Short-chain fatty acids (SCFAs) were determined by gas chromatogr

Short-chain fatty acids (SCFAs) were determined by gas chromatography (GC-14B; Shimadzu, Kyoto, Japan). Succinate and d-/l-lactate were measured by commercial assay kits (Megazyme, Wicklow, Ireland). To monitor the growth of each bacterial strain in culture, copy number of 16S rRNA gene was quantified by real-time PCR. Repeated bead beating plus column method

(Yu & Morrison, PS-341 in vitro 2004) was employed for DNA extraction and purification from 1 mL of inocula and cultures at 48 and 96 h. PCR targeting the 16S rRNA gene was performed with a LightCycler 480 system (Roche Applied Science, Mannheim, Germany) and a KAPA SYBR FAST qPCR kit (KAPA Biosystems, Woburn, MA). Primer sets specific to each bacterial strain were used as follows: U2_Fw (5′-CTAGGTGTAGGGGGTATC-3′) and U2_Rv (5′-GCTGCCCTCTGTCGTTG-3′) for strain R-25 (Koike et al., 2010), 193f (5′-GGTATGGGATGAGCTTGC-3′) and 654r (5′-GCCTGCCCCTGAACTATC-3′) for F. succinogenes S85 (Tajima et al., 2001) and Sele.rumi_Fw (5′-TGCTAATACCGAATGTTG-3′) and Sele.rumi_Rv (5′-TCCTGCACTCAAGAAAGA-3′) for S. ruminantium S137 (Tajima et al., 2001). All other quantification procedures, including the standard plasmids, PCR conditions, and calculations, were according to Koike et al. (2007, 2010). To measure the fibrolytic activity in culture, fibrolytic find more enzyme assays were carried out for extracellular and intracellular fractions.

Culture supernatant and bacterial cells from strains R-25 and F. succinogenes S85 monocultures and their coculture were separated by centrifugation (16 000 g, 4 °C, 10 min). The supernatant was placed in dialysis tubing (12 000- to 14 000-Da cut-off, Seamless Cellulose Tubing, Sanko-junyaku, Tokyo,

Japan) in potassium phosphate buffer (50 mM, pH 6.8) overnight. The dialyzed fraction was condensed with polyethylene glycol (MW 20,000) and used in extracellular enzyme assays. Cell-free extract was obtained by ultrasonic disruption of the cell pellet (10 × 1 min on ice, 20 kHz, 25 watts) using a VC-70 O-methylated flavonoid Ultrasonic Processor (Sonics and Materials, Newton, CT) followed by centrifugation (16 000 g, 4 °C, 20 min) and was used in intracellular enzyme assay. The carboxymethylcellulase (CMCase) and xylanase activities were determined by monitoring the increase in reducing sugar formation from the substrates using dinitrosalicylic acid reagents, as described by Cotta (1988). Carboxymethylcellulose and oat spelt xylan were dissolved in 50 mM potassium phosphate buffer (pH 6.8) at 1% (w/v) and used as the substrates. The protein concentration was determined using Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA) with bovine gamma globulin as a standard. Enzyme activity was expressed as specific activity (formation of 1 nmol of sugar min−1 mg of protein−1) or total activity mL−1 culture (formation of 1 nmol of sugar min−1 mL−1 of original culture).

Mass spectra were acquired by a Finnigan™ LCQ™ DECA ion trap inst

Mass spectra were acquired by a Finnigan™ LCQ™ DECA ion trap instrument. An ionization device was used for sample analyses (sheath gas: 80 mL min−1, auxiliary gas: 20 mL min−1, spray voltage: 5 kV, capillary temperature: 300 °C, capillary voltage: 46 kV, and tube lens: −60 kV). The Xcalibur 2.0

