0 mL, including 4.6 mmol of NH2) was reacted with mild stirring using 1-bromooctadecane (4.56 g, 13.7 mmol) in the presence of Na2CO3 (1.45 g, 13.7 mmol) at 70°C for 69 h in dimethylacetamide under nitrogen atmosphere. Particles were recovered by filtration and washed Belnacasan mouse with water, warm ethyl acetate, ethanol, and water successively. Changes in the appearance and porous structure were not observed by SEM. In the IR spectra (KBr pellet), ν CH of CH2 2,925 and 2,850 cm-1 was observed. Ion-exchange capacity was 2.3 meq g-1 dry particles. Using this value, the colloidal equivalent of chitosan (5.0 meq g-1, pH 4.0) used for the preparation of the cross-linked porous chitosan and the

ion-exchange capacity of the cross-linked porous chitosan (3.8 meq g-1) which was cross-linked by 1,6-diisocyanatohexane, GlcN of the cross-linked porous chitosan, and GlcNC18 of the resulting directly alkylated porous supports were calculated as 69 and 47 mol%, respectively. Therefore, about half of the monosaccharide units of the support particles are deemed to be octadecylated. Column-wise adsorption of LPS from protein solutions Purified water and buffer solutions were sterilized using an autoclave at 115°C to 121°C for 15 min. Glass wares were AG-014699 concentration also sterilized using the autoclave at 250°C for 2 h. LPS aqueous solution, which was prepared by vortex

mixing and dilution with water, was added to HSA preparation. The solution was filtered with a filter disk having 0.2-μm-diameter pores and diluted with a buffer solution to a desired concentration for column-wise experiments. Phosphate buffer was used for experiments at pH 7.0 and 8.0, and acetic acid buffer was used for those at pH 4.3 and 5.3. Buffer solutions were prepared by adding NaOH solution of a predetermined concentration to phosphoric acid or acetic acid to obtain the desired ionic strength (μ). Column-wise adsorption was done at 20°C. Adsorbents were suspended in water and fed into a glass column (8 mm i.d. × 100 mm length) using a LC-6A pump (Shimadzu Corp.,

Kyoto, Japan). 17-DMAG (Alvespimycin) HCl The length of the gel bed was between 930 and 980 mm. The resulting column was washed with 0.5 M NaOH, at 10 mL h-1 for 3 h, and left overnight filled with 0.5 M NaOH to decompose LPS in the column (depyrogenation). After washing with water for 3 h, 0.1 M acetic acid was passed through for 1.5 h to convert amino groups of N-octadecylchitosan immobilized on the supports to their ammonium forms. After the buffer solution was passed through for 6.5 h, HSA solution was passed through at 5 mL h-1 for 15 to 16 h. The eluted solution was collected immediately as ten fractions of 7.5 mL each. Fractions of 2, 4, 6, 8, and 10 were analyzed for the concentrations of LPS and HSA. Chemical stability Porous supports bearing lipid membranes of N-octadecylchitosan were immersed in 0.5 M NaOH or 0.1 M HCl at ambient temperature overnight and then washed with water and subjected to IR spectroscopic analysis.

23144, P = 0.0615, n = 66).

In both a and b, the regression lines for normotensive and hypertensive patients were not considered to be identical, with different R428 cell line y-intercepts, since there was a significant difference (P < 0.01, F test) in the y-intercept of the two regression lines under the null hypothesis that the y-intercept of two lines was identical. There was no significant difference (P = 0.6061 in a or P = 0.6079 in b, F test) in the slope of the two lines under the null hypothesis that the slope of the two lines was identical Table 4 shows that in young adult patients aged <36 years, eGFR was lower and TKV was larger in the hypertensive group than in the normal blood pressure group. Table 4 Comparison of eGFR and TKV between normal and high blood pressure groups in young adults (≤35 years)   Normotensive group Hypertensive group P value N 36 27   Initial BPa  Systolic (mmHg) 117.9 ± 15.1 148.1 ± 14.2 <0.0001  Diastolic (mmHg) 68.5 ± 6.9 85.9 ± 13.7 0.0001 Post-Tx BPb  Systolic (mmHg) 115.8 ± 14.4 128.4 ± 12.9 0.0030  Diastolic (mmHg) 70.5 ± 11.6

