Special thanks to Dr. Andrea Savarino for his kind assistance in photographing the biofilm, and for his invaluable suggestions for our future project. Thanks Dr. G. Mandarino and Dr. Anna Marella for their help in manuscript preparation and to Prof. Antonio Cassone for critical reading of the manuscript and suggestions. We also wish to thank Maurice Di Santolo for the English revision of the manuscript. Electronic supplementary material Additional file 1: Figure S1: Biofilm analysis of the mp65Δ mutant in Spider

medium. Cells of the wild type (wt), mp65Δ mutant (hom) and revertant (rev) strains were visualized before (Panel 1) and after (Panel 2) staining and then captured by using Gel Doc system (Bio-Rad). (PDF 3 MB) References 1. Cassone A: Fungal vaccines: real progress from real challenges. Lancet Infect Dis 2008, 8:114–124.PubMedCrossRef 2. Angiolella L, Stringaro AR, Tipifarnib datasheet De Bernardis F, Posteraro B, Bonito M, Toccacieli L, Torosantucci A, Colone M, Sanguinetti M, Cassone A, Palamara AT: Increase of virulence and its phenotypic traits in drug-resistant strains of Candida albicans . Antimicrob Agents Chemother 2008, 52:927–936.PubMedCrossRef 3. Morgunova E, Saller S, Haase I, Cushman M, Bacher A, Fischer M, Ladenstein R: Lumazine synthase from Candida albicans as an anti-fungal

target enzyme: structural and biochemical basis for drug design. J Biol Chem 2007, 282:17231–17241.PubMedCrossRef 4. Ram AF, Klis FM: Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and

selleck chemical Congo red. Nat Protoc 2006, 1:2253–2256.PubMedCrossRef 5. Norice CT, Smith FJ Jr, Solis N, Filler SG, Mitchell AP: Requirement for Candida albicans Sun41 in biofilm formation and virulence. Eukaryot Cell 2007, 6:2046–2055.PubMedCrossRef 6. Torosantucci A, Chiani P, Bromuro C, De Bernardis F, Palma AS, Liu Y, Mignogna G, Maras B, Colone M, Stringaro A, Zamboni S, Feizi T, Cassone A: Protection by anti-beta-glucan antibodies is associated with restricted Olopatadine beta-1,3 glucan binding specificity and inhibition of fungal growth and adherence. PLoS One 2009, 4:e5392.PubMedCrossRef 7. Brown JA, Catley BJ: Monitoring polysaccharide synthesis in Candida albicans . Carbohydr Res 1992, 227:195–202.CrossRef 8. de Groot PW, de Boer AD, Cunningham J, Dekker HL, de Jong L, Hellingwerf KJ, de Koster C, Klis FM: Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins. Eukaryot Cell 2004, 3:955–965.PubMedCrossRef 9. de Groot PW, Ram AF, Klis FM: Features and functions of covalently linked proteins in fungal cell walls. Fungal Genet Biol 2005, 42:657–675.PubMedCrossRef 10. Ecker M, Deutzmann R, Lehle L, Mrsa V, Tanner W: Pir proteins of Saccharomyces cerevisiae are attached to beta-1,3-glucan by a new protein-carbohydrate linkage.

The

book can be used as a reference work both for medical advice beyond occupational dermatoses and for an adequate professional dialogue with colleagues in the field of dermatology.”
“Introduction Hairdressers often complain of work-related airway symptoms. They are exposed to several irritating and sensitizing agents, but they often relate their symptoms to bleaching powder (Albin et al. 2002; Brisman et al. 2003). Persulphates found in bleaching powder have often been blamed because they are irritating and sensitizing agents causing both rhinitis and asthmatic symptoms. Specific challenge to persulphate has been suggested as an useful tool in diagnosis of occupational asthma in hairdressers (Muñoz et al. 2004). However, specific IgE antibodies against persulphates are seldom found (Parra et al. 1992) and another immunologic mechanism not yet elucidated has been suggested (Moscato see more et al. 2005; Muñoz et al. 2004). Furthermore, the clinical picture is quite complex as hairdressers reacting to bleaching powder very often complain of symptoms associated with exposure to other hairdressers chemicals. In a previous study, we found that hairdressers with

