Figure 1 Typical interconnect scheme of an α-Si:H module in superstrate configuration. P1, P2 and P3 indicate the different patterning steps. P1 is performed using an infrared laser to remove the front TCO. P2 and P3 use a green laser to cut the Si solar absorber layer and the rear electrode, respectively. In this letter, we demonstrate how the

energy density threshold for the scribing of the transparent contacts can be significantly reduced by replacing the standard thick AZO single layer with a 10 times thinner AZO/Ag/AZO multilayer structure with better electrical and optical properties. More specifically, for the lowest used pulse Quisinostat cell line energy, we measure a separation resistance for the AZO/Ag/AZO structure 8 orders of magnitude higher compared to much thicker AZO, currently used in thin film solar cells.

The experimental results and the numerical simulations provide clear Sotrastaurin nmr evidences of the key role played by the silver interlayer to steep temperature increase at the DMD/glass interface, leading to a more efficient P1 scribing through a reduction of the fluence in a single laser pulse. These results could open great opportunities for the implementation of thin AZO/Ag/AZO electrodes Ruxolitinib cell line on large-area modules liable to segmentation, such as for α-Si:H solar panels. Methods AZO/Ag/AZO multilayers were sequentially deposited on conventional soda lime glass substrates by RF magnetron sputtering at room temperature in argon atmosphere with a working pressure of 1 Pa. A ceramic AZO target containing 2 wt.% Al2O3 and a pure Ag target were employed as source materials. The sputtering powers were 225 and 30 W for AZO and Ag, respectively. The deposition times were set in order to obtain 40 nm for both top and bottom AZO films and an optimum thickness of 10 nm for the Ag interlayer. This

value was selected to fabricate a DMD structure that has high optical transparency in the visible range and good electrical conductivity [5]. The thicknesses of the films were verified by Rutherford backscattering spectrometry (RBS; 2.0-MeV He+ beam) measurements in normal detection mode. Laser treatments were performed in air by a single O-methylated flavonoid pulsed (12 ns) Nd:YAG laser operating with an infrared (λ = 1,064 nm), Gaussian-shaped (FWHM = 1 mm) beam. The laser power was varied to obtain fluences in the range from 1.15 to 4.6 J/cm2. The morphologies of the AZO/Ag/AZO multilayer after the laser irradiation process were investigated by field emission scanning electron microscopy (SEM) using a Zeiss Supra 25 microscope (Oberkochen, Germany). Electrical sheet resistance (R sh) of about 8 Ω/sq was measured on the as-deposited DMD electrode using a four-point terminal method by employing an HL5560 system (Bio-Rad, Hercules, CA, USA), while the change of the conductivity due to laser ablation process has been mapped by lateral current–voltage characteristics acquired with a Keithley 4200 semiconductor characterization system (Cleveland, OH, USA).

From a practical standpoint, we measured muscle performance by

calculating total work performed during the final 3 sets of knee extension exercise. Despite some positive findings for our non-exercise performance measures, we failed to note any difference in exercise performance between pre and post intervention. In fact, values were actually lower following MSM supplementation as compared to before supplementation. We have no explanation for these findings other than recognizing TPX-0005 research buy our small sample size and the potential for day-to-day variance in knee extension “muscle endurance” performance, as has been noted for isokinetic testing [24]. Also noteworthy, motivation is paramount when asking subjects to perform repetitions to exhaustion. In retrospect we believe that find more our chosen protocol may not have been ideal to discern performance differences between groups and across time. Although subjects performed a total of 18 sets of knee extension exercise, the first 15 sets were standardized in terms of repetition number. Hence, subjects were only provided a total of 3 performance

sets (16–18) to generate usable data for performance comparison. Future work may include a different exercise protocol, with the possible addition of isometric and dynamic force, as well as power data as done previously [25], in addition to actual volume load (reps x load). This would provide for a more complete assessment of muscle performance—as well as greater potential for observed differences in muscle soreness and oxidative stress related parameters. Moreover, the “damaging” exercise protocol may be altered to include a more robust model for inducing damage (e.g., pure eccentric loading using 1-RM