78.4 ± 6.5 0.0066 eGFR (ml/min/1.73 m2) 113.6 ± 42.5 86.6 ± 24.2 0.0044 N 10 12   TKV (ml) 826.3 ± 319.2 1713.2 ± 675.6 0.0011 Data are the mean ± SD. P values were calculated by Student’s t test aInitial BP is blood Selleck Fulvestrant pressure without anti-hypertensive treatment in hypertensive group and blood pressure at initial visit in normotensive group bPost-Tx BP is blood pressure at the study time. In hypertensive group, all patients were receiving antihypertensive medication Discussion ADPKD is the most common hereditary kidney disease. The disease is characterized by the formation of numerous kidney cysts and their development, leading to kidney enlargement and failure, reaching end-stage renal failure in up to about 50% by age 70 [16]. Polycystic kidney disease animal model studies suggested that earlier intervention resulted in more effective prevention of disease progression [17, 18]. The potential candidates

clinically examined so far seem Anacetrapib to attenuate progression but not to reverse progressed renal disease [6–8, 11]. Thus, it is a crucial issue when to start treatment intervention. The present study confirmed that renal function decreased progressively as a function of age [1, 3, 16, 19, 20]. In 196 patients with a mean age >30 years, the mean eGFR slope was −3.4 ± 4.9 ml/min/1.73 m2/year. In 46 patients with mean TKV >1500 ml, the TKV slope was 86.8 ± 161.6 ml/year (5.6 ± 8.8%/year) (Table 1). The present data of eGFR and TKV slopes are compatible with previous findings [3, 10]. The slopes of GFR (measured by iothalamate clearance) and TKV were analyzed according to TKV and age groups in the CRISP study [3]. Analysis of variance revealed that the slopes of GFR differed among subgroups with different initial TKV (P = 0.005), whereas the slopes of GFR did not differ significantly among subgroups with different initial ages (P = 0.20); there was no significant interaction between TKV and age (P = 0.

lactis IL1403/Streptococcus pneumoniae TIGR4 b ++ Genes detected in both alignments, L. lactis subsp. lactis IL1403 array probes vs S. pneumoniae TIGR4 genome, and S. pneumoniae TIGR4 array probes vs L. lactis subsp. lactis IL1403

genome; + positive in one of the two cases. c Only the results for the negative genes in BLAT80 are shown. d Only the results for the negative genes in both see more BLAT80 and BLAT70 are shown. After combined analysis of the results obtained in silico and in vitro, we established, under the hybridization conditions buy Liproxstatin-1 used in this study, a detection threshold based on a sequence similarity of ≥ 70% for alignments longer than 100 bp. This was established as the reference framework for the inter-species CGH assays. In vitro microarray CGH experiments with L. garvieae CECT 4531 vs reference microorganisms L. lactis subsp. lactis IL1403 and S. pneumoniae TIGR4, and in silico analysis of available

sequences from L. garvieae The microarray CGH experiments identified 267 genes in L. garvieae that had analogues in L. lactis CYTH4 and/or S. pneumoniae (Additional file 1). Of these, 111 genes (41.6%) were identified only with the L. lactis microarray, 70 genes (26.2%) only with the microarray of S. pneumoniae, and 86 genes (32.2%) were identified with both microarrays. These genes belong to diverse functional groups (Table 2). Most of the genes (96.6%) have been documented for the first time in L. garvieae.

Only nine genes (four present in both reference microorganisms: atpD/SP1508, pfk/SP0896, tig/SP0400, tuf/SP1489; three present in L. lactis: als, ddl, galK; two present in S. pneumoniae: SP0766, SP1219) out of the 267 genes detected have been either identified or sequenced before in diverse strains of L. garvieae (Tables 3 and 4). In silico analysis of these previously sequenced genes (n = 9) of L. garvieae were performed to assess the efficacy of the methodology. Alignments of these available sequences with the genomes of the corresponding reference microorganism and their respective array probes showed nucleotide identities ranging between 70% and 86% (Tables 3 and 4).