nasal symptoms from bleaching powder reacted to a nasal challenge with potassium persulphate in the same way as atopics without earlier exposure to bleaching powder (Kronholm U0126 nmr Diab et al. 2009). Phosphoprotein phosphatase This reaction was associated with a Th1 cell activation, which may be a part of the process of hyper reactivity from low irritant exposure (Banauch et al. 2005; Van Loveren et al. 1996). In an earlier study (Kronholm Diab 2002), hairdressers claimed that their work-related symptoms increased during periods of exposure and also that they became more sensitive to other stimuli as well, indicating an increasing reactivity in the nasal mucosa. They felt that the reactivity decreased considerably during time away from work. For this reason, frequent periods without

exposure were necessary for the hairdressers to be able to continue work. Health-related quality of life (HRQoL) has been introduced late in occupational medical research compared to care health research in general. HRQoL and working life are linked and must be of concern to occupational health researchers (Blanc 2004). Data indicate that allergic rhinitis may have an important impact on productivity because of symptoms as tiredness, poor concentration and headache (Blanc et al. 2001). The mechanisms of hairdressers’ nasal symptoms and the consequences for their HRQoL are not clear. This is problematic when hairdressers ask for medical advice concerning continued work as a hairdresser. To clarify this issue, further research about the symptom mechanism and the influence of the symptoms on HRQoL during exposure periods is of great need.

5.0.39 Software. The 2nd derivate method was used for all amplicons to determine Cp values. The standard curve method was used for relative gene expression quantification, and the transcript accumulation of each gene was normalized to 16S rRNA. The amplification efficiency and linear range of amplification were followed for each amplicon on each plate by analyzing a reference sample pool in four dilution steps of cDNA with two replicate wells per dilution step. Each sample was analyzed in two dilutions and two replicates per dilution step. Only samples where the ΔCp between two dilutions of target gene did not deviate

by more than 0.5 from ΔCp of the reference gene were used for relative quantification. The fold changes for each selleck screening library experimental point were calculated as a quotient of average transcript abundances between treated and control samples from three independent biological replicates in each time point. Microarray dataset accession number Microarray data analyzed in this study have been deposited in the Gene Expression Omnibus database with accession

number GSE15394. Acknowledgements The authors would like to acknowledge Dr Ron Peterson (Novartis Institutes for BioMedical Research) for help Tamoxifen with microarray hybridizations and Dr Roger Pain for language revision. The work was supported by Slovenian Research Agency (Grant Nos. P4-0165 and Z4-9697), the European Union FP6 Integrated Project EUR-INTAFAR (Project No. LSHM-CT-2004-512138) under the thematic priority Life Sciences, Genomics and Biotechnology for Health and Lek Pharmaceuticals d.d. Electronic supplementary material Additional file 1: Summary table for differentially expressed genes. Excel spreadsheet file

summarizing the transcriptional data from our study and publicly available transcriptional profiling results Aldehyde dehydrogenase from SAMMD. (XLS 3 MB) Additional file 2: Pathway Studio metabolic network. File containing the representation of S. aureus metabolic network (gpc format). The file can be viewed by Pathway Studio software http://​www.​ariadnegenomics.​com/​products/​pathway-studio/​. (GPC 19 MB) Additional file 3: Gene sets used for GSEA. Excel spreadsheet file containing gene sets generated from TIGRFAM ontology that were used to run GSEA. (XLS 90 KB) References 1. El Zoeiby A, Sanschagrin F, Levesque RC: Structure and function of the Mur enzymes: development of novel inhibitors. Mol Microbiol 2003,47(1):1–12.PubMedCrossRef 2. Freiberg C, Brotz-Oesterhelt H, Labischinski H: The impact of transcriptome and proteome analyses on antibiotic drug discovery. Curr Opin Microbiol 2004,7(5):451–459.PubMedCrossRef 3. Nagarajan V, Elasri MO: SAMMD: Staphylococcus aureus microarray meta-database. BMC Genomics 2007, 8:351.PubMedCrossRef 4. Becker SA, Palsson BO: Genome-scale reconstruction of the metabolic network in Staphylococcus aureus N315: an initial draft to the two-dimensional annotation. BMC Microbiol 2005,5(1):8.PubMedCrossRef 5.

