Additionally, magnesium sulphate or choline chloride at final con

Additionally, magnesium sulphate or choline chloride at final concentrations of 40 mM also failed to dequench the fluorescence (data not shown). Control assays conducted with inverted vesicles that contained the dysfunctional MdtM D22A

mutant did not exhibit any fluorescence dequenching in response to the addition of any of the cations tested (Figure 8; grey traces), thereby providing further robust evidence that the dequenching observed upon the addition of Rb+ and Li+ to vesicles generated from TO114 cells transformed with pMdtM was Selleck ALK inhibitor due to a process mediated by the functionally expressed recombinant transporter. Figure 8 MdtM-catalysed Rb + /H + , Li + /H + and Ca 2+ /H + exchange at alkaline pH. Exchange was determined by the fluorescence dequenching of acridine orange in inverted vesicles derived from antiporter-deficient E. coli TO114 cells that overexpressed recombinant wild-type MdtM (black traces) or the dysfunctional MdtM D22A mutant (grey traces). A ΔpH across the vesicle membrane was established by addition of lactate as indicated and once the fluorescence quench of acridine orange achieved a steady state, 40 mM Rb2SO4 (A), 40 mM Li2SO4 (B) or 40 mM CaSO4 (C) was added to the vesicles. Addition

of 100 μM CCCP abolished the ΔpH. The fluorescence intensity SPTLC1 of each measurement is represented as a percentage learn more of the initial acridine orange fluorescence signal prior to addition of lactate. The fluorescence measurements were conducted at pH 9.0 and the traces shown are representative of experiments performed in triplicate on at least two separate preparations of inverted vesicles. MdtM-catalysed K+/H+ and Na+/H+ antiport is electrogenic Generally, cation/proton antiporters involved in alkaline pH homeostasis are required to mediate

an electrogenic antiport that is energized by the transmembrane electrical potential, Δψ [5]. Therefore, to probe whether MdtM catalyses electrogenic antiport, inverted vesicles were generated from TO114 cells transformed with pMdtM and assayed for electrogenicity in a chloride-free and potassium-free buffer using the Δψ–sensitive fluorophore Oxonol V. Inverted vesicles produced from TO114 cells transformed with pD22A were used as a negative control. In all the assays, energization of the vesicles by lactate resulted in a rapid quench of Oxonol V fluorescence indicating the generation of respiratory Δψ (Figure 9). To ensure the suitability of the experimental conditions for detection of electrogenic antiport, a positive control (Figure 9F) was performed using inverted vesicles produced from E.

Atarashii Ganka 2006; 23(2): 237–43 19 Kakimaru A, Kawaguchi A,

Atarashii Ganka 2006; 23(2): 237–43 19. Kakimaru A, Kawaguchi A, Mihara E. Suture abscess of levofloxacin-resistant Corynebacterium spp. [in Japanese]. Atarashii Ganka 2004; 21: 801–4 20. Mitsui Y, Kitano S, Uchida Y, et al. Standardization of the evaluation of antimicrobial ophthalmic solutions and ointments, 1985 [in Japanese].

Nippon Ganka Gakkai Zasshi 1986; 90(3): 511–5PubMed 21. Kameyama K. Dacryocystitis. In: Usui M, Ohashi Y, Tagawa Y, et al., editors. Ocular infections clinic [in Japanese]. Tokyo: Igaku-shoin Ltd., 2000: 138–9 22. Japanese Association for Ocular Infection. Guidelines for the clinical management of infectious keratitis [in Japanese]. Nippon Ganka Gakkai Zasshi 2007; BAY 11-7082 in vitro 111: 769–809 23. Kanda Y, Kayama T, Okamoto S, et al. A post-marketing surveillance of 0.5% levofloxacin ophthalmic solution for external ocular infections. Rinsho Ganka 2008; 62(13): 2007–17″
“Dear Reader, As we reach the final issue of Drugs in R&D for 2012, we hope you have found the articles published throughout the year to be interesting and informative. The editors and publishing staff have appreciated the high quality of content contributed to the journal this year

and look forward to keeping you up to date with topical issues in the field of research and development in 2013. Following the acquisition of the Adis journals at the end of last year by Springer Science+Business Media, we have been working to transition the production and fulfillment of the Adis journals into Springer processes.

