The new continuous flux approach (Fig. 4) was conceived to monitor the initial rate of ECS decay during repetitive ms dark-intervals under steady-state as well as changing ECS conditions. Therefore, this new probe can also be used in the investigation of charge fluxes during dark-light induction of photosynthesis, which have played an important role in Pierre Joliot’s recent work on the role of cyclic PS I (CEF1) (reviewed in Joliot and Joliot 2006, 2008; Joliot et al. 2006). We have shown that the new continuous flux signal provides practically identical

information during dark-light induction as point by point assessment of the initial slopes of ECS decays in particular dark-intervals defined along an induction curve of ECS (Fig. 7). Major advantages of the new probe are the continuity of signal monitoring and the ease of operation.

Using the double-modulation approach, click here with microprocessor controlled signal processing, ambiguities in the assessment of initial slopes are eliminated. Hence, this approach can be even applied reliably by non-experts in absorbance spectroscopy. We have demonstrated that both the original P515 (ECS) signal and the P515 indicated continuous flux signal (“P515 flux”) can be measured simultaneously with gas Vismodegib datasheet exchange (Figs. 8, 9, 10) using a special cuvette developed for parallel measurements of CO2 uptake with the GFS-3000 and optical changes (chlorophyll fluorescence, P700, ECS, etc.) with the Dual-PAM-100 and KLAS-100 measuring systems. While in the range of low-to-moderate light intensities the rates of “P515 flux” and CO2 uptake were found to be almost linearly correlated, a relative decline of “P515 flux” was observed when saturating light intensities were approached (Fig. 8). It remains to be investigated whether this decline

reflects a decrease of H+/e − due to saturation of an alternative light-driven pathway that does not involve CO2-reduction. This pathway could consist in CEF1 (Heber and Walker 1992; Joliot and Joliot 2006; Laisk et al. 2010), but a participation of the MAP cycle (water–water cycle) may be envisaged as well (Schreiber et al. 1995; Asada 1999; Miyake selleck inhibitor 2010). At high light intensity and low CO2 substantial “P515 flux” was observed that was not paralleled by corresponding CO2 uptake (Fig. 9). Again, this finding argues for an alternative, ECS-generating pathway that could be CEF1 or MAP-cycle or both, but at low CO2 some contribution of photorespiration cannot be excluded, even at 2.1 % O2. Upon sudden increases of CO2- or O2-concentration, pronounced oscillations in CO2 uptake (with period of about 60 s) were found to be paralleled by corresponding oscillations in “P515 flux” and in the original P515 signal (Fig. 10). Interestingly, while oscillations in CO2 uptake and P515 flux were almost synchronous, the changes of the original P515 signal were delayed by about 10–15 s with respect to the former two signals.

These primers included restriction enzyme sites that enabled the cloning of these fragments into pGADT7AD. Competent yeast cells AH109 were transformed

with the cloned fragments and used for mating with Y187 containing plasmid pGBKT7 with the SSG-1 coding insert using the small scale mating protocol as described by the manufacturer. After mating the cells were plated in TDO and them transferred to QDO with X-α-gal. All colonies that grew in QDO and were blue were tested for the presence of both plasmids and the SsSOD AZD1208 ic50 and SsGAPDH inserts were sequenced for corroboration of the sequence and correct insertion. For all other Co-IP’s the original yeast two-hybrid clones were grown in QDO. Co-Ip and Western blots were used to confirm the interaction of proteins identified in the yeast two-hybrid analysis with SSG-1 as described previously [26]. S. cerevisiae diploids obtained in the selleck chemicals yeast two hybrid assay were grown in QDO, harvested by centrifugation and resuspended in 8 ml containing phosphate buffer saline (800 μl) with phosphatase (400 μl), deacetylase (80 μl) and protease inhibitors (50 μl), and PMSF (50 μl). The cells were broken as described previously [77]. The cell extract was centrifuged and the supernatant

used for Co-IP using the Immunoprecipitation Starter Pack (GE Healthcare, Bio-Sciences AB, Bjorkgatan, Sweden). Briefly, 500 μl of the cell extract were combined with 1-5 μg of the anti-cMyc antibody (Clontech, Corp.) and incubated