6 V, and the rectifying ratio is 24 at a voltage of 3 V. The revealed p-n junction-like I-V characteristic also demonstrates the successful integration of Sb in the ZnO microrod array. Figure 7 shows Wnt inhibitor the measured photocurrent at various biases. At a reverse bias of -3 V, the reverse currents are 990 and 25 μA with and without the illumination of ultraviolet light, respectively. A nearly 40-fold current gain was demonstrated

on this device. Figure 6 I – V measurement of the ZnO homojunction device. The inset shows characterization of the ohmic contacts for the ZnO homojunction device. Figure 7 Photocurrent measurement of the ZnO homojunction device. Finally, the photoresponsivity of the ZnO homojunction device is shown in Figure 8. At a wavelength

shorter than 380 nm, the ZnO homojunction device behaves DAPT nmr like a photodetector when a negative voltage between -1 and -3 V was applied. The responsivity of the ZnO p-n diode increases when more negative voltage was applied. Our results therefore suggest that the ZnO homojunction device has an application in photodetectors in the ultraviolet region [23, 24]. Figure 8 Photoresponsivity as a function of wavelength of the incident light at different reverse biases. Conclusions In this work, a high-quality Sb-doped ZnO microrod array was synthesized by electrodeposition. In Sb-doped ZnO, the shift of the XRD peak from that of the intrinsic ZnO was attributed to the increase of the lattice constant due to mafosfamide the replacement of a Zn atom by the Sb atom. In the case of the Sb-doped ZnO microrod array, the PL measurement indicated an acceptor-related photoemission. Strong violet luminescence at room temperature was observed since the Sb dopants would substitute Zn sites, instead of O sites, (SbZn) to form a complex with two VZn, which is the

SbZn-2VZn complex. This SbZn-2VZn complex has lower formation energy and acts as a shallow acceptor, which can induce a strong violet luminescence. In the I-V measurement, the diode-like behavior of the ZnO homojunction device indicated the successful integration of antimony atoms by electrodeposition. The nearly 40-fold current gain of the photoresponsivity of the ZnO homojunction device, acting like a p-n diode, indicates a potential application in photodetectors operating at the ultraviolet wavelength region. Acknowledgements This work was funded by the NSC, Taiwan (grant no. NSC 100-2112-M-002-017-MY3). References 1. Chu S, Lim JH, Mandalapu LJ, Yang Z, Li JL: Sb-doped p-ZnO/Ga-doped n-ZnO homojunction ultraviolet light emitting diodes. Appl Phys Lett 2008, 92:152103.CrossRef 2. Mandalapu LJ, Yang Z, Xiu FX, Zhao DT, Liu JL: Homojunction photodiodes based on Sb-doped p-type ZnO for ultraviolet detection. Appl Phys Lett 2006, 88:092103.CrossRef 3.

2004; Bloom and Chatterji 2009; Chowdhury and Santos 2010; Dees 2009; Smith and Stevens 2010). The latter define upscaling as increasing the impact produced by a social-purpose organization to better match the magnitude of the social need or problem it seeks to address. They distinguish upscaling and deep Opaganib order scaling. Upscaling refers to the growth in social value by expanding a current program to other geographic locations. This involves effort and costs in terms of building infrastructure, organizing and developing an ecosystem, obtaining licenses, and educating customers in a new region. Deep scaling refers to focusing energies and resources on achieving greater impact in the

same location where the enterprise was started by engaging in activities like improving the quality of services, achieving greater penetration of the target population, RAD001 price finding new ways to serve people, extending services to new people, and developing innovative financial management approaches. Karamchandani

