The fitting results for the different samples resulted in a PL decay time in the range of 19 to 23 μs and a constant β in the range of 0.85 to 0.95. The PL results are discussed in detail in the ‘Discussion’ section. The differences in the PL behavior of the different samples can be explained by taking into account

that the studied samples constitute very complicated systems of nanowires composed of nanocrystals of different sizes and different surface chemical compositions that, in addition, present different structural defects at their surface. Depending on the chemical treatment, the mean size of the nanocrystals composing the nanowires and their surface chemical composition are different. Moreover,

the number and nature of the structural defects change. Both surface composition and structural defects introduce states in the nanocrystal energy bandgap that influence the PL selleckchem recombination mechanism. In addition, the porous Si layer underneath the SiNWs contributes to the PL signal. The above will be discussed in detail for each sample in the ‘Discussion’ section. FTIR analysis The surface composition of the four different samples was characterized by FTIR find more Saracatinib transmittance analysis. The results are depicted in Figure 5. The spectra of the as-grown and the piranha-treated samples are similar, showing the characteristic asymmetric stretching signals of the Si-O-Si bridge between 1,000 and 1,300 cm−1, with a strong band at 1,080 cm−1 and a shoulder at 1,170 cm−1[22]. Furthermore, a strong broad signal between 3,000 and 3,650 cm−1 is present, attributed to the stretching signal of the SiO-H bond [22]. Finally, the peak at 626 cm−1 is in general attributed to the Si-H bond [22]. However, since no other vibrations of the Si-H bond are present, this peak can be attributed to the wagging vibration mode of the OSi-H bond. On the other hand, the FTIR transmittance spectra after the

first and the second HF dip (Figure 4, spectra 2 and 4) do not show any significant surface oxide signature, since the surface oxide has been removed by the HF. The characteristic asymmetric stretching signals of the Si-O-Si bridge between 1,000 and 1,300 cm−1 and the wagging and stretching points of O3Si-H at 847 and 2,258 Fossariinae cm−1 are too weak. Instead, the transmittance peaks due to different vibration modes of the SiHx bond (the wagging and stretching vibration modes of Si-H bond at 623 and 2,112 cm−1, and the wagging, scissors, and stretch vibration modes of Si-H2 bond at 662, 908, and 2,082 cm−1) respectively [22] are too strong, corresponding to the hydrogen signature at the SiNW surface. These results are exactly what one could expect from a Si surface after the above chemical treatments. Figure 5 FTIR transmittance spectra of SiNWs.

Ågren J, Sundström A, Håfström T, Segerman B: Gegenees: fragmented alignment

of multiple genomes for determining phylogenetic distances and genetic signatures unique for specified target groups. PLoS One 2012,7(6):e39107.PubMedCentralPubMedCrossRef AZD0156 nmr 32. Sota M, Endo M, Nitta K, Kawasaki H, Tsuda M: Characterization of a class II defective transposon carrying two haloacetate dehalogenase genes from Delftia acidovorans plasmid pUO1. Appl Environ Microbiol 2002,68(5):2307–2315.PubMedCentralPubMedCrossRef 33. Tsuda M, Iino T: Genetic-analysis of a transposon carrying toluene degrading genes on a TOL plasmid pWWO. Mol Gen Genet 1987,210(2):270–276.PubMedCrossRef 34. Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M: ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res 2006,34(Database issue):D32-D36.PubMedCentralPubMedCrossRef 35. Didelot X, Barker M, Falush D, Priest FG: Evolution of pathogenicity in the Bacillus cereus group. Syst Appl Microbiol 2009,32(2):81–90.PubMedCrossRef 36. Hu X, Hansen BM, Yuan Z, Johansen JE, Eilenberg J, Hendriksen NB, Smidt L, Jensen GB: Transfer

