In fact, the cellular pathways of DNA repair more involved in the

In fact, the cellular pathways of DNA repair more involved in the response to radiation injury are DSBR and BER In vitro and in vivo studies have shown that polymorphisms of genes involved in these two mechanisms of DNA repair may influence the cellular sensitivity to RT [48–50]. Our results showed no significant association between XRCC3 C18067T and radio-sensitivity in agreement with studies by Andreassen et al. and Chang-Claude et al. [51–53] in breast cancer patients or by Alsbeish et al. in head and neck cancer patients [54]. An association between wild type XRCC3 C18067 and an increased rate of late toxic effects, such as subcutaneous

fibrosis, were found in breast cancer [55] and prostate [56]. No statistical significant association between XRCC1 Arg399Gln and radio-sensitivity was found in our study, as well as in other studies [17, 19, 57]. However, Forest Epigenetics Compound Library research buy plot showed a behaviour as toxic agent of mut/het XRCC1 Arg399Gln in agreement with an increased rate of lung effects in non small cell lung cancer patients. [58]. Finally, no correlation was found between late toxicity mut/het XRCC3 A4541G and mut/het RAD51. Our low correlation between incidence of G2 or more fibrosis or fat necrosis and alleles/patient is probably due to the low number of patients with G2 or more fibrosis or fat necrosis. Another issue to consider is

that in comparison of other findings some differences are www.selleckchem.com/products/poziotinib-hm781-36b.html expected due to the types of adverse reactions studied, the length of follow-up for observing side effects, as well as, the additional patient-related factors. Conclusions The presence of some SNPs involved in DNA repair or response to oxidative stress seem to be able to predict late toxicity. This study, although affected by a limited number of patients, has a power of the study statistically L-NAME HCl sufficient to suggest that SNP in GSTP1 gene could

be useful to predict late toxicity in BC patients who underwent SSPBI. Further data are needed to confirm these preliminary results. Moreover, future research will focus on the performance of many additional SNPs in other genes that are associated with the development of radiation toxicity. Acknowledgements The Authors wish to thank Mrs. Tania Merlino for the English revision References 1. Veronesi U, Marubini E, Mariani L, Galimberti V, Luini A, Veronesi P, Salvadori B, Zucali R: Radiotherapy after breast-conserving surgery in small breast carcinoma: long-term results of a randomized trial. Ann Oncol 2001, 12:997–1003.PubMedCrossRef 2. Fisher ER, Anderson S, Redmond C, Fisher B: Ipsilateral breast tumor recurrence and survival following lumpectomy and irradiation: pathological findings from NSABP protocol B-06. Semin Surg Oncol 1992, 8:161–166.PubMed 3. Veronesi U, Luini A, Del Vecchio M, Greco M, Galimberti V, Merson M, Rilke F, Sacchini V, Saccozzi R, Savio T, et al.: Radiotherapy after breast-preserving surgery in women with localized cancer of the breast.

J Dent Res 2009,88(1):34–38 PubMedCrossRef 37 Shi X, Hanley SA,

J Dent Res 2009,88(1):34–38.PubMedCrossRef 37. Shi X, Hanley SA, Faray-Kele MC, Fawell SC, Aduse-Opoku J, Whiley RA, Curtis MA, Hall LM: The rag locus of Porphyromonas gingivalis contributes to virulence in a murine model of soft tissue destruction. Infect Immun 2007,75(4):2071–2074.PubMedCrossRef 38. Bagaitkar J, Williams LR, Renaud DE, Bemakanakere MR, Martin M, Scott DA, Demuth DR: Tobacco-induced alterations to Porphyromonas gingivalis -host interactions. Environ Microbiol 2009,11(5):1242–1253.PubMedCrossRef 39. Dufresne A, Ostrowski M, Scanlan DJ, Garczarek L, Mazard S, Palenik

BP, Paulsen IT, de JPH203 Marsac NT, Wincker P, Dossat C, et al.: Unraveling the genomic mosaic of a ubiquitous genus of marine cyanobacteria. Genome Biol 2008,9(5):R90.PubMedCrossRef 40. Fischer W, Windhager L, Rohrer S, Zeiller BIRB 796 M, Karnholz A, Hoffmann R, Zimmer R, Haas R: Strain-specific genes of Helicobacter pylori : genome evolution driven by a novel type IV secretion system and genomic island transfer. Nucleic Acids Res 2010, in press. 41. Foote SJ, Bosse JT, Bouevitch AB, Langford PR, Young NM,

