Methods Fungal strains and culture conditions P chrysogenum NRRL

Methods Fungal strains and culture conditions P. chrysogenum NRRL 1951, the natural isolate obtained from an infected cantaloupe [43] was used as wild-type strain. P. chrysogenum Wis54-1255, which contains a single copy of the penicillin gene cluster [6], was used as parental strain. P. chrysogenum npe10-AB·C [11], a derivative of the npe10 pyrG- strain (Δpen) [9, 10] complemented with the pcbAB and pcbC genes was used in the molecular analysis of IAT. P. chrysogenum DS54465 strain, a derivative of DS17690 [28] wherein the P. chrysogenum Cabozantinib mouse KU70 homologue has been deleted (Marco A. van den Berg, unpublished results), were used in the ial

gene deletion experiments. Fungal spores were collected from plates in Power medium [44] grown for 5 days at 28°C. P. chrysogenum liquid cultures were initiated by inoculating fresh spores in complex medium CIM (20 g/l corn steep solids, 10 g/l yeast extract, Sirolimus ic50 58 mM sucrose, 50 mM calcium

carbonate, pH 5.7) or defined DP medium [44] without phenylacetate. After incubation at 25°C for 20 h in an orbital shaker (250 rpm), aliquots were inoculated in complex penicillin production CP medium (4 g/l potassium phenylacetate, 20 g/l pharmamedia, 50 g/l lactose, 0.03 M ammonium sulphate, 0.05 M calcium carbonate, pH 6.6) or in defined DP medium with or without phenylacetate (4 g/l). Spores of the ial null mutant were used to inoculate shake flasks with synthetic media supporting β-lactam production [45]. To verify the validity

of the findings, two different penicillin side chain precursors were added to the media, phenyl acetic acid and adipate, at 0.3 and 0.5 g/l respectively. Cultivation was for 168 hours at 25°C and 280 rpm. As controls both parent strains, DS17690 and DS54465, were used. Plasmid constructs To completely block the transcription of the ial gene, 1500 base pairs of the promoter and the ORF were PCR amplified (for oligonucleotides see the Appendix) and fused to the amdS selection marker, obtained from pHELY-A1 [46] by DOK2 PCR amplification (Fig. 2). To block eventual read trough from any unconventional transcription start sites in the amdS gene, the trp terminator was PCR amplified from plasmid pAMPF21 [47] and inserted between the amdS gene and the ial ORF (Fig. 2). Plasmid p43gdh-ial was constructed to overexpress the ial gene in P. chrysogenum starting from plasmid pIBRC43BglII, a derivative of pIBRC43 [48] that contains the NcoI restriction site mutated to BglII. The ial gene was amplified from genomic P. chrysogenum Wis54-1255 DNA using the primers DElikeF and DElikeR (see the Appendix) and was cloned in the BglII-StuI sites of plasmid pIBRC43BglII, between the A. awamori gdh gene promoter (a very efficient promoter in ascomycetes) and the Saccharomyces cerevisiae cyc1 transcriptional terminator.

Here we demonstrated that truncated Scl1 fused with OmpA was dire

Here we demonstrated that truncated Scl1 fused with OmpA was directed to the outer membrane fraction of E. coli by western blot analysis, and likely exposed on the surface of E.

coli by FACS analysis. While ectopic expression of Scl1 on the heterologous bacteria E. coli is an alternative approach to reduce the potential interference of other factors on the surface of S. pyogenes, there are some limitations in our study. For example, it can not be ruled out that Scl1 protein was secreted to the periplasmic space, because Scl1 was constructed after the OmpA signal sequence. To avoid this problem, we performed FACS analysis on whole bacteria using Scl1 antibodies to detect the location of Scl1 in/on E. coli. FACS www.selleckchem.com/products/Everolimus(RAD001).html analysis has been widely used in identification of cell surface molecules in many immunologic and hematologic studies. Furthermore, we isolated proteins from the outer membrane fraction and confirmed the existence of Scl1 by western blot analysis with antibodies DNA Synthesis inhibitor against Scl1 and its fusion protein OmpA. However, the proper folding of ectopically expressed Scl1 and the integrity of the outer membrane of E. coli account for other issues influencing our interpretation of Scl1 in adhesion. Nevertheless, our findings concerning the adherence of Scl1-expressed E. coli to human epithelial cells unequivocally show that Scl1 contributes significantly to the adhesion of bacteria to human

