Re-suspended biofilm and planktonic susceptibility Selleck Roscovitine The antibiotic susceptibility of log phase (OD600 0.030 – 0.08) and re-suspended biofilms of P. aeruginosa was determined. One milliliter of an overnight culture of P. aeruginosa PAO1 was sub-cultured into 29 ml of PBM (1 g l-1 glucose)

and grown overnight with agitation (37°C, 200 rpm) prior to exposure to antibiotics. One milliliter aliquots from the overnight cultures were mixed with 29 ml of fresh PBM (1 g l-1 glucose) containing antibiotics (tobramycin at 10 μg ml-1 or ciprofloxacin at 1.0 μg ml-1) to start treatment. Biofilms (72 h) scraped from coupons, were homogenized in phosphate buffer for 1 minute using a tissue homogenizer and re-suspended in 30 ml of PBM (1 g l-1 glucose) with antibiotics (as above), to yield a cell density of 3.0 × 107 cells ml-1. After suspension in antibiotic containing media, cultures were placed in an orbital shaking incubator at 37°C and sampled over the course of 12 hours. The resulting cell suspensions were serially diluted and viable bacterial numbers were determined by plating on TSA. Preparation of biofilms for RNA extraction Biofilms were grown in the drip flow reactor for either 72 h (n = 3) or 84 h (n = 3). Data from these two time points were pooled. Biofilms were scraped directly into

1 ml of RNAlater ® (Ambion). Clumps were dispersed by repeated pippetting with a micro-pipette and the recovered biofilms were stored at 4°C for one day prior to removal of the RNAlater ® by centrifugation LEE011 solubility dmso (15 min, 4°C, and 14000 g) and freezing of the biofilm cells at -70°C. RNA extraction Biofilm cells were thawed on ice and re-suspended in 300 μl of 1 mg lysozyme ml-1 Tris-EDTA buffer (TE; 10 mM Tris, 1 mM EDTA, pH 8.0) and divided into three aliquots. Each aliquot was sonicated for 15 s, and incubated at room temperature for 15 minutes. RNA was extracted with an RNeasy® mini Ponatinib nmr kit (Qiagen

Sciences) with on column DNA digestion (DNA Free kit; Ambion) the three aliquots were combined onto a single column. RNA concentrations and purity were determined by measuring the absorbance at 260 nm, 280 nm and 230 nm using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). RNA quality was evaluated using the RNA 6000 NanoChip assay on a 2100 Bioanalyzer (Agilent Technologies). The 23 s:16 s rRNA ratio for all samples used exceeded 2.0. Microarray hybridization Isolated total RNA (10 μg) was reverse-transcribed, fragmented using DNAseI and biotin end-labeled according to Affymetrix’s Prokaryotic Target Labeling Protocol (GeneChip Expression Analysis Technical Manual; November, 2004). For each Pseudomonas genome array (#900339, Affymetrix), 4.5 μg of labeled fragmented cDNA was hybridized to the arrays at 50°C for 16 h with constant rotational mixing at 60 rpm. Washing and staining of the arrays was performed using the Affymetrix GeneChip Fluidics Station 450.

Control (cells without propofol exposure). Activation of Nrf2 by propofol stimulation We then evaluated the effect of propofol stimulation on activation of Nrf2 in mRNA and protein levels. The results showed that 3-Methyladenine nmr exposing to propofol (20 μmol/L) for 48 h up-regulated the expression of Nrf2 at mRNAs levels (Figure 2A). Besides, exposing to propofol (20 μmol/L) for 48 h also up-regulated the protein expression of both HO-1 and Nrf2 (Figure 2B). Moreover, cells exposed