et al. (2009) and Klein (2008) have a somewhat different view. They refer to upscaling as the capacity of the enterprise to expand quickly, effectively, and efficiently. Upscaling can also mean expanding the capacity of the existing business, in the sense of developing resources, building a knowledge base, employing people, developing management systems, and even developing a culture. According to them, upscaling, thus, includes serving more people with the same product within the same region, as well as extending into new markets, i.e., different geographies. In a given situation, the meaning

of upscaling, to a large extent, depends on the motivation of the entrepreneur. Some enterprises may focus on developing a specific region in terms of new products and services before scaling geographically, while others may choose to scale into new geographies before venturing into new products and services. According to Dees et al. (2004), choosing the right path towards broader social impact is a complex matter, since it involves judgment, experimentation, and continuous learning. They develop an approach towards upscaling based on following five Rs, i.e., Readiness, Resources, Receptivity, Risk, and Return. Bloom and Chatterji (2009) ADP ribosylation factor suggest the SCALERS model, i.e., Staffing, Communicating, Alliance-building, Lobbying, Earnings-generation, Replicating, and Stimulating market forces. Chowdhury and Santos (2010) suggest that successful upscaling can be achieved by disseminating information through the use of best-practice blueprints or intermediaries such as multilateral organizations and consulting firms. Since our study is set in an emerging economy with deep-rooted social inequality and poverty in addition to environmental problems, it is pertinent to also examine the literature about development projects, program, and non-governmental organizations (NGOs) for possibly useful insights about upscaling.

Methyl (+/−)-2-(2-benzyl-2-amino-2-oxo-1-phenylethylamino)-acetate

rac -2f From rac -1f (0.59 g, 1.60 mmol) and BF3·2CH3COOH (5 mL); FC (gradient: PE/AcOEt 4:1–1:2): yield 0.40 g (80 %) of rac -2f. White powder; mp 147–149 °C; TLC (AcOEt): R f = 0.63; IR (KBr): 700, 741, 1204, 1454, 1558, 1682, 1734, 2844, 2951, 3030, 3182, 3418; 1H NMR this website (CDCl3, 500 MHz): δ 3.07 (d, 2 J = 17.5, 1H, PhCH 2), 3.40 (d, 2 J = 17.5, 1H, Ph\( \rm CH_2^’ \)), 3.61 (s, 3H,

OCH 3), 3.66 (d, 2 J = 13.5, 1H, CH 2), 3.85 (d, 2 J = 13.5, 1H, \( \rm CH_2^’ \)), 4.75 (s, 1H, H-1), 5.85 (bs, 1H, CONH), 7.26–7.42 www.selleckchem.com/screening/mapk-library.html (m, 10H, H–Ar), 7.63 (bs, 1H, CONH′); 13C NMR (CDCl3, 125 MHz): δ 51.7 (OCH3), 51.8 (PhCH2), 56.8 (CH 2), 69.9 (C-1), 127.7, 128.4 (C-4′, C-4″), 128.64, 128.65 (C-2′, C-6′, C-2″, C-6″), 129.0, 129.6 (C-3′, C-5′, C-3″, C-5″), 134.7, 137.5 (C-1′, C-1″), 172.3 (CONH), 174.4 (COOCH3); HRMS (ESI+) calcd for C18H20N2O3Na: 335.1360 (M+Na)+ found 335.1372. Synthesis of compounds 3 by base-induced intramolecular cyclocondensation To a stirred solution of appropriate amidoester 2 in absolute EtOH (5 mL/1 mmol of amidoester), sodium hydroxide (1 equiv.) was added at room temperature. After dissolution Avelestat (AZD9668) of the hydroxide, the mixture was quenched with saturated aqueous solution of ammonium chloride (100 mL). The resulting cloudy solution was extracted with CH2Cl2 (3 × 30 mL). The combined organic phase was washed with water (20 mL), dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was purified by FC. (3S,5R)- and (3S,5S)-3-isopropyl-5-phenylpiperazine-2,6-dione

