Results Bioinformatics analysis of B. pseudomallei SDO A SDO amino-acid (aa) sequence of B. pseudomallei strain K96243 was retrieved from selleck chemicals GenBank

(NCBI Reference Sequence: YP_112245.1; locus_tag = “BPSS2242” [14]). It was composed of 271 aa with a calculated molecular weight of 28,766 Dalton. BLAST [15] sequence analysis [16] revealed that B. pseudomallei SDO was categorized into short-chain dehydrogenases/reductases (SDRs), which shared a 24% amino-acid sequence identity with Bacillus megaterium glucose INCB018424 price 1-dehydrogenase (PDB ID: 1GCO) (Figure 1A). Therefore, the SWISS-MODEL [17] was used to construct a structural model of B. pseudomallei SDO, using B. megaterium glucose 1-dehydrogenase as a template for homology modeling. The resulting model was validated by PROCHECK [18]. The structural model of B. pseudomallei SDO revealed a catalytic triad active site, consisting of Ser149, Tyr162, and Lys166, together with a NAD+ cofactor domain (Figure 1B). This suggests that the SDO of B. pseudomallei may have an enzymatic function similar to B. megaterium glucose 1-dehydrogenase. Figure 1 Protein sequence and structural comparison between B. pseudomallei SDO and B. megaterium glucose 1-dehydrogenase. mTOR inhibitor A) Sequence alignment

between B. pseudomallei SDO and B. megaterium glucose 1-dehydrogenase. B) Structural model of B. pseudomallei SDO (left) and structure of B. megaterium glucose 1-dehydrogenase (right), with bound NAD (yellow) RNA Synthesis inhibitor shown in both surface (top) and cartoon representations (bottom). B. pseudomallei SDO and B. megaterium glucose 1-dehydrogenase shared structural similarities with conserved catalytic triad, consisting of Tyr (green), Thr (pink) and Lys (orange).

Figures were generated by Discovery Studio Visualizer – Accelrys. Among available genomes of Burkholderia spp., BLAST analysis demonstrated that all species harbor the SDO protein. The amino-acid identities of pathogenic B. pseudomallei, B. mallei, B. oklahomensis, B. multivorans, B. vietnamiensis, and B. cenocepacia range from 83% to 100%, whereas those of non-pathogenic B. thailandensis are less than 36%. The high identity among pathogenic strains might indicate a common pathogenesis that is mediated by Burkholderia SDO. Mutagenesis of B. pseudomallei SDO mutant To identify the function of SDO in B. pseudomallei, we constructed a mutant defective in SDO production using a pEXKm5-based allele replacement system [19]. PCR analysis using primers flanking deleted alleles confirmed the deletion of the SDO gene on the B. pseudomallei chromosome (Additional file 1). As expected, a 566 bp DNA fragment was detected in the SDO mutant, whereas a 1,197 bp DNA fragment was detected in the wild type K96243, indicating a homologous recombination by deletion of 631 bp of the SDO gene on the chromosome of the B. pseudomallei mutant. B. pseudomallei SDO complement strain was constructed using the same strategy.

Targets of the CpxR homologue selleck in S. meliloti are completely unknown, but ABT-263 price expression of genes encoding DegP proteases (degP1P3P4) and peptidyl-prolyl isomerase Ppi (ppiABD) were significantly increased in tolC mutant. A search for the E. coli CpxR binding site GTAAAN5GTAAA consensus sequence in the upstream coding regions of S. meliloti using the RSA-tools web interface revealed that this sequence matched the putative promoter region upstream of the predicted operon SMb21562/SMb21561/SMb21560. In a recent study, the CpxR protein from Yersinia enterocolitica was shown to negatively affect transcription of gene rpoE, coding for the extracytoplasmic sigma-E factor [29]. We also observed decreased

expression of rpoE2 and rpoE8 genes. Our data suggest that in the absence of a functional 3-Methyladenine mouse TolC, cells trigger a Cpx instead of an RpoE-mediated

response. A very different situation was observed in wild-type S. meliloti cells grown under different stress conditions such as osmotic shock [30, 31], high metal ion concentration [32], acidic pH [33], heat shock and entry in stationary phase [34] where an rpoE2-mediated response was induced. This seems to indicate that the external stress imposed on the cells triggers a well defined extracytoplasmic response. When perturbations to the cell envelope, such as the absence of a functional outer membrane protein occur, cells seem to activate a distinct

