PubMedCrossRef 41 Sainte-Marie G: A paraffin embedding technique

PubMedCrossRef 41. Sainte-Marie G: A paraffin embedding technique for studies employing immunofluorescence. J Histochem Cytochem 1961, (10):250–256. Competing interests The authors declare that they have no competing interests. Authors’ contributions NAC carried out the microbiological work and the animal studies. GP and AdMdL conceived of the study. NAC,

AdMdL and GP designed the experiments. NAC performed the statistical analyses and prepared the figures. NAC and AdMdL wrote the draft of the manuscript. GP revised it for significant intellectual Talazoparib concentration content. All authors read and approved the final version of the manuscript.”
“Background Brucella is a genus of bacteria causing brucellosis, a zoonosis that affects a large variety of mammals and that is readily transmitted to humans. The VS-4718 research buy genus includes several classical species that can be distinguished by their preferential host range, surface structure, biochemical and physiological features, and genetic markers. This classification is reflected in some degree of genetic polymorphism,

one of the main sources of which is the copy number and distribution of IS711 (IS6501) [1, 2]. B. melitensis and B. suis contain seven complete IS711 copies [3]. B. abortus carries six complete and one truncated IS711 copies [4], B. ceti and B. pinnipedialis more than 20 copies [5, 6] and B. ovis 38 copies [7]. IS711 is very stable: its mobility has been demonstrated only by using a “”transposon trap”" in vitro in B. ovis and B. pinnipedialis, selleck compound but not in B. melitensis Phosphoglycerate kinase and B. abortus [3]. Based on this stability, polymorphism at the alkB locus [8] is used to differentiate B. abortus from B. melitensis, B. ovis and B. suis in the AMOS multiplex PCR assay [9]. IS711 stability is not only relevant for Brucella typification: its mobility is implicated in the generation of genetic diversity and speciation, as shown by the distribution of IS711 among the extant Brucella species. Here we report that IS711 transposition and the generation of the associated polymorphism takes place in B. abortus under natural conditions, when genetic drift should be limited by the selective pressure imposed by the host. Results and discussion In a previous

work with 46 B. abortus strains, it was found that two isolates (B12 and B16) displayed IS711 profiles that were different from that typical of B. abortus field strains [10]. This is confirmed here by the genetic profiling summarized in Table 1, and by the IS711 Southern blot presented in Figure 1. The latter shows that, while the reference strain B. abortus 544 presented seven IS711-carrying fragments, isolates B12 (x-B12), and B16, B49 and B50 (x-B16) displayed an additional one. It is known that RB51, a lipopolysaccharide rough strain obtained from B. abortus 2308 by multiple in vitro passages on antibiotic containing media, harbors eight copies plus an additional one that transposed into the lipopolysaccharide wboA gene [11]. Similarly, B.

Mycol Res 110:527–536PubMedCrossRef Kwasna H, Kosiak B (2003) Lew

Mycol Res 110:527–536PubMedCrossRef Kwasna H, Kosiak B (2003) Lewia avenicola sp. nov. and its Alternaria anamorph from oat grain, with a key to the species of Lewia. Mycol Res 107:371–376PubMedCrossRef Kwasna H, Ward E, Kosiak B (2006) Lewia hordeicola sp. nov. from barley grain. Mycologia 98:662–668

Leonard KJ, Suggs EG (1974) Setosphaeria prolata, the ascigerous state of Exserohilum prolatum. Mycologia 66:281–297CrossRef Leuchtmann A (1984) Über Phaeosphaeria Miyake und andere bitunicate Ascomyceten mit mehrfach querseptierten Ascosporen. Sydowia 37:75–194 Leuchtmann A (1985) Kulturversuche mit einigen Arten der Gattung Lophiostoma Ces. & de Not. Sydowia 38:158–170 Liew ECY, Aptroot A, Hyde Selleckchem EVP4593 KD (2000) Phylogenetic significance of the pseudoparaphyses in Loculoascomycete taxonomy. Mol Phylogenet Evol 16:392–402PubMedCrossRef Liew ECY, Aptroot A, Hyde KD (2002) An evaluation of the monophyly of Massarina based on ribosomal DNA sequences. Mycologia 94:803–813PubMedCrossRef