The aim of the study presented here was to analyze which MCF7 signaling pathways were affected by the placenta. Methods: MCF7-eGFP cells cultured on matrigel with or without placental explants were separated from the placenta

by cell sorting and their mRNA was subjected to microarray analysis. ERa/STAT3/mTOR expression and phosphorylation levels were analyzed in these cells. Results: Trophoblast cells differentiate into extravillous trophoblast cells (EVT) which migrate into the matrigel. The effects (apoptosis/proliferation/detachment) were mainly observed in MCF7 cells that were located near the EVT cells. Microarray results have demonstrated significant changes in genes related to cell adhesion, glycan, breast carcinoma estrogen and JAK-STAT pathways.

Decreased ERa and elevated pmTOR and R428 cost STAT3 proteins detected by immunobloting confirmed our findings at the transcript level. Moreover, promoter analysis of significantly affected genes in this array demonstrated an enrichment of motifs located at the transcription start site that match annotation for ISRE (interferon-stimulated response element), suggesting the activation of interferon (INF) signaling. Conclusion: Our results suggest that the placenta attenuates MCF7 growth in its vicinity by modulating INF/STAT and inducing detachment/migration and apoptosis. Published data demonstrated that the above pathways may indeed stimulate these processes. The involvement of these signaling pathways in buy Fulvestrant cell migration/apoptosis and the fate of the detached MCF7 cells will be further studied. Poster No. 113 Identification of Secretory Stromal Gene Signature of the Ovarian

Anacetrapib Tumor Microenvironment and Implications for Ovarian Cancer Progression Melissa Thompson 1 , Lina Albitar2, Kwong-Kwok Wong1, Michael Birrer3, Samuel Mok1 1 Department of Gynecologic Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA, 2 Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women’s Hospital, Boston, MA, USA, 3 Department of Medicine, Massachusetts General Hospital, Boston, MA, USA The stromal microenvironment provides structural support and myriad signaling cues, which can significantly affect cell growth and development. The tumor-associated stromal microenvironment has been shown to play a key role in many cancers by influencing tumor initiation, invasion and metastasis. Our objective was to identify specific genes that are differentially regulated in the stromal component of high-grade late-stage serous ovarian cancer that may contribute to ovarian cancer progression.

PubMedCrossRef 15. Luo P, Morrison

DA: Transient association of an alternative sigma factor, ComX, with RNA polymerase during click here the period of competence for genetic transformation in Streptococcus pneumoniae . J Bacteriol 2003,185(1):349–358.PubMedCrossRef 16. Chaillou S, Champomier-Vergès MC, Cornet M, Crutz-Le Coq AM, Dudez AM, Martin V, Beaufils S, Darbon-Rongère E, Bossy R, Loux V, et al.: The complete genome sequence of the meat-borne lactic acid bacterium Lactobacillus sakei 23 K. Nat Biotechnol 2005,23(12):1527–1533.PubMedCrossRef 17. Jones RJ, Wiklund E, Zagorec M, Tagg JR: Evaluation of stored lamb bio-preserved using a three-strain cocktail of Lactobacillus sakei . Meat Sci 2010,86(4):955–959.PubMedCrossRef 18. Vermeiren L, Devlieghere F, Debevere J: Evaluation of meat born lactic acid bacteria as protective cultures for the biopreservation of cooked meat products. Int J Food Microbiol 2004,96(2):149–164.PubMedCrossRef 19. Plewniak F, Jeanmougin F, Higgins DG, Thompson JD, Gibson TJ: The CLUSTAL

X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997,25(24):4876–4882.PubMedCrossRef 20. Chaillou S, Daty M, Baraige F, Dudez AM, Anglade P, Jones R, Alpert CA, Champomier-Vergès MC, Zagorec M: Intraspecies genomic diversity and natural population structure of the meat-borne lactic acid bacterium Lactobacillus sakei . Appl Environ Microbiol 2009,75(4):970–980.PubMedCrossRef 21. Wydau S, Dervyn R, Anba J, Dusko Ehrlich S, Maguin E: Conservation of key elements of natural competence in Lactococcus lactis Palbociclib in vitro ssp. FEMS Microbiol Lett 2006,257(1):32–42.PubMedCrossRef 22. Morikawa K, Ohniwa RL, Kumano M, Okamura H, Saito S, Ohta T: The sigH gene sequence can subspeciate staphylococci. Diagn Microbiol Infect Dis 2008,61(4):373–380.PubMedCrossRef 23. Stentz R, Loizel C, Malleret C, Zagorec M: Development of genetic tools for Lactobacillus sakei : disruption of the ß-galactosidase gene