values that are RANTES far greater than those used in the present design) [16]. In addition to performance, we used two distinct questionnaires to determine the extent of either muscle soreness or fatigue, before and following exercise, both pre and post intervention. Although preliminary, MSM did provide some evidence of effect at attenuating both muscle soreness and fatigue (Figures 1 and 2, respectively). As with other measures, additional larger scale studies are needed to corroborate these findings. If future work agrees with these initial findings, MSM may serve a useful purpose in enhancing post-exercise recovery. Conclusion Our data indicate that supplementation with MSM, specifically at a daily dosage of 3.0 grams, may favorably influence selected BVD-523 markers of exercise recovery. In particular, to our knowledge, this was the first study to observe an effect of MSM on antioxidant capacity, as measured by blood TEAC. While this study was small in scope, it is suggested that more research be done to extend these findings. Specifically, future studies should include a larger sample size, a placebo group for comparison, the inclusion of additional markers of recovery and exercise performance (e.g.

Figure 5 Effective index and figure of merit. 3D FDTD simulation this website of (a) real part of n eff, (b) imaginary part of n eff, and (c) figure of merit for the different phases of the Bi2Se3 dielectric layer, where the light source is p polarization at normal incidence angle. The refractive index is expressed in terms of the real and imaginary parts of the

permeability μ eff and permittivity ϵ eff. However, the sign of the real part of the permeability μ eff: Real(μ eff) determines the relative magnitudes of the imaginary and real parts of the refractive index [41]. To achieve a negative index with a small loss, a negative Real(μ eff) is required. Therefore, we have simulated μ eff and ϵ eff for the structure as shown in Figure  6. For the trigonal and orthorhombic phases of Bi2Se3, Real(μ eff) has a Fano-type line shape and Im(μ eff) has a Lorentzian line shape in the region of the negative index. Moreover, a double-negative MM can be BAY 1895344 cost achieved when Real(μ eff) and Real(ϵ eff) simultaneously reach negative values over a wide frequency range

and thus a reduced loss. The maximum negative Real(μ eff) decreases with the phase transition from the trigonal to orthorhombic, hence resulting in the smaller value of the maximum negative Real(n eff) at the orthorhombic phase. Figure 6 Permittivity and permeability. 3D FDTD simulation of (a) the real part of Paclitaxel nmr μ eff, (b) the imaginary part of μ eff, (c) the real part of ϵ eff, and (d) the imaginary part of ϵ eff for the different phases of the Bi2Se3 dielectric

layer, where the light source is p polarization at normal incidence angle. This magnetic negative response can be explained looking at the current and field distribution at the resonance wavelengths. Figure  7 shows the current and total magnetic field intensity for the magnetic resonant wavelengths of 2,140 and 1,770 nm at the β plane shown in Figure  1. In the field maps of Figure  7, the mTOR inhibitor arrows show the currents, whereas the color shows the intensity of the magnetic field. It clearly shows that the antiparallel currents are excited at opposite internal metallic interfaces, closed by an electric displacement current J D. Therefore, these virtual current loops between two Au layers on the β plane give rise to magnetic resonant responses of negative Re(μ eff) that interact strongly with the incident magnetic field at which the total magnetic field intensity H is strongly localized in the Bi2Se3 dielectric layer between the top and bottom Au layers [42]. Figure 7 Magnetic field intensity and displacement current.