and expression of the mosquitocidal learn more plasmid pBtoxis in Bacillus cereus group strains. FEMS Microbiol Lett 2005,245(2):239–247.PubMedCrossRef 37. Yuan Y, Zheng D, Hu X, Cai Q, Yuan Z: Conjugative transfer of insecticidal plasmid pHT73 from Bacillus thuringiensis to B. anthracis and compatibility of this plasmid with pXO1 and pXO2. Appl Environ Microbiol 2010,76(2):468–473.PubMedCentralPubMedCrossRef 38. Rasimus S, Mikkola R, Andersson MA, Teplova VV, Venediktova N, Ek-Kommonen C, Salkinoja-Salonen M: Psychrotolerant Paenibacillus tundrae isolates from barley grains produce new cereulide-like depsipeptides (paenilide and homopaenilide) that are highly toxic to mammalian cells. Appl Environ Microbiol 2012,78(10):3732–3743.PubMedCentralPubMedCrossRef 39. Van der Auwera GA,

Feldgarden M, Kolter R, Mahillon J: Whole-genome sequences of 94 environmental isolates of Bacillus cereus sensu lato . Genome Announc 2013.,1(5): 40. Hu XM, Van der Auwera G, Timmery S, Zhu L, Mahillon J: Distribution, diversity, and potential mobility of MM-102 extrachromosomal Thiamet G elements related to the Bacillus anthracis pXO1 and pXO2 virulence plasmids. Appl Environ Microbiol 2009,75(10):3016–3028.PubMedCentralPubMedCrossRef 41. Eickbush TH: Mobile introns: Retrohoming by complete reverse splicing. Curr Biol 1999,9(1):R11-R14.PubMedCrossRef 42. Ferat JL, Michel F: Group II self-splicing introns in bacteria. Nature 1993,364(6435):358–361.PubMedCrossRef 43. Jia KZ, Zhu Y, Zhang YP, Li Y: Group II intron-anchored gene deletion in Clostridium . PLoS One 2011.,6(1): 44. Belhocine K, Yam KK, Cousineau B: Conjugative transfer of the Lactococcus lactis chromosomal sex factor promotes dissemination of the Ll.LtrB group II intron. J Bacteriol 2005,187(3):930–939.PubMedCentralPubMedCrossRef 45.

Comparison of individual libraries The Shared OTUs and Similarity (SONS) program [24] was used to compare the unfractioned sample with each of the %G+C fractions and with the combined sequence data from the fractions (Table 3). Using a 98% similarity criterion for the phylotypes, at least 80% of sequences from %G+C fractions Evofosfamide price 30–35 and 35–40 were shared with the unfractioned sample (Vobs values). However, for two of the high %G+C content fractions with %G+C content from 55 to 65, the Vobs values were considerably lower (32–33%). When comparing the combined sequence data from the fractioned sample with the unfractioned sample, a higher percentage of sequences

and OTUs in the unfractioned were shared. Table 3 Results from library comparisons with SONS [24]. Library A Unfractioned Uobs a Vobs b Aotu_shared c Botu_shared d Library B Fr G+C 25–30% 0.41 0.40 0.22 0.34 Library B Fr G+C 30–35% 0.59 0.83 0.40 0.56 Library B Fr G+C 35–40% 0.67 0.82 0.44 0.64

Library B Fr G+C 40–45% 0.72 0.75 0.45 0.51 Library B Fr G+C 45–50% 0.62 0.63 0.33 0.40 Library B Fr G+C 50–55% 0.34 0.64 0.20 0.40 Library B Fr G+C 55–60% 0.18 0.33 0.13 0.34 Library B Fr G+C 60–65% 0.44 0.32 0.17 0.36 Library B Fr G+C 65–70% 0.68 0.53 0.39 0.39 Library B Fr G+C 70–75% 0.69 0.67 0.42 OSI-906 clinical trial 0.47 Library B Fr G+C 25–75%e 0.92 0.60 0.81 0.26 a. Fraction of sequences observed in shared OTUs in library A b. Fraction of sequences observed in shared OTUs in library B c. Fraction of shared OTUs in library A d. Fraction of shared OTUs in library B e. The combined