Nash JH: The complete genome sequence of Actinobacillus pleuropneumoniae L20 (serotype 5b). J Bacteriol 2008,190(4):1495–1496.PubMedCrossRef 42. Rasmussen TB, Danielsen M, Valina O, Garrigues C, Johansen E, Pedersen MB: Streptococcus thermophilus core genome: comparative genome hybridization study of 47 strains. Appl unless Environ Microbiol 2008,74(15):4703–4710.PubMedCrossRef 43. Touchon M, Hoede C, Tenaillon O, Barbe V, Baeriswyl S, Bidet P, Bingen E, Bonacorsi S, Bouchier C, Bouvet O, et al.: Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths. PLoS Genet 2009,5(1):e1000344.PubMedCrossRef

44. Waterhouse JC, Swan DC, Russell RR: Comparative genome hybridization of Streptococcus mutans strains. Oral Microbiol Immunol 2007,22(2):103–110.PubMedCrossRef 45. Wu J, Yu T, Bao Q, Zhao F: Evidence of extensive homologous recombination in the core genome of rickettsia . Comp Funct Genomics 2009, 510270. 46. Hosogi Y, Duncan MJ: Gene expression in Porphyromonas gingivalis after contact with human epithelial cells. Infect Immun 2005,73(4):2327–2335.PubMedCrossRef 47. Yoshimura M, Ohara N, Kondo Y, Shoji M, Okano S, Nakano Y, Abiko Y, Nakayama K: Proteome analysis of Porphyromonas gingivalis cells placed in a subcutaneous chamber of mice. Oral Microbiol Immunol 2008,23(5):413–418.PubMedCrossRef 48. Bourgeau G, Lapointe H, Peloquin P, Mayrand D: Cloning, expression, and sequencing of a protease gene ( tpr ) from Porphyromonas gingivalis W83 in Escherichia coli . Infect Immun 1992,60(8):3186–3192.PubMed 49. Rumpf RW, Griffen AL, Leys EJ: Phylogeny of Porphyromonas gingivalis by ribosomal intergenic spacer region analysis. J Clin Microbiol 2000,38(5):1807–1810.PubMed 50.

The precipitated DNA was collected by centrifugation (15000 g, 10

The precipitated DNA was collected by centrifugation (15000 g, 10 min at 4°C),

followed S3I-201 chemical structure by phenol-chloroform extraction and ethanol precipitation as described [11]. DNA manipulation Restriction enzymes (EcoRI, XhoI, NotI and AvrII), T4 DNA ligase and Taq DNA polymerase were purchased from New England Biolabs (Frankfurt, Germany). All enzymes were used under the conditions specified by the manufacturer. Plasmids were isolated using a QIAprep Spin Miniprep Kit (QIAGEN, Hilden, Germany), and the PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). PCR reactions were performed in a (total volume of 50 μL) Mastercycler ep gradient S (Eppendorf, Hamburg, Germany). The recovered PCR fragments and plasmids were sequenced by Eurofins MWG Operon (Ebersberg, Germany). Plasmids were transformed into E. coli and P. pastoris KPT-8602 using a Multiporator (Eppendorf, Hamburg, Germany), according to the supplier’s protocol. Total RNA isolation To obtain the full-length cDNA of MCAP gene, total RNA was isolated from solid-state culture of the M. circinelloides as follows: 250 mL Erlenmeyer flasks containing 10 g of wheat bran moistened with 200 mM HCl, up to a water content of 120% on a dry basis, and autoclaved at 121°C

for 20 min, were inoculated with 5×106 spores of M. circinelloides. Cultured for four days at 24°C, 100 mg of the mycelium were collected with tweezers and immediately used for total RNA extraction using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). The concentration and quality of the total RNA was determined by

using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc. Wilmington, Delaware, USA). First-strand cDNA synthesis, 5′-RACE cDNA and 3′-RACE cDNA Two microgram of total RNA were used for the synthesis of the first strand of 5′-RACE-Ready cDNA and 3′-RACE-Ready cDNA. The synthesized first strand cDNA was used as a template for the 5′-RACE cDNA and 3′-RACE cDNA using the gene specific reverse primer GSP-Mucor-2R and forward primer GSP-Mucor-1 F, check respectively (Table 2). In these cases, the conditions for PCR reactions were as described by Clontech (SMART RACE cDNA Amplification Kit User Manual). The amplified RACE fragments were separated by agarose gel electrophoresis and recovered using NucleoTrap Gel Extraction Trial Kit (Takara Europe-Clontech, Saint-Germain-en-Laye, France). Using this technique, the sequences of the extreme ends of the MCAP gene (5′and 3′) were obtained. Finally, the full-length cDNA sequence of the aspartic proteinase of M. circinelloides (deposited in GenBank under accession number JQ906105) was amplified from the 5′-RACE-Ready cDNA while the genomic MCAP of the aspartic proteinase (deposited in GenBank under accession number JQ906106) was amplified from genomic DNA of M. circinelloides using the forward primers APMC-Met-F and the reverse primer APMC-stop-R (Table 2).