and use of lacZ as a reporter gene to study regulation of the putative copper ATPase, AtkB. Appl Environ Microbiol 2000,66(10):4272–4278.PubMedCrossRef tuclazepam 24. Weir J, Predich M, Dubnau E, Nair G, Smith I: Regulation of spo0H , a gene coding for the Bacillus subtilis sigma H factor. J Bacteriol 1991,173(2):521–529.PubMed 25. Desroche N, Beltramo C, Guzzo J: Determination of an internal control to apply reverse transcription quantitative PCR to study stress response in the lactic acid bacterium Oenococcus oeni . J Microbiol Methods 2005,60(3):325–333.PubMedCrossRef 26. Tao L, Wu X, Sun B: Alternative sigma factor sigmaH modulates prophage integration and excision in Staphylococcus aureus . PLoS Pathog 2010,6(5):e1000888.PubMedCrossRef 27. Crutz-Le Coq AM, Zagorec M: Vectors for lactobacilli and other Gram-positive bacteria based on the minimal replicon of pRV500 from Lactobacillus sakei . Plasmid 2008,60(3):212–220.PubMedCrossRef 28.

7 10.4 7.1 3.8 4.4 3.8 5.5 21.3 4.4 3.8 3.8 0.5 2.2 2.2 2.7 0 PtoSSB 5.3 5.3 4.6 6.0 2.6 6.0 7.3 10.6 2.6 5.3 9.9 5.3 4.6 3.3 9.3 2.0 1.3 3.3 3.3 2.0 EcoSSB 7.3 2.8 4.5 7.3 3.4 16.3 6.7 3.4 5.6 4.5 5.6 10.1 4.5 5.6 5.0 0.6 2.2 2.2 2.2 0 TteSSB3 4.0 5.3 7.3 8.7 2.0 6.0 6.0 5.3 6.0 10.7 8.0 1.3 4.0 6.7 8.0 0.7 2.0 6.0 1.3 0 TmaSSB 5.0 4.3 5.7 9.2 2.8 4.3 7.1 3.5 10.6 6.4 12.8 0.7 2.1 5.0 10.6 0 0.7 7.8 1.4 0 The glycine content in psychrophilic SSBs, particularly in the DpsSSB, at 11.3%, ParSSB,

at 16.4%, PcrSSB, at 16.9%, and PprSSB, at 10.4%, and in the mesophilic EcoSSB, at 16.3%, is much higher than in the thermophilic SSBs, at 6.0% and 4.3% for TteSSB3 and TmaSSB, respectively. This accords with the known tendency of thermostable proteins to have a preference for a decrease in the Gly content in positions of low structural importance for fold conservation [36, 37]. The high content of glutamine Selleck Paclitaxel and asparagine residues observed in the ParSSB, at 20.0%, PcrSSB, selleck compound at 23.0%, PinSSB, at 24.93, and PprSSB, at 25.4% is one and a half times greater than that of the EcoSSB, at 14.5% and much higher than for the thermophilic SSBs, at 5.3% and 2.8% for the TteSSB3

and TmaSSB, respectively. Of the 39 glutamine residues in the PinSSB and PprSSB, 34 are located in the C-terminal fragment of the former and 29 in that of the latter, which represents, respectively, 30.4% and 38.2% of that domain. At up to 9 rests side by side, the glutamine residue repetitions in the C-terminal fragment of the PprSSB are extremely numerous, endowing the domain with a highly hydrophilic character. This area is reminiscent of the ‘glutamine-rich

(Q-rich) regions’ in proteins other than SSBs, which form a ‘polar zipper’ and with which different protein subunits interact in a specific manner. The ratio of polar to non-polar amino acid residues is one of the major determinants of protein stability and increasing the fraction of polar and charged residues leads to protein disorder Rutecarpine [29]. The content of polar amino acid residues N, Q, S, T, and Y in the DpsSSB, FpsSSB, ParSSB, PcrSSB, PinSSB, PprSSB, and PtoSSB is 30.2%, 31.5%, 33.3%, 37.4%, 36.5%, 36.0% and 25.8%, respectively. With the exception of PtoSSB, this is considerably more than that found in the mesophilic EcoSSB, at 27.4%, and very much more than that found in the thermophilic SSBs, at 21.3% and 19.8% for TteSSB3 and TmaSSB, accordingly. Russell [35] and Zuber [38] noticed that psychrophilic proteins appear to have more polar residues than thermophiles or mesophiles do, which is consistent with our research. As mentioned previously, a lower Pro content is one of the features of cold-adapted proteins.