to propofol showed translocation of Nrf2 into the nucleus (Figure 2C). Figure 2 Activation of Nrf2 by propofol stimulation. (A) After stimulating by propofol, Nrf2 mRNA levels were quantified by real-time PCR analysis. Data were normalized by using GAPDH as an internal standard. * P < 0.05 vs. Control (cells without propofol exposure). These experiments were performed in triplicate. (B) After stimulating by propofol, HO-1 and Nrf2 protein level was analyzed by western blot. β-actin expression was monitored as the internal standard. (C) After stimulating by propofol, subcellular location of Nrf2 was detected by immunofluorescence assay. Propofol stimulation increased translocation of Nrf2 into the nucleus. Knock-down of Nrf2 by specific shRNAs In order to knock down Nrf2,

we constructed Talazoparib Nrf2-shRNA recombinant plasmids and transfected them into GBC-SD cells to knockdown the expression of Nrf2. qRT-PCR and western blot showed that Nrf2 expression was dramatically down-regulated at both the mRNA and protein levels Phosphoprotein phosphatase in GBC-SD cells compared with parental cells and Sh-NC (Figure 3A and Figure 3B). Among the four recombinant plasmids, ShRNA-1118 and ShRNA-2019 has the highest suppression efficiency, so both of them were used to process

the following experiments. Figure 3 Knock-down of Nrf2 by specific shRNAs. Forty-eight hours after transfection, cells were harvested. (A) Nrf2 mRNA levels were quantified by real-time PCR analysis. Data were normalized by using GAPDH as an internal standard. *P < 0.05 vs. Control (parental cells). (B) Nrf2 protein level was analyzed by western blot. β-actin expression was monitored as the internal standard. Loss of Nrf2 reverses the effects of propofol on cell proliferation, apoptosis, and invasion Finally, we examined whether loss of Nrf2 reversed the effects of propofol on cell proliferation, apoptosis, and invasion. Results showed that propofol alone and propofol plus sh-NC significantly promoted proliferation, stimulated invasion and inhibited apoptosis compared to parent cells. In contrast, propofol with ShRNA-1118 and ShRNA-2019 reversed these effects (Figure 4A to Figure 4D). Figure 4 Regulation of loss of Nrf2 for the effects of propofol on cell proliferation, apoptosis, and invasion. After transfected by different vectors, GBC-SD cells were incubated with propofol (20 μmol/L).

4 h),

and the median hours sprayed in the last year by the Malaysian females was 1,560 h compared with 60 h for all users. A higher proportion of the Malaysian female plantation workers had experienced a serious or moderate health incident in the last year than the full group of users (13.7 vs. 7.9%). Nevertheless, the proportions of Malaysian female users experiencing a serious incident or an incident of any severity were close to the average for the survey and reflected their generally good working practices. Although the survey collected a considerable amount of information PF-01367338 chemical structure about the KAP of users, information about exposure to pesticides is not very specific. Nevertheless, the logistic regression models to predict which farmers would experience incidents and the negative binomial regression models for incidence rates were informative and consistent. Farmers who experienced agricultural equipment and livestock incidents were much Proteasome inhibitors in cancer therapy more likely to experience agrochemical-related incidents and this was a much stronger predictor than the practices adopted by the user when measuring, mixing and spraying agrochemicals. It was an especially strong factor

in a number of countries and in Korea only 1 out of 50 users who had experienced an agrochemical-related incident had not had an incident involving agricultural equipment in the last 12 months. In some cases, the agricultural equipment incidents may have involved the spraying equipment, but the association Nutlin 3 with livestock incidents suggests that the association indicates a failure

to exercise caution. Younger farmers were more likely to experience agrochemical-related incidents than older users, a finding also reported by Yassin et al. (2002), but this factor was less important in models for the number of incidents, although close to significance. The confidence of the user was a key factor, especially the confidence of users about their practices when spraying. Those who felt that their practices were the safest were much less likely to experience incidents even if their practices were not the best. Users who sprayed more than the median insecticide spraying hours were at a significantly increased risk of agrochemical-related incidents but, a stronger association might have been expected given that most of the brands that users stated had caused incidents were insecticides. The regression modelling was able to confirm the value of some of the steps in the five key steps approach towards safe handling of pesticides, such as caution (demonstrated by not experiencing machinery or livestock incidents) and equipment maintenance. Personal hygiene (the user and their spraying clothes) had been expected to be more strongly associated with incidents, but cleaning contamination immediately after spillages was an important factor.