Lindau G (1897) Pyrenomycetineae, Laboulbeniineae. In: Engler A, Prantl K (eds) Die Natürlichen Pflanzenfamilien 1. Verlag von Wilhelm Engelmann, Leipzig, pp 321–505 Lindemuth R, Wirtz N, Lumbsch HT (2001) Phylogenetic analysis of nuclear and mitochondrial rDNA sequences supports the view that loculoascomycetes (Ascomycota) are not monophyletic. Mycol Res 105:1176–1181 Ruboxistaurin manufacturer Liu YX (2009) Biological characteristics of a bamboo fungus, Shiraia Silibinin bambusicola, and screening for hypocrellin high-yielding isolates. Dissertation, Suranaree University of Technology Locquin MV (1972)

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Am Chem Soc 2001,123(31):7723–7724 CrossRef 5 Caruso F: Nanoengi

Am Chem Soc 2001,123(31):7723–7724.CrossRef 5. Caruso F: Nanoengineering of particle surfaces. Adv Mater 2001, 13:11–22.CrossRef 6. Tissot I, Reymond JP, Lefebvre F, Bourgeat-Lami E: SiOH-functionalized polystyrene latexes.

A step toward the synthesis of hollow silica GSK2879552 ic50 nanoparticles. Chem Mater 2002, 14:1325–1331.CrossRef 7. Liu SQ, Lu LC, Sui XY, Vera M, Pegie C, Etienne FV: Fast fabrication of hollow silica spheres with thermally stable nanoporous shells. Microporous Mesoporous Mater 2007, 98:41–46.CrossRef 8. Chen YW, Kang ET, Neoh KG, Grener A: Preparation of hollow silica nanospheres by surface-initiated atom transfer radical polymerization on polymer latex templates. Adv Funct Mater 2005,15(1):113–117.CrossRef 9. Darbandi M, Thomann R, Nann T: Silica coated nanocomposites. Chem Mater 2007,19(7):1700–1703.CrossRef 10. Deng ZW, Chen M, Zhou S, You B, Wu LM: A novel method for the fabrication of monodisperse hollow silica spheres. Langmuir 2006, 22:6403–6407.CrossRef 11. Chen M, Wu L, Zhou S, You B: A method for the fabrication of monodisperse hollow silica spheres. Adv Mater 2006, 18:801–805.CrossRef 12. Tsai MS, Li MJJ: A this website novel process to prepare a hollow silica sphere via chitosan-polyacrylic acid (CS-PAA) template. Non-Cryst Solids 2006,352(26–27):2829–2833.CrossRef

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controlled large-scale synthesis of hollow silica spheres with tunable sizes and wall thicknesses. Phys Chem C 2008,112(10):3641–3647.CrossRef 16. Chen Y, Chen HR, Guo LM, He QJ, Chen F, Zhou J, Feng JW, Shi JL: Hollow/rattle-type mesoporous nanostructures by a structural difference-based selective etching strategy. ACS Nano 2010, 4:529–539.CrossRef 17. Lou XW, Archer LA, Yang ZC: Hollow micro-/nanostructures: synthesis and applications. Adv Mater 2008, 20:3987–4019.CrossRef 18. Ding SJ, Chen JS, Qi G, Duan X, Wang Z, Giannelis EP, Archer LA, Lou XWJ: Formation of SnO 2 hollow spheres inside silica nanoreactors. Am Chem Soc 2011, 133:21–23.CrossRef 19. Zhang Q, Ge JP, Goebl J, Hu YX, Lu ZD, Yin YD: Rattle-type silica colloidal particles prepared by a surface-protected etching process. Nano Res 2009, 2:583–591.CrossRef 20. Tan LF, Chen D, Liu HY, Tang FQ: A silica nanorattle with a mesoporous shell: an ideal nanoreactor for the preparation of tunable gold cores. Adv Mater 2010, 22:4885–4889.CrossRef 21.