MHCC-97H-PDCD4, MHCC-97H-vector or MHCC-97H cells were suspended in 100 μl DMEM containing 10% FBS and plated at 1 × 105 cells/well onto the upper compartment of the chamber. The lower chambers were filled with 600 μl NIH3T3-CM, which was obtained by a 24 h incubation of NIH3T3 cells with 50 μg/ml ascorbic acid in serum-free DEME media [16]. Cells were cultured at 37°C in a humidified, 5% CO2 atmosphere for another 24 hours. The filters were then washed with PBS, fixed with 95% methanol for 20 min and stained with hematoxylin and eosin 3-MA ic50 solution.

Cells on the upper surface of the filters were gently removed with cotton swabs. The number of cells that had migrated to the lower surface of the filter membrane was counted in five randomly chosen fields under a light microscope (× 200). The average number of migrated cells per microscopic field was analyzed[28]. Statistical Analyses Data were reported as means ± SD of the combined experiments. Student’s two-tailed t test for independent means was employed to determine significant differences (P < 0.05). Analyses were performed using SPSS16.0 statistical program. Results Expression of PDCD4 The expression of PDCD4 in

three different metastatic potential HCC cell lines was detected. The positive immunocytochemical staining for PDCD4 was brownish and localized in cytoplasm (Fig. 1A). HSCORE Linsitinib cost for MHCC-97H cells, MHCC-97L cells and Hep3B cells was 0.85 ± 0.17, 1.46 ± 0.36 and 1.97 ± 0.29, respectively

(Fig. 1B). Difference between Group1 and Group2 (n = 5, P < 0.05) or Group1 and Group3 (n = 5, P < 0.01) or Group2 and Group3 (n = 5, P < 0.05) was significant. Figure 1 Expression of Farnesyltransferase PDCD4 in HCC cells. A: Immunocytochemical staining. The positive staining(×200) was brownish and localized in cytoplasm. D: Western blot assay. Representative figures are shown from one of three individual experiments. B, C or E shows statistical analysis for immunocytochemical staining, real – time PCR or western blot assay, respectively. In A, a, b or c represents cells of MHCC-97H, MHCC-97L or Hep3B, respectively; d shows cell staining without the primary antibody. In B, C and E, Group1, Group 2 or Group3 represents cells of MHCC-97H, MHCC-97L and Hep3B, respectively. Bars represent the means ± SD. The difference between Group1 and Group2 (P < 0.05) or Group1 and Group3 (P < 0.01 in B; P < 0.05 in E) was significant. The quantitative assay by real time PCR was reported in RQ units as compared with the noninvasive Hep3B cells (Fig. 1C). RQ for MHCC-97H cells and MHCC-97L cells was 0.126 ± 0.023 and 0.385 ± 0.084, respectively. The mean RQ for Group1 and Group2 was 0.126 ± 0.023 and 0.385 ± 0.084, respectively. The difference between Group1 and Group2 was significant (n = 3, P < 0.05). Western blots for PDCD4 expression display a band of 54 kD (Fig. 1D). The relative densities (RD) of PDCD4 for Group1, Group2 and Group3 were 0.053 ± 0.045, 0.

