The MI

The endurance training protocol used in this study was a modification of a widely used protocol in the literature [23, 25, 26]. As shown in Figure 1, distance run increased with time. These data suggest that the training workload was well adjusted, since a plateau in the training volume is a sign of overtraining [27]. No difference was found in the average daily distance run between the QT and PT groups. VO2 peak values in rats vary depending on the methodological test used or on their weight [28]. Our results show that six weeks of quercetin supplementation

did not increase VO2 peak or VO2 at exhaustion in sedentary or trained rats. It must be noted that our protocol did no alter inclination in order to examine the maximum speed achieved. Protocols that do not use an incline are known to induce a lower VO2 peak than others with 15°-20° inclination [28, 29]. However, our results were similar to those recently reported [17], but were in contrast with the ones that reported an increase of VO2 peak by quercetin in sedentary humans [19]. Speed at VO2 peak was also analyzed in this experiment, with no change reported in the quercetin groups. We hypothesized that quercetin would increase VO2 peak due to its ability

to increase mitochondrial biogenesis in mice (6). However, as described above, no differences were observed in any groups on IGF-1R inhibitor measures related to oxygen uptake by quercetin supplementation. These results are similar to those obtained by Bigelman et al [30]. There are several potential reasons MI-503 supplier for these results: firstly, VO2 peak is influenced by muscle mitochondrial oxidative capacity, but relative to endurance capacity, it is limited to a greater extent by oxygen delivery via the cardiovascular system [31]. Secondly, larger doses over extended periods using added flavonoids such as eppigallocatechin G protein-coupled receptor kinase gallate (EGCG) may augment quercetin’s effects on mitochondrial biogenesis. This could be a more appropriate supplement to increase oxygen consumption [16]. However, previous work did not find any ergogenic effect of quercetin and EGCG supplementation in a moderately

trained sample [30]. To examine additional ergogenic effects of quercetin in rats, oxygen consumption and carbon dioxide production were measured during the incremental exercise test. This enabled the calculation of RQ. In all groups of rats, the average RQ remained fairly constant and did not differ between groups (data not shown). When VCO2 is greater than VO2 (RQ>1.0), this point of inflection is correlated with blood lactate accumulation [32]. QT group showed a trend to run longer before reaching an RQ of 1.0 (Figure 4B) indicating that these rats were able to use oxidative metabolism for a longer period. Fatigue in the endurance test is thought to arise primarily from limitations in the periphery, like the cardiovascular system and muscles [6].

pseudomallei [32],


pseudomallei [32],

are AZD3965 also found in B. thailandensis but are absent in the B. oklahomensis strains. BprP activates the expression of TTSS genes, and a bprP mutant in B. pseudomallei does not secrete TTSS effector proteins and is unable to kill macrophages [32]. The absence of this activator in B. oklahomensis might therefore explain the low virulence of this species. In this study we have not tested Burkholderia mallei, another species closely related to B. pseudomallei, for virulence in cell culture or Galleria models. It is known that B. mallei is able to click here infect and grow in macrophages [33] and to kill G. mellonella larvae [19]. However, the pathogenesis of B. mallei infection in G. mellonella may be quite different from the pathogenesis of B. thailandensis or B. pseudomallei infection see more we report here. Whereas we recorded larval

death by 24 hrs post challenge with typical B. pseudomallei isolates, larval deaths occurred over the period 24 – 144 hrs post challenge with B. mallei [19]. This might be explained by the restricted host range of the obligate intracellular bacterium B. mallei compared to B. pseudomallei with its much more versatile genome [34]. Conclusions Our findings indicate that murine macrophage cell culture or Galleria infection models can be used to discriminate B. pseudomallei, B. thailandensis and B. oklahomensis isolates on the basis of their virulence. In general, our results support the proposal that the virulence of isolates in these models reflects virulence in murine models of disease. However, some important exceptions merit further investigation which is not within the scope of this study. Our finding that virulence of three