stress response pathway. Genes involved in transcription and translation It is possible that under the cytoplasmic and extracytoplasmic stress conditions experienced by the tolC mutant, many proteins and cofactors become inactive and need to be synthesized de novo or protected from denaturation. It is then not surprising that many genes encoding proteins involved in transcription and translation were found to have significantly increased expression in the tolC mutant strain. This was the case for genes encoding all RNA polymerase subunits (rpoABCZ), genes nusA and Cell press nusG involved in transcriptional pausing, termination, and antitermination, and the gene encoding transcription termination factor Rho. RNA degradation is mediated by the RNA degradosome, a multiprotein complex involving RNase E, polynucleotide phosphorylase (PNPase), helicase RhlB, and enolase [35]. In S. meliloti, those components are encoded by the genes rne, pnp, deaD, and eno, respectively, all of them showing increased expression in tolC mutant suggesting that, besides increased expression of genes encoding products involved in transcription, the mutant also increases expression of genes encoding products participating in RNA degradation. Of the 105 genes differentially expressed and involved in translation and ribosome biogenesis only three had a decreased expression in the tolC mutant.

For control assays, incubation with the primary antiserum was omitted. Results and discussion Bioinformatic analysis of kinetoplast-associated proteins in trypanosomatid species As previously stated, originally five distinct kinetoplast-associated

proteins were described in C. fasciculata, named CfKAP1–5 [12, 13]. However, the CfKAP5, also designated p15, was never characterized. Since little is known about kDNA-associated proteins in T. cruzi [18, 19] and other trypanosomatids, we sought initially to address this problem by examining genome database of the T. cruzi [34], T. brucei [35], Leishmania major [36], L. infantum and L. braziliensis Idasanutlin in vivo [37]. In a BLASTp search, using as query the available CfKAP protein sequences, we have identified 35 protein sequences related to CfKAPs: 11 in T. cruzi; 7 in L. braziliensis; 6 in L. major and L. infantum; and 5 in T. brucei. A phylogenetic analysis including these 35 sequences and the five CfKAPs used as query was performed, in order to construct a phylogenetic tree (figure 1). Additionally, a synteny conservation analysis was performed, where chromosome location was highly correlated with tree topology, allowing us to infer the homology relationships

between the trypanosomatid KAPs [see additional file 1]. Figure 1 Phylogenetic analysis of trypanosomatid KAPs proteins with confidence values PRKACG shown as percentages. Lb, Leishmania braziliensis; Li, Leishmania infantum; Lm, Leishmania major; Tb, Trypanosoma brucei; Tc, Trypanosoma cruzi. In the Sapanisertib solubility dmso T. cruzi genome, we were able to identify the KAP3 and KAP4 genes, but not the KAP1 and KAP2 genes, which were only identified in Leishmania

spp. and C. fasciculata. Furthermore, we were able to identify two other genes that are similar to the CfKAPs, herein named KAP6 and KAP7. They have not been characterized in Crithidia, as the available sequence information for this genome is limited (227 nucleotide sequences in the current version of GenBank). The KAP6 gene whose size is compatible to others KAPs, is more related to KAP4 (figure 1) and was selleck screening library annotated in all five genomes analyzed as “”kinetoplast DNA-associated protein”". The KAP7 gene, also present in all trypanosomatid genomes, has been annotated as “”hypothetical protein, conserved”". Although it is clustered with the KAP1 gene (figure 1), the lower bootstrap value of this clade reinforces the uncertainty of KAP7 relationship to other KAPs. The KAP genes of T. cruzi are present as two copies, with the exception of TcKAP4c, probably due to the hybrid nature of the CL Brener strain [34]- [see additional file 1]. Characterization of TcKAPs In this work, we cloned and expressed two KAPs in T. cruzi: TcKAP4 and TcKAP6.