Two subjects dropped out, as they found the procedure to be overl

Two subjects dropped out, as they found the procedure to be overly burdensome.

Data from the CIS and SF-36 questionnaires were available for all 25 subjects. The SHC yielded usable data from 24 subjects. Data on the HRV parameters (SDNN and RMSSD) for both conditions (reclining and cycling) were available for 24 subjects. For the cycling condition, RR data were available from 25 subjects; for the reclining condition, data were available from 23 subjects. Questionnaires Table 1 shows the Milciclib nmr number of subjects completing the questionnaires as well as the means and the RGFP966 manufacturer standard deviations of the total score on the CIS, the scores on four subscales of the MOS 36-item Short-Form Health Survey (SF-36) and the score on the subscale PN of the SHC questionnaire. Table 1 Number of subjects (N) completing the questionnaires and the means and standard deviations of the total score on the Checklist Individual Strength (CIS), the scores on four subscales

of the MOS 36-item Short-Form Health Survey (SF-36) and the score on the subscale Pseudoneurology (PN) of the Subjective Health Complaint Selleck Vactosertib (SHC) questionnaire   CIS SF-36 SHC PF SF RLP RLEP PN N 25 25 25 25 25 24 Mean (standard deviation) 100.7 (22.5) 75.8 (14.6) 41.0 (21.2) 16.0 (23.8) 46.7 (43.0) 15.7 (9.7) PF physical functioning, SF social functioning, RLP role limitations due to physical problems, RLEP role limitations due to emotional problems The mean total score of all subjects on the CIS was 100.7. The scores on the four

SF-36 subscales ranged from 16.0 to 75.8. Finally, the mean score on the subscale PN of the SHC was 15.7. Parameters The number of subjects is presented in Table 2, along with the means and standard deviations of the HRV parameters SDNN, RMSSD and RR. Table 2 Number of measurements (N) used for analysis and the means and standard deviations for heart rate variability [SDNN (ms) and RMSSD (ms)] and respiration rate [RR (breaths/min)] required at measurement 1 (T1) and measurement 2 (T2)   Cycling Reclining N Mean (standard deviation) N Mean (standard deviation) SDNN (ms) for  T1 24 17.79 (8.89) 24 40.88 (19.77)  T2 24 19.08 (8.20) 24 42.75 (22.19) RMSSD (ms)  T1 24 6.67 (3.14) 24 15.33 (7.56)  T2 24 6.67 (2.68) 24 16.46 (8.67) RR (breaths/min)  T1 25 18.63 (5.11) 23 9.40 (3.07)  T2 25 17.94 (5.22) 23 9.67 (3.10) The mean SDNN was approximately 18 ms for the cycling condition and approximately 41 ms for the reclining condition. The mean values for RMSSD were approximately 7 ms for cycling and approximately 16 ms for reclining. The mean RR values were approximately 18 breaths/min while cycling and approximately 9 breaths/min while reclining. Reproducibility The number of measurements used for analysis, ICC, ICC 95% LoA and SEM values for both HRV parameters (SDNN and RMSSD) and for RR are presented in Table 3.

925 × 1011 particles/mL (16-nm AuNPs) and 1 689 × 1011

pa

925 × 1011 particles/mL (16-nm AuNPs) and 1.689 × 1011

particles/mL (26-nm AuNPs), we estimated the total surface area simply based on the diameters of the uncoated AuNPs. Thus, the total available surface area in the suspensions was estimated as approximately 6.37 × 10−4 m2/mL (16-nm AuNPs) and 3.59 × 10−4 m2/mL (26-nm AuNPs). We then calculated the Veliparib nmr amount of PEG needed to cover all nanoparticles with a single monolayer of four typical PEG samples (APEG 400, 600, 6,000, and 20,000) occupying areas dictated by their R h (Ro 61-8048 chemical structure Additional file 1: Tables S1 and S2). These numbers were then compared to the total concentration of PEG available in the solution for the bulk concentration used (11.25 mg/mL). This concentration is considered to ensure that there are at least 5 orders of magnitude more PEG molecules than necessary as selleck inhibitor needed to saturate the nanoparticle surfaces, based on the above calculations. The Debye length (κ −1) is the measure of a charge carrier’s net electrostatic effect in the solution and the distance over