B. pseudomallei isolates with high, intermediate and low virulence in mice is reflected in their virulence in cell culture or Galleria infection models indicates the potential value of these models for the identification of virulence-associated genes. Our findings support the proposal that B. oklahomensis isolates are of low virulence and indicate that these isolates are defective in growth in macrophages and in actin-based motility within cells. Methods Bacterial strains and growth conditions Selleckchem Ponatinib The Burkholderia strains used in this study are summarised in Table 1. All strains were grown in LB broth with aeration or on LB agar plates at 37°C unless otherwise stated. When appropriate, antibiotics (Sigma-Aldrich) were used at the following concentrations, unless otherwise stated: kanamycin, 50 μg/ml; chloramphenicol, 25 μg/ml; and gentamicin, 50 μg/ml. Cell lines J774A.1 mouse macrophage cell lines were maintained at 37°C under 5% CO2 atmosphere in DMEM (Hyclone) supplemented with 10% fetal bovine serum (Hyclone), 1% L-glutamine (250 mM) (Hyclone) and 1% Penicillin/Streptomycin solution (Hyclone).

Metabolic capabilities are indicated next to representative pictu

Metabolic capabilities are indicated next to representative pictures of symbiont growth in vitro: Plus or minus indicate the ability (+) or inability (−) to grow on the corresponding media. In order to visualize the phylogenetic placement of symbionts and highlight their metabolic capabilities, symbiont strains were connected to their respective hosts with colored lines:

Red lines correspond to strains unable to grow on medium with peptones; green lines correspond to strains unable to grow on ammonium as the only source of nitrogen. Characterization of ‘S. philanthi’ biovars In all antennal samples used for isolation, buy NVP-BSK805 the symbiotic Streptomyces showed a characteristic “antennal” phenotype: bacteria looked like individual or relatively short-chained cells, unbranched or with very short side branches, while no well-developed long mycelium was Erismodegib manufacturer observed (exemplified by biovar ‘triangulum’ in Figure 1A). In culture, the vast majority of biovars developed typical

mycelium. However, two biovars were clearly distinguishable from all other symbiotic Streptomyces due to their “antennal” phenotype also in culture: in liquid medium, the actively growing biovar ‘elongatus’ formed micro-colonies, but in late stage of logarithmic growth or in stationary phase they tended to fall apart into short, often poorly branched fragments. A similar pattern was also observed for the biovar ‘loefflingi’, which could express one or the other phenotype over several transfers and change find more it by the next passage, although conditions triggering such phenotypic changes remained unknown (Additional file 6: Figure S2). Although all beewolf-associated symbionts were originally assigned to biovars of the same species ‘Ca. S. philanthi’ [21], the morphology of even closely related biovars growing

on the same medium varied strongly (Figure 4). On Grace’s medium, bacteria from the clade ‘S. philanthi’ formed pigmented (yellow or beige) opaque colonies of round or irregular form, flat or gibbous with wave, broken or smooth border, and the surface varied from matte to slightly shiny, from smooth Reverse transcriptase to rimmed and rugose. Only biovar ‘multimaculatus’, when grown on the Grace’s insect medium, formed white colonies with well-developed aerial mycelium typical for Streptomyces (Figure 4). Since all isolates were obtained on rich medium (supplemented with the full set of amino acids) imitating insect hemolymph, the next step was to assess the nutrient requirements of the isolated biovars by testing whether they could grow on media containing either an organic (peptones) or inorganic source of nitrogen (ammonium).

One milliliter of

this suspension was dropped into each w

One milliliter of

this suspension was dropped into each well of a 12-well microplate (Corning) and incubated at 33°C for 7 days. The microplate, prepared as described above, was used for culturing the mycobacteria. Each well of the microplate was inoculated with a final concentration of 106 mycobacteria/ml (MOI = 10). The inoculum was sonicated for 5 min at 234 watts (BRANSON 2210; Branson Ultrasonics Corporation, Danbury, CT, USA) in order to limit mycobacteria cell clumping. The microplate was SHP099 nmr centrifuged at 1,000 g for 30 min and incubated at 33°C under a humidified, 5% CO2 atmosphere. This microplate was examined daily for 15 days for cytopathic effects and the presence of intra-amoebal organisms by shaking, cytocentrifugation at 200 g for 10 min and buy Momelotinib Ziehl-Neelsen staining. Encystment and excystment of infected amoeba In