Soft Latin-style cheeses like queso fresco typically are not aged,

have a short shelf-life (about 2 weeks), and have a high moisture content (41/59%) [5]. The lack of an aging step as well as high moisture content and the moderate pH level of Latin-style cheeses can all contribute to pathogen growth and increases the likelihood of pathogens surviving and possibly multiplying to the levels necessary to cause GDC-0980 illness [6]. For this reason, the US FDA prohibits the interstate sale of this cheese type if it is manufactured using raw milk [5]. However, for some the taste of Latin-style cheese made with raw milk is preferable. Between 1998 and 2009, 56 cheese-associated disease outbreaks occurred in the United States resulting in 1,377 illnesses,

171 hospitalizations, and 2 deaths [7–9]. Eighteen of these occurrences (32%) specifically involved Latin-style cheeses and a variety of pathogens, resulting in 212 illnesses (15% of total), 95 hospitalizations (55%), 2 deaths (100%), and at least 7 stillbirths [10]. Individuals making homemade cheese (i.e. bathtub cheese) sold in grocery stores accounted for 85 illnesses [7–9, 11]. selleck compound The most serious outbreak involving Latin-style cheeses occurred in 1985; 142 cases of listeriosis caused 48 deaths, of which 30 involved neonates or fetuses [10]. In response to a foodborne outbreak, suspect samples are analyzed according to standardized methods including those described in the FDA Bacteriological Analytical Manual (BAM). One goal of analysis is to recover isolated colonies click here of the pathogenic bacteria that can assist in matching any recovered clinical, food, and environmental isolates to

determine the source(s) of illness. Most methods described in the FDA BAM begin with enriching the suspected food product in a universal or microbe-specific enrichment broth for up to 24 hours. The sample is then plated onto selective agar specific for the target bacteria to obtain isolated colonies. The initial enrichment step is designed to recover and propagate bacterial pathogens in the product facilitating downstream detection efforts. However, enrichment can also influence levels of background microflora. A food sample may consist of a complex consortium of bacteria that can out-compete and otherwise hinder efforts to recover human pathogens. With improved characterization of the microbial taxonomy and abundance associated with a given enriched food product, broths and agar formulations can be vastly improved in terms of culture selectivity. Several studies have attempted to describe the full range of microbes present in cheeses as well as in various steps along the manufacturing and maturation process to understand temporal microflora changes [12–18]. The most widely-used approach begins with the plating of cheese samples on agar and picking isolated colonies for subsequent identification using biochemical analyses or molecular characterization.

Purified protein aliquots containing 10% glycerol were stored at−80°C. Infusion ESI-Q-TOF experiment ElectroSpray Ionization coupled with a quadrupole-time of flight tandem was used in the positive ion mode using a Q-TOF Ultima Instrument (Waters). The SpdA protein was dissolved in water with 0.05% formic acid and directly introduced into

the source at a flow rate of 5 μl/min. Capillary entrance voltage was set to 2.7 kV, and dry gas temperature to 150°C. Voltages: Cone: 80 V, Rf lens: 40 V. MS profile [500 (10%), 1500 (60%), 2500 (20%), ramp 10%]. Scanning domain: m/z 1000-3000. Calibration was performed with orthophosphoric clusters. Continuum spectra exhibiting multicharged ions were transformed into molecular mass envelops using the MaxEnt 1 software. Electromobility Selleckchem C646 selleck compound library shift assay A set of DNA probes covering the predicted Clr binding palindrome were obtained

by annealing two complementary oligonucleotides. The annealing reactions were performed in water with 25 μM strand + (WTN8+ or MN8+ (see Additional file 10)) and 25 μM strand–(WTN8-or MN8-(see Additional file 10)) for each probe in a total reaction volume of 100 μl. Mixes were incubated at 95°C during 5 min following by slow cooling to 25°C. 175 nM double-strands probes were end labelled using 20 μCi of [ATPγ-32P] and 10 U of T4 polynucleotide kinase (Promega). Probes (1.75 nM each) were incubated in binding buffer (10 mM Tris [pH 8.0], 1 mM EDTA, 1 mM DTT, 10 μg/ml bovine serum albumin, 100 mM KCl) containing 50 μg/ml poly(2′-deoxyinosinic-2′-deoxycytidylic acid) selleck compound (Sigma) and 10% glycerol for 30 min at room temperature with purified Clr and 3′, 5′cAMP or 2′, 3′cAMP added to the concentrations indicated in the figure legends in a final reaction volume of 15 μl. Samples were subjected to electrophoresis on a 10% polyacrylamide TBE 0.5 X gel containing 4% PEG-8000. Electrophoresis was conducted in TBE 0.5 X buffer at 80 V at room temperature. Gels were dried and analysed by autoradiography.