which those electrostatic effects persist. It is also appropriately termed the electrostatic ‘screening length,’ beyond which the charges are electrically screened [13]. For a single symmetrical electrolyte in water at room temperature (25°C), it can be readily calculated in the form [13]: (5) where C is the electrolyte concentration (M) and z PRKD3 is the valence of the electrolyte. In this study, we added varying amounts of 10.0% NaCl solution (40, 50, or 60 μL, w/v) to each PEG-coated AuNP solution (1 mL) to screen the electrostatic repulsion between nanoparticles. The electrostatic repulsion originates from the surface underlying the adsorbed polymer layer. The resulting NaCl concentrations were 65.8, 81.5, and 96.9 mM, respectively. The corresponding values of κ −1 were determined to be 1.19, 1.07, and 0.98 nm, which were calculated using the above data and Equation 5. The amount of the salt present in the added 40 μL of 10.0% (w/v) NaCl solution does not ensure complete screening of the electrostatic repulsion. This may be attributed to the fact that

the R h of APEG 400 is 0.568 nm (2R h < κ −1 = 1.19 nm) and the zeta potentials of the fully coated nanoparticles range from −13.4 (APEG 400, 16-nm AuNPs) to −9.5 mV (APEG 20,000, 16-nm AuNPs) and from −12.6 (APEG 400, 26-nm AuNPs) to −8.4 mV (APEG 20,000, 26-nm AuNPs) after adding NaCl solution. The salt added in a 50-μL amount of 10.0% (w/v) NaCl solution can more adequately screen the electrostatic repulsion as a result of the relatively shorter κ −1 with the zeta potentials ranging from −8.3 (APEG 400, 16-nm AuNPs) to −4.8 mV (APEG 20,000, 16-nm AuNPs) and from −7.8 (APEG 400, 26-nm AuNPs) to −4.4 mV (APEG 20,000, 26-nm AuNPs) after NaCl addition. Likewise, the amount of salt for the addition of 60 μL of 10.0% (w/v) NaCl solution can also screen the electrostatic repulsion.

Phys Rev Lett 2006, 97:155701 CrossRef 35 Singh A, Tsai AP: Melt

Phys Rev Lett 2006, 97:155701.CrossRef 35. Singh A, Tsai AP: Melting behaviour of lead and bismuth nano-particles in quasicrystalline matrix – the role of interfaces. Sadhana 2003, 28:63–80.CrossRef 36. Hadjisavvas G, SBE-��-CD Kelires PC: Structure and energetics of Si nanocrystals embedded in a-SiO2. Phys Rev Lett

2004, 93:226104.CrossRef 37. Soulairol R, Cleri F: Interface structure of silicon nanocrystals embedded in an amorphous silica matrix. Solid State Sci 2010, 12:163–171.CrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions GZ, AP, and JM carried out the spectroscopic measurements as well as calculations. JC and FG designed and deposited the investigated samples. All authors read and

approved the final manuscript.”
“Background Nowadays, electronic devices invade strongly our daily life. In the race to efficiency, they have to be faster and faster, smaller and smaller, and with better and better performance [1–4]. One way to reach this goal is to integrate supercapacitors in their microelectronic circuit. Supercapacitors are commonly used to complete batteries whenever pulse power, long term cycling, and high charge/discharge are required [5–9]. Many studies are currently dedicated to the design of micro-ultracapacitors with different types of carbons [5–7] or pseudo-capacitive materials LY411575 cell line (RuO2, MnO2 …) [8, 9]. However, their integration in microelectronic circuit is still a challenge. Elaborate silicon based micro-ultracapacitors should facilitate it. Moreover, such devices could directly be manufactured on chips. Recently, porous silicon nanowires (SiNWs) [10], porous silicon coated with gold [11, 12], SiNWs coated with NiO [13, 14], or SiC [15] have been studied as potential materials for supercapacitor electrodes. Si/SiC core-shell nanowires-based electrodes Oxalosuccinic acid show the most promising performances and cycling stability, but no studies have been performed in the two electrode devices. More recently, we proved that chemical vapor deposition (CVD)-grown, SiNWs-based electrodes show a promising cycling stability