This integration will be complete by the end of 2012, such that production and delivery of all Adis journals will be fully transitioned for the first issues of 2013. Adis journal content will be available on a new and improved online platform. We are pleased that Springer has decided to keep the Adis brand and recognizes the core values that the brand represents. We are confident that the association with Springer selleck will lead to increased awareness and usage of Adis content, further driving impact factors and recognition. Collectively, we feel that the future for Adis looks bright, and we are happy with the acquisition by Springer. The high quality of Adis Journals was acknowledged in the new ISI impact factors for 2011, with the majority of our titles making gains over 2010. The most impressive gains were made by Clinical Pharmacokinetics (5.398), with a 19.6% increase, Drugs (4.226), which increased by 13%, and Clinical Drug Investigation (1.822), an increase of 12.3% over 2010. Two of our journals received their first impact factors in 2011 — Pediatric Drugs, with an impact factor of 1.786, and The Patient (0.571). We would like to say a big thank you to all of the authors who have contributed articles to Drugs in R&D within the last 12 months. Without their hard work and diligence, we would not have been able to publish the journal.

(PDF 34 KB) Additional file 2: Table S2 Expression levels of SAP

(PDF 34 KB) Additional file 2: Table S2. Expression levels of SAP genes in biofilms grown in the various model systems. (PDF 30 KB) Additional file 3: Table S3. Expression levels of PLB and LIP genes in biofilms grown in the various model systems. (PDF 39 KB) References 1. Odds FC: Meeting MM-102 solubility dmso Candida and Candidiosis. 2nd edition. Bailliere Tindall London UK; 1988. 2. Calderone RA, Fonzi WA: Virulence factors of Candida albicans . Trends in Microbiology 2001, 9:327–335.PubMedCrossRef 3. Hube B: From commensal to pathogen: stage- and tissue-specific gene expression of Candida albicans . Current Opinion in Microbiology 2004, 7:336–341.PubMedCrossRef 4. Hoyer LL: The ALS

gene family of Candida albicans . Trends in Microbiology 2001, 9:176–180.PubMedCrossRef 5.

Staab JF, Bradway SD, Fidel ARS-1620 price PL, Sundstrom P: Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1. Science 1999, 283:1535–1538.PubMedCrossRef 6. Hoyer LL, Green CB, Oh SH, Zhao X: Discovering the secrets of the Candida albicans agglutinin-like sequence ( ALS ) gene family–a sticky pursuit. Medical Mycology 2008, 46:1–15.PubMedCrossRef 7. Ghannoum MA: Potential role of phospholipases in virulence and fungal pathogenesis. Clinical Microbiology Reviews 2000, 13:122–143.PubMedCrossRef 8. Hube B, Stehr F, Bossenz M, Mazur A, Kretschmar M, Schäfer W: Secreted lipases of Candida albicans : cloning, characterization and expression analysis of a new gene family with at least ten members. Archives of Microbiology 2000, 174:362–374.PubMedCrossRef 9. Naglik JR, Challacombe SJ, Hube B: Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiology and Molecular Biology 2003, 67:400–428.CrossRef 10. Schaller M, Borelli C, Korting HC, Hube B: Hydrolytic enzymes as virulence factors of Candida albicans . Mycoses 2005, 48:365–377.PubMedCrossRef

11. Douglas LJ: Candida biofilms and their role in infection. Trends in Microbiology 2003, 11:30–36.PubMedCrossRef 12. Kojic EM, Darouiche RO: Candida infections of medical devices. Clinical Microbiology Reviews ALOX15 2004, 17:255–267.PubMedCrossRef 13. Kumamoto CA, Vinces MD: Alternative Candida albicans lifestyles: growth on surfaces. Annual Review of Microbiology 2005, 59:113–133.PubMedCrossRef 14. Kumamoto CA: Candida biofilms. Current Opinion in Microbiology 2002, 5:608–611.PubMedCrossRef 15. Blankenship JR, Mitchell AP: How to build a biofilm: a fungal perspective. Current Opinion in Microbiology 2006, 9:588–594.PubMedCrossRef 16. Schaller M, Zakikhany K, Naglik JR, Weindl G, Hube B: Models of oral and vaginal candidiasis based on in vitro reconstituted human epithelia. Nature Protocols 2006, 1:2767–2773.PubMedCrossRef 17. Hawser SP, Douglas LJ: Biofilm formation by Candida species on the surface of catheter material in vitro. Infection and Immunity 1994, 62:915–921.PubMed 18.