Interestingly, size exclusion chromatography showed that PA(FLAG)p is only in fractions www.selleckchem.com/products/lonafarnib-sch66336.html that contain Ssa1p indicating that nearly all of the detectable PA(FLAG)p was complexed with Ssa1p (Figure 6B). This PA(FLAG)p-Ssa1p Sapitinib nmr complex is

quite stable since treatment with reducing agents liberated some, but not all PA(FLAG)p from the Ssa1p complex. Furthermore, in a strain with SSA1 deleted, different chaperone proteins, Ssb2p, or Hsp60 (both detected in our analysis) tightly complexed with the PA(FLAG)p (Additional file 1: Figure S7, Additional file 2: Table S2). We note that several Hsp70 proteins, including both Ssa1p and Ssb2p, assist in protein folding [28] and have been observed to interact with aggregating proteins [29, 30]. Therefore, it appears that Ssa1p and Ssb2p/Hsp60 effectively bind to the PAp incompatibility factor when it is overexpressed in yeast. Figure 6 High-level expression of the PA incompatibility domain results in an interaction with Hsp70 protein concomitant with remediation of aberrant PA-associated

phenotypes. A) Proteins were extracted under reducing conditions from PA-expressing and control yeast grown in YPRaf/Gal. Immunoblotting using anti-FLAG antibody reveals that over-expressed PA(FLAG)p forms a complex (P-S) with another protein that was identified by mass spectroscopy as Ssa1p (Additional file 1: Table S1). The weak PA(FLAG)p signal (P) demonstrated that most PA(FLAG)p is sequestered into this PA(FLAG)p-Ssa1p complex. The position

of control (FLAG) protein is indicated (H). B) When overexpressed, virtually all of the PA(FLAG)p interacts with learn more Ssa1p. Cells were grown overnight at 30°C in YPD, washed in PBS, resuspended in YPRaf/Gal and grown with shaking until mid-log phase. Proteins were then extracted and subjected to size exclusion chromatography as described in the main text. The control (FLAG) protein was detected in fractions 3–8. In contrast, the PAp monomer check was detected only in the presence of the Ssa1p-PA(FLAG)p complex (fractions 3–5). This indicates that the majority of PA(FLAG)p was bound to Ssa1p and that treatment with reducing agents prior to immunoblotting dissociated some but not all of the PA(FLAG)p from the complex. Duplicate Coomassie blue stained protein gels were used to verify equal loading across lanes. Positions of molecular weight markers are shown at left. For both panels, similar trends were observed in two independent extractions and immunoblots. Discussion We define a protein domain with incompatibility function in RNR from N. crassa and demonstrate it can elicit an incompatibility-like reaction in yeast. Previous studies have examined trans-species expression of fungal nonself recognition genes in closely related filamentous fungi [31–33]. In particular, expression of N. crassa tol results in mat-associated heterokaryon incompatibility in Neurospora tetrasperma[34], and PA alleles of N.

For device C, the situation is similar to device B, as indicated in Figure 3c. However, there is a 0.3-eV barrier at the [LUMO]EML/[LUMO]BCP interface, and electrons are confined in the LUMO energy level of BCP. Meanwhile, the larger barrier of 0.7 eV at the interface of [HOMO]EML/[HOMO]BCP results in holes confined in the HOMO energy level of EML. Since electrons and holes are confined in different organic layers, which

increase the probability of excitons disassociation and decrease the AZD1480 price recombination efficiency of carriers [23], device C presents inferior EL performances. Therefore, the different level alignments both for [LUMO]EML/[LUMO]PBL and [HOMO]EML/[HOMO]PBL for devices A, B, and C lead to the different Nutlin 3a distributions and recombination efficiencies of carriers. That is also proven by their different EL spectra as shown in Figure 4. From the emission

spectra, we note that device A with type-I MQW structure offers a larger blue emission than the reference device PCI-32765 supplier which makes better CIE coordinates (see Table 1). For devices B and C with type-II MQW structure, there is a low possibility of carrier recombination due to the fact that only a single carrier could be confined in the EML, while another carrier is confine in PBL, which results in poor EL performances. It is a fact that strong blue emission and week red emission present in device C resulted from the accumulation of holes at the interface of [HOMO]blue-EML/[HOMO]BCP and that there are less holes in potential wells of green EML and red EML, especially in potential wells of red EML. Conclusions In conclusion, WOLEDs with type-I MQW structure offer higher EL performances