(b) A schematic drawing of the rhombohedral unit cell. The shaded plane is the (001) plane. Within the plane, orange lines represent the three in-zone directions: HM781-36B mouse [100], [010], and , along which

planar defects can be observed. Blue lines represent the three off-zone directions: [001], , and , from which the planar defects cannot be seen. (c) A roadmap consisting of simulated diffraction patterns of major low index zone axes within the (001) plane. During TEM examination, the roadmap helps the operator to determine whether it is possible to tilt to the desired zone axes. A roadmap consisting of simulated diffraction patterns of major low index zone axes within the (001) plane is shown in Figure 2c. During TEM examination, this roadmap can help us judge if it is possible to tilt to the next zone axis according to the calculated angle between different zone axes. For example, it is nearly impossible to obtain results from both and [010] zone axes on the same nanowire because the calculated inter-axial angle (57.1°) is close to the tilting limit of our TEM specimen holder (60°). In the roadmap, there are four independent patterns such as those from , , [010], and [110] directions, as grouped in four colors. During TEM examination, planar defects can be seen along directions

of , , and [010] whose diffraction patterns are asymmetric and with streaks in them. While AICAR research buy viewing along the [110] direction, BAY 80-6946 ic50 the layered faults feature is hidden because of the mirror symmetry. In addition, planar defects are more distinctive when viewing along directions of and [010] than that of (see Additional file 1 for comparison between experimental results obtained from the aforementioned four different zone axes). Therefore, in our real TEM practice, only results from the two independent directions: and [010] are recorded and analyzed. There are a total of six equivalent -type and [010]-type Megestrol Acetate directions in the rhombohedral system, as drawn in orange and blue lines in Figure 2b. Characteristic features of planar defects can be observed by TEM when the viewing direction is along the rhombohedral axes or the short

diagonal within the (001) plane, i.e., the directions of [100], [010], and . These three directions (outlined in orange) are denoted as in-zone directions. Meanwhile, the other three directions: [001], , and , located out of the (001) plane (marked in blue), are denoted as off-zone directions, due to the fact that planar defects are invisible from them. Now the difficulty to visualize planar defects in boron carbide nanowires becomes obvious. If the viewing direction is not parallel to planar defects, the defects will be invisible. In addition, even if the viewing direction is parallel to planar defects, depending on the initial orientation of the viewing direction, planar defects may also not be observed. For example, if the initial viewing direction (i.e.

PubMedCrossRef 45. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M: Blast2GO:

a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005,21(18):3674–3676.PubMedCrossRef 46. Gotz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talon M, Dopazo J, Conesa A: High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 2008,36(10):3420–3435.PubMedCrossRef 47. Stekel DJ, Git Y, Falciani F: The comparison of gene expression from multiple cDNA libraries. Genome Res 2000,10(12):2055–2061.PubMedCrossRef 48. Al-Shahrour F, Diaz-Uriarte R, Dopazo J: FatiGO: a web tool for finding significant associations of Gene Ontology terms with groups of genes. Bioinformatics 2004,20(4):578–580.PubMedCrossRef Capmatinib chemical structure 49. Vallier A, Vincent-Monegat AG-120 cell line C, Laurencon A, Heddi A: RNAi in the cereal weevil Sitophilus spp: systemic gene knockdown in the bacteriome tissue. BMC Biotechnol 2009, 9:44.PubMedCrossRef 50. Pfaffl MW, Horgan GW, Dempfle L: Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002,30(9):e36.PubMedCrossRef 51. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP: Determination of stable

housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 2004,26(6):509–515.PubMedCrossRef 52. Richards S, Gibbs RA, Weinstock GM, Brown SJ, Denell R, Beeman RW, Gibbs R, Bucher G, Friedrich M, Grimmelikhuijzen CJ, et al.: The genome

of the model beetle and pest Tribolium castaneum. Nature 2008,452(7190):949–955.PubMedCrossRef 53. Zhang G, Ghosh S: Negative Amisulpride regulation of toll-like receptor-mediated signaling by Tollip. J Biol Chem 2002,277(9):7059–7065.PubMedCrossRef 54. Lemaitre B, Kromer-Metzger E, Michaut L, Nicolas E, Meister M, Georgel P, Reichhart JM, Hoffmann JA: A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense. Proc Natl Acad Sci U S A 1995,92(21):9465–9469.PubMedCrossRef 55. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA: The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 1996,86(6):973–983.PubMedCrossRef 56. Kopp E, Medzhitov R, Carothers J, Xiao C, Selleckchem Savolitinib Douglas I, Janeway CA, Ghosh S: ECSIT is an evolutionarily conserved intermediate in the Toll/IL-1 signal transduction pathway. Genes Dev 1999,13(16):2059–2071.PubMedCrossRef 57. Akira S, Takeda K: Toll-like receptor signalling. Nat Rev Immunol 2004,4(7):499–511.PubMedCrossRef 58. Wood KW, Sarnecki C, Roberts TM, Blenis J: ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell 1992,68(6):1041–1050.PubMedCrossRef 59.