monocytogenes

monocytogenes Y-27632 mouse cytoxicity in protozoa. Our observations on the reduced growth of the hly gene deficient mutant in the co-culture with T. pyriformis compared to isogenic wild type bacteria are in line with a previous report that a hly gene deletion prevented L. monocytogenes from A. castellanii phagosome escaping [8]. Phagosome escaping is prerequisite for L. monocytogenes replication in mammalian but not insect cells [27]. It is not clear at present how the failure to escape the phagosome impairs intracellular growth in protozoan cells. However, the improved intracellular survival in synergy with rapid reduction of trophozoite concentration might be responsible

for the advantages that LLO exerts on bacterial survival in the presence of actively grazing protozoa. Considering the natural environment, LLO production seems to increase L. monocytogenes survival compared to non-haemolytic bacteria. Obtained results demonstrated higher counts for wild type L. monocytogenes than for the isogenic LLO deficient mutant during first days of co-cultivation supposing that wild type bacteria better survived upon initial interactions with the predator than non-haemolytic counterparts. Furthermore, prolonged bacterial survival might be supported by bacterial maintenance in protozoan cysts forming due to LLO activity.

It GSK3235025 price is generally accepted that entrapped bacteria may benefit from the protective coat conferred by protozoan [28–30]. It has been demonstrated

previously that encysted bacteria could survive sewage water treatment, which is fatal to free living bacteria [31]. Survival PtdIns(3,4)P2 of human pathogens inside protozoan cysts was demonstrated previously for Vibrio cholerae, L. pneumophila, Mycobacterium spp and an avirulent strain of Yersinia pestis [32–34]. However, to our knowledge active stimulation of protozoan encystment by bacteria was demonstrated only in case of L. monocytogenes ([7]; and this work). Maintenance of pathogenic bacteria within cysts not only protects them from unfavorable environmental conditions but as well can preserve at the first stages of interactions with the macroorganism. That might be an important mechanism for bacterial spreading in the natural ecosystems when cyst protection not only supports pathogen survival in the hostile environment but as well increases its chance to multiply upon host invasion. Involvement of LLO in different aspects of interactions between L. monocytogenes and protozoa has a striking similarity with its multiples roles during infection in mammals. Phagosome membrane disruption is the major role for LLO in intracellular parasitism in mammalian cells [2, 14]. However, LLO input in L. monocytogenes virulence is not limited to phagosome escaping: LLO generates a calcium flux into cells, promotes bacterial invasion in certain epithelial cells, and causes apoptosis in dendritic cells and T lymphocytes [13, 17, 18].

To fabricate the samples used for this work, DNA

To fabricate the samples used for this work, DNA Ferrostatin-1 ic50 strands were deposited on a silicon nitride grid surface. These DNA strands were used as biomolecular templates for the self-assembly of gold nanoparticles [4].

These samples were acquired from Dune Sciences (Eugene, OR, USA). The fabrication process was described elsewhere, and it is not included here because this process is not the aim of this work. Results and discussion Figure 1 shows the results of the LSPR analysis performed on a 26-nm gold spherical nanoparticle linked through DNA strands to a silicon nitride membrane. The top-right corner inset in (a) shows a high-angle annular dark-field (HAADF) image of the area where the SI was acquired including the gold spherical nanoparticle. Two representative EELS spectra marked by the two colored dots are displayed in the chart. The raw data extracted from the SI are displayed