25 cm3 culture flasks (Corning), 10 ml of amoeba that had been infected for 48 hours were rinsed once with encystment buffer adapted from [21] (0.1 M KCl, 0.02 M Tris, 8 mM MgSO4, 0.4 mM CaCl2, 1 mM NaHCO3). After centrifugation at 500 g, the pellet was resuspended in 10 ml of fresh encystment buffer and incubated for 3 days at 32°C. The excystment of the cysts selleck inhibitor was examined by light microscopy. Amoebal cysts were pelleted by centrifugation at 1,000 g for 10 min and treated with 3% (vol/vol) HCl as previously described [21]. Treated cysts were then washed three times with PAS buffer. Half of the sample was processed for electron microscopy (see above), and the other part was incubated for 7 days in PYG medium at 33°C. Intra-amoebal mycobacteria were released by lysing the monolayer with 1 ml of 0.5% sodium dodecyl sulfate, followed

by two successive passages through a 27-gauge needle [3]. The presence of viable mycobacteria was documented by detecting colonies on GPX6 Middlebrook 7H10 agar inoculated with 200 μl of the cell lysate and incubated at 30°C for 15 days. The identities of the mycobacteria were confirmed by Ziehl-Neelsen staining and partial rpoB gene sequencing using primers Myco-F (5′-GGCAAGGTCACCCCGAAGGG-3′) and Myco-R (5′-AGCGGCTGCTGGGTGATCATC-3′) [34]. All experiments were repeated three times. Electron microscopy Non-ingested mycobacteria were eliminated by rinsing the amoebal monolayer twice with sterile PBS. The amoeba monolayer that was previously infected by MAC species was then fixed in 2% glutaraldehyde and 0.1 M cacodylate buffer overnight. After this first fixation, the bacteria were fixed in 2% glutaraldehyde and 0.33% acroleine in a 0.07 M cacodylate buffer for 1 hour. After washing in 0.2 M cacodylate buffer, the bacteria were post-fixed in 1% osmium bioxide in 0.1 M potassium ferrycyanure for 1 hour and dehydrated in an ascending series of ethanol concentrations, and after 100% ethanol, the dehydration was finished in propylene oxide, and the samples were embedded in an Epon 812 resin.

005, P TrxB, HCl = 0 009, P Cj0706, Ac = 0 016, P MogA, HCl, Ac <

005, P TrxB, HCl = 0.009, P Cj0706, Ac = 0.016, P MogA, HCl, Ac < 0.03). Volume% of bacterioferritin (Dps) during HCl stress was higher compared with the control, but probably due to the variation of the control this difference was not significant (P 11168, Dps, HCl = 0.061). For the acid-robust strain 305, Dps, p19, MogA and TrxB were significantly induced (P Dps, HCl = 0.0028, P p19, HCl = 0.0008, P MogA, HCl = 0.018, P TrxB, HCl = 0.017). Fewer proteins were induced in the acid-sensitive

strain 327, which was also reduced during the acid stress (Figure  2B). Only induction of Cj0706 and MogA was observed during HCl acid stress (P Cj0706, HCl = 0.0037, P MogA, HCl = 0.04). In the case of NCTC 11168 and 305, the two proteins alkyl hydroperoxide reductase (AhpC) and superoxide dismutase (SodB) had higher% Bafilomycin A1 concentration volume intensity than for the control indicating induction; however the differences were not significant. A reference profile of proteins separated by 2D-electrophoresis for C. jejuni 305 exposed to HCl stress (pH 5.2) GS-7977 is shown in Figure  3. Table 3 Induced proteins (% volume intensity) during HCl (pH 5.2) and acetic acid (pH 5.7) exposure in C. jejuni NCTC 11168, C. jejuni 305 and C. jejuni 327 at 37°C in chemically defined broth    

    Campylobacter jejuni strains3 Protein/(NCBInr 1 ) Mw (kDa) Score 2   NCTC 11168 305 327 Dps (NP282665) 17.4 222 Vol% p19 (CAA73983) 17.0 255 Vol% AhpC (NP281525) 22.0 668 Vol% SodB (NP281379) 25.0 241 Vol% TrxB (NP281357) 33.5 204 Vol% Cj0706 (NP281878) 28.0 431 Vol% MogA (YP_178829) 20.3 318 Vol%   C HCl Ac C HCl Ac C HCl Ac Dps: Bacterioferritin,