Patients with grade 1a,1b or 2a, 2b open abdomen, as classified by Bjorck et al. [7] (Table 1) were suitable for inclusion. The following exclusion criteria were also applied: <18

years, pregnant, malignancy in wound bed, unexplored fistulas, high risk for imminent death (as determined by the treating surgeon), pre-existing large ventral hernia, significant loss of abdominal wall fascia as a result of trauma or infection, patients with grade 4 open abdomen (Bjorck et al. classification, see Table 1), patients with a known history of poor compliance with medical treatment and any patients who had previously been withdrawn from the study. The trial was approved by local ethics boards at both selleck chemicals llc institutions and was carried out in strict accordance with the Helsinki declaration. Informed consent was obtained where possible from the patient, but if the patient was incapable, the patient’s legal representative was asked to provide consent on the patient’s behalf. If this was not possible then independent physician consent was considered acceptable as approved by the local ethics committee. All patient information was anonymised at source. Patients suitable for inclusion underwent initial damage

control laparotomy, where initial control of haemorrhage and contamination was performed. This was followed by intra-peritoneal packing when required and TAC. Further buy GDC-0068 resuscitation to near normal physiology in the intensive care unit (ICU) was continued. Re-laparotomy was performed at 48 hours or earlier if indicated. Negative pressure wound therapy (CB-839 ic50 RENASYS-AB Abdominal

Dressing and RENASYS EZ pump Smith & Nephew; St Petersburg, FL, USA) was applied to the wound in the following way. A fenestrated non adherent film was placed directly over the exposed viscera but under the rectus sheath. Polyurethane foam was then reduced along pre-cut perforations to the appropriate size and placed on top of the film within the open abdomen. A transparent film then covered the foam and the surrounding peri-wound skin before a suction port was connected over to the NPWT pump. Negative pressure was delivered at a continuous -80 mmHg. The trial comprised a maximum of 20 days of treatment with the NPWT system with an additional 8 day post-treatment initiation follow up. Dressing changes usually took place at 48 hours during re-laparotomy for removal of packs and re-establishment of bowel continuity. Full medical and wound assessments were made. Wound closure was carried out when possible and at the discretion of the attending trauma surgeon. The primary objective was to determine the number of days taken to achieve delayed primary fascial closure.

7% and 1.5%. No significant Dibutyryl-cAMP molecular weight effect of oscillating MNPs in killing cancerous cells was observed. At the concentration of 100 μg/mL, the corresponding decreasing rates were 11.7% and 30.9%, proving the morphological effect of MNPs. While at concentration of 500 μg/mL, 12.5% and 13.9% HeLa cells were killed by spherical MNPs and rod-shaped MNPs, respectively, but no significant difference was observed as well. The details of cell viability relative to AMF treatment time were shown in Figure 5. For the three concentrations of MNPs in this study, only the medium concentration was demonstrative of the morphological effect. For the interesting phenomenon

that medium concentration was more suitable than higher or lower ones, we assume that it could be explained by the following two aspects. Firstly, the power of the device used in this study was too low to drive MNPs in high concentrations to oscillate inside cells or tissue efficiently and simultaneously, and

https://www.selleckchem.com/products/obeticholic-acid.html too many particles in AMF had mutual restraint effect if they Daporinad datasheet assembled in clusters, especially for rod-shaped MNPs. On the contrary, with low intake of MNPs, it was hard to effectively influence cell viability by mechanical oscillations. Figure 4 SEM images of rMNP-loaded cells membrane before (upper) and after (lower) 2 h AMF treatment. Figure 5 Cell viability of MNP-loaded HeLa cells after AMF treatment for a while. The corresponding morphologies and MNPs concentration in microgram per milliliter are listed on the right. It is supposed that MNPs embedded into the cell membranes mainly contributed to cell old death by destroying the membranes. Cell dyeing is indicative of cell membrane damage. In this study, trypan blue assay, which was sensitive to permeability of membranes, was further used to verify the observed morphological effect at the concentration