high/intermediate) and between subjective change of symptoms (unchanged vs. relieved). These analyses did not yield any relevant and consequential additional information on the relation of texture features to grouping parameters. Discussion The goals

of this study were show that a) MRI texture analysis can be used in NHL chemotherapy response evaluation b) statistical tests Wilcoxon paired test and R&R can be used to evaluate the separability of texture parameters used to describe textural changes in NHL. Limitations of our study may be the non-standardized MRI sequence protocols within intra and inter patient images and the use of different slice thickness due check details to imaging in clinical practice, where patient’s clinical stage and the size of the tumor were taken into account when setting imaging parameters. However, multicenter studies on MRI TA have shown transferability of TA parameters achieved from MRI images Osimertinib obtained

at different MRI centers with own acquisition parameters [16, 38]. To achieve new clinical relevant information by means of texture analysis, the texture changes should come out at the same or earlier timepoint as other quantitative measures of tumor response, for example decrease in tumor volume. The RECIST and WHO criteria for evaluating tumor response in one- or two-dimensional (diameter and product) tumor size is equivalent to a 65% decrease in tumor volume [1]. In this study we calculated tumor size decrease in

a short time period: before and after the first cycle of chemotherapy. There are no commonly used criteria for early response assessment using volumetric analysis for use as early in the therapy course as our volumetric evaluation was performed. Considering this, we can use the volumetric results as indicative of early imaging based evaluation of response, not to meet response, and also accept tumor volume decrease percentages smaller than 65% as consequential decrease in tumor size. However, in lymphomas, final clinical response evaluation should include other clinical tests according Methocarbamol to [5, 6]. Wilcoxon test showed encouraging values in the analyses of E1 and E3, including transferability of feature sets between T1- and T2-weighted images. This confirms our recent results with smaller patient data MaZda texture analysis of combination of T1- and T2-weighted images in single analysis [32]. Our study show that the statistical and autoregressive model texture parameters of MRI data can be successfully tested one by one with Wilcoxon paired test and Gage Repeatability and Reproducibility test to assess the impact of parameter separability in evaluating chemotherapy response in lymphoma tissue. Our results strengthen the applicability of Fisher and POE+ACC methods used in MaZda for automatic feature selection, and also confirm the suitability of the raw parameters in statistical tests.

Thus, our results indicate that macrophages are an important PLX4032 manufacturer component of the bone marrow stromal cells and may contribute to myeloma cell survival and resistance to chemotherapeutic treatment in vivo. O79 Blockade of TNFα Signaling in Tumor-associated Macrophages: a New Radiosensitizing Strategy Yuru Meng1, Michael A. Beckett1, Hua Liang1, Nico

van Rooijen2, Helena J. Mauceri1, Kenneth Cohen3, Ralph R. Weichselbaum 1 1 Department of Radiation and Cellular Oncology, The University of Chicago Medical Center, Chicago, IL, USA, 2 Department of Molecular Cell Biology, Vrije Universiteit, VUMC, Amsterdam, Netherlands, 3 Department of Medicine, Section of Hematology/Oncology, The University of Chicago Medical Center, Chicago, IL, USA Radiotherapy is an important anti-cancer treatment and approximately 60% of all cancer patients receive radiotherapy during the course of their disease. However, improvements in the therapeutic index of radiation therapy have been mostly based on physical improvements in radiation delivery. Radiosensitizer development targeting tumor cells has not yielded effective agents. Recent investigations in several

BGJ398 laboratories have focused on the tumor stroma as a potential target for radiosensitization. Here we report that depletion of tumor associated macrophages prior to radiotherapy increases the anti-tumor effects of ionizing radiation (IR) following both systemic and local injection of macrophage depleting Liposomal Clodronate Glutamate dehydrogenase (Lip-Clod). These anti-tumor effects were noted following large single dose (20 Gy) and low dose (2 Gy) fractionated radiation. Co-implantation of tumor cells with BM-derived macrophages (BMDMφ) resulted in increased tumor resistance to IR. Experiments using animals with germ line deletions of TNF receptors 1,2 (TNFR1,2-/-) or TNFα (TNF-/-) demonstrated that the radioprotective effect of BMDMφ required intact TNFα signaling.