in contrast with the reference device with traditional three-layer structure. WOLEDs with TPBi as PBL exhibits a peak current efficiency and a power efficiency of 16.4 cd/A and 8.3 lm/W at about 1,000 cd/m2, which increase by 53.3% and 50.9% over the reference device, AMP deaminase respectively; meanwhile, a maximum luminance of 17,700 cd/m2 is achieved, which keeps a similar luminance with the reference device. The achievement of high EL performance with type-I MQW structure WOLEDs would be attributed to the uniform distribution and rigorous confinement of carriers and excitons within EMLs. However, when Bphen or BCP acts as PBL instead of TPBi, low EL performances (especially for BCP) are obtained, which are attributed to poor level alignment at the interface of EML/PBL for type-II MQW structure; thus, incomplete confinement and low recombination efficiency of carriers occur. In terms of the results, we find that type-I MQW is a promising structure design for improving white EL performance by choosing the suitable PBL.

0.9.0. Reference sequences were downloaded from GenBank and the software program GARLI [Genetic Algorithm for Rapid Likelihood Inference] was used to generate the maximum likelihood (ML) tree [14]. Development of ISSR Fingerprinting Method The ISSR primers were designed to flank di-, tri- and tetra-nucleotide repeats.

A total of ten repeat primers were synthesized: two di-nuclotide [DDB(nn)8], five trinucleotide [DDB(nnn)5], and three tetranuclotide [DDB(nnnn)4] (capital letters denote degenerate sites: B denotes nucleotides c, g, or t; D denotes a, g, or t; subscripts indicate the number of repeats) and 5′ labeled with 6-carboxyfluorescein dye (6-FAM) at the Centers for Disease Control and Prevention Biotechnology Core Facility (VX-689 ic50 Atlanta, CA-4948 chemical structure GA) (Table 1). Table 1 ISSR primers designed for this study Primer Sequence Repeat Type ISSR_7 DDB(agg) 5 Trinucleotide ISSR_8 DDB(cag)5 Trinucleotide ISSR_9 DDB(gag)5 Trinucleotide ISSR_10 DDB(ctc) 5 Trinucleotide ISSR_11 DDB(gtg)5 Trinucleotide ISSR_12 DDB(aacg)4 Tetranucleotide ISSR_13 DDB(cgca) 4 Tetranucleotide ISSR_14 DDB(gcca)4 Tetranucleotide ISSR_15

DDB(ct)8 Dinucleotide ISSR_16 DDB(ca)8 Dinucleotide Capital letters in ISSR primer sequences denote degenerate sites: B denotes nucleotides c, g, or t; D denotes a, g, or t. Subscripts indicate the number of repeats. Bold lettering indicates the primers used for fingerprinting. Initially, ten ISSR primers were tested for their ability to generate reproducible, complex fingerprinting patterns on a panel of 40 A. terreus isolates randomly selected AZD1390 price from the global isolate collection. For PCR amplification, 3-5 μl of genomic DNA was used as the template in a final reaction volume of 25 μl consisting of PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3); 0.2 mM each of dATP, dGTP, dCTP, and dTTP; 2 pmol of a single primer; and 1.3 U of Taq DNA polymerase (Roche Applied Science, Mannheim, Germany). Amplification

was performed in a GeneAmp PCR system 9700 thermocycler (Applied Biosystems, Carlsbad, CA). Initial denaturation at 95°C for 5 min was followed by 36 cycles of 95°C for 30 s, 50°C for 45 s, and 72°C for 2 Protein kinase N1 min. The last cycle was followed by a final extension at 72°C for 7 min. Fluorescently labeled PCR products were separated by capillary electrophoresis on an ABI 3130 DNA analyzer (Applied Biosystems, Carlsbad, CA). Briefly, 0.5 μl of a 1:10 dilution of PCR product was added to 0.25 μl GeneScanTM 1200 LIZ internal size standard and 9.25 μl Hi-Di formamide (Applied Biosystems, Carlsbad, CA). The 10 μl samples were denatured by heating to 95°C for 3 min., cooled and run on a 50 cm array in the POP-7 polymer matrix using the 1200LIZ run module. Four of the ten primers tested produced complex, reproducible, banding patterns over multiple PCR reactions and a series of DNA concentrations, and these four ISSR primers were therefore selected for the analysis of the remaining sequence-confirmed A. terreus isolates.