epithelial cells. Collagen is a triple-helical, elongated protein structure the that is the main structural component

of the extra-cellular matrix in all multicellular organisms. Collagen-like sequences are found not only in proteins of multicellular organisms but also in proteins of microorganisms, such as a pullulanase in Klebsiella pneuminiae [28] and a platelet aggregation-associated protein in S. sanguis [29, 30]. Moreover, collagens interact with several macromolecules in a specific manner, suggesting that the collagen-like repeat sequences not only play a basic structural role, but also have a functional significance. Many eukaryotic cells bind collagen through integrins expressed on their surface [11]. Studies have demonstrated that the recombinant Scl1.41 protein interacted with α2β1 and α11β1 integrins, induced intracellular signaling in host cells, and promoted the internalization of S. pyogenes [9, 12, 13]. While the hypothesized region mediating the binding to α2β1 and α11β1 integrins in the recombinant Scl1.41 is in a motif called the GLPGER motif [9, 12, 13], Scl1 protein of S. pyogenes M29588 strain in our study does not contain the GLPGER motif. The novel aspect of this study is the observation that, in this Scl1 sequence type, the GLPGER motif is absent, yet adherence is maintained. Nevertheless, our results indicate that protein receptors, α2 and β1 integrins, contribute to Scl1-dependent binding to the surface of human epithelial cells.

Detection of anti-MtsA antibodies in sera from Kunming mice that

Detection of anti-MtsA antibodies in sera from Kunming mice that were experimentally infected with S. iniae HD-1 To detect the presence of specific anti-MtsA antibodies in the sera from Kunming mice, 10 male Kunming mice (20 ± 2 g) were purchased from Guangdong Laboratory Animals Research Center, and approval from the Animal Ethics Committee

of Life Sciences Institute was obtained prior to using the animals for research. The experiments were performed as stipulated by the China State Science and Technology Commission [47]. Mice were acclimatized at the SPF animal center and fed twice daily for 2 weeks in the laboratory Roscovitine solubility dmso of the Life Science Institute prior to use. Each mouse was injected with 100 μl of 6.2 × 108 CFU ml-1 S. iniae HD-1 cells, and the infected sera were collected 10 days post infection. The infected sera and purified MtsA were used in dot-blot and western-blot assays. The sera from 10 Kunming mice injected with PBS were used as the negative control. Statistical analysis The nucleotide and deduced amino acid homology analysis of mtsABC was carried out by ClustalX 1.83 and NCBI BLAST http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi.

The presumed signal sequence was predicted by the signalP 3.0 Server http://​www.​cbs.​dtu.​dk/​services/​SignalP/​. The theoretical pI/MW was analyzed by the ExPASy Compute pI/MW tool http://​www.​expasy.​org/​tools/​pi_​tool.​html. Small molecule library in vitro The main domains of mtsABC were detected by the SMART software http://​smart.​embl-heidelberg.​de/​. The amino acid sequences DNA Methyltransferas inhibitor were aligned using the SECentral Align Multi 4 program. To determine