in an organic electrolyte and a quasi-ideal pure capacitive behavior, i.e., the energy that is stored thanks to electrolyte ions Selleckchem Defactinib accumulation at the polarized electrode/electrolyte interface [16]. As pure capacitive supercapacitor capacitance is proportional to the developed surface area on the electrode, increasing the SiNWs length should improve the device capacitance. SiNWs length and doping level can easily be tuned by CVD, thanks to the vapor–liquid-solid (VLS) mechanism [17, 18], using a metal catalyst as seed to the SiNWs growth [19–21]. The SiNWs diameter and density can also be monitored. This work underlines the importance of HCl use during the SiNWs growth by CVD to obtain very long nanowires and investigates the influence of SiNWs length on SiNWs/SiNWs micro-ultracapacitors devices capacitance.

J ApplPhys 1966, 37:2775–2782 CrossRef 26 Švorčík V, Slepička P,

J ApplPhys 1966, 37:2775–2782.CrossRef 26. Švorčík V, Slepička P, Švorčíková J, Špírková M, AZD5582 Zehentner J, Hnatowicz V: Characterization of evaporated and sputtered

thin Au layers on PET. J Appl Polym Sci 2006, 99:1698–1704.CrossRef 27. Jacobs T, Morent R, Geyter ND, Dubruel P, Leys C: Plasma surface modification of biomedical polymers: ON-01910 manufacturer influence on cell-material interaction. Plasma Chem Plasma Process 2012, 32:1039–1073.CrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions AR carried out the AFM analysis, evaluated the surface morphology and roughness, and wrote and designed the study. ZN analyzed the electrical and optical properties, carried out gravimetry and goniometry measurements, and calculated the number of VSMCs Epigenetics inhibitor of gold-coated glass samples. NSK performed the cytocompatibility tests. VS participated in the study coordination and paper correction.

All authors read and approved the final manuscript.”
“Background Platinum (Pt) is a noble metal with unique physiological and chemical properties widely used in chemistry, physics, biology, and medicine. Regarding the biological activities of Pt, it is known that Pt compounds have the ability to arrest the cell cycle [1, 2] and cause DNA strand breaks. The DNA damage is caused by Pt ions, which attach to N7 sites of DNA guanine bases and, after hydrolysis of Pt-Cl bonds, form adducts with the DNA double helix [2, 3]. These properties of Pt are exploited in cancer therapy in the form of antineoplastic drugs to treat different types of cancer such as head, neck, brain [4], testicular, bladder, ovarian, or uterine cervix carcinomas [5]. However, toxic side effects of Pt-based drugs are major drawbacks in cancer therapy [6, 7]. Nanotechnology has introduced possibilities for using alternate forms of elements – nanoparticles. Nanoparticles have unique physiochemical features

because of their small size (<100 nm), large surface-to-mass ratio, exceptional quantum characteristics [8], and consequently unique biological properties. Smaller nanoparticles can move across cellular and also nuclear Anacetrapib membranes and are able to penetrate cells and intracellular structures, and target defined points within the body [9, 10]. Platinum nanoparticles (NP-Pt) have recently elicited much interest because of their physicochemical properties such as catalytic activity and high reactivity [11]. NP-Pt, as metal structures (Pt0), differ significantly from platinum salts and have quite different chemical properties when administered to an organism. They are a very limited source of ions, and consequently, the process of forming platinum salts is very slow and restricted. However, the solubility and, consequently, the bioavailability of NP-Pt depend on their size [12].