p19: 19 kDa periplasmic protein, AhpC: Alkyl hydroperoxide reductase, SodB: Superoxide dismutase Montelukast Sodium (Fe), TrxB: Thioredoxin-disulfide reductase, Cj0706: hypothetical protein, MogA: Molybdenum cofactor biosynthesis protein. Columns: light grey: control (C), dark grey: HCl stressed cells (HCl), white: Acetic acid stressed cells (Ac). 1 Identification was based on Mascot MS/MS Ion Search using sequence data from the database NCBInr. 2 Mowse Score (Score). 3 The intensity of the induced proteins was estimated by Image MasterTM 2D Platinum and % volume intensity was calculated. The intensity of the protein spots was analyzed using the Image MasterTM 2D Platinum (version 5.0, Amersham Biosciences, Melanie). Three biological independent replicates was performed and % volume intensity was calculated as: % volume intensity control (protein x) = volume intensity /(volume intensity control + volume intensity HCl + volume intensity acetic acid). Figure 3 Reference map of proteins from C. jejuni 305 separated by 2D-gel-electrophoresis. The strain was grown in modified chemically defined broth modified (CDB) containing 0.01 mM methionine at 37°C to late exponential phase and until the cell level was 1 × 108 CFU/ml.

2005; Gomelsky et al 2008) The data indicate that the LHII ante

2005; Gomelsky et al. 2008). The data indicate that the LHII antenna complexes are severely diminished relative to the wild type. The correlation between MK0683 the reduction or lack of LHII and the presence of

tubular structures has been noted by Selleck GSI-IX others (Kiley et al. 1988; Hunter et al. 1988; Sabaty et al. 1994; Siebert et al. 2004). But we believe this is the first report of such aberrant structures in regulatory gene mutants. Importantly, the available information regarding regulation of PS gene expression by PrrA and PpsR does not explain why LHII is absent while LHI and RC are present (Gomelsky et al. 2008). It implies that other genes necessary for proper ICM development, such as assembly factors required for LHII formation, are also inappropriately (not) expressed in the absence of PrrA and PpsR. Ultrastructure of R. sphaeroides and R. capsulatus wild type and fnrL mutant bacteria FnrL belongs to the Fnr–Crp protein family (Zeilstra-Ryalls and Kaplan 1995). All members are characterized by the presence of an effector

domain located within the N-terminal SN-38 manufacturer region and a DNA binding domain located within the C-terminal region. For FnrL, the effector domain is thought to contain an oxygen-labile 4Fe-4S cluster whose presence is required for the protein to be properly configured for DNA binding. Thus, the protein regulates gene transcription when oxygen is limiting. While FnrL is essential for all anaerobic growth of R. sphaeroides 2.4.1, both in the light and in the dark with DMSO (Zeilstra-Ryalls and Kaplan 1995), the reason for this is not yet resolved (detailed in Gomelsky and Zeilstra-Ryalls 2013). Thin sections of cells cultured under low-oxygen conditions, which are permissive for growth of FnrL null mutant bacteria but also support some FnrL regulatory activity (Roh and Kaplan 2002), were examined using TEM (Fig. 4A). In contrast to the typical high density of ICM observed in the thin sections of wild type

cells, approximately 3-oxoacyl-(acyl-carrier-protein) reductase 5–10 ICM-like structures per cell were seen in the sections of the fnrL null mutant JZ1678. While the number of these structures is approximately the same as that seen in sections of the PrrA− mutant bacteria cultured under low-oxygen conditions (Fig. 1A), spectral complexes are detectable in cells lacking FnrL (Zeilstra-Ryalls et al. 1997), which correlates with regulation of different sets of genes by these two transcription factors (Gomelsky and Zeilstra-Ryalls 2013), even though both are indispensable for phototrophic growth. Fig. 4 TEM micrographs of thin sections of wild type and mutant strains of R. sphaeroides (A) and R. capsulatus (B) bacteria that had been cultured under low-oxygen conditions. The strains used are as explained in the legends, with details provided in Table 1 Although both R. sphaeroides and R. capsulatus require FnrL for anaerobic–dark growth with DMSO, R.