SR2 software (copyright Thermo Electron Corporation 1998–2006) was used. Morphological and cultural studies of the most productive isolate containing the ts gene, SBU-16, including conidial morphology, the mechanism PI3K Inhibitor high throughput screening of conidia production, and growth characteristics on PDA, potato-carrot agar (PCA), and on the firm base of an alfalfa stem were carried out according to Simmons (2001). The isolate of SBU-16 was grown on the media in a culture chamber under check details a 10-h photoperiod provided by 56 W cool-white fluorescent lamps (Philips Master, Holand) at 22 °C. Anamorph and telomorph populations were examined at 4–5 days and 2–6 weeks, respectively. The size and morphology of 100 mature conidia and 50 conidiophores

in lactic acid were recorded by light microscopy at 100× magnification and photographed. A total of 25 isolates separated from the inner bark of T. baccata were screened for the presence of the ts gene. Based on the conserved region of the ts gene, the specific primers were designed and synthesised for the amplification of the core DNA fragment of ts from 25 isolated endophytic fungi. Following PCR amplification, a 334-bp product was obtained. Of 25 isolates, 4 (SBU-16, SBU-17, SBU-69 and SBU-71) showed PCR positive for the conserved sequence of the ts gene (Fig. 1). Taxol and 10-DAB III were extracted from culture filtrates and mycelia of the four ts PCR positive fungi and then analyzed selleck products by HPLC-DAD. Under the same analysis conditions, the samples containing chemical reference substances of 10-DAB III and taxol were also compared with fungal extracts (Fig. 2). Further convincing evidence for the identity of 10-DAB III and taxol was obtained by high-performance

liquid chromatography-mass spectrometry (LC-MS). Characteristically, standard 10-DAB III and taxol yielded both an [M + H]+ peak at a molecular weight of 854 and an [M + Na]+ peak at a molecular weight of 876, respectively (see Fig. 3a and b). By comparison, fungal taxol also produced peaks, [M + H]+ at m/z 854 and [M + Na]+ at m/z 876. The peaks corresponding to taxol exhibited mass-to-charge (m/z) ratios corresponding to the molecular ions (M + H)+ of standard taxol (at 854), confirming the presence of taxol in the fungal extracts. It was evident that taxol was much more complex because its molecular weight (from high-resolution mass spectrometry) was 854, which corresponds to a molecular formula of C47H51NO14 as reported earlier (McClure & Schram, 1992). The results of the quantification analysis among the four ts PCR positive isolates showed that SBU-16, which was isolated for the first time in our laboratory, produces taxol (6.9 ± 0.2 μg L−1) and its intermediate compound, 10-DAB III (2.

Mass spectra were acquired by a Finnigan™ LCQ™ DECA ion trap inst

Mass spectra were acquired by a Finnigan™ LCQ™ DECA ion trap instrument. An ionization device was used for sample analyses (sheath gas: 80 mL min−1, auxiliary gas: 20 mL min−1, spray voltage: 5 kV, capillary temperature: 300 °C, capillary voltage: 46 kV, and tube lens: −60 kV). The Xcalibur 2.0

SR2 software (copyright Thermo Electron Corporation 1998–2006) was used. Morphological and cultural studies of the most productive isolate containing the ts gene, SBU-16, including conidial morphology, the mechanism Pexidartinib clinical trial of conidia production, and growth characteristics on PDA, potato-carrot agar (PCA), and on the firm base of an alfalfa stem were carried out according to Simmons (2001). The isolate of SBU-16 was grown on the media in a culture chamber under CB-839 concentration a 10-h photoperiod provided by 56 W cool-white fluorescent lamps (Philips Master, Holand) at 22 °C. Anamorph and telomorph populations were examined at 4–5 days and 2–6 weeks, respectively. The size and morphology of 100 mature conidia and 50 conidiophores

in lactic acid were recorded by light microscopy at 100× magnification and photographed. A total of 25 isolates separated from the inner bark of T. baccata were screened for the presence of the ts gene. Based on the conserved region of the ts gene, the specific primers were designed and synthesised for the amplification of the core DNA fragment of ts from 25 isolated endophytic fungi. Following PCR amplification, a 334-bp product was obtained. Of 25 isolates, 4 (SBU-16, SBU-17, SBU-69 and SBU-71) showed PCR positive for the conserved sequence of the ts gene (Fig. 1). Taxol and 10-DAB III were extracted from culture filtrates and mycelia of the four ts PCR positive fungi and then analyzed ADAMTS5 by HPLC-DAD. Under the same analysis conditions, the samples containing chemical reference substances of 10-DAB III and taxol were also compared with fungal extracts (Fig. 2). Further convincing evidence for the identity of 10-DAB III and taxol was obtained by high-performance