G+C fractions Shannon entropies of clone libraries of the %G+C profiled sample The %G+C fractions 50–55 and 55–60 had comparatively low Shannon entropies (Additional file 2), indicating lower diversity, and were abundant with bifidobacteria (Figure 2, Additional file 1). The peripheral %G+C fractions and the %G+C fraction 45–50 with sequences affiliating mainly with Clostridium clusters IV and XIV had comparatively higher diversity according to Shannon entropies. The peripheral fraction from the low %G+C end (25–30% G+C content) contained a substantial proportion of Firmicutes that do not belong to the Clostridum clusters IV and XIV. It had the highest Shannon entropy (Additional file 2), indicating rich diversity, and did Chloroambucil not reach a plateau in the rarefaction curves (data not shown), which means that more OTUs would have been likely to appear after further sequencing. Discussion For a comprehensive evaluation of the human intestinal microbiota, 16S rRNA gene clone libraries were selleck compound constructed from a %G+C fractioned pooled faecal DNA sample of 23 healthy subjects followed by a sequence analysis of 3199 clones. Previously, only selected fractions of such profiles have been sequenced and analysed.

One isolate per patient was analyzed, and each isolate represented a single case. Isolates were cultured in Luria-Bertani (LB) broth and stored at -80°C until use. Medical records were reviewed and information related to clinical manifestations and underlying diseases was collected. Clinical research was conducted according to the human experimentation guidelines of Chung-Shan Medical University. Ethical approval was not needed for the present study. Determination of the hypermucoviscosity (HV) phenotype and detection of HV-related genes The HV phenotype display was examined with a string-formation test as described by Fang et al [14]. Bacterial strains to be tested

were inoculated onto 5% sheep blood plates and incubated at 37°C for 16 h. Positive of hypermucoviscosity see more phenotype was defined as the formation of viscous strings > 5 mm in length when a standard inoculation loop was used to stretch the colony on blood agar plates. K. CFTRinh-172 order pneumoniae isolates, capable of displaying

the HV-phenotype from three independent tests were described as HV-positive and those that were unqualified in string forming were HV-negative. Induction of diabetes in mice Six-week-old male C57BL/6J mice were purchased from the National Laboratory Animal Center (NLAC, Taiwan) and allowed to NVP-BSK805 molecular weight acclimatize in the animal house for one week before experiments. Mice (25-30 g body weight) were randomly divided into two groups. One group received intraperitoneal injection of the pancreatic β-cell toxin streptozotocin (STZ; Sigma) for five days (55 mg/kg per day in 0.05 M citrate PTK6 buffer, pH 4.5) [16]. The other group received injections of citrate buffer as the control. The serum glucose concentrations and body weights of the mice were determined at indicative time points after the multi-injection of STZ. Pneumonia or KLA infection models To recapitulate a

pneumonia infection, thirty-week-old mice were anesthetized with isoflurane and intratracheally inoculated with 104 CFU of K. pneumoniae by intubation with a blunt-ended needle [28]. At 20 h post-inoculation, lungs and blood were retrieved, homogenized, and plated onto M9 agar for enumerating bacterial counts. Based on the KLA infection model established in our previous study [17], groups of two to four thirty-week-old diabetic or naïve mice were orally inoculated with 105 or 108 CFU of K. pneumoniae, respectively. Twenty microliter of blood was retrieved from the retroorbital sinus of infected mice at 24, 48, and 72 h post-inoculation for enumeration of bacterial counts. Survival of the infected mice was monitored daily for seven days. For histological examination, livers retrieved from mice were fixed in 4% paraformaldehyde, paraffin embedded, and stained with haematoxylin and eosin. All the animal experiments were performed according to NLAC guidance and the Institutional Animal Care and Use Committee approved protocols.