at 4°C for 4 h, followed by the addition of protein G beads and incubated at 4°C overnight in a rotary shaker. The suspension was centrifuged and the supernatant discarded, 500 μl of the wash buffer added followed by re-centrifugation. This was repeated 4 times. The pellet was resuspended in Laemmeli buffer (20 μl) Loperamide and heated for 5 min at 95°C, centrifuged and the supernatant used for 10% SDS PAGE at 110 V/1 h. Electrophoretically separated proteins were transferred to nitrocellulose membranes using the BioRad Trans Blot System® for 1 h at 20 volts and blocked with 3% gelatin in TTBS (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5) at room temperature for 30-60 min. The strips were washed for with TTBS and incubated overnight in the antibody solution containing 20 μg of antibody, anti-cMyc or anti-HA (Clontech, Corp.). The bait protein (SSG-1) is expressed with a c-myc epitope tag and is recognized by the anti c-myc antibody. The prey proteins are all expressed with an HA epitope tag that is recognized by the anti HA antibody. Controls where the primary antibody was not added were included. The antigen-antibody reaction was detected using the Immun-Star™ AP chemiluminescent protein detection system from BioRad Corporation (Hercules, CA, USA) as described by the manufacturer.

One can see the presence of several endothermic processes on the thermograms, which

confirm the existence of different structural formations in OIS bulk and correspond to their glass transition temperatures. The temperatures of the glass transitions are shown in Table  2. For OIS with reactivity R = 0.04, in which the organic component consists of only high-molecular-weight MDI, one glass transition process T g1 near −50°C can be found and corresponds to elastic hybrid organic-inorganic network MDI/SS that was formed in reactions between the NCO groups of the MDI BGB324 cell line and OH groups of SS. Figure 1 DSC curves of OIS with different organic component reactivities R . R is varied from 0.04 to 0.32. Table 2 DSC studies: temperatures of the relaxation Luminespib price processes Compositions Glass transition temperatures Reactivity (R) MDI (%) PIC (%) T g1(°C) T g2(°C) 0.04

100 0 −50 – 0.1 80 20 −48 39 0.14 65 35 −53 54 0.16 58 42 −58 55 0.18 50 50 −63 59 0.22 35 65 −70 67 0.26 20 80 −76 74 Compositions and glass transition temperatures of OIS obtained from DSC investigations, depending on the reactivity R of the organic component of OIS. The increase of the organic component reactivity R by adding PIC in the reactive mixture leads to the appearance of the second glass transition process T g2 near 40°C. Thus, it can be referred to the more rigid hybrid organic-inorganic network PIC/SS that is formed in reactions between the NCO groups of PIC and the OH DOCK10 groups of SS. Further increase of R shifts T g1 to lower temperatures due to the presence of a low-molecular-weight

product that appeared during polymerization and plays the role of plasticizer for elastic network MDI/SS. At the same time, the rise of T g2 is observed since the plasticizing effect is weak as compared with a strong impact of growing and cross-linking of rigid hybrid network PIC/SS. DMTA results The DMTA results show the presence of two (Figure  2) and three (Figure  3) relaxation processes, depending on the composition of OIS. The temperatures of these relaxation processes are noted in Table  3. The relaxation temperatures T r1 and T r2 relate to the glass transition temperatures T g1 and T g2 and correspond to the hybrid networks MDI/SS and PIC/SS, respectively. A good correlation between values and shifts of relaxation temperatures (DMTA results) and glass transition temperatures (DSC results) is revealed. The third weak relaxation process T r0 near −90°C (Figure  3) corresponds to the relaxation of a low-molecular-weight product that plays the role of plasticizer for hybrid networks. The rise of R leads to the increase of a low-molecular-weight product in OIS bulk and, correspondingly, to the increase of its relaxation temperature and plasticizing effect.