This has been demonstrated by persistent elevation of pro-inflammatory cytokines like IL-6 among infertile women [12] and in tear fluid from post-scarring trachoma populations [13]. One study identified IL-6 secretion via the TLR2 signaling pathway after C. trachomatis infections [14]. This TLR2 pathway has been shown to be associated with fallopian-tube pathology, potentially contributing to the immunopathogenesis associated with C. trachomatis infection [14]. The chemokine monocyte chemoattractant protein-1 (CCL2) has also been Akt inhibitor identified in chronic chlamydial infections demonstrating elevated levels in post-scarring

trachoma populations [13]. Due to the high prevalence of worldwide trachoma, the World Health Organization (WHO) established and supports selleck compound the use of the SAFE (surgery, antibiotics, facial cleanliness, and environmental improvements) strategy to reduce disease transmission in endemic areas. Mass antibiotic Regorafenib nmr therapy has been a mainstay in this program resulting in

diminished prevalence of active chlamydial infections [15–17]. However, heightened recurrence rates of infection 6-24 months after termination of antibiotic therapy were evident in multiple studies [18–21]. Additionally, Burnham et al. saw an increase in chlamydia-associated STI Reinfection after a control program with antibiotic treatment was established [22]. The mass administration of antibiotics may lead to the development of antibiotic resistance in chlamydial species as well as other pathogenic bacteria. It is apparent pentoxifylline that research into alternative treatments is warranted, and the use of phototherapy may be an attractive option. Phototherapy utilizing low power lasers or light emitting diodes (LEDs) has been

shown to reduce pain and chronic inflammation, and to promote tissue regeneration via a photochemical mechanisms (reviewed in [23]). Additionally, anti-bacterial effects due to the increased production of reactive oxygen species resulting in membrane instability and DNA damage have been evident with phototherapy [23–27]. Its use with several discrete wavelengths exhibits anti-bacterial activity requiring short treatment times without inducing anti-bacterial resistance subsequent to multiple treatment sessions [28]. In this study, we analyzed the effect of low-level 405 nm and 670 nm LED irradiation on the growth of C. trachomatis and the ensuing secretion of pro-inflammatory cytokines IL-6 and CCL2 from C. trachomatis-infected epithelial cells. Results Inhibition of chlamydial growth post – 405 nm irradiation This study assessed the use of 405 nm and 670 nm LEDs as an alternative treatment against chlamydial infections. In Figure 1A, HeLa cells were infected with C. trachomatis at a multiplicity of infection (MOI) of 5. Irradiation treatment with violet 405 nm LEDs demonstrated chlamydial growth inhibition at energy densities as low as 5 J/cm2 (Figure 1B, P < 0.005).

The stromatolites can be classified as close laterally linked hemispheroid (LLH-C) type. Maximum and minimum thickness of laminaes is between 0.55 and 4.93 mm, respectively. Laminaes are wavy in nature, show low synoptic relief

and high inheritance. In profile section, the laminaes are gently convex. This finding has a tremendous bearing on the evolution of hitherto unknown early life forms in the selleck compound Archean Bundelkhand craton vis-à-vis central Indian shield. https://www.selleckchem.com/products/VX-809.html Pati, J. K. (2005). The Dhala Structure, Bundelkhand craton, Central India—a new large Paleoproterozoic impact structure (abstract), Meteoritics & Planetary Science 40 (Supplement): A121. Pati, J. K., Reimold, W.U., Koeberl, C. and Pati, P. (in press).