The radioprotective effect of TNFα was mediated by the upregulation of VEGF production in tumor associated macrophages (TAMφ). Treatment of experimental tumors with a neutralizing antibody to TNFα (EnbrelR) improved tumor regression with IR compared to IR alone without an increase in host toxicity. These data provide a mechanistic basis for targeting macrophage populations generally and TNFα induced macrophage VEGF specifically to improve radiotherapy outcomes. Y.M., M.A.B., and R.R.W. contributed equally to this work. O80 The Role of Microenvironment on the Regulation of Epstein-Barr Virus Latent Gene Expression Eva Klein 1 , Lorand L. Kis1, Daniel Salamon1 1 Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden Depending on the differentiation of EBV-carrying cells, the virally encoded proteins are expressed in various combinations. These determine the fate of the viral genome harbouring cells.

5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 1 mM EDTA] supplemented with protease-inhibitor mix (Roche), resolved on precast NuPAGE 4-12% gels (Invitrogen), and transferred onto nitrocellulose membranes (Bio-Rad). The following antibodies were employed for immunedetection: rabbit anti-ATM (Santa Cruz), EPZ6438 mouse anti-α-tubulin (Immunological Sciences), HRP-conjugated goat anti-mouse and anti-rabbit (Cappel). Immunoreactivity was determined using the ECL-chemiluminescence reaction (Amersham Corp) following the manufacturer’s instructions. Ionizing radiation (IR) When indicated, cells were

irradiated using a 137Cs source (IBL-437-C irradiator, CIS bio International) at a dose rate of 6.8 Gy/min. Citotoxicity and BrdU assays Cells (5 × 104/ml) were seeded in 96-well plates in growth medium and incubated 24 hrs at 37°C in 5% CO2 atmosphere. Drugs were added at the indicated concentrations and for the indicated times before incubation with reagents of XTT, WST-1, and BrdU

(all from Roche Applied Science), following the manufacturer’s instructions. The absorbance at 450 nm (XTT and WST-1) or at 370 nm (BrdU) were measured by the microplate reader Infinite F200 (Tecan). Each experiment was performed in triplicate. The survival fraction for a given dose was calculated as the plating efficiencies for that dose divided by the plating efficiencies of solvent-treated cells. Cell cycle profiles Treated and untreated cells (5 × 105) were washed in PBS 1X and resuspended in 300 μl hypotonic fluorochrome solution [50 μg/ml propidium

Selleck Birinapant iodide, 0.1% sodium citrate, 0.1% Triton-X-100 (all from Sigma)] for 30 min at room temperature. DNA content was measured by a FACScan flow cytometer (Becton Dickinson). Colony forming assays Cells were treated with drugs at the indicated doses for 24 hrs, then plated at low density in 60 mm Petri dishes and grown for twelve days in the absence of drugs. Surviving colonies were fixed and stained with Cristal Violet (0.5% in methanol) (Sigma), air-dried, and counted. Statistics The Wilcoxon test for paired samples has been used for repeated measurements. A p-value less than 0.10 (*) and less than 0.05 (**) were considered statistical significant. Results and discussion Effects of ATM-depletion in breast cancer MCF-7 cell line To assess the influence of ATM in breast cancer susceptibility nearly to PARP inhibitors, we genetically repressed ATM expression by RNA interference in MCF-7 cells. We chose the MCF-7 breast cancer cell line because it is ER positive, HER2 negative, and wild-type for the BRCA1, BRCA2, and TP53 genes [25], features we observed in breast tumors arising in our A-T heterozygotes [23]. Stable interference of ATM was obtained by MCF-7 transfection with shATM-carrying vectors (MCF7-ATMi) and its siR5 negative control (MCF7-ctr) (see Materials and methods). Stable-transfected cells were selected in the presence of puromycin for ten days and maintained as polyclonal populations.