Plant assays and plant extracts preparation Seeds of M. sativa cv. Europe were surface sterilized, germinated, and allowed to grow in 12-cm2 plates containing slanting nitrogen-free Fahraeus agar medium for 3 days at 22°C with day/night cycles of 16/8 h. The plants were inoculated with 2.103 bacteria per plant. Nodules were counted every day during 8 days then every 2 days until 35 days post-inoculation (dpi). At 35 dpi, shoots were collected and dried overnight at 65°C for weight measurements. Plant extracts were prepared as previously described [3]. β-Galactosidase assays S. meliloti strains carrying the pGD2178, pXLGD4 or pGD2179 plasmids were grown at 28°C in VGM. Overnight cultures were diluted to an OD600 of 0.1 in VGM and grown for an additional 2 h. 5 ml-cultures supplemented with 3′, 5′cAMP (2.5 mM or 5 mM), 2′, 3′cAMP (7.5 mM) or 5 mM 3′, 5′cGMP were grown for an additional 5 hours at 28°C. Overnight incubation was used for other potential inducers listed in Additional file 3.

Extracellular bacteria were removed by extensively washing MØ with warm HBSS. Infected MØ were directly used in tests (day 0) or cultured for 1, 2 or 6 days, as indicated in Figures.

Ingestion of bacteria Resting MØ and IFN-γ-activated MØ (1 × 105 cells/well) were prepared in 8-well Permanox learn more chamber slides (Nunc, Denmark) and then infected with FITC-labeled Mtb strains at an MOI of 10. After infection, MØ were fixed by incubating with 3% FA for 15 minutes (37°C, 5%, CO2) and washed twice with HBSS. The number of infected MØ and the number of bacteria engulfed by one MØ were determined by fluorescence microscopic examination (Nikon ECLIPSE TE 2000 U). In all cases, 200 MØ were counted. Intracellular growth of bacteria Resting MØ and IFN-γ-activated MØ (1 × 105 cells/well) were prepared in 24-well plates (Nunc). MØ were then treated with 10 μM IRAK1/4 inhibitor or with a saturating concentration of anti-TLR2 blocking mAbs (35 μg/ml) for 1 hour or LY294002 nmr left untreated. Afterwards, MØ were infected with Mtb strains at an MOI of 1. After infection, fresh CM and IRAK1/4 inhibitor or anti-TLR2 blocking mAb (when required) were added, and cells were cultured for 6 days. On the day of infection (day 0) and 6 days post-infection, MØ were lysed with 1 ml of 0.2% Triton X-100 and appropriate dilutions of cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC.

After 21 days of culture, CFUs were counted. The data were presented as fold-increase in CFUs, calculated as CFUs on day 6 divided by CFUs on day 0. NO production

Resting MØ and IFN-γ-activated MØ (1 × 105 cells/well) were prepared in 96-well plates (Nunc) and treated with IRAK1/4 inhibitor or left untreated (as described above). Next, MØ were infected with Mtb strains at an MOI of 10 and cultured for 2 days with or without IRAK1/4 inhibitor. The presence of nitrite (stable metabolite of NO) in the culture supernatants was determined using the Griess reagent. OD was determined using a Multiscan RC ELISA reader (Labsystem, Finland). Nitrite concentration was calculated from a standard curve prepared using sodium nitrite as a reference. ROS production Glutathione peroxidase Resting MØ and IFN-γ-activated MØ (1 × 105 cells/well) were prepared in 96-well plates (Nunc) and then infected with Mtb strains at an MOI of 10. After culturing for 1 day, 1 μg/ml of PMA (to initiate ROS production) as well as 40 U of HRP and 1 mM luminol (to enhance chemiluminescence) were added to the cells. Chemiluminescence (CL) was recorded over 4 hours at 5-minute intervals using Fluoroscan Ascent FL (Labsystem, Finland). Data were acquired as relative light units (RLUs), and the area under the curve of CL versus assay time (total RLUs) was calculated. Data were presented as percent inhibition of ROS production calculated according to the formula, 1 – (total RLUs for cells infected with bacteria and stimulated with PMA/total RLUs for cells stimulated with PMA) × 100.