liquid chromatography-mass spectrometry (LC-MS). Characteristically, standard 10-DAB III and taxol yielded both an [M + H]+ peak at a molecular weight of 854 and an [M + Na]+ peak at a molecular weight of 876, respectively (see Fig. 3a and b). By comparison, fungal taxol also produced peaks, [M + H]+ at m/z 854 and [M + Na]+ at m/z 876. The peaks corresponding to taxol exhibited mass-to-charge (m/z) ratios corresponding to the molecular ions (M + H)+ of standard taxol (at 854), confirming the presence of taxol in the fungal extracts. It was evident that taxol was much more complex because its molecular weight (from high-resolution mass spectrometry) was 854, which corresponds to a molecular formula of C47H51NO14 as reported earlier (McClure & Schram, 1992). The results of the quantification analysis among the four ts PCR positive isolates showed that SBU-16, which was isolated for the first time in our laboratory, produces taxol (6.9 ± 0.2 μg L−1) and its intermediate compound, 10-DAB III (2.

These effects occur whether the neuron is excited or inhibited by

These effects occur whether the neuron is excited or inhibited by Sp5 stimulation alone. Our results demonstrate that multisensory Carfilzomib mw integration in DCN alters spike-timing representations of acoustic stimuli in pyramidal cells. These changes likely occur through synaptic modulation of intrinsic excitability or synaptic inhibition. “
“Extended periods of deafness have profound effects on central auditory system function and organization. Neonatal deafening results in loss of the normal cochleotopic organization of the primary

auditory cortex (AI), but environmentally-derived intracochlear electrical stimulation, via a cochlear implant, initiated shortly after deafening, can prevent this loss. We investigated whether such stimulation initiated after an extended period of deafness selleck can restore cochleotopy. In two groups of neonatally-deafened cats, a multi-channel intracochlear electrode array was implanted at 8 weeks of age. One group received only minimal stimulation, associated with brief recordings at 4–6-week intervals, over the following 6 months to check the efficacy of the implant. In the other group, this 6-month period was followed by 6 months of near-continuous

intracochlear electrical stimulation from a modified clinical cochlear implant system. We recorded multi-unit clusters in the auditory cortex and used two different methods to define the region of interest in the putative AI. There was no evidence of cochleotopy in any of the minimally stimulated animals, confirming our earlier finding. In three of six chronically Adenosine stimulated cats

there was clear evidence of AI cochleotopy, and in a fourth cat in which the majority of penetrations were in the anterior auditory field there was clear evidence of cochleotopy in that field. The finding that chronic intracochlear electrical stimulation after an extended period of deafness is able to restore cochleotopy in some (but not all) cases has implications for the performance of patients implanted after an extended period of deafness. “
“The basal ganglia have a local renin–angiotensin system and it has been shown that the loss of dopaminergic neurons induced by neurotoxins is amplified by local angiotensin II (AII) via angiotensin type 1 receptors (AT1) and nicotinamide adenine dinucleotide phosphate (NADPH) complex activation. Recent studies have revealed a high degree of counter-regulatory interactions between dopamine and AII receptors in non-neural cells such as renal proximal tubule cells. However, it is not known if this occurs in the basal ganglia.