Thus, 1D nanostructure exhibits a superior sensitivity to light and

chemical molecules compared to the thin film and bulk. Due to these properties, electronic devices fabricated using 1D nanostructure have been extensively adapted in photodetectors [5], gas sensors [6], and dye-sensitized solar cells [7], respectively. Of these application fields, photodetectors or switches based on semiconductor materials have been the focus of considerable attention in recent years because of their high MLN0128 mw sensitivity and high quantum efficiency. Furthermore, the different energy band gaps imply that photodetectors can be applied flexibly on various wavelengths. To date, photodetectors based on 1D semiconductor nanostructures, such as SnO2 nanowires [8], ZnO nanowires [9], ZnSe nanobelts [10], CdS nanoribbons [11], and CuO nanowires [12], have been reported. These 1D nanostructure photodetectors exhibit outstanding

performance; however, the detection range that has been investigated so far falls primarily between the infrared and ultraviolet region. In fact, 1D nanostructure photodetectors of the mid- to long-wavelength infrared (IR) region have seldom been reported because only a few other materials can be used in this region. Indium antimony (InSb), one of the III-V compounds with a face-centered cubic structure of the zincblende type, is a useful material for producing mid- to long-wavelength IR photodetectors because of the smallest band gap (E g = 0.17 eV, at 300 K). In addition,

learn more owing to the small effective mass (m*e = 0.014 m o) and the ballistic length (up to 0.7 μm at 300 K), InSb has an extremely high carrier mobility (i.e., electron mobility of 77,000 cm2V-1s-1) [13]. Therefore, InSb is a highly promising material for device applications involving high-speed-response electronic nanodevices, optical communication devices, and optical detectors [13, 14]. Owing to the aforementioned unique characteristics, now, many groups use different synthesis methods to produce InSb nanowires, i.e., chemical beam epitaxy [15], chemical else vapor deposition [16], and pulsed laser deposition (PLD) [17]. Meanwhile, the electrical transport characteristics are also widely investigated [18, 19]. However, only few groups study on the IR detectors, particularly on the mid- to long-wavelength region [20, 21]. This work shows that InSb nanowires can be successfully synthesized at room temperature by applying electrochemical method with an anodic aluminum oxide (AAO) template. The synthesizing process was simple, fast, and straightforward in fabricating large-area InSb nanowires at low temperature compared to other thermal reactive processes. Moreover, individual InSb nanowires based on a metal–semiconductor-metal (M-S-M) structure were fabricated into the photodetectors.

Altschul SF, Gish W, Miller W, Myers EW, DJ L: Basic local alignment search tool. J Mol Biol 1990,215(3):403–10.PubMed 47. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 1994, 22:4673–4680.CrossRefPubMed 48. Felsentein J: Phylip; Phylogeny Inference Package Version 3.2. Cladistics 1989, 5:164–166. 49. Creevey CJ, McInerney JO: Clann: investigating phylogenetic information through supertree analyses. Bioinformatics 2005, 21:390–392.CrossRefPubMed Authors’ contributions OOS Primaryauthor, experimental design and contributed to all experiments. JOC reviewed, sugar

metabolism work and intellectual Sirolimus research buy contribution to the manuscript. ASV Contributed to

experiments. OMcA contributed to experiments and reviewed manuscript. LS contributed to experiments. PK contributed to experiments. MC Selleckchem Bioactive Compound Library experimental design and intellectual input. GF Principal investigator and intellectual input RPR Principal investigator and intellectual input. TB Principal investigator and intellectual input. All authors have read and approved the final manuscript.”
“Background Cryptococcus neoformans is an encapsulated yeast that is a facultative intracellular pathogen and a frequent cause of human disease in immunocompromised patients [1, 2]. Macrophages are essential for effective host defense against C. neoformans in humans [3, 4]. However, murine macrophages have been shown to be permissive for intracellular replication of C. neoformans, which can subsequently be extruded from or lyse the macrophages [2, 5–8]. In this regard, C. neoformans has a unique intracellular pathogenic strategy that involves cytoplasmic accumulation of polysaccharide-containing