whether mtsABC is a Lipoprotein, its sequence was assessed by the ScanProsite analysis software http://​www.​expasy.​ch/​tools/​scanprosite/​. All statistical analyses were performed using the SPSS 16.0 software (SPSS Inc., USA). Acknowledgements Project support was provided in parts by grants from Key Projects in the National Science & Technology Pillar Program in the Eleventh Five-year Plan Period (2007BAD29B05) to Dr. An-Xing Li. Project support was provided in parts by grants from Chongqing Engineering Technology Research Centre of Veterinary Drug (CSTC, 2009CB1010) to Dr. Lili Zou. We thank Prof. Shaoping Weng and Drs. Lichao Huang, Xiangyun Wu, Yangsheng Wu, Jianfeng Yuan, and Suming Zhou for their helpful technical advice. We also thank Dr. Shenquan Liao for providing plasmid pet-32a-c (+) used in this study, and the professional copyediting service from the International Science Editing. Electronic supplementary material Additional file 1: Tables 1-7. Microsoft word file containing Tables 1-7 as individual tab-accessible tables within a single file (Supplemental Tables 1-7). (DOC 128 KB) Additional file 2: Figures 1-4. Microsoft word file containing Figures 1, 2, 3, 4 as individual tab-accessible figures within a single file (Supplemental Figures 1-4). (DOC 358 KB) References 1.

Approved standard, NCCLS document M2-A8 8 Edition NCCLS, Wayne, P

Approved standard, NCCLS document M2-A8 8 Edition NCCLS, Wayne, Pa 2003. 19. Ward LR, de Sa JD, Rowe B: A phage-typing scheme for Salmonella enteritidis. Epidemiol Infect 1987,99(2):291–294.CrossRefPubMed 20. Anderson ES, Ward LR, Saxe MJ, de Sa JD: Bacteriophage-typing designations of Salmonella typhimurium. J Hyg (Lond) 1977,78(2):297–300.CrossRef 21. Ribot EM, Fair MA, RAD001 Gautom R, Cameron DN, Hunter SB, Swaminathan B, Barrett TJ: Standardization of pulsed-field gel electrophoresis

protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis 2006,3(1):59–67.CrossRefPubMed 22. Lindstedt BA, Vardund T, Aas L, Kapperud G: Multiple-locus variable-number tandem-repeats analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and multicolor capillary electrophoresis. J

Microbiol Methods 2004,59(2):163–172.CrossRefPubMed Authors’ contributions ND and MC conceived of and participated in MK-1775 cost the design of the study. ND drafted the manuscript. ND, JOC, GMD and GD carried out the serotyping, AST, PFGE and VNTR. MC helped to draft the manuscript. All authors read and approved the final manuscript.”
“Background Salmonella enterica serovar Enteritidis (SE) is one of the leading etiologic agents of non-typhoid fever [1]. The disease usually manifests as a self-limiting enteritis, although systemic spread of the infections accompanied by mortalities occurs in young and immunocompromised human patients [2]. Epidemiological studies suggest that poultry flocks may serve as a major reservoir for SE organisms implicated in human clinical cases [3]. Salmonella enterica silently colonizes the intestinal and reproductive tracts of chickens, which can provide a mechanism for SE-contamination of chicken meat, shell-eggs, and hatchery eggs if proper Oxalosuccinic acid processing and handling are not observed [4]. Recent investigations have shown that SE utilizes its type three secretion systems (T3SS) encoded by Salmonella pathogenicity island-1 and -2 (SPI-1

and SPI-2), respectively, to promote intestinal and reproductive tract colonization [5–7]. The T3SS of Salmonellae functions as a needle-like apparatus that injects an array of effector proteins into host cells. The T3SS-1 effectors act in concert to modulate host cell cytoskeleton rearrangement, thereby facilitating bacterial entry into host epithelial cells [8]. The T3SS-2 effectors promote bacterial survival or replication within host phagocytes [9]. The T3SS effectors also shape the type of pathological changes associated with Salmonella infection via modulating host cytokine and chemokine expressions [10]. It has been commonly accepted that the outcomes of microbial infections, including salmonellosis, are largely determined by the type and magnitude of host systemic and local immune responses.