Finally, we received 24 completed T3 questionnaires of the 41 we had sent out (response 59%, or 44% of the original 54 patients). The characteristics of the participants at baseline are presented

in Table 1. The average age was 42 years, and 48% of the patients were women. Table 2 presents the baseline measurements (T0) of the perceived severity, the general BVD-523 datasheet quality of life as measured with a visual analogue scale and with the SF-36, the level of current health, the disease-specific functional impairment and the sickness absence. All of the subscale scores on the SF-36 and the DASH were statistically significant lower than the reference values of the general population. Table 1 Baseline measurements of participants with work-related upper extremity disorders (N = 48) Variable Number (%) Mean (SD) Age   42.4 (10.2) Sex  Women 23 (48%) Smad inhibitor   Education level  Primary school 3 (6%)    Lower vocational education

15 (31%)    Intermediate vocational education 17 (35%)    Higher vocational education/university 4 (8%)    Other 9 (19%)   Working hours per week   33.7 (7.8) Table 2 Baseline values of perceived severity, quality of life as measured with a visual analogue scale and the SF-36, the level of current health, the disease-specific functional Impairment (DASH) and sickness absence in the work-related upper extremity disorder patient population (N = 48) Variable Mean (SD/95% CI) Patients Mean general population p value Perceived severity (VAS 0-100) selleck compound 68 (SD: 24) na   General quality of life

(VAS 0-100) 84 (SD: 14) na   Current health (VAS 0-100) 57 (SD: 23) na   Quality of life (SF-36)  Physical functioning 74.2 (70.4–78.1) 89 <0.001*  Physical role functioning 20.8 (12.3–29.3) 82 <0.001* cAMP  Bodily pain 38.9 (33.5–44.2) 75 <0.001*  Social functioning 73.2 (66.4–80.0) 84 0.003*  Mental health 68.1 (62.7–73.5) 76 0.005*  Emotional role functioning 68.8 (57.1–80.5) 86 0.005*  Vitality 52.3 (46.9–57.7) 68 <0.001*  General health perceptions 65.0 (59.2–70.7) 74 0.003* Functional impairment (DASH) 43.8 (37.6–49.9) 13 <0.001* Percentage of days absent due to sickness in previous 2 weeks 32 (SD: 38) na   Number of days absent due to sickness in previous 3 months 28 (SD: 29) na   The results of the SF-36 and DASH measurements were compared with the reference values in the general population (one sample t test) na not available, * statistically significant Perceived severity of the disorder Measurements over time showed that in 67% of the patients the perceived severity of the disorder declined more than 10 points (scale 0-100) during 1 year of follow-up after notification. The average perceived severity of the disease declined statistically significant during the follow-up period from 68 at T0 to 40 at 1-year follow-up (p < 0.001).

When A549 cells were grown to approximately 60-70% confluence, they were washed five times with SFM to remove albumin

and other elements contained in FBS. Cells were then either infected with 10 CFU/cell of M. pneumoniae in SFM or left untreated for further conditioned media (CM) collection. Cell viability in SFM was assessed by MTT test and trypan blue exclusion assay, and the cell death was assessed ATPase inhibitor by apoptosis assay using the Annexin V-FITC/PI Kit (Multiscience, Hangzhou, China). Sample preparation The CM was harvested 24 h after infection by centrifugation at 9,000 g for 15 min to remove floating cells and cellular debris, and filtered through a 0.22 μm filter (Chemicon, Millipore, MA, USA). After the ABT-263 addition of protease inhibitors (Inhibitor cocktail complete, Roche Diagnostics, Mannheim, Germany), the media was concentrated

using the Amicon Ultra-15 (Millipore) centrifugal filter devices with a 3,000-nomina-weight limit (NMWL). The supernatants were subsequently precipitated by acetone at -20°C overnight, and harvested by centrifugation at 16,000 g for 20 min. The protein pellets were dried in air and then resuspended in an appropriate volume of reducing solution containing 6 M urea, 2 M thiourea and 25 mM ammonium bicarbonate (Sigma, St Louis, MO). The protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA). 100 μg of each sample was reduced with 10 mM DTT (Sigma) at 37°C for 2.5 h, and then carbamidomethylated with 50 mM iodoacetamide (IAA) (Sigma) at room temperature in the dark for 40 min. Subsequently, digestion was performed by sequencing grade trypsin (Promega, Madison, Idelalisib ic50 WI) using a 1:50 enzyme:protein