The Dhala Structure, Bundelkhand Craton, Central India—eroded remnant of a large Paleoproterozoic impact structure. To appear in the Meteoritics & Planetary Science. Schopf, J. W., Kudryavtsev, A. B., Czaja, A. D., Tripathi, A. B. (2007). Evidence of Archean life: Stromatolites and microfossils. Precambrian Research, 158:141–155 E-mail: jkpati@yahoo.​co.​in The Minimal Size of Cells: An Experimental Approach Based on Liposomes Tereza Pereira de Souza1, Pasquale Stano1, Pier Luigi Luisi1 Biology Department, University of RomaTre, Viale G. Marconi 446; 00146 Rome, Italy In the last few years the notion of the “minimal Selonsertib order cell”, as a form of minimal life, has gained considerable attention both from the theoretical and experimental OSBPL9 point of views (Luisi, 2006; Luisi et al. 2006). This concept is important for assessing the minimal and sufficient conditions for cellular life, and also to gain an insight of the early cells, conceivably much simpler than the modern cells. There are two sides to the notion of minimal cell: one side is the question of the minimal genome, namely the minimal number of expressed genes that permit the functioning of the cell (usually seen in terms of the triad self-maintenance, reproduction,

and evolvability). The other side to it concerns the minimal physical dimension of the cell the question, namely, on the dimension that still permits a cellular life. These two aspects minimal genome and minimal size are obviously connected to one another, being also related to evolutionary paths and to the environment composition. Here we propose to examine the question of the minimal physical size of cells by using liposomes with entrapped the complete ribosomal machinery for protein expression (enhanced green fluorescence protein, EGFP). Liposomes are formed by film or ethanol injection method. The synthesis outside vesicles was inhibited using the EDTA, RNAse or protease, with the inhibitor being added just after vesicles formation. The system with the addition of inhibitor inside and outside of vesicles formed our negative control.

influenzae on sBHI plates supplemented with bacitracin (0.3 g/L) and either streptomycin (4 mg/L) or nalidixic acid (5 mg/L). Infant Rat Model Although neonatal rats do not naturally carry S. aureus, S. pneumoniae and H. influenzae, they can be reproducibly colonized with these species. All animal experiments were performed under the guidelines approved by the Emory Institutional Animal Care and Use Committee. Three-day-old pups, born of timed-pregnant Sprague-Dawley rats (Charles River Laboratories), were randomly reassigned to dams. At 3 or 5 days of age, rats were intranasally inoculated by touching a drop of 102 – 108 bacteria of either S. aureus, S. pneumoniae

or H. influenzae (that had been spun down and re-suspended LY3039478 in 5 μl PBS supplemented with 0.1% gelatin (PBS-G)) to the right and then another 5 μl to the left external nares [45]. The nasal flora of un-inoculated neonatal rats, buy VX-689 determined

by colony morphology on blood plates, appeared to consist primarily of non-hemolytic streptococci and coagulase-negative staphylococci. No S. aureus, S. pneumoniae and H. influenzae colonies were isolated from un-inoculated neonatal rats and all of these strains colonized in spite of the presence of this nasal flora. Two days after the innoculation, nasal wash was collected from 200 μl of PBS-G instilled into a 5 cm intramedic polyetylene tubing (PE50, intramedic, Clay Adams) placed into the trachea, and nasal epithelium was scraped from the nasal passages after a second wash of 200 μl of PBSG and removal of the frontal bones. 3 sequential nasal washes of 200 μl of PBS-G contained no significant decrease in the bacteria density compared to the first wash. The nasal epithelium was homogenized in 1 ml of PBS-G. In all experiments, 100 μl of the nasal wash and nasal epithelium samples were plated directly and serially diluted onto selective plates. The limit for detection was 10 cfu/ml. Nasal wash densities were converted to cfu in rat by multiplying cfu/ml by 5 (200 uL total vol.) and nasal epithelium by multiplying by 1 (1 ml total vol.). With the exception of the H. influenzae -S. pneumoniae Endonuclease interaction, data from the nasal wash and