2 0 (TAKARA, Dalian, China) The entire coding regions of aac(3)-

2.0 (TAKARA, Dalian, China). The entire coding regions of aac(3)-II, aac(6′)-Ib, aac(6′)-II, ant(3″)-I, aph(3′)-VI, armA and rmtB were amplified individually from the positive control isolates with the specific primer listed in Table 3. PCR conditions for the amplifications were as follows: 5 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 56°C and 1 min at 72°C and a

final extension of 5 min at 72°C. PCR products were cloned using the pMD18-T vector (TAKARA, Dalian, China), into E. coli JM109 and positive clones were selected using an X-Gal/IPTG LB agar plate containing ampicillin (100 mg/L). Recombinant plasmids were buy Vorinostat purified with QIAGEN Plasmid Mini Kit (Qiagen, Hilden, Germany), treated with the RNAse to eliminate residual RNA and subjected to DNA sequencing using T7 and SP6 sequence primers on an AB SOLiDTM 4.0 System (Applied Biosystems, USA). The obtained DNA sequences were compared with relevant sequences in the GenBank database by using the BLAST algorithm (http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi?​PROGRAM=​blastn&​BLAST_​PROGRAMS=​megaBlast&​PAGE_​TYPE=​BlastSearch&​SHOW_​DEFAULTS=​on&​LINK_​LOC=​blasthome).

Table 3 Primers for the entire coding regions of 7 aminoglycoside-Small molecule library manufacturer resistance genes Primer Sequence (5′→3′) Reference or source Size (bp) aac(3)-II F: ATATCGCGATGCATACGCGG [31] 877 R: GACGGCCTCTAACCGGAAGG aac(6’)-Ib F: TTGCGATGCTCTATGAGTGGCTA [32] 472 R: CTCGAATGCCTGGCGTGTTT aac(6’)-II F: CGACCATTTCATGTCC Janus kinase (JAK) This study* 542 R: GAAGGCTTGTCGTGTTT ant(3″)-I F: CATCATGAGGGAAGCGGTG [33] 787 R: GACTACCTTGGTGATCTCG Ruboxistaurin in vivo aph(3’)-VI F: ATGGAATTGCCCAATATTATT [34] 780 R: TCAATTCAATTCATCAAGTTT armA F: CCGAAATGACAGTTCCTATC [13] 846 R: GAAAATGAGTGCCTTGGAGG rmtB F: ATGAACATCAACGATGCCCTC [13] 769 R: CCTTCTGATTGGCTTATCCA *The primers have been validated with referenced strains (GU944731.1 and HQ880255.1).

Primers In this study, a total of one pair of universal primers (Tag-F/Tag-R) and seven pairs of chimeric primers (specific primers linked to the 3’ end to the universal primers) were designed (Table 4). Tag-F (AGGTGACACTATAGAATA) and Tag-R (GTACGACTCACTATAGGGA) were quasi-T7 sequences and selected by default using the GeXP eXpress Profiler software. The gene-specific sequences of the primers for aac(3)-II, aac(6′)-II, ant(3″)-I were previously reported [15, 20]. The gene-specific sequences of other four pairs of primers were designed by NCBI Primer-Blast and GeXP eXpress Profiler softwares. The primer for aac(6′)-Ib also covered the variant gene aac(6′)-Ib-cr which not only resulted in aminoglycosides resistance but also mediated quinolone resistance [28]. The 5’ ends of the forward and reverse universal primers were labeled with fluorescent dye Cy5 and purified with high pressure liquid chromatography. All chimeric primers were purified by polyacrylamide gel electrophoresis.

B) Unwinding

of 1 nM Fork 3 by 2 nM PriA in the presence

B) Unwinding

of 1 nM Fork 3 by 2 nM PriA in the presence of wild type N. gonorrhoeae PriB (circles) or PriB:K34A (squares). Measurements are reported in triplicate and error bars represent one standard deviation of the mean. When we examined PriA helicase activity on Fork 3 in the presence of PriB:K34A, we found that levels of DNA unwinding are similar to those seen when wild type PriB is used to stimulate PriA (Figure 5B). Based on the value of the apparent dissociation constant for the interaction of PriB:K34A with ssDNA, and assuming that it is a reliable selleck kinase inhibitor indicator of the affinity of PriB:K34A for DNA in the context of a ternary PriA:PriB:DNA complex, we would not expect the PriB:K34A variant to be interacting with DNA to a significant degree under the conditions of this DNA unwinding assay. It is particularly noteworthy that in E. coli, a PriB variant with severely weakened ssDNA binding GF120918 clinical trial activity (the W47,K82A double mutant) fails to stimulate the DNA unwinding activity of its cognate PriA to a significant degree [7]. Therefore, unless formation of a PriA:PriB:DNA ternary complex significantly enhances the DNA binding activity of N. gonorrhoeae PriB, our results suggest that ssDNA binding by N. gonorrhoeae PriB does not play a major role in N. gonorrhoeae PriB stimulation of its cognate PriA helicase. PriB activates PriA’s ATPase activity PriA helicase