ratio at 37°C for 20 h. After digestion, samples were lyophilized under vacuum and kept at -80°C until use. Three independent experiments were performed and samples were prepared individually for further study. Total cell lysates from the A549 cells were prepared as previously described [3]. Briefly, cells were washed and detached on ice in phosphate-buffered saline (PBS), and lysed in cell lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, and 0.2% biolyte (Bio-Rad). The lysates were frozen and thawed with liquid nitrogen three times, and then centrifuged for 1 h at 10,000 g to remove cellular debris. The supernatant was then collected for further Western blot analysis. LC-MS/MS All of the mass analyses were performed using a nano-LC-MS/MS system, which consisted of a nano-HPLC system (the Ettan MDLC system; GE Healthcare, Piscataway, NJ) and a linear trap quadruple (LTQ) mass spectrometer (LTQ VELOS; Thermo Finnigan, San Jose, CA) equipped with a nano-ESI source. A RP trap column (Zorbax 300SB-C18 peptide traps, Agilent Technologies, Wilmington, DE) was used for desalting of samples, and a C18 reverse-phase column (150 μm i.d., 150 mm length, Column Linsitinib mw Technology Inc., Fremont, CA) was used for separation. Mobile phase A consisted of HPLC-grade water containing 0.

From the wealth of available data (see Additional Files 2, 3, 4, 5), we highlight in this report the most relevant conclusions. First, our study reinforces the idea that cell permeation is not the only mechanism required to fully describe the effect of, and response to, AMP in microorganisms [8–12]. We have also shown that PAF26 and melittin have common but also differential effects on yeast. Finally, a previously overlooked observation is that a significant part of the response relies on genes of unknown function, or with poorly informative GO terms associated to them. A remarkable example

of uncharacterized genes uncovered in our study is YLR162W, the only gene not related to ribosome biogenesis among the seven induced by melittin and repressed by PAF26 (Figure 2). It is a predicted gene QNZ in vivo of unknown function that codes for a small protein with potential transmembrane domains [49]. An independent study has shown that over expression of YLR162W this website confers resistance to the plant antimicrobial peptide MiAMP1 in a susceptible yeast strain [49]. Strikingly, our study indicates (in a different yeast genotype) that YLR162W reacts distinctly to different AMP, and thus highlights the

interest of studying its function since it might have an important and Enzalutamide molecular weight distinctive role in the response to AMP. BLAST searches do not show any homolog of this gene in known fungal sequences (data not shown). The role of the fungal cell wall in susceptibility to AMP The most obvious shared response is related to reinforcement of the cell wall. Among the 43 genes that were co-expressed in the peptide treatments (Figure 2), the only GO significant annotations were related to the fungal CW (Additional File 4.3). Additional studies found altered genes involved in CW maintenance in response to other antifungal agents or CW perturbants as well [38, 61, 62]. Among the previous genomic studies of the response to AMP in yeast, only the one that used the esculentin 1-21 peptide highlighted Ribonuclease T1 CW responses at the transcriptomic level [30],

while others did not [32, 33]. In addition, six genes (different from those found herein) were identified whose deletions confer increased sensitivity to either dermaseptin S3 or magainin 2 [33]. Our observations sustain that the improvement of CW integrity is a common response of S. cerevisiae to AMP. Further support arises from the data on BWG7a strain, which has a weakened CW phenotype related to a dysfunctional SSD1 allele [47] that compromises viability in the presence of AMP and at higher incubation temperatures (Additional File 1). Yeast cells are capable of reinforcing their CW when subjected to stress or damage conditions [64], and our study contributes to demonstrate that this is also the case after AMP treatment.

Biotechnol Lett 2006,28(4):207–213.PubMedCrossRef 9. Curley JM, Lenz RW, Fuller C: Sequential production of two different polyesters in the inclusion bodies of Pseudomonas oleovorans . Int J Biol Macromol 1996, 19:29–34.PubMedCrossRef 10.