A growth analysis with this strain was carried out in vz0825 supp

A growth analysis with this strain was carried out in vz0825 supplemented LB-medium and in T-medium with different potassium and sodium ion concentrations (Figure  4). Overall, growth of the T283M mutant was much less effected by vz0825 in comparison to the wild type strain. Sensitivity of the T283M mutant against compounds vz0500 and 1541–0004 did not differ from the wild type strain NM06-058 (data not shown). Figure 4 Growth determination. Growth of V. cholerae wild type strain NM06-058 (A) and the T283M exchange mutant (B) in the presence of vz0825 in media with different K+ and Na+ concentrations. Attempts to construct a kdpD knockout mutant For a further elucidation of the

effect of vz0825, the construction of a V. cholerae kdpD knockout Idasanutlin molecular weight mutant was attempted. If KdpD is a major target of compound vz0825, the V. cholerae kdpD knockout mutant should be insensitive to the compound, unless the protein itself and its function are essential for the viability of the bacteria. The cloning procedure delivered the expected plasmid construct according to sequencing. The plasmid was successfully transformed into the

E. coli strain S17-1, according to the acquirement of ampicillin resistance, which is located on the plasmid pEX18Ap and also according to PCR amplification of the construct. The conjugation of the transformed E. coli with V. cholerae and the following selection on LB agar Selleck GSK2118436 plates supplemented with carbenicillin (Carb) and Km did not lead to clones with Nirogacestat a deleted VC_A0531 gene, even after several modifications of the protocol. A possible explanation is that the gene product KdpD is indeed essential for V. cholerae, in agreement with KdpD being a prime target of vz0825. Discussion A HTS assay for small molecule inhibitors

of V. cholerae was developed and validated using a viability phenotype of V. cholerae that constitutively expresses green fluorescence. The assay is reliable, reproducible and simple to perform. Etofibrate During the development of the reporter strain, two reference strains of O1 serogroup belonging to biotypes O395 (classical) and N16961 (El Tor) were included along with the O139 strain MO10. The green fluorescence producing plasmid pG13 was electroporated into the three strains. During initial standardization experiments it was observed that the strain MO10 pG13 produced much greater level of green fluorescence as compared to other two strains (data not shown). For this reason strain MO10 pG13 was used in the screening experiments. A data bank search in SciFinder for the most active compounds vz0825 and vz0500 did not reveal pre-described antibacterial activities of the compounds with structural similarities above 70%. Compound 1541–0004, stemming from the commercial CDI collection, belongs to the group of styryl dyes, which have already in 1966 been shown to possess antimicrobial effects against the plant pathogen Xanthomonas oryza[16].

Other examinations of the association between plant characteristi

Other examinations of the association between plant characteristics and rarity have generally categorized rarity on only a single axis, or have used IUCN red list criteria (Bekker and Kwak 2005). Single-axis approaches have AZD4547 mouse either (1) categorized species as either “abundant” or not, utilizing

the axes of GR and LA interchangeably (Kunin and Gaston 1993; Hegde and Ellstrand 1999), (2) developed a single rarity index utilizing endemism, GR, and endangerment status (Farnsworth 2007), or (3) used GR (Thompson et al. 1999; Lester et al. 2007; Gove et al. 2009; Leger and Forister 2009). The IUCN red list combines population size, growth rate, population fluctuation, habitat 4SC-202 fragmentation, and range size into an endangerment index (IUCN 2001). A previous, trait-based meta-analysis combining the three rarity axes (Murray et al. 2002) found a very limited number of studies that encompassed more than one axis of rarity. Although the separation of rarity into different types is controversial (Kunin and Gaston 1993; Hegde and Ellstrand 1999), we conducted this study to determine if the research resulting from the widespread use of this matrix 3-Methyladenine cell line supports the separation of rarity into different syndromes. While plant species distributions may reflect basic

demographic processes of seed production, dispersal, and establishment, the distribution of species may also in itself be Amino acid a selective force and affect evolutionary trajectories. For example, species that grow in locally abundant populations may evolve to tolerate intraspecific competition better than interspecific competition (Rabinowitz et al. 1984; Rabinowitz and Rapp 1985). Species of locally sparse populations may be highly dependent on pollinators to ensure reproduction when non-autogamous. Species with large GR have been found