using dotted lines. After applying PCA, the results are shown using dashed lines with long dashes. The result after ZLP subtraction is shown as dashed lines with medium-sized dashes. The difference between the data after PCA reconstruction and the ZLP fit is displayed in the chart using dashed lines with small dashes. The Gaussian fit function is shown with solid lines. Energy loss and amplitude maps are shown in Figure 1b,c. The chart in (b) uses a color-scale that goes from blue as the lowest energy value to red as the highest one. The chart in (c) uses a color-scale that ranges from black, through red and yellow to white BAY 11-7082 ic50 as the highest amplitude value for the fitted Gaussian. Figure 1 Electron energy loss spectra (a) and energy loss (b) and amplitude (c) maps. (a) Electron energy loss spectra of a 26-nm gold nanosphere linked through DNA strands to a Si3N4 membrane; the inset shows an HAADF image of the nanoparticle. The spectrum marked as (curve i) shows the energy loss along the trajectory marked with a red dot where a resonance peak can be clearly seen at 2.4 eV, the one marked as (curve ii) shows the peak at 2.5 eV approximately corresponding to the Sclareol trajectory

through the nanoparticle marked with the blue dot. (b) Energy loss map displaying the value of the center of the fitted Gaussian to the LSPR peak. (c) Amplitude map with the intensity value of the center of the fitted Gaussian to the LSPR peak. Both the energy map and the spectrum labeled in red as (curve i) show a very distinct peak at 2.4 eV, this is the typical value for a dipolar LSPR mode in a gold nanoparticle of this size in air [15, 16]. To validate the results, the Mie theory has been used to solve the Maxwell equations using both the quasistatic approximation and solving the full Maxwell equations. A 26-nm gold sphere standing in vacuum was considered yielding both approximations a result of 2.44 eV for the extinction of light with the absorption as the main contribution over scattering which corresponds for a metal nanoparticle of this size [1].

PubMedCrossRef 4 Gallegos MT, Marques S, Ramos JL: Expression of

PubMedCrossRef 4. Gallegos MT, Marques S, Ramos JL: Expression of the TOL plasmid xylS gene in Pseudomonas putida occurs from a σ 70 -dependent promoter or from σ 70 – and σ 54 -dependent tandem promoters according to the compound used for growth. J Bacteriol 1996, 178:2356–2361.PubMed 5. Dominguez-Cuevas P, Marin P, Busby S, Ramos JL, Marques S: Roles of effectors in XylS-dependent transcription activation: intramolecular domain derepression and DNA binding. J Bacteriol 2008, 190:3118–3128.PubMedCrossRef 6. Ruiz R, Marques S, Ramos JL: Leucines 193 and 194 at the N-terminal domain of the XylS protein, the positive transcriptional regulator of the TOL meta-cleavage pathway, are involved in dimerization. J Bacteriol

find more 2003, 185:3036–3041.PubMedCrossRef 7. Schleif R: AraC protein, regulation of the L-arabinose

operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol Rev 2010, 34:779–796.PubMed 8. Schleif R: AraC protein: a love-hate relationship. Bioessays 2003, 25:274–282.PubMedCrossRef 9. Dominguez-Cuevas P, Marin P, Marques S, Ramos JL: XylS-Pm promoter interactions through two helix-turn-helix motifs: identifying EPZ015666 research buy XylS residues important for DNA binding and activation. J Mol Biol 2008, 375:59–69.PubMedCrossRef 10. Vee Aune TE, Bakke I, Drablos F, Lale R, Brautaset T, Valla S: Directed evolution of the transcription factor XylS for development of improved expression systems. Microb Biotechnol 2010, 3:38–47.PubMedCrossRef 11. Michan C, Kessler B, De Lorenzo V, Timmis KN, Ramos JL: XylS domain interactions Carnitine palmitoyltransferase II can be deduced from intraallelic dominance in double mutants of Pseudomonas putida. Mol Gen Genet 1992, 235:406–412.PubMedCrossRef 12. Ruiz R, Ramos JL: Residues 137 and 153 at the N terminus of the XylS protein influence the effector profile of this transcriptional regulator and the sigma factor

used by RNA polymerase to stimulate transcription from its cognate promoter. J Biol Chem 2002, 277:7282–7286.PubMedCrossRef 13. Gallegos MT, Marques S, Ramos JL: The TACAN 4 TGCA motif upstream from the -35 region in the σ 70 -σ S -dependent Pm promoter of the TOL plasmid is the minimum DNA segment required for transcription stimulation by XylS regulators. J Bacteriol 1996, 178:6427–6434.PubMed 14. Gonzalez-Perez MM, Ramos JL, Gallegos MT, Marques S: Critical nucleotides in the upstream region of the XylS-dependent TOL meta-cleavage pathway operon promoter as deduced from analysis of mutants. J Biol Chem 1999, 274:2286–2290.PubMedCrossRef 15. Gonzalez-Perez MM, Marques S, Dominguez-Cuevas P, Ramos JL: XylS activator and RNA polymerase binding sites at the Pm promoter overlap. FEBS Lett 2002, 519:117–122.PubMedCrossRef 16. Dominguez-Cuevas P, Ramos JL, Marques S: Sequential XylS-CTD binding to the Pm promoter induces DNA bending prior to activation. J Bacteriol 2010, 192:2682–2690.PubMedCrossRef 17.