J Bacteriol 2003,185(13):3853–3862.PubMedCrossRef 52. Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, et al.: CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 2011,39(Database issue):D225-D229.PubMedCrossRef 53. Galibert F, Finan TM, Long SR, Pühler A, Abola P, Ampe F, Barloy-Hubler F, Barnett MJ, Becker A, Boistard P, et al.: The composite genome of the legume symbiont Sinorhizobium meliloti. Science 2001,293(5530):668–672.PubMedCrossRef

54. Becker A, Barnett MJ, Capela D, Dondrup M, Kamp PB, Krol E, Linke B, Ruberg S, Runte K, Schroeder BK, et al.: A portal for rhizobial genomes: RhizoGATE integrates a Sinorhizobium meliloti genome annotation update with postgenome data. J Biotechnol 2009,140(1–2):45–50.PubMedCrossRef 55. Barloy-Hubler F, Cheron A, Hellegouarch CDK assay A, Galibert F: Smc01944, a secreted peroxidase induced by oxidative stresses in Sinorhizobium meliloti 1021. Microbiology 2004,150(Pt 3):657–664.PubMedCrossRef Ivacaftor cost 56. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997,25(17):3389–3402.PubMedCrossRef 57. Watt SA, Tellstrom

V, Patschkowski T, Niehaus K: Identification of the bacterial superoxide dismutase (SodM) as plant-inducible elicitor Unoprostone of an oxidative burst reaction in tobacco cell suspension cultures. J Biotechnol 2006,126(1):78–86.PubMedCrossRef 58. Davies BW, Walker GC: Disruption of sitA Compromises Sinorhizobium meliloti for

Manganese Uptake Required for Protection Against Oxidative Stress. J Bacteriol 2006,189(5):2101–2109.PubMedCrossRef 59. Kobayashi H, De Nisco NJ, Chien P, Simmons LA, Walker GC: Sinorhizobium meliloti CpdR1 is critical for co-ordinating cell cycle progression and the symbiotic chronic infection. Mol Microbiol 2009,73(4):586–600.PubMedCrossRef 60. Pobigaylo N, Wetter D, Szymczak S, Schiller U, Kurtz S, Meyer F, Nattkemper TW, Becker A: Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti. Appl Environ Microbiol 2006,72(6):4329–4337.PubMedCrossRef 61. Colombatti A, Bonaldo P, Doliana R: Type A modules: interacting domains found in several non-fibrillar collagens and in other extracellular matrix proteins. Matrix 1993,13(4):297–306.PubMedCrossRef 62. Barnett MJ, Toman CJ, Fisher RF, Long SR: A dual-genome Symbiosis Chip for coordinate study of signal exchange and development in a prokaryote-host interaction. Proc Natl Acad Sci U S A 2004,101(47):16636–16641. Epub 12004 Nov 16612PubMedCrossRef 63. Krol E, Becker A: Global transcriptional analysis of the phosphate starvation response in Sinorhizobium meliloti strains 1021 and 2011. Mol Genet Genomics 2004,272(1):1–17.

Experimental design and data analyses Randomized block design with two factor factorial arrangement was adopted for conducting the experiments. The data were subjected to one-way analysis of variance (ANOVA) and the mean of treatments compared by Duncan’s Multiple Range Test at p ≤ 0.01 using SPSS Software version 7.5. Cluster analysis based on the organic acid profiles

was performed using STATISTICA data analysis software system version 7 (StatSoft® Inc. Tulsa, USA, 2004). Results Production of organic acids HPLC analysis of the culture filtrates was done to identify and quantity the organic acids produced during the solubilization Birinapant cell line of TCP, MRP, URP and NCRP by Pseudomonas fluorescens strain, three Pseudomonas poae strains, ten Pseudomonas trivialis strains, and five Pseudomonas spp. strains (Fig. 1). During TCP solubilization all strains

showed the production of gluconic and 2-ketogluconic acids (Table 2). Apart from one Pseudomonas sp. strain no other strain showed oxalic acid production. All strains exhibited the production of malic acid excepting one Pseudomonas sp. strain and succinic acid excluding one Pseudomonas sp. strain. The production of lactic acid was restricted to one strain learn more of both P. trivialis and Pseudomonas sp., formic acid to six P. trivialis, P. fluorescens and two Pseudomonas spp. strains, and citric acid to three P. trivialis strains and one strain each of P. poae and Pseudomonas sp., and P. fluorescens strain.