Hybridization and washing procedures were carried out as describe

Hybridization and washing procedures were carried out as described previously (Tobino et al., 2011). Chemiluminescent detection was performed using an antidigoxigenin antibody conjugated with alkaline phosphatase and CSPD (both Roche) according to the instruction manual (DIG Application Manual for Filter Hybridization, Roche), and the signal was GSK126 mouse recorded by LAS-4000 mini (Fujifilm, Tokyo, Japan) using a 10 min exposure. Signals were background corrected and considered positive when the signal to noise ratio was > 3 in all the replicated

spots. Partial sequences from both ends (60–700 bp) of each probe were read using SP6 and T7 primers as described previously (Tobino et al., 2011). The full probe sequence was defined as the segment that was on and within both end sequences in the genome, found using the blastn tool from the National Center for Biotechnology Information (NCBI). INK 128 concentration The full probe sequences were then searched against the target genome sequences using

blastn in NCBI under the default settings. The match that had the least e-value was selected as the representative similarity pair between the probe and the target genome. To eliminate short alignments and anomalous high signals, caused by the high gene copy number, those pairs that had < 500 bp alignment or significant multiple hits were rejected in the subsequent analysis. Specific responses were observed from probes corresponding to the target genome at all hybridization temperatures tested (Fig. S2). Visible signals were also found from some probes whose origins were different from the target genome, Phosphoprotein phosphatase indicating the occurrence

of cross-hybridization (i.e. false positives). As shown in Table 1, the level of false positive signals was 64.7% (216 of 334) at 55 °C but decreased steadily to 22.5% (75 of 334) at 70 °C and was almost completely absent (1.5%; 5 of 334) at 75 °C. In contrast, very few probes (0.6%; 1 of 167) corresponding to the target genome fell in negative and were only found at hybridization temperatures above 70 °C. These results suggest that randomly generated genomic fragments (~ 2000 bp) can function as specific probes to discriminate species in the genus Pseudomonas under highly stringent conditions. Sequence similarity searches between the fragment probes and target genomes produced a total of 496 similarity pairs (Fig. S3). With the exception of probes that originated from the target genome (resulting in 100% similarity), most of the pairs had < 90% similarity, while only two pairs sharing a partial sequence of rrn operon were found to have > 90% similarity of > 500 bp.

We used a freely available algorithm to perform spectral rotation

We used a freely available algorithm to perform spectral rotation on the musical stimuli (http://www.fil.ion.ucl.ac.uk/~jcrinion/rotation/blesser3.m). This method has been described in previous works (Blesser, 1972; Scott et al., 2000; Warren et al., 2006; Abrams et al., 2012). The center frequency for spectral rotation was 5512 Hz. This center frequency was chosen so that the rotated frequencies would be within the frequency response range of the fMRI-compatible headphones (20–10 000 Hz). Phase-scrambling was performed by applying

a Fourier transform to each of the four symphonies that constitute the Natural Music stimulus and then randomizing its Ibrutinib solubility dmso phase response by adding a random phase shift at every frequency

(Prichard & Theiler, 1994). The phase shifts were obtained by randomly sampling in the interval (0, 2π). This process preserves the power spectrum of each of the four symphonies. Note that, by design, the Phase-Scrambled control stimulus preserves spectral density but not time-dependent fluctuations. We preferred this design as it facilitates a simple and interpretable result: brain structures that show greater ISS for Natural Music compared with the Phase-Scrambled condition are sensitive to the temporal structure of music. Our design therefore forms a necessary starting point for future investigations of more complex time-dependent attributes of musical structure that lead to synchronized responses among subjects, perhaps using a wavelet transform that preserves

both the Nutlin-3a clinical trial spectral density and the time-dependent fluctuations in that density. Brain images were acquired on a 3T GE Signa scanner using a standard GE whole head coil (software Lx 8.3). For the Natural Music, Spectrally-Rotated and Phase-Scrambled conditions, images were acquired every 2 s in two runs that lasted 9 min 42 s. The sequence of these stimulus conditions was consistent across listeners: the Natural Music condition was presented first, the Phase-Scrambled condition CYTH4 was presented second and the Spectrally-Rotated condition was presented third. While it would have been preferable to have randomized the stimulus presentation order across subjects to control for attention and fatigue, we do not believe that this had a significant effect on the results given that there was vastly greater ISS for the final stimulus condition (Spectral-Rotation) relative to the penultimate stimulus condition (Phase-Scrambled), which would not have occurred had fatigue and attention negatively affected ISS results. Subjects were instructed to attend to all the music and music-like stimuli. To allow for a natural listening experience, we did not provide any additional instructions to the subjects. A custom-built head holder was used to prevent head movement. Twenty-eight axial slices (4.0 mm thick, 0.