of 100 μg/mL. As shown in Figure 4, the HeLa cells that were incubated with 100 μg/mL rod-shaped MNPs appeared to have a loose cell structure after 2 h AMF treatment. For the 2-h groups, 39.47% of the rMNP-loaded cells were stained, while only 15.13% of sMNP-loaded cells were stained. Details of trypan blue staining were shown in Figure 6. This result is consistent with the observed decreases in cell viability. In a previous research, the concentration- and time-dependent damage of iron oxide MNPs to cell membrane injury was observed as well [23], supporting the concentration dependence of this study. The morphological effect was fully shown in this situation: rod-shaped MNPs pre-incubated with 100 μg/mL and placed in AMF for 2 h or more. Figure 6 Percentage of trypan blue-stained cells. These cells had been pre-cultured in 100 μg/mL MNPs suspended culture medium and exposed to an AMF for up to 2 h. Mechanisms of morphological effect The results showed that MNP morphology and concentration have an important influence on the cell inactivation effects of AMF-assisted forced vibration of MNPs.

Restriction enzymes with a single recognition site are given in bold. (TIFF 314 KB) Additional file 2: Schematic presentation of AggL and MbpL proteins. Boxes indicate domains of Selleckchem PS-341 proteins and arrows selleck kinase inhibitor indicate repeats. (TIFF 238 KB) References 1. Gasson MJ, Swindell S, Maeda S, Dodd HM: Molecular rearrangement of lactose plasmid DNA associated with high-frequency transfer and cell aggregation in Lactococcus lactis 712. Mol Microbiol 1992, 6:3213–3223.PubMedCrossRef 2. Gajic O: Relationships between MDR proteins, bacteriocin production and proteolysis

in Lactococcus lactis . PhD thesis. University of Groningen; 2003. 3. Anderson DG, McKay LL: Genetic and physical characterization of recombinant plasmids associated with cell aggregation and high frequency conjugal transfer in Streptococcus lactis ML3. J Bacteriol BAY 63-2521 molecular weight 1984, 158:954–962.PubMed 4. Gasson MJ, Davies FL: High-frequency conjugation associated with Streptococcus lactis donor cell aggregation. J Bacteriol 1980, 143:1260–1264.PubMed 5. Walsh PM, McKay LL: Recombinant plasmid associated with cell aggregation and

high-frequency conjugation of Streptococcus lactis ML3. J Bacteriol 1981, 146:937–944.PubMed 6. Bringel F, van Alstine GL, Scot JR: Transfer of Tn916 between Lactococcus lactis subsp . lactis strains is nontranspositional: evidence for a chromosomal fertility function in strain MG1363. J Bacteriol 1992, 174:584–5847. 7. Stentz R, Jury K, Eaton T, Parker M, Narbad A, Gasson M, Shearman C: Controlled expression of CluA in Lactococcus lactis and its role in conjugation. Microbiology

2004, 150:2503–2512.PubMedCrossRef 8. Stentz R, Gasson M, Shearman C: The Tra domain of the lactococcal CluA surface protein is a unique domain that contributes to sex factor DNA transfer. J Bacteriol 2006, 188:2106–2114.PubMedCrossRef 9. Kojic M, Strahinic I, Fira D, Jovcic B, Topisirovic L: Plasmid content and bacteriocin production by five strains of Lactococcus lactis isolated from semi-hard cheese. Can J Microbiol 2006, 52:1110–1120.PubMedCrossRef 10. Fischetti VA, Pancholi V, Schneewind O: Conservation of a hexapeptide sequence Atorvastatin in the anchor region of surface proteins from Gram-positive cocci. Mol Microbiol 1990, 4:1603–1605.PubMedCrossRef 11. Navarre WW, Schneewind O: Surface proteins of Gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol Mol Biol Rev 1999, 63:174–229.PubMed 12. Jenkinson HF: Cell surface protein receptors in oral streptococci. FEMS Microbiol Lett 1994, 121:133–140.PubMedCrossRef 13. Jenkinson HF, Demuth DR: Structure, function and immunogenicity of streptococcal antigen I/II polypeptides. Mol Microbiol 1997, 23:183–190.PubMedCrossRef 14. Kolenbrander PE, London J: Adhere today, here tomorrow: oral bacteria adherence. J Bacteriol 1993, 175:3247–3252.PubMed 15. Jett BD, Atkuri RV, Gilmore MS: Enterococcus faecalis localization in experimental endophtalmitis: role of plasmid-encoded aggregation substance.