vesicles and intracellular replication leading mafosfamide to the formation of large phagosomes where multiple Cryptococcal cells are present [5]. Our group and others have recently reported that after C. neoformans ingestion by macrophages, the yeast replicates and is subsequently extruded, in a process whereby both the yeast and macrophages survive [8, 9]. Moreover, it was also recently discovered that C. neoformans can spread from an infected to an uninfected murine macrophage cell [9, 10]. Here we further extend our extrusion studies to human peripheral blood monocytes (HPBMs) and report that as in murine macrophages, the interaction between human monocytes and C. neoformans leads to ingestion, intracellular replication, and polysaccharide shedding of C. neoformans, followed by cell to cell spread and extrusion of C. neoformans. The occurrence of phagosomal ‘extrusion’ in human peripheral blood monocytes suggests a central role for this phenomenon in the propagation and dissemination of this fungal pathogen. C. neoformans has a novel intracellular strategy that, to date has no precedent in other well-characterized intracellular pathogens. Since C.

J Appl Physiol 1998, 85:883–889.PubMed 7. Graham TE, Spriet LL: Performance and metabolic responses to a high caffeine dose during prolonged exercise. J Appl Physiol 1995, 78:867–874.PubMed Sirolimus solubility dmso 8. Hoffman JR, Kang J, Ratamess NA, Jennings PF, Mangine G, Faigenbaum AD: Effect of Nutritionally Enriched Coffee Consumption on Aerobic and Anaerobic Exercise Performance. J Strength Cond Res 2007, 21:456–459.PubMed 9. Hogervorst E, Bandelow S, Schmitt J, Jentjens R, Oliveira M, Allgrove J, Carter T, Gleeson M: Caffeine improves physical and cognitive performance during exhaustive exercise. Med Sci Sports Exerc 2008, 40:1841–1851.CrossRefPubMed

10. Kalmar JM: The influence of caffeine on voluntary muscle activation. Med Sci Sports Exerc 2005, 37:2113–2119.CrossRefPubMed 11. Woolf K, Bidwell WK, Carlson AG: Effect of caffeine as an ergogenic aid during anaerobic exercise performance in selleck products caffeine naïve collegiate football players. J Strength Cond Res 2009, 1363–1369. 12.

Hoffman JR, Ratamess NA, Ross R, Shanklin M, Kang J, Faigenbaum AD: Effect of a Pre-Exercise ‘High-Energy’ Supplement Drink on the Acute Hormonal Response to Resistance Exercise. J Strength Cond Res 2008, 22:874–882.CrossRefPubMed 13. Ratamess NA, Hoffman JR, Ross R, Shanklin M, Faigenbaum AD, Kang J: Effects of an Amino Acid/Creatine/Energy Supplement on Performance and the Acute Hormonal Response to Resistance Exercise. Int J Sport Nutr Exerc Metab