(3 S ,5 S )-3a and (3 S ,5 R )-3a From (2 S ,1 S )-2a (1.86 g, 7.04 mmol) and NaOH (0.28 g, 1 equiv.); FC (gradient: PE/EtOAc 6:1–1:1): yield 1.34 g (82 %): 0.72 g (44 %) of (3 S ,5 S )-3a, 0.32 g (19 %) of (3 S ,5 R )-3a and 0.30 g (19 %) of diastereomeric mixture. (3 S ,5 S )-3a: white powder; mp 103–105 °C; [α]D = −152.1 (c 1, CHCl3); IR (KBr): 756, 1030, 1099, 1180, 1234, 1331, 1454, 1497, 1701, 2932, 1963, 3225; TLC (PE/AcOEt 3:1): R f = 0.35; 1H NMR (CDCl3, 500 MHz): δ 0.99 (d, 3 J = 7.0, 3H, CH 3), 1.09 (d, 3 J = 7.0, 3H, \( \rm CH_3^’ \)), 2.18 (bs, 1H, NH), 2.49 (2 sp, 3 J 1 = 6.5, 3 J 2 = 5.0, 1H, CH), 3.26 (d, 3 J = 4.5, 1H, H-3), 4.90 (s, 1H, H-5), 7.32–7.46 (m, 5H, H–Ar), 8.34 (bs, 1H, CONHCO); 13C NMR (CDCl3, 125 MHz): δ 17.1 (CH 3), 19.3 (CH 3), 27.7 (CH), 58.7 (C-3), 59.8 (C-5), 127.1 (C-2′, C-6′), 128.5 (C-4′), 129.0 (C-3′, C-5′), 134.6 (C-1′), 172.3 (C-6), 173.

From the experiment of incubation time, it is deduced that to discriminate with accuracy the susceptible strains from the rest it is enough, in a practical clinical approach, to assess the control 0 dose and the CLSI cut-off dose for susceptibility, incubating with the antibiotic for 60 min in case of cultures growing 24 h in agar plate, as usual in the standard clinical microbiology laboratory. If the cultures were exponentially growing in liquid medium, the incubation time with the antibiotic may be decreased for 30 min. We have observed that the greater the ageing of

the culture in agar plate, or when the culture is achieving the stationary phase of growth, Selleckchem SCH727965 the longer the incubation time necessary to observe the effect of the antibiotic, even several hours. To evaluate clinical strains using the technique to assess

the integrity of the cell wall, it is mandatory to simultaneously process a sensitive, an intermediate and a resistant strain as controls of the activity of the antibiotic and the efficacy of the technique. Sensitive strains from gram-negative bacteria assayed showed a background www.selleckchem.com/products/obeticholic-acid.html of extracellular microgranular-fibrilar material, its concentration being dose and time dependent. This material corresponded to DNA fragments released by the bacteria, since it was digested by DNase I and hybridized with a specific whole genome probe, being clearly visualized with high sensitive DNA dyes, i.e. SYBR Gold. It is interesting to note that this background of DNA fragments was practically undetectable in gram-positive strains, despite being susceptible to β-lactams or vancomycin. Methane monooxygenase Moreover, it was also undetected in the same bacteria after quinolone treatment in susceptible strains, as evidenced in our previous works with the procedure [15, 16]. This fact suggests that the release of DNA fragments could be specific to cell wall directed antibiotics

or β-lactams at least. This interesting phenomenon requires a deeper study in future works, to address the mechanisms and kinetics of production. DNA fragmentation must be a secondary effect, after cell wall damage. It could be a passive result of attack by DNases or reactive species of oxygen (ROS) liberated in the affected bacteria, or it could be active, a consequence of an apoptotic-like process triggered after cell wall damage. Considering to the first possibility, it has been recently reported that, unlike bacteriostatic antibiotics, β-lactams induce the formation of ROS in gram-negative and gram-positive microorganisms [18]. Hydroxyl radicals should attack proteins and DNA, possibly inducing DNA breakages, resulting in death of the bacteria. This response was also found with other bactericidal antibiotics, like fluoroquinolones. Possibly, the increased permeability of the cell wall that would result after impairment of peptidoglycan biosynthesis by the β-lactams, would allow the release of DNA fragments to the medium.