Figure 1 HPLC chromatograms of authentic organic acids (a) and culture supernatant of Pseudomonas trivialis strain BIHB 747 grown for 5 days at 28°C in NBRIP broth with tricalcium phosphate (b), Udaipur rock phosphate (c), Mussoorie rock phosphate (d), North Carolina rock phosphate (e), and North Carolina rock phosphate spiked with OA (f). OA = oxalic acid, GA = gluconic acid, TA http://www.selleck.co.jp/products/Adrucil(Fluorouracil).html = tartaric acid, FA = formic acid, MA = malic acid, MalA = malonic acid, LA = lactic acid, 2-KGA = 2-ketogluconic acid, SA = succinic acid, CA = citric acid and PA = propionic acid. Table 2 Organic acid production by fluorescent Pseudomonas during tricalcium phosphate solubilization.       Organic acid (μg/ml)   Strain P-liberated (μg/ml) Final pH Oxalic Gluconic 2-KGA Lactic Succinic Formic Citric Malic Total organic acids (μg/ml) P. trivialis                       BIHB 728 771.3 ± 1.2 3.63 ND 18350.0 ± 5.8 257.0 ± 4.9 49.3 ± 1.8 987.7 ± 3.0 ND 30.5 ± 2.8 2051.8 ± 5.2 21726.3 BIHB 736 778.7 ± 2.4 3.90 ND 18035.3 ± 9.0 177.0 ± 2.6 ND 583.7 ± 4.1 96.0 ± 2.3 ND 1042.0 ± 3.8 19934.0 BIHB 745 827.4 ± 1.8 3.65 ND 18054.3 ± 8.1 210.0 ± 2.9 ND 2249.0 ± 4.4 ND 65.2 ± 2.6 1654.5 ± 3.8 22233.0 BIHB 747 743.0 ± 1.7 3.52 ND 18216.7 ± 3.5 330.7 ± 2.9 ND 1307.7 ± 4.6 ND 25.5 ± 2.1 667.0 ± 3.2 20547.6 BIHB 749 801.0 ± 2.1 3.42 ND 17745.3 ± 7.2 193.7 ± 3.3 ND 797.6 ± 1.9 117.5 ± 2.0 ND 1236.0 ± 6.2 20090.1 BIHB 750 774.3 ± 1.9 3.82 ND 18624.0 ± 4.6 172.3 ± 3.7 ND 509.9 ± 2.7 93.5 ± 1.

Lysostaphin and LytM185-316 bind peptidoglycan or cell walls differently The involvement of different regions of lysostaphin in peptidoglycan binding has been investigated earlier. The results show that lysostaphin has affinity for the pentaglycine crossbridges themselves [34], but also binds cell

walls via the cell wall targeting domain [35]. In contrast, almost nothing is known about the role of different LytM fragments in peptidoglycan binding. Therefore, we investigated this question by the pulldown assay (Figure 4A). Comparing the amounts of protein in the pulldown and supernatant fractions, we found that the full length protein (LytM26-316) did not efficiently bind to peptidoglycan. Mutation of the Zn2+ ligand Asn117 to alanine, which should weaken the binding of the occluding region

to the catalytic domain, did not significantly change the situation. The SCH772984 concentration isolated N-terminal domain of the enzyme also failed to bind to peptidoglycan, whereas LytM185-316 bound efficiently. When the buy RXDX-106 two Zn2+ ligands His210 and Asp214 were separately mutated to alanine, the binding was lost again. Changing the third Zn2+ ligand, His293 of the HxH motif to alanine, made the protein insoluble as reported earlier [12], so that peptidoglycan binding could not be tested. The first histidine of the HxH motif, His291, is likely to act as a general base in catalysis [11]. When this residue was mutated to alanine, peptidoglycan binding was reduced, but not fully abolished. Figure 4 Pulldown assay of various LytM fragments and inhibitors with purified peptidoglycans from S. aureus . (A) Full length LytM and various fragments were analyzed by denaturing gel electrophoresis and Thiamet G Coomassie straining either directly (control, C) or after separation into peptidoglycan binding (PG) and supernatant (S) fractions. (B) LytM185-316 was incubated with peptidoglycan in the presence of various protease inhibitors and the pellet fraction after pulldown analyzed by denaturing gel electrophoresis and Western blotting. The requirement of an intact active site for peptidoglycan binding was

also supported by inhibitor studies. We had previously shown that EDTA and 1,10-phenanthroline blocked activity, presumably by chelating Zn2+ ions. We now observed that both metal chelators also abolished binding of LytM185-316 to peptidoglycan (Figure 4B, lanes 1–2). In contrast, the weak Zn2+ ion chelator glycine hydroxamate and other small molecules and protease inhibitors did not interfere with peptidoglycan binding (Figure 4B, lanes 3–6). We conclude from these experiments that the accessibility and integrity of the active site is essential for the binding of the protein to peptidoglycan (Figure 4). Lysostaphin and LytM185-316 activities depend differently on pH Peptidoglycan hydrolase activities were assayed in a turbidity clearance assay, using S. aureus cells.