As the host range of E amylovora also includes pear trees, we fu

As the host range of E. amylovora also includes pear trees, we further investigated selleck screening library the virulence of the wild type and its acrD-deficient mutant on immature pear fruits (cv. ‘Bartlet’) with the conclusion that AcrD is not involved in the interaction of the fire

blight pathogen with this host. Additionally, we studied the expression levels of the AcrAB and AcrD efflux pumps in vitro and in planta, respectively. The activity of the acrA promoter was lower in planta than in LB medium (Table 3). However, it is possible that growth of the bacteria in LB broth may increase expression of the AcrAB pump. A similar induction of the RND-type efflux system MexAB-OprM in Pseudomonas syringae was observed during growth in complex King’s B medium [40]. Specific components of the complex media might induce the expression of these RND efflux systems. Alternatively, the efflux pumps may play a role in the secretion of metabolites during exponential growth of bacteria in complex medium. PP2 The level of acrD expression was low during growth in LB medium (Figure 1B), whereas it was slightly induced in planta (Table 3) indicating that plant-derived compounds are able to induce the AcrD pump. The nature of these

compounds remains to be elucidated. Several multidrug transporters are induced in response to the presence of toxic substances [18]. We identified the substrates deoxycholate, naringenin, tetracycline, novobiocin, fusidic acid, tannin and zinc as inducers of acrD in E. amylovora. In prokaryotes, the expression of drug transporter genes is frequently mediated by transcriptional regulatory proteins, whose genes are often located adjacent to those encoding

the transport system. However, no local transcriptional regulator was identified flanking the acrD gene in E. amylovora, suggesting that expression of acrD may be subject to regulation at the global level. The acrD gene belongs to the regulon of the envelope stress response, two-component system BaeSR in E. coli and Salmonella enterica. A baeSR-deficient mutant of E. amylovora Ea1189 has previously been Org 27569 evaluated for virulence on immature pears, and exhibit full-virulence, as that of wild type, on immature pear fruits [41]. The core regulon of BaeSR consists of spy, encoding a protein chaperon, and the RND efflux pump genes acrD and mdtABC[42]. Interestingly, we identified a partial overlap between the compounds inducing expression of acrD in E. amylovora and baeR in E. coli, e.g., flavonoids (naringenin), zinc, and tannin [24, 42]. Accordingly, the contribution of the two-component system BaeSR to regulation of the acrD gene in E. amylovora became of particular interest to us. In E. coli and S. enterica, BaeR, upon activation by phosphorylation through BaeS, binds to the upstream promoter region of mdtA and acrD[19, 35].

Two-dimensional high-performance

Two-dimensional high-performance

KPT-8602 manufacturer liquid chromatography-mass spectrometry analysis Trypsinized peptides with or without iTRAQ label were separated in the first dimension using an Agilent 1100 Series HPLC system (Agilent Technologies, Wilmington, DE). Samples were injected onto a C18 X-Terra column (1 × 100 mm, 5 μm, 100 Å; Waters Corporation, Milford, MA, USA) and eluted with a linear water-acetonitrile gradient (20 mM ammonium formate, pH 10, in both eluents A and B, 1% acetonitrile/min, 150 μL/min flow rate). A concentrated 200 mM solution of ammonium formate at pH 10 was prepared as described TSA HDAC in vivo by Gilar et al.[43]. Buffers A and B for first-dimension separation were prepared by a 1/10 dilution of this concentrated buffer with water and acetonitrile,