is thought to couple the energy released from hydrolysis of ATP to the unwinding of duplex DNA. Thus, we wanted to determine if N. gonorrhoeae PriB stimulation of PriA helicase activity involves PriA’s ability to hydrolyze ATP. To examine PriA’s ATPase activity, we used a spectrophotometric assay that couples PriA-catalyzed ATP hydrolysis to oxidation of NADH. This assay allowed us to measure steady-state PriA-catalyzed

ATP hydrolysis rates in the presence and absence of PriB. As expected, PriA’s ATPase activity is negligible in the absence of DNA (Figure 6A). The DNA dependence of PriA’s ATPase activity has been observed in E. coli as well [30], and likely reflects a mechanistic coupling of ATP hydrolysis and duplex DNA unwinding. Figure 6 PriA’s ATPase activity is Casein kinase 1 stimulated by DNA and by PriB. A) DNA-dependent ATP hydrolysis catalyzed by 10 nM PriA in the presence (circles) or absence (squares) of 100 nM PriB (as GSK2245840 monomers). The DNA substrate is Fork 3. Measurements are reported in triplicate and error bars represent one standard deviation of the mean. B) Effect of ATP concentration on rates of ATP hydrolysis catalyzed by 10 nM PriA in the presence of 100 nM Fork 3 and in the presence (circles) or absence (squares) of 100 nM PriB (as monomers). Measurements are reported in triplicate and error bars represent one standard deviation of the mean. With 10 nM PriA and in the absence of PriB, near maximal rates of ATP hydrolysis are observed with 10 nM Fork 3 (Figure 6A).

References 1 Kane CL, Mele EJ: Z2

References 1. Kane CL, Mele EJ: Z2 topological order and the quantum spin Hall effect. Phys Rev Lett 2005, 95:146802.CrossRef 2. Bernevig BA, Zhang SC: Quantum spin

Hall effect. Phys Rev Lett 2006, 96:106802.CrossRef 3. Fu L, Kane CL, Mele EJ: Topological insulators in three dimensions. Phys Rev Lett 2007, 98:106803.CrossRef 4. Zhang H, Liu C-X, Qi XL, Dai X, Fang Z, Zhang S-C: Topological insulators in Bi2Se3, Bi2Te3 and Sb2Te3 with a single Dirac cone on the surface. Nat Phys 2009, 5:438–442.CrossRef 5. Qi X-L, Zhang S-C: The quantum spin Hall effect and topological insulators. Phys Today 2010, 63:33–38.CrossRef 6. Hasan MZ, Kane CL: Colloquium topological insulators. Rev Mod Phys 2010, 82:3045–3067.CrossRef 7. Ando Y: Topological insulator materials. selleckchem J Phys Soc Jpn 2013, 82:102001.CrossRef 8. Hong SS, Cha JJ, Kong D, Cui Y: Ultra-low carrier concentration and surface-dominant transport in antimony-doped Bi2Se3 topological insulator nanoribbons. Nat Commun 2012, 3:757.CrossRef 9. Chen YL, Chu J-H, Analytis JG, Liu ZK, Igarashi K, Kuo H-H, Qi XL, Mo SK, Moore RG, Lu DH, Hashimoto M, Sasagawa T, Zhang S-C, Fisher IR, Hussain Z, Shen ZX: Massive Dirac fermion on the surface of a magnetically doped topological insulator. Selleck STI571 Science 2010, 329:659–662.CrossRef 10. Lee CH, He R, Wang ZH, Qiu RLJ, Kumar A, Delaney C, Beck B, Kidd TE, Chancey CC,

Sankaran RM, Gao XPA: Metal-insulator transition in variably doped (Bi1−xSbx) 2Se3 nanosheets. Nanoscale 2013, 5:4337–4343.CrossRef 11. Cha JJ, Kong D, Hong S-S, Analytis JG, Lai K, Cui Y: Weak antilocalization in Bi2 (SexTe1−x)3 nanoribbons and nanoplates. Nano Lett 2012, 12:1107–1111.CrossRef 12. Wang L-L, Johnson DD: Ternary tetradymite compounds as topological insulators. Phys Rev B 2011, 83:241309.CrossRef 13. Ren Z, Taskin AA, Sasaki S, Segawa K, Ando Y: Large bulk resistivity and surface quantum triclocarban oscillations in the topological insulator Bi2Te2Se.