The expression of tppB was examined in mycelium from wild-type, Δ

The expression of tppB was examined in mycelium from wild-type, ΔtppB and tppB+. In the deletion mutant, no expression was detected, whereas in the complemented strain, the levels were in the same range as in the wild-type

(Figure 8B). From these experiments we concluded that the deletion of tppB causes the lowered trehalose levels in ΔtppB. However, since the plasmid carrying the wild-type version of the gene was lost in most conidia, the tppB+ strain was not included in the following experiments. Figure 8 Trehalose content of mycelium (A) and relative expression selleck chemicals of tppB (B). Error bars show standard error of the mean, based on three biological replicates, and for qPCR each biological replicate was calculated as the average of three technical replicates. To evaluate the importance of trehalose as a stress protectant, the trehalose contents of the ΔtppB mutant and the control strains were analyzed in early stages of germination, and were subjected to lethal and sub-lethal heat and oxidative stress as well as sub-lethal salt and acid stress. The trehalose levels in ΔtppB followed the same pattern of breakdown and re-synthesis as in the control strains, but they were consistently

lower in accordance with the lower initial value (Figure 9). Dormant conidia of ΔtppB were significantly less tolerant to heat stress compared to the control strains; After 60 min of heat stress, the survival of ΔtppB was 35% compared to 78% in wild-type. After an additional 60 min, Astemizole the survival of IWR1 ΔtppB further decreased to 2%

compared to 38% in wild-type. (Figure 10). These experiments were repeated with the new independent deletion mutant, ΔtppB2, and the results were identical to those for ΔtppB (data not shown). For the other stressors tested, benzoic acid, NaCl and H2O2, as well as long-term viability where conidia were stored in water at 4°C for a total of 8 weeks, no significant differences between the mutant and the control strains could be detected (data not shown). Figure 9 Concentration of trehalose during outgrowth of wild-type, pyrG +  and ΔtppB conidia. Note the scale break between 12 and 72 h and that pyrG + observations are horizontally offset to avoid visual overlap. The error bars represent the standard error of the mean. The level of trehalose in ΔtppB was significantly different compared to wild-type for all time points except 3 h (two-way ANOVA, P < 0.0001 at 0, 6 and 12 h, and P < 0.01 at 72 h). Figure 10 Viabilities of dormant A. niger conidia after subjection to heat stress. Conidia were held at 55°C for 20, 60, 90 and 120 min. For all strains, the numbers of counted colonies were normalized to 25 at time = 0 min to avoid differences in numbers of assayed spores. Note that pyrG + observations are horizontally offset to avoid visual overlap. There were no significant differences between the control strains (N402 and pyrG+).

7% in athletes during caloric restriction

lasting four to

7% in athletes during caloric restriction

lasting four to eleven weeks resulted in reductions of fat mass of 21% in the faster weight loss group and 31% in the slower loss group. In addition, LBM Pexidartinib increased on average by 2.1% in the slower loss group while remaining unchanged in the faster loss group. Worthy of note, small amounts of LBM were lost among leaner subjects in the faster loss group [13]. Therefore, weight loss rates that are more gradual may be superior for LBM retention. At a loss rate of 0.5 kg per week (assuming a majority of weight lost is fat mass), a 70 kg athlete at 13% body fat would need to be no more than 6 kg to 7 kg over their contest weight in order to achieve the lowest body fat percentages recorded in

competitive bodybuilders following a traditional three month preparation [4, 6, 17–20]. If a competitor is not this lean at the start of the preparation, faster weight loss will be required which may carry a greater risk for LBM loss. In a study of bodybuilders during the twelve weeks before competition, male competitors reduced their caloric intake significantly during the latter half and subsequently lost the greatest amount of LBM in the final three weeks [21]. Therefore, diets longer than two to four months yielding weight loss of approximately 0.5 to 1% of bodyweight weekly selleck screening library may be superior for LBM retention compared to shorter or more aggressive diets. Ample time should be allotted to lose body fat to avoid an aggressive deficit and the length of preparation should be tailored to the competitor; those leaner dieting for shorter periods than those with higher body fat percentages. It must also be taken into consideration that the leaner the competitor becomes the greater the risk for LBM loss [14, 15]. As the availability of adipose tissue declines the likelihood of muscle loss increases, thus it may be best to pursue a more gradual approach to weight loss towards the