nasal epithelium data are in agreement and only the nasal epithelium data are presented; as nasal epithelium likely represents the Napabucasin research buy persistent colonizing population [22]. Experimental Design For the population dynamics of nasal colonization, groups of 4-16 5-day-old rats were intranasally inoculated with either 104 or 107 cfu bacteria of S. aureus, S. pneumoniae or H. influenzae and sampled 12-144 hours after inoculation. Inoculum independence was confirmed by inoculating groups of 7-16 5-day-old rats with 102- 108 cfu bacteria of S. aureus, S. pneumoniae or H. influenzae and sampling at 48 hours. For intra-species invasion, one marked variant of a particular strain was intranasally inoculated into two groups of 24-36 3-day-old rats.

diphtheriae protein DIP1281 was, as its homologs Ce1659, Cg1735, and JK0967 in Corynebacterium efficiens, Corynebacterium glutamicum, and Corynebacterium jeikeium, previously annotated as hypothetical invasion-associated protein. Generation and analyses Smad inhibitor of mutant strains indicate that DIP1281 is predominantly involved in the organization of the outer surface protein layer of C. diphtheriae rather than in the

separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface of bacteria. Specific interaction partners for DIP1281 and its homologs in other corynebacteria are unknown and might be the focus of further studies to unravel the specific functions and targets of these proteins on a molecular level. Methods Bacterial strains and growth Strains used in this study are listed in check details Table 2. Escherichia coli DH5αMCR was grown in Luria Bertani (LB) medium at 37°C, C. diphtheriae in Heart Infusion (HI) broth at 37°C. If appropriate, kanamycin was added (30 μg/ml for E. coli; 50 μg/ml for C. diphtheriae). Table 2 Bacterial strains and eukaryotic cells used in this study. Strains Description Reference C. diphtheriae     DSM44123 non-toxigenic isolate, type strain DSMZ (Braunschweig) ISS3319 C. diphtheriae var. mitis, non-toxigenic isolate [9] ISS4060

C. diphtheriae var. gravis, non-toxigenic isolate [9] Lilo1 ISS3319 DIP1281::JNK-IN-8 purchase pK18mob’DIP1281” This study Lilo2 ISS4060 DIP1281::pK18mob’DIP1281” This study E. coli     DH5αMCR endA1 supE44 thi-1 λ- recA1 gyrA96 relA1 deoR Δ(lacZYA-argF) U196 φ80ΔlacZ ΔM15 mcrA Δ(mmr hsdRMSmcrBC) [28] Cell lines     Detroit562 human hypopharyngeal carcinoma cells [29] Preparation of C. diphtheriae protein extracts To prepare surface proteins, bacteria

were grown in 20 ml HI broth (with kanamycin added for the mutant strains) for approximately six hours and used to inoculate 250 BCKDHA ml HI broth for overnight growth. Bacteria were harvested by centrifugation at 5,000 × g for 20 min, washed twice with pre-cooled (4°C) 50 mM Tris-HCl buffer (pH 7.2), resuspended in 50 mM Tris-HCl (pH 7.2) containing 2% 3-[(3-choamidopropyl)-dimethylammonio] propanesulfonate (CHAPS) and incubated on ice overnight, followed by centrifugation at 3,500 × g and 4°C for 30 min to separate the cell surface proteins. After filtration of the protein solution using 0.45 μm pore-size filters (SARSTEDT, Nümbrecht, Germany), further preparation of the surface proteins by phenolic acid extraction and methanol precipitation followed a protocol described by Watt and co-workers [23]. The precipitated proteins were harvested by centrifugation at 3,500 × g and 4°C for 30 min. The pellet was washed twice with 3 ml of 70% ethanol (-20°C) and once with 3 ml of acetone (-20°C). Finally, the protein pellet was dried on ice and solubilised in 450 μl of dehydration buffer (8 M urea, 20 mM DTT, 2% CHAPS).