Phys Rev B 2010, 82:241306.CrossRef 14. Bao L, He L, Meyer N, Kou X, Zhang P, Chen Z-G, Fedorov AV, Zou J, Riedemann TM, Lograsso TA, Wang KL, Tuttle G, Xiu F: Weak Selleck Entospletinib anti-localization and quantum oscillations of surface states in topological insulator Bi2, Se2Te. Sci Rep 2012, 2:726.CrossRef 15. Wang G, Zhu X-G, Sun Y-Y, Li Y-Y, Zhang T, Wen J, Chen X, He K, Wang LL, Ma X-C, Jia J-F, Zhang SB, Xue Q-K: Topological insulator thin films of Bi2Te3 with controlled electronic structure. Adv Mat 2011, 23:2929–2932.CrossRef 16. Yan Y, Liao Z-M, Zhou Y-B, Wu H-C, Bie Y-Q, Chen J-J, Meng J, Wu X-S, Yu D-P: Synthesis and quantum transport properties of Bi2Se3 topological insulator nanostructures. Sci Rep 2013, 3:1264. 17. Peng H, Lai K, Kong D, Meister S, Chen YL, Qi XL, Zhang S-C, Shen ZX, Cui Y: Aharonov-Bohm interference in topological insulator nanoribbons. Nat Mater 2010, 9:225–229. 18.


In SGC-CBP30 supplier the last ten years, the greatest insights into the human intestinal microbiome have come about as a result of the application of metagenomics approaches to faecal samples, as attested by more than 1294

scientific publications found under the terms “human faecal microbiome” and “human fecal microbiome ” in PubMed. Metagenomics approaches in biomedicine seek to provide a comprehensive picture of the diversity and abundance of dominant and subdominant microbial species in health [2, 3] and in diseased states such as inflammatory bowel disorders (IBDs), irritable bowel syndrome (IBS) and other functional bowel disorders (FBD) [4–7]. During the course of these diseases, stool consistency is altered, varying from very hard (in

constipation) to entirely liquid (in diarrhoea), as determined by the Bristol stool scale [8]. Diarrhoea is defined as an abnormally frequent discharge of semi-solid or fluid faecal matter from the bowel. As such, it usually implies a large percentage of water. A normal stool sample is considered to have a water content of about 75%, while that of a diarrhoeic stool is > 85% [9]. The freezing of specimens containing water causes the formation of ice crystals, which damage the microbial cell wall. Consequently, there is an increased release of cellular components such as DNase and RNase, which in turn may degrade nucleic acids at the beginning of the DNA extraction procedure. In intestinal disorders, Cilengitide such as IBD, IBS, and infectious diseases, the sampling of diarrhoeic stools is find more common [10, 11]. However, how the water content of these samples affects the integrity of microbial DNA, and therefore the analysis of microbial

composition, is unclear. Steps such as stool homogenisation during collection or mechanical cell wall breaking during DNA extraction may affect the analysis of the microbial community. To date, no study on stool homogenisation or mechanical cell wall breaking using high-throughput sequencing technique has been reported. An appropriate Dolutegravir concentration collection protocol, together with a better understanding of the characteristics of a stool, is critical for downstream microbial community analysis. Here we tested various factors that may affect microbial community analysis during stool sample collection and DNA extraction steps using gel electrophoresis and pyrosequencing of the 16S rRNA gene. In this regard, we examined the effect of homogenising the stool before freezing, the addition of a physiological solution to the stools to simulate a diarrhoeic condition before freezing, and the use of beads to breakdown the microbial cell wall during DNA extraction. Results and discussion Experimental design Faecal samples were collected from healthy volunteers (n = 8) who had not taken antibiotics during the previous three months. Fresh samples were aliquoted as described below.