Microbial Ecol 1986, 12:65–78.CrossRef 56. Selvam K, Vishnupriya B, Subash Chandra Bose V: Screening and quantification of marine actinomycetes producing industrial enzymes

amylase, cellulase and lipase from South cost of India. Int J Pharma Biol 2011, Gefitinib 2:1481–1487. 57. Jang H-D, Chen K-S: Production and purification of thermostable cellulases from Streptomyces transformant T3–1. World J Microbiol Biotechnol 2003, 19:263–268.CrossRef Competing interests The authors declare that they have no competing interest. Authors’ contribution Research concept and the experiments were performed by BM and LAR, NVV and RK analyzed the data and reviewed the manuscript. All authors approved the final manuscript.”
“Background Pseudomonas aeruginosa is the major pathogen involved in the decline of lung

function in patients with cystic fibrosis (CF) [1–5]. Its presence in the lungs is associated with an increased mortality and morbidity of Akt inhibitor CF patients [6]. Early detection of this bacterium from respiratory tract is determinant because it ensures effective patient management [5, 7, 8]. Indeed, after intermittent colonization by different strains, once acquired, chronic P. aeruginosa colonization by mucoid and biofilm-growing isolates is difficult to eradicate [2, 4, 9, 10]. Thus, the earlier the treatment toward P. aeruginosa onset, the higher the chance to efficiently control P. aeruginosa [5, 7, 8]. However, accurate identification of this bacterium in CF sputum by conventional microbiology techniques is known

to be limited. This can be explained by a large phenotypic diversity of P. aeruginosa isolates recovered from CF patients such as loss of pigment production or exopolysaccharide production. Moreover, Singh et al. demonstrated that P. aeruginosa can form biofilms in the airways of CF patients [11]. Biofilms contain bacterial cells that are in a wide range of physiological states. One of the mechanisms find more contributing to this physiological heterogeneity includes the adaptation to the local environmental conditions. For instance, bacterial cells from the deep layers of biofilm depleted of oxygen [12] can grow in anaerobic conditions. Therefore, the CF patients isolates obtained from biofilms, i.e. in anaerobic conditions, grow hardly in aerobic conditions on a conventional culture medium [13]. Another limitation of conventional culture is that P. aeruginosa can be easily misidentified with closely related Gram-negative bacilli in CF sputum [14–19]. The use of molecular techniques such as PCR could improve accurate identification of P. aeruginosa [14–19], and consequently, its early detection in CF sputum patients [20–24]. To date, there is no consensus for a universal protocol for the molecular detection of P. aeruginosa. Indeed, its genome is known to be highly polymorphic. Changes that can occur at the genetic level could compromise the reliability of molecular identification techniques.

Representative sections of each specimen were stained with haematoxylin-eosin to confirm the diagnosis of endometriosis. Atezolizumab molecular weight For immunohistochemistry 5-7 μm specimen sections embedded in paraffin, were cut, mounted on glass and dried overnight at 37°C. All sections were then deparaffinized in xylene, rehydrated through a graded alcohol series and washed in phosphate-buffered saline (PBS). PBS was used for all subsequent washes and for antiserum dilution. Tissue sections were quenched sequentially in 3% hydrogen peroxide in aqueous solution and blocked with PBS-6% non-fat

dry milk (Biorad, Hercules, CA, U.S.A.) for 1 h at room temperature. Slides were then incubated at 4°C overnight at 1:100 dilution with a rabbit polyclonal antibody for AMH (Abcam, Cambridge, UK). After three washes in PBS to remove the excess of antiserum, the slides were