, 2008), as assessed using a checklist completed prior to testing

, 2008), as assessed using a checklist completed prior to testing. The protocol was in accordance with the Declaration of Helsinki and was approved by the Human Research Ethics Committee at The University of Western Australia. All subjects provided informed written consent prior to testing. No subjects reported adverse effects to the tDCS procedure, other than the reddening of skin under the

electrode, and none withdrew from the study. All testing took place in a sound-attenuated room. The acoustic stimuli were generated NVP-LDE225 purchase with a Creative SoundBlaster Live! Soundcard in Experiment 1 and with an ASUS Xonar Essence ST soundcard in Experiments 2A and 2B. Stimuli were presented monotically to the left ear by Sennheiser 280 Pro headphones. The same Rucaparib in vivo procedure was used for all reported experiments, with anodal tDCS being delivered by a constant-current battery-driven stimulator (Dupel Iontophoresis System, MN) through two 6 × 4 cm electrodes in saline-soaked pouches placed on the scalp. The anode was placed 1 cm inferior to the midpoint of C4 and T4 in the International 10-20 system, corresponding to the right auditory cortex (Mathys et al., 2010) and the cathode was placed on the contralateral supraorbital region. This electrode montage has been shown to increase excitability in auditory

cortex (Zaehle et al., 2011). Right auditory cortex was stimulated as frequency discrimination appears to be at least partially lateralized to this hemisphere (Lauter et al., 1985; Hyde et al., 2008). For anodal stimulation, the current was ramped up to 1 mA over 30 s, maintained at this level for 20 min, and then ramped off over 30 s. For sham stimulation, the current

was ramped up to 1 mA over 30 s and immediately ramped off over 30 s. There is no ongoing sensation of stimulation after the initial ramp-up period so that sham stimulation produces the sensation of stimulation without inducing changes Protirelin in cortical excitability (Ladeira et al., 2011; Kessler et al., 2012), making subjects blind to the stimulation condition. Subjects began the psychophysical procedures 30–60 s after stimulation had commenced. We trained Naïve subjects for 2 days on a frequency discrimination task. To assess the effects of tDCS stimulation on rapid learning, we applied either anodal or sham tDCS stimulation during the first day of testing. The psychophysical procedure was repeated on the second day without tDCS to assess the effects of stimulation on retention of learning from the first day. The task followed that used by Hawkey et al. (2004) as they showed that the rapid decreases in frequency difference limens (DLFs) with training were genuine perceptual learning. A baseline measure could not be taken because this would prevent examination of rapid auditory learning that occurs during the early trials.

Fraction D3 was then dialyzed against 10 mM NH4OAc buffer (pH 51

Fraction D3 was then dialyzed against 10 mM NH4OAc buffer (pH 5.1) before chromatography on a 2.5 × 20 cm column of carboxymethyl (CM)-cellulose (Sigma) in 10 mM NH4OAc buffer (pH 5.1). After elution of unadsorbed proteins, the adsorbed proteins were eluted successively

using 10 mM NH4OAc buffer (pH 5.1) containing 50, 150 and 1000 mM NaCl. Fraction C3 eluted with 150 mM NaCl was dialyzed against 10 mM phosphate buffer (pH 7) before chromatography on a 1 × 15 cm column of Q-Sepharose (GE Healthcare) in 10 mM phosphate buffer (pH 7). After removal of unadsorbed proteins (fraction Q1), adsorbed proteins were desorbed with a 0–0.4 M NaCl gradient in 10 mM phosphate buffer (pH 6). The first adsorbed fraction (Q2) was then subjected to BIBW2992 molecular weight gel filtration on a Superdex 75 HR 10/30 column (GE Healthcare) in 0.2 M NH4HCO3 buffer (pH 8.5) using an AKTA Purifier (GE Healthcare). The second fraction (SU2) with a molecular mass of 29 kDa constituted purified hemolysin, which was designated as schizolysin. The assay was carried out as follows: to 0.1 mL of a 2% suspension of rabbit erythrocytes were added 250 μL 0.15 M NaCl and 50 μL test sample. After incubation in a water bath at 37 °C for 15 min, the mixture was centrifuged at 900 g for 5 min. The A540 nm was