Huisman GW, https://www.selleckchem.com/products/Vorinostat-saha.html Wonink E, De Koning GJM, Preusting H, Witholt B: selleckchem Synthesis of poly (3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains. Appl Microbiol Biotechnol 1992, 38:1–5.CrossRef 11. Stuart ES, Foster LJR, Lenz RW, Fuller RC: Intracellular depolymerase functionality and location in Pseudomonas olevorans inclusions containing polyhydroxyoctanoate. Int J Biol Macromol 1996, 19:171–176.PubMedCrossRef 12. Jurasek L, Marchessault RH: The role of phasins in the morphogenesis of poly(3-hydroxybutyrate) granules. Biomacromolecules 2002,3(2):256–261.PubMedCrossRef 13. Prieto MA, Bühler B, Jung buy PD173074 K, Witholt B, Kessler B: PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes. J Bacteriol 1999,181(3):858–868.PubMed 14. Ruth K, de Roo G, Egli T, Ren Q: Identification of two acyl-CoA synthetases from Pseudomonas putida GPo1: One is located at the surface of polyhydroxyalkanoates granules. Biomacromolecules 2008,9(6):1652–1659.PubMedCrossRef

15. Huisman GW, Wonink E, Meima R, Kazemier B, Terpstra P, Witholt B: Metabolism of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans . J Biol Chem 1991, 266:2191–2198.PubMed 16. García B, Olivera ER, Minambres B, Fernández-Valverde M, Canedo LM, Prieto MA, García JL, Martínez M, Luengo JM: Novel biodegradable aromatic plastics from a bacterial source. J Biol Chem 1999,274(41):29228–29241.PubMedCrossRef 17. de Eugenio LI, Garcia P, Luengo JM, Sanz JM, San Roman J, Garcia JL, Prieto MA: Biochemical evidence that phaZ gene encodes a specific intracellular medium-chain-length polyhydroxyalkanoate depolymerase in Pseudomonas putida KT2442 – Characterization of a paradigmatic enzyme. J Biol Chem 2007,282(7):4951–4962.PubMedCrossRef 18. Steinbüchel A, Aerts K, Babel W, Follner C, Liebergesell M, Madkour MH, Mayer F, Pieper-Fürst U, Pries A,

Valentin HE, et al.: Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions. Can J Microbiol 1995, 41:94–105.PubMedCrossRef 19. Ren Q, de Roo G, Ruth K, Witholt Branched chain aminotransferase B, Zinn M, Thöny-Meyer L: Simultaneous accumulation and degradation of polyhydroxyalkanoates: Futile cycle or clever regulation? Biomacromolecules 2009,10(4):916–922.PubMedCrossRef 20. Doi Y, Segawa A, Kawaguchi Y, Kunioka M: Cyclic nature of poly(3-hydroxyalkanoate) metabolism in Alcaligenes eutrophus . FEMS microbiol Lett 1990, 67:165–170.CrossRef 21. de Roo G, Ren Q, Witholt B, Kessler B: Development of an improved in vitro activity assay for medium chain length PHA polymerase based on CoenzymeA release measurements. J Microbiol Meth 2000, 41:1–8.CrossRef 22.

All tested strains, namely three urease positive streptococci [19] and LbGG, proved to be able to utilize N-acetyl-D glucosamine, but not D-glucuronic acid as well as HA. LbGG is a probiotic strain able to PRT062607 clinical trial survive to 30 min of exposure BTSA1 mouse to simulated gastric juice but not to 90 min [20]. Strain’s survival, evaluated

in presence of increasing concentration of HA (0.0125-1.6 mg ml-1) to simulated gastric juice for 90 min, highlighted a weak positive gastro-protective effect that appeared directly correlated to HA concentration: 1) At 1.6 and 0.8 mg ml-1 HA a five Log of reduction (from 7 to 2 CFU ml-1) was recorded; 2) At 0.4 and 0.2 mg ml-1 HA a 5.5 Log reduction (from 7 to 1.5 CFU ml-1) was recorded; 3) At HA concentration lower than 0.1 mg ml-1 no strain survival was detected. At the used concentrations, HA is not able to protect the probiotic