incubated with diluted goat anti-rabbit biotinylated antibody (Vector Laboratories, Burlingame, CA, U.S.A.) at 1:200 dilution in PBS-3% non-fat dry milk (Biorad) for 1 h. All the slides were then processed by the ABC method (Vector Laboratories) for 30 min at room temperature. Diaminobenzidine (Vector Laboratories) was used as the final chromogen and hematoxylin was used as VX-809 research buy the nuclear counterstain. Negative controls for each tissue section were prepared by leaving out the primary antiserum. All samples were processed under the same conditions. Experiments were performed in compliance with the Helsinki Declaration and the protocols were approved by the ethics committee of the Fondazione Italiana Endometriosi. Cell lines and primary cells Human endometriosis stromal and epithelial cells were described elsewhere [13]. Cells

were grown following standard procedures and were propagated in DMEM/F12 (1:1) with 10% Fetal Bovine Serum (FBS) (Gibco, Life Technologies Italia, Monza, Italy), 2 mM L-Glutamine (Euroclone S.p.a, Piero, Italy) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin and 250 ng/mL amphotericin-B). In vitro treatment with AMH Cultured human endometrial stromal and epithelial cells were treated with Recombinant Human Mullerian-Inhibiting Substance (rhMIS)/anti-Mullerian hormone (AMH) – E-Coli derived (R&D Systems) Selleck AZD9291 and Purified recombinant protein of Homo sapiens AMH (OriGene Technologies, Rockville, MD, USA) at three different final concentrations (10-100-1000 ng) for three different time (24-48-72 hrs). Plasmin-cleaved AMH was used instead of the full-length molecule for incubation times indicated. AMH was digested by Plasmin from human plasma (Sigma-Aldrich, Italia) 1 h at 37°C in a ratio of 25 to 1, as described [14]. The effect of AMH on the activity of cytochrome P450 aromatase (CYP19) was measured through the P450-Glo assays (Promega Italia, Milano, Italy) [15].

PCR products were subsequently electrophoresed on a 1.5% agarose gel, and visualized under a UV transilluminator. Western blot analysis Cells were lysed in buffer containing 20 mmol/L HEPES, 1 mmol/L EGTA, 50 mmol/L β-glycerophosphate, 2 mmol/L sodium orthovanadate, 100 mL/L glycerol, 10 mL/L

Triton X-100, 1 mmol/L DTT, and 1 × Protease Inhibitor Cocktail (Roche, Mannheim, Germany). The lysate was centrifuged at 13 000 g and 4°C for 10 min. The supernatant was the total cell lysate. Protein concentration was measured using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL, USA). Thirty micrograms of protein was loaded per lane, separated by 100 g/L SDS-PAGE, and transferred onto equilibrated polyvinylidene difluoride membrane by electroblotting. Membranes Copanlisib manufacturer were blocked with 5% non-fat milk in 1% TBS-T buffer for 2 h at room temperature. AhR, CYP1A1, and GAPDH were detected for 2 h using antibodies against AhR (SC-5579, Santa Cruz Biotechnology, USA, working dilution 1:150), CYP1A1 (AB1258, Chemicon International, USA, working dilution 1:500), and GAPDH (2118, Cell Signaling Technology, USA, working dilution 1:1000). After secondary antibody incubation (7074,Cell Signaling Technology, USA, working dilution 1:2000) for 2 h, protein bands were detected using ECL system (Pierce Biotechnology, Inc., USA). Cell viability assay The Lumacaftor in vitro effect of DIM on the proliferation of gastric

cancer cells was determined by MTT assay. Briefly, A total of 1 × 104 trypsin-dispersed cells in 0.1 mL culture medium were seeded into each well of a 96-well plate and cultured for 24 hours. Next, cells were treated with DIM as described above. Then, 20 μL of MTT (5 g/L) was added to each well and the incubation was continued for 4 h at 37°C. Finally, the culture medium was removed and 150 μL of DMSO was added to each