end of the preparation diet compared to the beginning to avoid LBM loss. Determining macronutrient intake Protein Adequate protein consumption during contest preparation is required to support maintenance of LBM. Athletes require higher protein intakes to support increased activity check details and strength athletes benefit from higher intakes to support growth of LBM [5, 22–28]. Some researchers suggest these requirements increase further when athletes undergo energy restriction [13, 16, 22, 28–33]. Furthermore, there is evidence that protein requirements are higher for leaner individuals in comparison to those with higher body fat percentages [7, 33, 34]. The collective agreement among reviewers is that a protein intake of 1.2-2.2 g/kg is sufficient to allow adaptation to training for athletes whom are at or above their energy needs [23–28, 35–38]. However, bodybuilders during their contest preparation period typically perform resistance and cardiovascular training, restrict calories and achieve very lean conditions [2–6, 17–21].

Drug resistance in tuberculosis (TB) is a matter of great concern

Drug resistance in tuberculosis (TB) is a matter of great concern for TB control programs since these strains could spread in the community, stressing the need for early detection of drug resistance and subsequently initiation Nutlin-3a nmr of adjusted therapy. Conventional diagnosis of drug-resistance in MTB strains relies heavily upon mycobacterial culture and drug susceptibility testing in liquid or solid media. Usually, results are only obtained

after weeks to months of incubation and many developing countries lack the resources to establish the stringent laboratory conditions needed for these growth-based methods. From a clinical perspective, the existing growth-based diagnostics are too slow as patients undergoing treatment with drugs to which they are resistant, remain contagious, and those with XDR-TB and HIV often die before they are even diagnosed [6]. Major advances in molecular biology and the availability of new information generated after deciphering

the complete genome sequence of M. tuberculosis[7], Ibrutinib cell line have led to the development of new tools for rapid detection of drug resistance [8, 9]. Molecular methods are based on assigning the presence or absence of certain mutations in specific positions or genetic locations which are known to be associated with resistance [10]. About 95% of rifampicin (RIF) -resistant strains have mutations in the 81-bp core region of the rpoB gene encoding the β-subunit of the RNA polymerase, named RIF-Resistance Determining Region (RRDR) Silibinin [11]. In contrast to RIF, the situation for isoniazid (INH) is much more complex. Resistance mutations have been reported in at least 4 different genes including katG, inhA, ahpC and oxyR[10]. Meanwhile, resistance

against streptomycin (SM) has been reported to be associated with mutations in rrs gene, which codes for 16S ribosomal RNA, and rpsL coding for the ribosomal protein S12 [12] and these mutations are found in a limited proportion of clinically isolated SM-resistant M. tuberculosis strains. Recently, Okamoto et al. [13] found that mutations within the gidB gene which encodes a conserved 7-methylguanosine (m7G) methyltransferase specific for the 16S rRNA, is associated with low-level SM-resistance and are an important cause of resistance found in 33% of resistant M. tuberculosis isolates. Resistance to ethambutol (EMB) is primarily mediated by mutations in the embB gene, coding for an arabinosyltransferase participating in mycobacterial cell wall synthesis, with codon 306 being most frequently affected [14]. Furthermore, mutations in the embA[15, 16] and upstream of embC[16, 17] are also involved in EMB -resistance.