strain Lb. rhamnosus Selleck Napabucasin GG during a 90 minutes long exposition to simulated gastric juice, but further studies would be useful to understand if results may be improved by considering higher concentration of HA. A widely accepted in vitro system, which allows simultaneous evaluation of several HA doses, was compared with an innovative method based on the old concept of dynamic light scattering. By these two approaches comparable kinetic curves were obtained. Firstly, tests were performed on three selected urease positive strains belonging to Streptococcus (St.) thermophilus species in presence of growing concentrations of HA, until 48 h. As shown in Figure 1, each strain displayed a recurrent trend in the O.D. kinetics. In detail, curve profiles dropped after 24 h in all cases, showing a higher marked decrease

when HA concentration was higher. When lower concentrations Selleckchem Sorafenib of HA were used, O.D. decrease was limited. Strain 82A behaved as 247 and therefore was not shown. Figure 1 Effects of HA on St. thermophilus strains 309 and 247 until 48 h. Bacteria were employed at a starting concentration of 1 × 106 CFU mL-1. Lower panel: statistical significance between HA-treated and untreated strains. **Highly significant (P < 0.01); *significant (P < 0.05); - not significant (P > 0.05). Streptococci were even employed for the same set of trials previously described, but in presence of both HA and Hy. According to obtained data (Figure 2), strains displayed after 24 h a completely different behavior: strains 309 and 247 exhibited an O.D. increase, above all in presence of higher concentrations of HA, indicating a bacterial growth enhancement. Figure 2 Effects of HA and Hy on St. thermophilus 309, 247 and 82A until 48 h. Bacteria were employed at a starting concentration of 1 × 106 CFU mL-1. Lower panel: statistical significance between HA-Hy-treated and untreated strains. **Highly significant (P < 0.01); *significant (P < 0.05); - not significant (P > 0.05).

Since protein kinase inhibitors are known to be promiscuous [53–55] and compound D7 could

BV-6 cost inhibit a kinase or other enzyme required for the growth of C. pneumoniae, a similar growth inhibition by compound D7 might be expected for other intracellular bacteria. Since compound D7 did not inhibit the growth of Chlamydia trachomatis serovar D or Salmonella enterica sv. Typhimurium SL1344, an effect of D7 on a common signaling pathway used by intracellular pathogens is not likely the mechanism of C. pneumoniae growth retardation. Our results show that compound D7 inhibits the autophosphorylation of PknD and subsequent phosphorylation of C. pneumoniae CdsD in vitro and significantly SRT2104 solubility dmso retards the growth of C. pneumoniae in HeLa cells. However, our data does not allow us to state unequivocally that the reduced rate of

growth in the presence of compound D7 is directly due to inhibition of PknD activity. Our SGC-CBP30 concentration attempts to detect phosphorylated CdsD in vivo by mass spectrometry have not been successful as it is technically difficult to harvest enough CdsD protein suitable for this method. We are exploring other methods for detecting CdsD phosphorylation in vivo as the detection of the phosphorylation status of PknD or CdsD in the presence of compound D7 would allow us to make a stronger link between PknD activity and growth rate. Since C. trachomatis contains

a PknD ortholog we might expect compound D7 to affect C. trachomatis but this is not the case as compound D7 did not affect the growth of C. trachomatis in HeLa cells. However, the limited homology between the catalytic domains of the PknD orthologs in C. trachomatis and C. pneumoniae might explain the differential effect of compound D7 on their respective growth rates. We are presently initiating experiments to assess whether compound D7 has any inhibitory effect on PknD orthologs of other chlamydial species and to determine effects on bacterial replication rates. Electron microscopic examination of Chlamydia-infected mafosfamide cells exposed to compound D7 revealed the presence of very small inclusions with significantly reduced numbers of bacteria. Inclusions contained all 3 developmental forms including RB, EB and IB and therefore both replication and differentiation of C. pneumoniae occurred in the presence of D7, albeit at a reduced rate. If inhibition of PknD is the mechanism by which compound D7 exerts its inhibitory effect on chlamydial replication, the presence of replicating RB in inclusions indicates that PknD activity is not essential for bacterial replication.