20 μg of total protein samples

extracted in the same cond

20 μg of total protein samples

extracted in the same conditions were separated in a 10 % tricine-SDS polyacrylamide gel and blotted to a nitrocellulose membrane. A non-specific band (Control) detected with the same antibodies was used as loading control. To check if the increment observed on the RNA levels would influence the final levels of protein in the cell, we analysed the expression of SmpB under the same conditions. SmpB expression was compared by Western blot in the wild type, the RNase R- mutant derivative and the RNase R- strain complemented LY3023414 nmr with RNase R expressed in trans. Analysis of SmpB levels with specific antibodies raised against purified TIGR4 SmpB showed a significant increase in the protein levels in the absence of RNase R (~13-fold at 15°C and ~7-fold

at 37ºC) (Figure 5b). This phenotype was partially restored in the strain complemented with RNase R, suggesting that RNase R is determinant for the final levels of SmpB in the cell. Discussion RNase R levels and activity are known to increase in stationary phase and under certain stress situations, namely cold-shock DNA Damage inhibitor and starvation [11, 12, 17]. RNase R is the unique exoribonuclease able to degrade RNA molecules with extensive secondary structures, and the increase of RNase R under multiple stress conditions may indicate a general modification of structured RNA in response to environmental changes. In fact this enzyme was shown to be important for growth and viability of several bacteria especially under cold-shock, a condition where RNase R levels are considerably increased [12, 18, 24, 33, 34]. Mutants lacking any of the trans-translation components (tmRNA and SmpB) also have a variety of stress phenotypes. These range from attenuated antibiotic resistance to problems in adaptation to oxidative stress, cold- and heat-shock [35, 36]. In this report we have studied the regulation of the RNase R expression and the interplay of this exoribonuclease with the components of the trans-translation Interleukin-2 receptor system in the human pathogen S. pneumoniae. Our results show that, as occurs in E. coli, pneumococcal RNase R is induced after a downshift from 37°C to 15°C. According to our data, both rnr mRNA and protein

levels are elevated after cold-shock treatment, which could suggest that the higher levels of protein would be directly related with the increased amount of mRNA molecules in the cell. However, the expression of RNase R seems to be also modulated by SmpB. In the absence of this protein the levels of RNase R are similar at 15°C and 37°C and the temperature-dependent regulation observed in the wild type seems to be lost. This result resembles the E. coli situation where RNase R was shown to be destabilized by SmpB during exponential phase in a tmRNA-dependent manner [28]. Interestingly, E. coli RNase II (a protein from the same family of RNase R) was reported to be destabilized by Gmr, which is encoded by a gene located immediately downstream [37].

27 ± 1 83* 18 22 ± 0 31 AEP

+ NS 20 14 ± 0 56 16 68 ± 1 9

27 ± 1.83* 18.22 ± 0.31 AEP

+ NS 20.14 ± 0.56 16.68 ± 1.96 Taurine + AEP 23.86 ± 1.73* 22.49 ± 2.09 GABA + AEP 23.16 ± 1.38* 21.97 ± 4.93 Data were shown as mean ± S.E.M. Statistical evaluation was carried out by one-way analysis of variance (ANOVA) followed by Scheffe’s multiple range tests: *P < 0.05, AEP + NS versus control + NS, taurine + AEP, or GABA + AEP. In the hippocampus of rat brains and cerebral cortex, the activity of GSH-Px is lowest in the AEP + NS group and close to each other in the taurine + AEP, GABA + AEP, and control + NS groups. When AEP groups are treated using taurine or GABA, the GSH-Px activity of the AEP + NS group shows significant difference (P < 0.05) relative to those of the GABA + AEP and taurine + AEP groups, but those among the taurine + AEP, GABA + AEP, selleck inhibitor and control + NS groups