then read. One hundred percent hemolysis was defined as OD540 nm of hemoglobin released from erythrocytes treated with 0.1% Triton X-100. One hemolysin unit (HU) was defined as the amount of hemolysin eliciting 50% hemoglobin release (Ngai & Ng, 2006). Schizolysin was subjected C59 wnt chemical structure to sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) (Laemmli & Favre, 1973) and gel filtration on a calibrated fast protein liquid chromatography (FPLC)-Superdex 75 HR 10/30 column (GE Healthcare) to determine its molecular mass. Its N-terminal sequence was determined by Edman degradation using a Hewlett-Packard amino acid sequencer. The sequence similarity analysis was performed using blast software against the NCBI protein database. The hemolysis inhibition tests to investigate inhibition of schizolysin-induced hemolysis by various carbohydrates were conducted in a similar manner to the hemolysis test. The results would indicate whether schizolysin interacts with any carbohydrate(s) on the erythrocyte membrane to exert its hemolytic action. A 20-μL aliquot of a water-soluble stock solution Dichloromethane dehalogenase of different carbohydrates (400 mM) was added to 250 μL of 0.15 M NaCl and 25 μL of schizolysin with 16 HU. The mixture was allowed to stand for 30 min at room temperature and then mixed with 100 μL of a 2% rabbit erythrocyte suspension. After incubation in water bath at 37 °C for 15 min, the remaining activity was detected. To investigate inhibition of schizolysin-induced hemolysis by various metal chlorides, the stock solutions of different metal chlorides were individually mixed with hemolysin solution and 250 μL of 0.15 M NaCl to achieve a final metal ion concentration of 5 and 10 mM, respectively.

(2001) 3xFLAG epitope tails were added to the ends of the sopA,

(2001). 3xFLAG epitope tails were added to the ends of the sopA, sopB and sopD gene. The 3xFLAG epitope is a sequence of three tandem FLAG epitopes (22 aa). A pair of primers was designed to amplify a 3xFLAG and http://www.selleckchem.com/products/obeticholic-acid.html kanR coding sequence using plasmid pSUB11 (Uzzau et al., 2001). The 3′-ends of these oligonucleotides were complementary to the first 20 nt of the pSUB11 3xFLAG coding region (GACTACAAAGACCATGACGG, forward primers) and to the 20 nt of the pSUB11 priming site 2 (CATATGAATATCCTCCTTAG, reverse primers). The 5′-ends

of the oligonucleotides were designed to be homologous to the last 40 nt of each tagged gene, not including the stop codon (forward primers), and to the 40 nt immediately downstream of the gene stop codon (reverse primers). For in vitro studies, bacteria were grown under different culture conditions. To mimic the intestinal environment (Miki et al., 2004) bacteria were grown overnight at 37 °C without aeration in a Luria–Bertani (LB) broth containing 0.3 M NaCl. An intracellular milieu was recreated by growing bacteria overnight in MgM minimal medium containing 0.1%

casaminoacids at 37 °C MS-275 with aeration (Miki et al., 2004) at different pH. Early and late intracellular conditions were mimicked by growing bacteria at pH 6 or 4.5, respectively. sopD∷3xFLAG cat∷FLAG strain was used as control for in vitro experiments; SopD is a dual effector because it is translocated into