respectively. Fifty 1-min fractions were collected (roughly 6.6 μg/fraction). Samples were concatenated (fraction 1 and 31, 2 and 32, etc.) into a total of 25 fractions as described by Dwivedi et al.   [44]. Each was lyophilized and re-suspended in 100 μL of 0.1% formic acid. A splitless nanoflow Tempo LC system (Eksigent, Dublin, CA, USA) with 20 μL sample injection via a 300 μm × 5 mm PepMap100 precolumn and a 100 μm × 150 mm analytical column packed with 5 μm Luna C18(2) (Phenomenex, Torrance, CA) was used in the second-dimension separation prior to tandem MS analysis. Both eluents A (2% acetonitrile in water) and B (98% acetonitrile) contained 0.1% formic acid

as ion-pairing modifier. A 0.33% acetonitrile/min linear gradient (0-30% B) was used for peptide elution, providing a total 2 hour run time per fraction in the second dimension. Mass spectrometry A QStar Elite mass spectrometer (Applied Biosystems, Foster City, CA) was used in standard MS/MS data-dependent acquisition mode with a nano-electrospray ionization source. The 1 s survey MS spectra were collected (m/z 400–1500) Adenosine followed by three MS/MS measurements on the most intense parent ions (80 counts/s threshold, +2 to +4 charge state, m/z 100–1500 mass range for MS/MS), using the manufacturer’s “smart exit” settings and iTRAQ settings. Previously targeted parent ions were excluded from repetitive MS/MS acquisition for 60 s (50 mDa mass tolerance). Database search, protein identification, and statistical analysis Raw spectra WIFF files of unlabeled peptides were treated using standard script (Analyst QS 2.0) to generate text files in Mascot Generic File format (MGF) [45] and ProteoWizard to generate mzML files [46].

Suboptimal vitamin D status, coupled with the unaccustomed physic

Suboptimal vitamin D status, coupled with the unaccustomed physical activities associated with military training, may have profound effects on bone health. During bone remodeling, resorption and formation are coupled; however, once resorption occurs, bone deposition may require up to 90 days for completion [23], and may induce temporary weaknesses at remodeling sites. Evans et al. [10] noted increases in both Pitavastatin cost markers of bone formation and resorption during military training, similar to the findings of the present study. Similarly, studies assessing the effects of resistance-type training have documented increases in markers of bone

formation, and a reduction in markers of bone resorption [24]. The increase in markers of both bone resorption and formation observed in the present study may indicate a mechanism to repair microdamage caused by repeated stress. If stress continues to affect bone, microdamage may further develop into stress RAAS inhibitor fractures. Stress fracture is of particular concern in military personnel, as up to 60% of female Soldiers that experience fracture

may attrite from military training [12, 25, 26]. Studies reviewing stress fracture risk in military personnel indicate that a number of factors not affected by diet, such as female sex, menstrual status, contraceptive use, or polymorphisms in the vitamin D receptor, may be strong predictors of fracture risk [8, 12, 25]. Other factors, such as optimizing vitamin D status, may provide the opportunity to limit fracture risk through intervention.

For example, Non-specific serine/threonine protein kinase Ruohola et al. [7] found that serum levels of 25(OH)D below the study population median (76 nmol/L) at the onset of military training was a significant risk factor for stress fracture in Finnish male military personnel. Burgi et al. [14] confirmed the relationship between 25(OH)D levels and stress fracture risk; in a case–control study with female Navy recruits it was determined that stress fracture risk was approximately double in volunteers who began training in the lowest quintile of 25(OH)D levels (35 nmol/L) as compared to those in the top quintile (124 nmol/L). In a recent randomized, placebo-controlled intervention trial, Lappe et al. [12] found that daily provision of supplements containing 20 μg of vitamin D and 2000 mg of calcium reduced stress fracture incidence by up to 20% in female Navy recruits during training. Although this nutritional intervention appears beneficial for the prevention of stress fracture, the study did not include biochemical or functional assessments of serum 25(OH)D levels, PTH or bone health. As such, it is difficult to draw definitive conclusions regarding the mechanism by which supplementation with vitamin D and calcium may have conferred protection.