to be better colonizers (Leger and Forister 2009), and colonization ability may in turn be selected for in these species. Assuming equilibrium conditions in species distributions, once there is a fitness advantage to reproducing and dispersing within the current distribution, it is reasonable to predict adaptation to the biological and ecological conditions of the distribution itself (Morris 2003). We presume species persist in their current distribution pattern because they have historically succeeded that distribution pattern. This presumption is heavily relied upon to predict trajectories of plant invasions (e.g. Higgins et al. 1999; Thuiller et al. 2005) and may be applicable to native short-lived species. Distributions of longer-lived species, such as trees and perennial grasses, may reflect land use history (e.g. Palo et al. 2008) or previous climate (Kruckeberg and Rabinowitz 1985). Factors that once determined establishment of these species may no longer be present although factors that affect mortality are very likely still in action.

After the construction, all the mutants remained sensitive to P22

After the construction, all the mutants remained sensitive to P22 and did not show any obvious defects when grown in nutrient rich LB medium or glucose minimal medium. The mutants were also as resistant as the wild-type strain to the action of blood serum, egg white, bile salts, polymyxin (as a representative of antimicrobial peptides), Protein Tyrosine Kinase inhibitor hydrogen peroxide or pH 4 (not shown). Table 3 List of strains used in this study. Strain SPI present SPI absent Reference S. MI-503 purchase Enteritidis 147 Nal wild type 1, 2, 3, 4, 5 none [25] S. Enteritidis 147 Nal ΔSPI1 2,3,4,5 1 this study S. Enteritidis 147 Nal ΔSPI2 1,3,4,5 2 this study S. Enteritidis

147 Nal ΔSPI3 1,2,4,5 3 this study Cyclosporin A order S. Enteritidis 147 Nal ΔSPI4 1,2,3,5 4 this study S. Enteritidis 147 Nal ΔSPI5 1,2,3,4 5 this study S. Enteritidis 147 Nal ΔSPI1-5 none 1,2,3,4,5 this study S. Enteritidis 147 Nal SPI1o 1 2,3,4,5 this study S. Enteritidis 147 Nal SPI2o 2 1,3,4,5 this study S. Enteritidis 147 Nal SPI3o 3 1,2,4,5 this study S. Enteritidis 147 Nal SPI4o 4 1,2,3,5 this study S. Enteritidis 147 Nal SPI5o 5 1,2,3,4 this study S. Enteritidis 147 Nal ΔSPI1&2 3,4,5 1,2 this study S. Enteritidis 147 Nal SPI1&2o 1,2 3,4,5 this study

Infection of chickens In the first experimental infection, day-old chickens (Ross breed, 10 birds/group) were infected orally with 5 × 107 CFU of either the wild-type strain or the SPI mutants. In the second infection, four groups, each of 10 chickens, were infected with the wild type strain, or ΔSPI1&2, Farnesyltransferase SPI1&2o and SPI1-5 mutants. Counts of the strains in caeca, liver and spleen were determined in 5 birds on day 5 and in remaining 5 birds on day 12 of life i.e. 4 and 11 days post infection, respectively. The last experimental infection was focused on cytokine signaling and in this case, besides 3 non-infected control chickens, three additional chickens per group were infected with wild type strain, ΔSPI1, ΔSPI2, and ΔSPI1&2 mutants. In

all euthanised birds, S. Enteritidis counts in the caeca, liver and spleen were determined after tissue homogenisation in peptone water and plating tenfold serial dilutions on XLD, BGA or Bromothymol-blue agars (Merck) supplemented with nalidixic acid. Samples negative after the direct plating were subjected to pre-enrichment in RV broth supplemented with nalidixic acid for qualitative S. Enteritidis determination. Counts of S. Enteritidis positive after the direct plating were logarithmically transformed. In the case of samples positive only after the pre-enrichment, these were assigned a value of 1 and the negative samples were assigned a value of 0. Samples from the caeca and liver were also fixed in 10% formaldehyde and subjected to haematoxylin and eosin staining.