Nature 2010,464(7285):59–65.PubMedCentralPubMedCrossRef 3. Human Microbiome Project: A framework for human microbiome research. Nature 2012,486(7402):215–221.CrossRef 4. Manichanh C, Rigottier-Gois

L, Bonnaud E, Gloux K, Pelletier E, Frangeul L, Nalin R, Jarrin C, Chardon P, Marteau P, Roca J, Dore J: Reduced diversity of faecal microbiota in Crohn’s disease revealed by a metagenomic approach. Gut 2006,55(2):205–211.PubMedCentralPubMedCrossRef 5. Versalovic J: The human microbiome and probiotics: implications for pediatrics. Ann Nutr Metab 2013,63(Suppl 2):42–52.PubMedCrossRef 6. Jeffery IB, O’Toole PW, Ohman L, Claesson MJ, Deane J, Quigley EM, Simren M: An irritable bowel syndrome subtype defined by species-specific alterations in faecal microbiota. Gut 2012,61(7):997–1006.PubMedCrossRef 7. Manichanh C, Eck A, Varela E, Roca J, Clemente JC, Gonzalez A, Knights BB-94 D, Knight R, Estrella S, Hernandez C, Guyonnet D, Accarino A, Santos J, Malagelada JR, Guarner F, JQEZ5 ic50 Azpiroz F: Anal gas evacuation and colonic microbiota in patients with flatulence: effect of diet . Gut 2013,63(3):401–408.PubMedCentralPubMedCrossRef 8. Lewis SJ, Heaton KW: Stool form scale as a useful guide

to intestinal transit time. Scand J Gastroenterol 1997,32(9):920–924.PubMedCrossRef 9. Fischer B, Hoh S, Wehler M, Hahn EG, Schneider HT: Faecal elastase-1: lyophilization of stool samples prevents false low results in diarrhoea. Scand J Gastroenterol 2001,36(7):771–774.PubMedCrossRef 10. Longstreth GF, Thompson WG, Chey WD, Houghton LA, Mearin F, Spiller RC: Functional bowel disorders. Gastroenterology 2006,130(5):1480–1491.PubMedCrossRef Tozasertib solubility dmso 11. Tang Y, Forsyth CB, Keshavarzian A: New Florfenicol molecular insights into inflammatory bowel disease-induced diarrhoea. Expert Rev Gastroenterol Hepatol 2011,5(5):615–625.PubMedCentralPubMedCrossRef

12. Wenzl HH, Fine KD, Schiller LR, Fordtran JS: Determinants of decreased fecal consistency in patients with diarrhoea. Gastroenterology 1995,108(6):1729–1738.PubMedCrossRef 13. Fujimoto S, Nakagami Y, Kojima F: Optimal bacterial DNA isolation method using bead-beating technique. Memoirs Kyushu Univ Dep Of Health Scis Of Medical Sch 2004, 3:33–38. 14. Cardona S, Eck A, Cassellas M, Gallart M, Alastrue C, Dore J, Azpiroz F, Roca J, Guarner F, Manichanh C: Storage conditions of intestinal microbiota matter in metagenomic analysis. BMC Microbiol 2012, 12:158.PubMedCentralPubMedCrossRef 15. Godon JJ, Zumstein E, Dabert P, Habouzit F, Moletta R: Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol 1997,63(7):2802–2813.PubMedCentralPubMed 16. Walters WA, Caporaso JG, Lauber CL, Berg-Lyons D, Fierer N, Knight R: PrimerProspector: de novo design and taxonomic analysis of barcoded polymerase chain reaction primers. Bioinformatics 2011,27(8):1159–1161.PubMedCentralPubMedCrossRef 17.