2007, 17:608–623.PubMed 14. Spriet LL: Caffeine and performance. Int J Sport Nutr 1995, 5:S84-S99.PubMed 15. Sawynok J: Pharmacological rationale for the clinical use of caffeine. Drugs 1995, 49:37–51.CrossRefPubMed 16. Hoffman JR, Kang J, Ratamess NA, Hoffman MW, Tranchina CP, Faigenbaum AD: Examination of a high energy, pre-exercise supplement on exercise performance. J Int Soc Sports Nutr 2009, 6:2.CrossRefPubMed 17. Scholey AB, Kennedy DO: Cognitive and physiological effects of an “”energy drink”": an evaluation of the whole drink and of glucose, caffeine, and herbal flavouring fractions. Psychopharm 2004, 176:320–330.CrossRef 18. Smit HJ, Cotton JR, Hughes SC, Rogers PJ: Mood and cognitive performance effects of “”energy”" drink constituents: fantofarone caffeine, glucose and carbonation. Nutr Neurosci 2004, 7:127–139.CrossRefPubMed 19. Smith A: Effects of caffeine on human behavior. Food Chem Toxicol 2002, 40:1243–1255.CrossRefPubMed 20. Miyazaki T, Matsuzaki Y, Ikegami T, Miyakawa S, Doy M, Tanaka N, Bouscarel B: Optimal and effective oral doses of taurine to prolong exercise performance in rat. Amino Acids 2004, 27:291–298.CrossRefPubMed 21. Zhang M, Izuma I, Kagamimori S, Sokejima S, Yamagami T, Liu Z, Qi B: Role of taurine supplementation to prevent exercise-induced oxidative stress in healthy young men. Amino Acids 2004, 26:203–207.CrossRefPubMed 22.

The RT reaction was performed at

50°C for 30 min. PCR amplification was performed at 94°C for 2 min for 1 cycle; 94°C for 30 s, 55–58°C for 30 s, and 72°C for 1.0 min for 20–28 cycles; and 72°C for 10 min for 1 cycle . Molecular biology techniques Routine techniques were performed using BGB324 in vitro standard protocols [69]. Genomic DNA of P. syringae pv. phaseolicola NPS3121 was isolated as described previously [70]. PCR products were amplified with Platinum supermix (Invitrogen). Primers were designed using Vector NTI Software (Invitrogen), with reference to the previously reported sequence of the 1448A strain (Gene Bank accession no. CP000058) [18]. The oligonucleotide primers used in this study are listed in Additional file 1. Motility assays To evaluate the motility of P. syringae pv. phaseolicola NPS3121 and the influence of temperature on this process, three strategies were used. The BAY 57-1293 concentration swimming and swarming motility of P. syringae pv. phaseolicola NPS3121 were assessed on semisolid KB plates containing 0.3% and 0.5% agar, respectively, as described in previous studies [41, 42]. The cells were grown in KB broth overnight

at 28°C, and harvested and resuspended in KB to OD600 = 1. 50 μL of bacterial suspensions were inoculated on filter disks (6 mm in diameter) and placed in the center of the plate. Plates were incubated for 24 h at 28°C and 18°C before photography. A second strategy was performed to evaluate the swimming and swarming motility of P. syringae pv. phaseolicola NPS3121. To ensure that the bacteria were in the same physiological condition as when the transcriptome analysis was performed, the P. syringae pv. phaseolicola NPS3121 strain was grown in M9 media at 28°C and 18°C until they reached the transition phase. Bacterial Fenbendazole suspensions (50 μL) were inoculated on filter disks (6 mm in diameter) and placed in the center of semisolid M9 plates containing 0.3%, 0.4%, and 0.5% agar. Plates were incubated for 48 h at 28°C and 18°C. Finally, motility was also evaluated using the stab technique in semisolid

KB and M9 media (0.3% and 0.5% agar) in glass tubes. The tubes were incubated at 28°C and 18°C for 48 h. As controls, we used the P. syringae strains pv. tomato DC3000 and pv. tabaci PTBR2004. Experiments were performed three times with three replicates per treatment. Quantification of siderophores Siderophore production into the culture supernatant by bacterial strains was determined using chrome azurol S (CAS) liquid assays as previously described [71]. Briefly, the P. syringae pv. phaseolicola NPS3121 strain was grown in M9 media at 28°C and 18°C until they reached the transition phase. The supernatant was recovered by centrifugation at 8,000 rpm for 15 min at 4°C and filtered through a 0.45-μm-pore-size filter (Millipore). For siderophore quantification, a standard curve was prepared with desferoxamine mesylate. Experiments were performed three times with four replicates per treatment.