At all time points (24, 48 and 72 hours) IC50 was greater than 10

At all time points (24, 48 and 72 hours) IC50 was greater than 100 μg/mL. The screening

test for the JC cells with doses of 1, 10 and 100 μg/mL measured for 1 μg/mL: after 24 hours showed cell viability of 98%; after 48 hours 97%; and after 72 hours H 89 chemical structure 70%; for 10 μg/mL: after 24 hours cell viability showed 85%; after 48 hours 84%; and after 72 hours 21%; for 100 μg/mL: after 24 hours cell viability showed 77%; after 48 hours 84%; and after 72 hours 8%. At the time points 24 and 48 hours IC50 was greater than 100 μg/mL and at 72 hours IC50 was 2.5 μg/mL (95% confidence interval (C.I.) 0.22 to 28 μg/mL). A similar type of biological assay was performed with the find more purified

compound EPD at final concentrations of 1, 5 and 10 μg/mL for 24, 48 and 72 hours (Table 1). Percent of cell reduction for normal fibroblasts at 72 hours at the highest dose (10 μg/mL) was approximately 30%, while IC 50 was greater than 10 μg/mL. Screening tests for OVCAR3 and SKOV3 cells showed that more than 50% and 80% of cells were killed at doses of 5 and 10 μg/mL, respectively. Table 1 Cell viability with EPD treatment of normal fibroblasts, OVCAR3 and SKOV3 cancer cells (average (AV) and standard deviation (SD))   % cell viability:

average and standard deviation EPD Conc 24 hours 48 hours 72 hours μg/mL AV SD AV SD AV SD   Normal fibroblasts 1 102 2.5 107 3.9 105 3.3 5 105 6.3 108 1.6 72 2.1 10 101 10.1 112 1.8 47 4.6   OVCAR 3 1 96 5.1 101 7.4 109 29.2 5 87 6.7 67 4.5 50 14.4 10 70 7.4 23 0.9 21 6.4   SKOV 3 1 103 5.0 123 CYTH4 8.2 119 6.0 5 102 4.0 96 18.2 69 16.5 10 86 11.6 31 36.0 23 1.8 IC50 for OVCAR3 at 24 hours was 13 μg/mL (95% C.I. 10 to 18 μg/mL), at 48 hours 6.4 μg/mL (95% C.I. 5.3 to 7.8 μg/mL) and at 72 hours 5.3 μg/mL (95% C.I. 4.3 to 6.5 μg/mL). IC50 for SKOV3 at 24 hours was 16 μg/mL (95% C.I. 9.4 to 27 μg/mL), at 48 hours 8.4 μg/mL (95% C.I. 6.7 to 11 μg/mL) and at 72 hours 6.5 μg/mL (95% C.I. 5.2 to 8.3 μg/mL). In vivo pilot experiment Control mice only injected with the OVCAR3 cells, were killed when the ascites became a burden. EPD (at final concentration of 20 mg/kg b.w.) was administered i.p. twice/week for six weeks and Cisplatin (at final concentration of 5 mg/kg b.w.) was administered i.p. during 4 weeks, once/week. In general a similar cytotoxic effect was observed between EPD and Cisplatin on the OVCAR3 cells.

As seen in Table 3, the rectification factor dropped to 2 and 3,

As seen in Table 3, the rectification factor dropped to 2 and 3, close to that of the expected as-made membranes. The disappearance of rectification effect provided

supportive evidence that the functional anionically charged dye played as gatekeeper to modulate the ionic flux through DWCNT membranes. Table 3 Summary of ionic this website rectification factor on DWCNT membrane after water plasma oxidation to remove gatekeepers Concentration Rectification factor (mM) Potassium ferricyanide NDS Sodium benzenesulfonate 10 3.2 ± 0.3 1.7 ± 0.2 2.4 ± 0.2 50 2.8 ± 0.3 1.5 ± 0.07 2.0 ± 0.2 100 2.4 ± 0.2 1.4 ± 0.0.02 2.0 ± 0.2 Ferricyanide has a well-known redox potential of 0.17 V (vs. Ag/AgCl), and thus, an important control experiment was AG-014699 cost done to make sure that the observed rectification was not due to faradic current; instead, it was due to transmembrane ionic current. Cyclic voltammetry scans (−0.6 to 0.6 V) showed no redox reaction on both as-made and one-step functionalized DWCNT membranes in 50-mM ferricyanide (Additional file 3: Figure S3). We also did not observe redox reaction on glassy carbon in 2-mM ferricyanide, as seen in the flat curve in Additional file 4: Figure S4A. The much larger conductive