well. The absorbance was determined with an ELISA reader at 490 nm. The cell viability percentage was calculated as: Viability percentage (%) = (Absorption value of experiment group)/(Absorption value of control group) × 100%. Flow cytometric analysis SGC7901 cells were plated on 60-mm diameter culture plates and treated with DIM at different concentration (10, 20, 30, 40, 50 μmol/L) for 48 h. The control contained 1 mL/L DMSO only. Prior to harvesting, the cells were washed twice with 0.01 mol/L PBS, trypsinized, and 17-DMAG (Alvespimycin) HCl pelleted. The cells were then fixed with 70% ice-cold ethanol at 4°C overnight. Finally, the cells were washed twice with PBS and dyed with PI. The DNA content was analyzed with a flow cytometer (Beckman-Coulter, Brea, USA). The cell cycle of SGC7901 cells were analyzed using MULTYCYCLE and winMDI2.9 software (Phoenix, AZ, USA). For cell apoptosis analysis, after incubation for 48 h, cells were stained with annexin V-FITC and PI. Cells with annexin V (−) and PI (−) were deemed viable cells. Cells with annexin V (+) and PI (−) were deemed early apoptotic cells.

PFGE patterns B1, D1, D3, D4 and E1 were found on several farms (table 1). The minimal similarity within the farms varied from 52% (farm 5) to 100% (farm 4) and the minimal similarity between the farms was 61% (data not shown). Figure 2 shows the PFGE results of farm 6 with 4 different PFGE patterns and from farm 9 which all had indistinguishable PFGE patterns. Table 1 Overview of transmission of ST398 MRSA on 9 farms (n = 40) Strain nr Farm spa-type Origin PFGE pattern Coefficient*

1110701181 1 t011 farmer B3 70 1110700844 1 t011 pig D7   1110701184 2 t011 farmer D4 86 1110700857 2 t011 pig D4   1110701182 2 t011 employee E1   1110701185 2 t011 relative E1   1110701429 3 t011 pig B1 87 1110701595 3 t011 relative B2   1110701592 3 t011 farmer D19   1110701192 4 t108 farmer D1 100 1110700908 4 t108 pig D1   1110701196 5 t567 farmer DAPT concentration D18 52 1110701197 5 t567 relative D18   1110700912 5 t567 pig I   1110701611 6 t108 dust D1 84 1110701614 6 t108 dust D1   1110701604 6 t108 pig D1   1110701200 6 t011 farmer D20   1110701612 6 t011 dust D4   1110701605 6 t011 pig D4   1110701201 6 t011 relative E1   1110701600 7 t2741 employee D14 95 1110701596 7 t011 farmer D14   1110701580 7 t011

pig D14   1110701601 7 t108 employee D21   1110701576 7 t011 pig D21   1110701577 7 t011 pig D21   1110700882 8 t011 pig B1 Inhibitor Library 66 1110700884 8 t108 pig D1   1110700876 8 t108 pig D3   1110700889 Mannose-binding protein-associated serine protease 8 t2330 dust D4   1110701188 8 t2330 relative D4   1110701191 8 t2330 relative D4   1110700890 8 t108 dust K   1110701791 9 t108 dust D1 86 1110701783 9 t108 pig D1   1110701788 9 t108 pig D1   1110703030 9 t108 relative D1   1110703031 9 t588 relative D1   1110703032 9 t108 relative D3   * Dice similarity coefficient, using UPGMA. Optimization 0,5%, position tolerance Figure 2 PFGE patterns of ST398 isolates digested with Cfr 9I restriction enzyme using NCTC 8325 as the reference standard. Lanes 6, 12, 18, and 24, NCTC 8325; Lanes 1-5, isolates from an outbreak in a residential care facility, all PFGE pattern J; Lanes 7-8, and 14-15, two pairs of a veterinarian and a close family member with distinct PFGE

patterns; Lanes 9-11, and 13, two pairs of a veterinarian and a close family member with identical banding patterns; Lanes 16-17, and 19-22, isolates of pig farm 6 with four different PFGE patterns; Lanes 23, and 25-28, isolates from pig farm 9 with identical banding patterns Discussion MRSA isolates belonging to the ST398 clonal lineage are hard to discriminate based on spa-typing and/or MLST, hampering the assessment of transmission and outbreaks. Therefore, other techniques such as a modified PFGE could provide a new opportunity to differentiate ST398 isolates. The restriction enzyme SmaI does not cut the DNA of NT SmaI -MRSA isolates, due to methylation of the SmaI site. However, Cfr9I, a neoschizomer of SmaI, can be used for generating PFGE profiles of the NT SmaI -MRSA isolates.