Elevated systolic BP has a continuous, graded, and independent association with risk of coronary heart

disease, stroke, and ESKD [21]. LVH selleck inhibitor might be a beneficial compensatory process in CKD patients, allowing the left ventricle to produce additional force to increase cardiac work and maintain constant wall tension [22]. Even though mean systolic BP was well controlled (132.4 ± 18.1 mmHg), systolic BP was higher in patients with LVH than in patients without LVH in the present study. According to multivariate logistic regression analysis, systolic BP was an independent variable associated with LVH. Recently, it was reported that systolic arterial hypertension and elevated pulse pressure are closely associated with LVH in pre-dialysis patients, suggesting that fluid overload and increased arterial stiffness play important roles in LVH before starting dialysis therapy [12]. Fluid volume management and maintenance of a near euvolemic state are crucial for the amelioration of LVH [23]. After adjusting for several potential confounders, multivariate logistic regression analyses showed that the presence of a previous

CVD was significantly associated with LVH. The potential explanations for how the CKD state can accelerate atherosclerosis Tofacitinib price and cause CVD have been of considerable interest in clinical practice. The 4 basic explanations are: (1) uncontrolled confounding, or the impact of comorbidities that occur in CKD patients, especially older age; (2) therapeutic nihilism, meaning CKD patients receive lesser degrees of cardioprotective therapies; (3) excess treatment toxicities, intolerances, or risks such that therapy cannot be used or offers a less favorable Selleck Pazopanib benefit-to-risk ratio; and (4) a unique vascular pathobiology that occurs in the CKD state [24]. By using the large sample size of the Kidney Early Evaluation Program (KEEP), McCullough

et al. [25] demonstrated in stratified analysis that the presence of CKD in young adults was clearly related to premature CVD. These findings suggest the biological changes that occur with CKD promote CVD at an accelerated rate that cannot be fully explained by conventional risk factors or older age. In accordance with the theory of non-hemodynamic LVH-promoting factors in our CKD patients, BMI was found to be a factor that was independently associated with LVH. Obesity is thought to be a risk factor independent of LVH, and heart disorders in obesity include structural adaptation with LVH and functional abnormalities [26]. Kotsis et al. [27] reported that obesity and daytime pulse pressure are predictors of LVH in true normotensive individuals.

The BF microscopy and FL microscopy images were obtained using a Keyence BZ-8000 microscope (Osaka, Japan). Red fluorescent images were taken using a 540-nm excitation. Results and discussion Figure 4 shows typical UV-visible absorption spectra of the ten-layered LB film of MS and Lumacaftor cost C20 with the molar mixing ratio of 1:2 before and after HTT (80°C, 60 min). The well-known J-band, which is located at

594 nm in the as-deposited state, shifts to 599 nm, as shown in Figure 4. The dichroic ratio R ≡ A // / A ⟂ = 1.81 at its peak around 594 nm before HTT but the anisotropy almost disappears (R = 1.03 at 599 nm) after HTT (80°C, 60 min), as shown in Figure 4. Furthermore, the band shape becomes appreciably sharper by HTT. These results are in good agreement with our previous works. Figure 4 Typical absorption spectra of a ten-layered MS-C 20 binary LB film. The thick solid and dashed lines represent A // and A ⟂of the as-deposited state, respectively; the thin solid and dashed lines represent