area of the glassy carbon electrode compared to 5% DWCNT membrane requires the use of more diluted (2 mM) ferricyanide solution. However, with the supporting 0.5-M electrolyte KCl solution, the oxidation and reduction peaks were observed at 0.29 and 0.06 V, which

were similar to those found in reports [30, 50]. The experiment was also repeated with both redox species. In Additional file 4: Methane monooxygenase Figure S4B, no redox peak was found on glassy carbon in 50-mM ferricyanide solution and 25-mM ferricyanide/ferricyanide solution. The control experiments of cyclic voltammetry on DWCNT membrane and glassy carbon ruled out the redox reaction of ferricyanide, which supports the ionic rectification on electrochemically grafted CNT membranes. The non-faradic (EIS) spectra indicated that the functionalized gatekeeper by a single step can be actuated to mimic the protein channel under bias. This functional chemistry was proven to be highly effective on the enhancement of ion rectification. The disappearance of rectification also supported its effectiveness after removing the grafted gatekeeper by plasma etching. Interestingly, no apparent change of rectification was seen for the two-step functionalization. The likely reason is that highly efficient functional density can be obtained by electrografting of amine in one step since the poor yield in the second step (carbodiimide coupling reaction) resulted in a significantly lower gatekeeper density on CNT membranes. To address this question, two- and one-step functionalizations were quantified using dye assay on glassy carbon due to its well-defined area and similar chemical reactivity to CNTs.

The average information depth of the present XPS measurement is l

The average information depth of the present XPS measurement is limited to approximately 8 to 10 atomic surface layers. One can see that with ongoing deposition, the concentration of silver increases, while the fluorine content decreases and becomes undetectable on the sample sputtered for 200 s. The decrease is due to the increasing masking effect of the growing Ag layer which at last becomes homogeneous and continuous. On the other hand, with decreasing thickness of Ag layer, its masking effect gradually declines, e.g., because of the appearance of cracks and discontinuities in the layer, and the chemical structure of the underlying PTFE becomes

visible in the XPS spectra. For the sputtering

time of 20 s, the measured fluorine concentration of 37.3 at.% FK506 is close to that of the pristine PTFE. The F/C ratio of silver-sputtered samples is markedly different from that of the pristine PTFE (F/C = 2:1) CP-690550 and may be due to the ability of silver to attract hydrocarbon contaminants from ambient atmosphere [27]. The thicker the sputtered layer, the lower the F/C ratio. This effect is most pronounced in the case of the thickest Ag layer (200-s sputtering time), where fluorine is not detected because of the masking effect of the silver coating. However, the concentration of carbon is still notable (54 at.%) in this case. The origin of carbon may completely be attributed to the contamination with hydrocarbons and other carbon-rich compounds from ambient atmosphere. XPS data (Table 1) also elucidate the processes in the course of the sample relaxation. During the 14 days of relaxation, the surface chemical composition changes significantly. A gradual decrease of the detected silver content, compared to that of the as-sputtered samples, occurs as a consequence of the tendency to minimize surface energy at the metal-polymer interface. This phenomenon has been frequently observed especially in the case of plasma-treated polymers,

where oxygen-containing groups reorient towards polymer volume in order to reduce surface energy in the contact with Nintedanib (BIBF 1120) ambient atmosphere [28]. Thus, the relaxation leads to segregation on the metal-polymer interface and boarding of cracks in the silver coating (Table 1, increase of fluorine content). This process favorably affects the surface wettability which finally stabilizes at a constant level (Figure 1). However, there are other concurrent processes that make the simple and straightforward explanation of the observed phenomena difficult (e.g., anomalous decrease of fluorine content for deposition time of 20 s, Table 1). This may particularly be caused by random, uncontrollable adsorption of hydrocarbons from ambient atmosphere during the relaxation process (see decrease of oxygen content at 100 and 200 s deposition times, Table 1).