host cells by both TTSSs (Brumell et al., 2003). For in vivo studies, bacterial inocula used to infect cells or animals were prepared by growing the tagged strains overnight under SPI-1 noninducing Phosphatidylinositol diacylglycerol-lyase conditions (LB at 28 °C) as described previously (Giacomodonato et al., 2009). In this way, the residual expression of SopB from in vitro bacterial growth was ruled out. Cultures were centrifuged, diluted in sterile saline and inoculated to cell cultures or mice. Viable bacteria in the inoculum were quantified by dilution and plating onto LB agar plates with appropriate antibiotics. For the isolation of cell-associated proteins, 1.5 mL of bacterial cultures were centrifuged and resuspended in 100 μL of H2O and immediately mixed with 100 μL of Laemmli buffer. For the isolation of proteins released into the culture supernatants (secreted proteins), bacteria were pelleted by centrifugation and 2 mL of supernatant was collected from each sample. Supernatants were then filtered (0.45 μm pore size), and the proteins were precipitated with 25% trichloroacetic acid and sedimented by high-speed centrifugation (14 000 g for 30 min). The pellet was washed in cold acetone and resuspended in phosphate-buffered saline (PBS) and Laemmli buffer. Four independent extractions for each sample were added together to minimize differences in protein recovery from sample to sample.

(2001) 3xFLAG epitope tails were added to the ends of the sopA,

(2001). 3xFLAG epitope tails were added to the ends of the sopA, sopB and sopD gene. The 3xFLAG epitope is a sequence of three tandem FLAG epitopes (22 aa). A pair of primers was designed to amplify a 3xFLAG and find more kanR coding sequence using plasmid pSUB11 (Uzzau et al., 2001). The 3′-ends of these oligonucleotides were complementary to the first 20 nt of the pSUB11 3xFLAG coding region (GACTACAAAGACCATGACGG, forward primers) and to the 20 nt of the pSUB11 priming site 2 (CATATGAATATCCTCCTTAG, reverse primers). The 5′-ends

of the oligonucleotides were designed to be homologous to the last 40 nt of each tagged gene, not including the stop codon (forward primers), and to the 40 nt immediately downstream of the gene stop codon (reverse primers). For in vitro studies, bacteria were grown under different culture conditions. To mimic the intestinal environment (Miki et al., 2004) bacteria were grown overnight at 37 °C without aeration in a Luria–Bertani (LB) broth containing 0.3 M NaCl. An intracellular milieu was recreated by growing bacteria overnight in MgM minimal medium containing 0.1%

casaminoacids at 37 °C www.selleckchem.com/products/Roscovitine.html with aeration (Miki et al., 2004) at different pH. Early and late intracellular conditions were mimicked by growing bacteria at pH 6 or 4.5, respectively. sopD∷3xFLAG cat∷FLAG strain was used as control for in vitro experiments; SopD is a dual effector because it is translocated into

host cells by both TTSSs (Brumell et al., 2003). For in vivo studies, bacterial inocula used to infect cells or animals were prepared by growing the tagged strains overnight under SPI-1 noninducing MYO10 conditions (LB at 28 °C) as described previously (Giacomodonato et al., 2009). In this way, the residual expression of SopB from in vitro bacterial growth was ruled out. Cultures were centrifuged, diluted in sterile saline and inoculated to cell cultures or mice. Viable bacteria in the inoculum were quantified by dilution and plating onto LB agar plates with appropriate antibiotics. For the isolation of cell-associated proteins, 1.5 mL of bacterial cultures were centrifuged and resuspended in 100 μL of H2O and immediately mixed with 100 μL of Laemmli buffer. For the isolation of proteins released into the culture supernatants (secreted proteins), bacteria were pelleted by centrifugation and 2 mL of supernatant was collected from each sample. Supernatants were then filtered (0.45 μm pore size), and the proteins were precipitated with 25% trichloroacetic acid and sedimented by high-speed centrifugation (14 000 g for 30 min). The pellet was washed in cold acetone and resuspended in phosphate-buffered saline (PBS) and Laemmli buffer. Four independent extractions for each sample were added together to minimize differences in protein recovery from sample to sample.