A // and A ⟂ after hydrothermal treatment (HTT) at 80°C for 60 min. Figure 5a shows a typical FL micrograph of the as-deposited MS-C20 LB film of ten layers with the schematic layered structure shown in Figure 5b. click here Intense red fluorescence is observed over the whole film area, and the intensity steps are clearly seen at monomolecular steps created by shifts of meniscus lines during the deposition process of the MS-C20 LB film, as shown by arrows in Figure 5a. It has been well known that MS and C20 are phase separated in MS-C20 binary LB system. Minari and coworkers estimated that the length of the MS J-aggregate as several hundred nanometers and that the MS J-aggregates are separated from the regions of matrix molecules of C20 based on the analytical model for characterizing the flow orientation effect during the transfer process of the LB deposition [27]. Kato and coworkers also indicated that the MS-C20 mixed system is phase separated

into MS-rich (dye-rich) regions and C20-rich (fatty acid-rich) ones and that the MS-rich (dye-rich) regions are further separated into dye monomer regions and J-aggregate crystallites based on characterization by atomic force microscopy (AFM) observation, FL microscopy, and PAK6 second harmonic generation (SHG) microscopy [9, 28]. Kato and coworkers further estimate that the size of J-aggregate is in the range of 0.5 to 10 μm based on SHG microscopy observation. We hypothesize a similar mesoscopic texture in which the mixed ultrathin film is separated into MS-rich regions and C20-rich ones and the MS-rich regions are further separated into the dye monomer regions and J-aggregate crystallites in the as-deposited MS-C20 mixed system because the dye monomer band and J-band coexist at 545 to 555 nm and 594 nm, respectively, as shown in Figure 4.

Mutated triplets selleck chemicals are underlined. The start codon of inlA is in italics. Production of electrocompetent Lactococcus lactis The protocol of Holo and Nes [19] was adapted

for the transformation of L. lactis MG1363 derivative NZ9000. A GM17 overnight culture of NZ9000 was diluted 1:100 into 5 ml of GM17 containing 500 mM sucrose and 2.5% glycine (GS-GM17). This culture was inoculated into 50 ml of fresh GS-GM17 and grown overnight. The 50 ml culture was inoculated into 400 ml of fresh GS-GM17, grown to OD600 of 0.3 and cells were subsequently harvested by centrifugation at 4,000 × g for 20 min at 4°C. The pellet was resuspended in 200 ml of ice cold SGB (500 mM sucrose and 10% (w/v)

glucose – filter sterilized), centrifuged, resuspended in 100 ml SGB and left on ice for 15 min. The cells were centrifuged, resuspended in 50 ml SGB and left on ice for 15 min Selleckchem IWR1 before a final centrifugation and re-suspension with 2 ml SGB. Cells were frozen at -80°C in 40 μl aliquots. To electroporate, cells were thawed on ice, mixed with 4 ul of pellet paint (Novagen) precipitated DNA and transferred to a 1 mm electroporation cuvette (Biorad). Cells were pulsed at 20 kV/cm, 200 Ω and 25 μF, regenerated in 1 ml GM17 containing 2 mM CaCl2/20 mM MgCl2 for 1.5 h and then plated onto GM17 agar containing 5 μg/ml chloramphenicol. An efficiency of 1 × 107 cfu/μg was routinely obtained with pNZ8048. Cloning of InlA into pNZB The unique BglII site up stream oxyclozanide of the nisA promoter in pNZ8048 was removed by linearization of the vector with BglII and ends blunted with T4 DNA polymerase. The vector was religated to

generate pNZB. The inlA gene was PCR amplified (primers IM194 and IM188) as described previously [20], digested with NcoI/PstI and ligated into the complementary digested pNZB. Ligations were directly electroporated into NZ9000 as described above and the sequence of the inlA gene was verified by DNA sequencing. QuikChange mutagenesis in L. lactis Primers for site directed mutagenesis (SDM) (Table 1) were designed according to the Quikchange SDM manual (Stratagene). All plasmid template isolated from NZ9000 strains was methylated with Dam methylase following manufacturer recommendations (New England Biolabs). The PCR thermocycling conditions were conducted as described previously [21]. Separate 50 μl KOD hotstart high fidelity polymerase PCR reactions were preformed with each primer for 10 cycles and an extension time of 5 min 30 sec.