfate uptake induced by cadmium itself [12]. These organisms rely on photosynthesis to generate reducing power that is essential for carbon fixation. If this is shunted towards sulfate assimilation, it would inhibit cellular metabolism and growth. By temporally displacing BAY 1895344 energy requirements to a pretreatment period, this is overcome and the cells are able to adequately cope with any stress imposed by subsequent exposure to Cd(II). The simultaneous sulfate and metal treated cells grew marginally better than the cells treated selleck products with metal alone in Cyanidioschyzon and Synechococcus (Figures 1B & C), but not in Chlamydomonas. Metabolic differences

might ac-count for this; i.e. the former species may have relatively more efficient sulfate assimilation. Interestingly, in a separate study it was revealed that Synechococcus is able to utilize elemental sulfur as a sulfur source resulting in enhanced metal tolerance (data not shown). These results point to the importance of sulfur nutrition in cadmium tolerance that has implications for other organisms [20, 21], including humans [22]. Nevertheless, this has not been well documented in the literature. The other treatment in which Synechococcus grew better than in cadmium alone was that in which cysteine was supplied both prior to and during metal exposure. However, this cannot be accounted for by a relatively high cysteine desulfhydrase activity in Synechococcus (Figure 4). Both eukaryotic species were not as adept at coping with this form of sulfur supplementation.

Langmuir 2010, 26:1354–1361 CrossRef 22 Noh SY, Sun K, Choi C, N

Langmuir 2010, 26:1354–1361.find more CrossRef 22. Noh SY, Sun K, Choi C, Niu M, Yang M, Xu K, Jin S, Wang D: Branched TiO 2 /Si nanostructures for enhanced photoelectrochemical PD0332991 water splitting. Nano

Energy 2013, 2:351–360.CrossRef 23. Ko SH, Lee D, Kang HW, Nam KH, Yeo JY, Hong SJ, Grigoropoulos CP, Sung HJ: Nanoforest of hydrothermally grown hierarchical ZnO nanowires for a high efficiency dye-sensitized solar cell. Nano Lett 2011, 11:666–671.CrossRef 24. Liu KW, Chen R, Xing GZ, Wu T, Sun HD: Photoluminescence characteristics of high quality ZnO nanowires and its enhancement by polymer covering. Appl Phys Lett 2010,96(023111):1–3. 25. Vanheusden K, Warren WL, Seager CH, Tallant DR, Voigt JA, Gnade BE: Mechanisms behind green photoluminescence in ZnO phosphor powders.

J Appl Phys 1996, 79:7983–7990.CrossRef 26. Holmes JD, Johnston KP, Doty RC, Korgel BA: Control of thickness BAY 57-1293 mouse and orientation of solution-grown silicon nanowires. Science 2000, 287:1471–1473.CrossRef 27. Zhou J, Huang Q, Li J, Cai D, Kang J: The InN epitaxy via controlling In bilayer. Nanosc Res Lett 2014,9(5):1–7. 28. Peng K, Wu Y, Fang H, Zhong X, Xu Y, Zhu J: Uniform, axial-orientation alignment of one-dimensional single-crystal silicon nanostructure arrays. Angew Chem Int Ed 2005, 44:2737–2742.CrossRef 29. Chern W, Hsu K, Chun IS, de Azeredo BP, Ahmed N, Kim KH, Zou J, Fang N, Ferreira P,

Li X: Nonlithographic patterning and metal-assisted chemical etching for manufacturing of tunable light-emitting silicon nanowire arrays. Nano Lett 2010, 10:1582–1588.CrossRef Cytidine deaminase 30. Fellahi O, Hadjersi T, Maamache M, Bouanik S, Manseri A: Effect of temperature and silicon resistivity on the elaboration of silicon nanowires by electroless etching. Appl Surf Sci 2010, 257:591–595.CrossRef 31. Chang SW, Chuang VP, Boles ST, Thompson CV: Metal-catalyzed etching of vertically aligned polysilicon and amorphous silicon nanowire arrays by etching direction confinement. Adv Funct Mater 2010, 20:4367–4370.CrossRef 32. Cheng C, Liu B, Yang H, Zhou W, Sun L, Chen R, Yu SF, Zhang J, Gong H, Sun H, Fan HJ: Hierarchical assembly of ZnO nanostructures on SnO 2 backbone nanowires: low-temperature hydrothermal preparation and optical properties. ACS Nano 2009, 3:3069–3076.CrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions SH designed and performed the experiments, analyzed the data, and drafted the manuscript. QY helped prepare and characterize the samples and analyze the data. BY, DL, and RZ participated in the preparation of the samples. SL and JK participated in the final data analysis and critical review of the manuscript. All authors read and approved the final manuscript.

glutamicum WT using the respective primer pairs A/B and C/D as in

glutamicum WT using the respective primer pairs A/B and C/D as indicated in CYC202 Additional file 3: Table S1. The PCR products were purified and linked by crossover PCR using the respective primer pair A/D (Additional file