have no statistical significance. GSH-Px activities of different groups are shown in Table 4. click here Table 4 Test result of GSH-Px activity of the hippocampus and cerebral cortex of every group Groups Hippocampus (U/mg protein) Cerebral cortex (U/mg protein) Control + NS 26.21 ± 1.30* 32.14 ± 10.97* AEP + NS 14.55 ± 2.07 13.90 ± 2.52 Taurine + AEP 28.17 ± 3.11* 36.68 ± 12.90* GABA + AEP 26.12 ± 2.97* 37.65 ± 8.47* Data were shown as mean ± S.E.M. Statistical evaluation was carried out by one-way analysis of variance (ANOVA) followed by Scheffe’s multiple range tests: *P < 0.05, AEP + NS versus control + NS, taurine + AEP, or GABA + AEP. Discussion Taurine is widely applied as an antioxidant or dietary supplement and is demonstrated to reduce significantly MDA levels in the serum and/or tissue [38]. GABA is widely applied as an additive [26]. Similarly, it is reported that Glu and Asp can prevent cardiac toxicity by alleviating oxidative Tau-protein kinase stress [30]. Our

results demonstrate that taurine or GABA reacts rapidly with MDA, and the reaction of Glu or Asp with MDA under supraphysiological conditions is difficult (Figures 1 and 2). The observations are consistent with the hypothesis that amino acids act as a sacrificial nucleophile, trapping reactive intermediates [36, 37]. Scavenging carbonyl function of four amino acids is shown in Figures 4 and 5. The strong inhibition effect of taurine and GABA on MDA and the fast formation of products show that taurine and GABA can react rapidly; however, the reaction of Glu or Asp with MDA is very weak under supraphysiological conditions due to its different chemical structures (Table 1, Figure 3). In addition, if it is thought of four amino acids in the context of the neural system, taurine and GABA are important inhibitory amino acid neurotransmitters, and Glu and Asp are significant excitatory amino acid neurotransmitters. Glu and Asp uptake induce excitotoxicity, thereby causing oxidative stress and further lipid peroxidation [6].

Our data showed that cd5, cd6, and cd7 loci did not decrease the

Our data showed that cd5, cd6, and cd7 loci did not decrease the congruency with PCR-ribotyping (Table 2; Additional File 2). The result may be due to that the 16S-23S intergenic spacer region, on which the PCR-ribotyping based on, was not as conserved as a housekeeping gene

that is used to construct the phylogenic tree [9, 38]. However, the variations from these incomplete repeat loci should be detected in our follow-up surveillance. PCR ribotyping is a standard technique used worldwide for epidemic clone detection, but the ambiguous Tipifarnib in vitro data generated by this technique is difficult for assessing inter-laboratory efficacy. MLVA is a fast and easy-to-use method, and its numerical profile output is more transferable than the standard PCR ribotyping technique. In our laboratory setting, the cost of PCR ribotyping, MLVA10, and TRST per isolate was $0.87, $2.53, and $13.60, respectively, and the cost of the most recent MLST is $24.65 according to Griffiths’ estimation [21]. In the current study, the cost of

MLVA10 was slightly higher than that of PCR ribotyping, but was still significantly less expensive than the TRST and MLST sequence-based typing techniques. Moreover, when analyzing a large number of isolates, it is simpler to perform one genotyping technique than multiple techniques. Taken together, the MLVA10 is recommended for the detection of C. difficile PCR-ribotype groups and for use in combination with the MLVA panel designed for the detection of outbreak strains. Future studies

will involve evaluation of MLVA10 for 17-AAG mw its phylogenetic information by comparison to MLST typing. Conclusions For the classification of C. difficile strains, the MLVA technique can result in a distinguishable data set that is more useful for comparison and is highly congruent with PCR-ribotype results. The MLVA10 panel may be used either to detect the PCR-ribotype groups or to overcome the drawbacks of the PCR ribotyping technique. In addition, the MLVA4 can be used to detect closely-related strains. These two MLVA panels can be combined and used for epidemiological studies of C. difficile. Methods Bacterial strains A total of 142 C. difficile strains that were either toxigenic or non-toxigenic Megestrol Acetate were used in this study. Five reference strains (NCTC11204, NCTC13366, NCTC13287, NCTC13404, and NCTC13307) were purchased from the National Collection of Type Cultures (NCTC, London, UK) and three reference strains (BCRC17900, BCRC17702, and BCRC17678) were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). One strain (NAP1/027) was kindly provided by Dr. Brandi Limbago from the United States Centers for Disease Control and Prevention (CDC), and 133 strains were isolated from clinical laboratory specimens in Taiwan.

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