3: Table S1). The either SmaI or BamHI restricted purified PCR product was cloned into pK19mobsacB resulting in the construction of the respective deletion vector (Additional file 3: Table S1). Targeted deletion of a carotenogenic buy Erastin gene via two-step homologous recombination using the respective deletion vector was carried out as described previously [26]. For the first recombination event integration of the vector into one of the flanking regions was selected via kanamycin resistance. Integration of the deletion vector into the genome results in a sucrose sensitivity because of the sacB gene product levansucrase. Selection for clones Compound C that have excised the deletion vector in a second recombination event was carried out via sucrose-resistance. Deletion of a carotenogenic gene was verified by PCR analysis of the constructed mutant using the respective primer pair E/F (Additional file 3: Table S1). Extraction of carotenoids from bacterial cell cultures To extract carotenoids

from the C. glutamicum strains 20 ml aliquots of the cell cultures were centrifuged at 10,000 × g for 15 min, and the pellets were washed with deionized H2O. The pigments were extracted with 10 ml methanol:acetone

mixture (7:3) at 60°C for 80 min with thorough vortexing every 20 min. When necessary, several extraction cycles were performed to remove all visible colors from the cell pellet. Analysis of carotenoids The extraction mixture was centrifuged 10,000 × g for 15 min and the methanol supernatant was transferred to a new tube. The absorption spectra of the various ex-tracts were measured at wavelengths between 400 and 800 nm using the UV-1202 spectrophotometer (Shimadzu, Duisburg, Germany). High performance liquid chromatography (HPLC) analyses of the C. glutamicum extracts were performed FAD on an Agilent 1200 series HPLC system (Agilent Technologies Sales & Services GmbH & Co. KG, Waldbronn), including a diode array detector (DAD) for UV/visible (Vis) spectrum recording. Quantification of carotenoids was performed using the extracted wavelength chromatogram at peak λmax, 450 nm for decaprenoxanthin and carotenoids with corresponding UV/Vis profiles and 470 nm for lycopene and corresponding carotenoids. Lycopene from tomato (Sigma, Steinheim, Germany) was used as standard. It was dissolved in chloroform according to its solubility and diluted in methanol. The HPLC protocol comprised isocratic elution for 25 min using a flow rate of 1.

Bacterial cultures growing in TY medium for 48 h to an OD600 of 0

Bacterial cultures growing in TY medium for 48 h to an OD600 of 0.6 were diluted 1000-fold in M1 minimal medium supplemented with Dilworth’s vitamins, and 100 μl of VS-4718 mw diluted cultures were added to each well and grown under static conditions at 28°C for up to 4 days. After 2 and 4 days, the contents of the wells were removed and each well was washed two times with 150 μl of sterile physiological saline solution, and then stained for 30 min with 100 μl of 25 μg/ml Calcofluor or 100 μl of 0.85% NaCl containing 5 μM Syto-9 and 30 μM propidium iodide. Next, dye solutions were removed and the wells were washed three times

with 150 μl of 0.85% NaCl, covered by 30 μl of fresh portion of physiological saline solution, and observed in a microscope. This experiment was repeated two times. To analyze different parameters of biofilm, 36 images from 3 wells of individual strain were collected. The ratio of live to dead cells was calculated using the ImageJ 1.43e software

(Wayne Rasband, NIH, USA). Images of biofilms stained with Syto-9 were analyzed to calculate several morphological parameters. The percentage of area covered by biofilm, a fractal dimension, and the length CA4P nmr of coastline were calculated using ImageJ 1.43e software according to [76, 77]. Three-dimensional images were reconstructed using the Laser Scanning Confocal Microscope LSC 5 PASCAL (Carl Zeiss, Germany) with 200x magnification. Plant tests Red clover (Trifolium pratense cv. Diana) seeds were surface sterilized, germinated and grown on Fåhraeus medium [66] slants. 5-day-old seedlings were inoculated with bacterial suspensions at an OD600 of 0.2 (200 μl/plant), and grown under natural light supplemented with artificial light (14 h day at 24°C and 10 h night at 18°C) in a greenhouse. The clover plants were inspected for root nodule formation and harvested after 4 weeks. Wet and dry masses of clover shoots were estimated. Plant competition assay For the competition assay, the Rt2472 and Rt2441 mutants, and the CYTH4 wild type Rt24.2 were collected from TY

agar medium into sterile water to an OD600 of 0.1. The mutants and wild type suspensions were mixed in 1:1, 10:1, 100:1, and 1000:1 ratios, and 200 μl of each mixture were added per plant. Twenty seedlings were used for each treatment. 28 days after infection, nodules were surface sterilized, crushed in 20 μl of saline solution, and 10 μl portions were plated on 79CA agar plates supplemented with nalidixic acid or kanamycin, and incubated at 28°C for 3 days. Ninety nodules per each mixture were examined. Bacteria growing exclusively on the medium supplemented with nalidixic acid corresponded to the wild type strain, and those growing on the medium supplemented with kanamycin corresponded to the rosR mutants. The competitive ability of rhizobia was expressed as the percentage of the particular strain in the analyzed nodules. Assays for root attachment and buy Idasanutlin growth on the root surface Root attachment of the Rt2472 and the Rt24.