DXA-based hip structure analysis (HSA), conducted as a subgroup o

DXA-based hip structure analysis (HSA), conducted as a subgroup of the Fracture Prevention Trial (DXA-HSA study) [9], also showed that periosteal apposition appeared to be reduced in patients receiving daily teriparatide in comparison with a placebo-treated group. On the other hand, some studies reported daily treatment with teriparatide

seemed to stimulate new bone formation on the Selleck S3I-201 periosteal and endosteal surfaces [14, 15]. Thus, periosteal and endosteal apposition may be stimulated within a certain time window or may vary depending on skeletal sites, such as weight bearing or non-weight bearing bone [13]. Bone generally expands in diameter with age [16, 17], as less bone density requires a wider bone to maintain bending strength. selleck compound It has been speculated that expansion is a homeostatic adaptation to a net bone loss in order to maintain bone strength [18, 19]. This age-related adaptive response was not seen in the placebo group of the current study. Once-weekly injection of teriparatide increased cortical thickness with no change in cortical perimeter at the femoral neck. Thus, it is tempting to speculate that as a result of increased cortical thickness (which improves bone strength), periosteal apposition may not be

required under once-weekly teriparatide treatment. Actually, a change in BR based upon improvement in cortical thickness was observed in the teriparatide group. The r 2 between percent change of cortical thickness and that of BR

in the teriparatide group ROS1 was higher than the placebo group. As illustrated in Fig. 4, teriparatide improved all geometry and biomechanical parameters, while maintaining their relationships with changes in cortical thickness (as in the placebo group). However, the distribution patterns of their relationships indicate that the effect of teriparatide is in the exact opposite direction of age-related skeletal changes. It is suggested, therefore, that compared with the changes in the placebo group, once-weekly teriparatide injection reverses age-related deteriorations in bone structure and strength by increasing cortical thickness/CSA and total vBMD, not increasing cortical perimeter, and improving biomechanical parameters. In our previous study which characterized femoral neck geometry in patients with hip versus trochanteric fractures and compared them with age-matched controls [7], patients with femoral neck fracture had a significantly longer hip axis length (HAL), lower cross-sectional moment of inertia (CSMI), and higher BR, while those with trochanteric fractures had a smaller cortical CSA of the femoral neck. Once-weekly teriparatide may improve all these geometric changes.

Comparing FGO-DDA/PS with pristine PS, all of the peaks from the

Comparing FGO-DDA/PS with pristine PS, all of the peaks from the FGO-DDA/PS composite have lower intensities, and the -CONH-

peak appeared in the same region as FGO-DDA [22], which prove that FGO-DDA was associated with the PS matrix. Figure 1 FT-IR spectra of GO, FGO-DDA, FGO-DDA/PS composites, and neat PS. The elemental analysis was further used to confirm the covalent functionalization of GO with DDA. The N contents selleck compound were determined to be 3.07, 3.17, 3.21, and 3.21 wt.% for reaction times of 6, 12, 18, and 24 h, respectively, while the Cgraphene/O ratios were in the range of 2.01 to 2.43. After 12 h of reaction, the Cgraphene/N ratio tended to saturate around 12.5, corresponding to one DDA molecule per six aromatic rings on the GO sheet. Cross-sectional images of freshly fractured pristine PS and FGO/PS composites were observed using SEM (Figure 2a,b,c,d,e). As shown in Figure 2a,b, even with a small amount of FGO, the FGO/PS composite exhibited noticeably increased wrinkles compared to pristine

PS. As the FGO content increased, the wrinkles became finer, which indicates a strong interaction AZD0156 between FGO and PS. It is interesting to note that all of the FGOs were homogeneously dispersed onto the PS matrix even at high loading (10 wt.%). When the chain length of the alkyl group of the FGOs was increased, the wrinkles of the FGO/PS composite became larger and wider (Figure 2d,e), which can be attributed to the effect of the increased aspect ratio of the alkylamines

[23].The dispersions obtained at a 10 wt.% loading of the FGOs over PS composites were also observed by TEM (Figure 2f,g,h). Because the FGOs are compatible with the PS matrix, the FGO sheets were uniformly dispersed on the PS matrix, which is consistent with the SEM images. Notably, Leukotriene-A4 hydrolase FGO-OA/PS showed a broad, plate-type dispersion on the transparent PS film, whereas FGO with a long length alkyl chain had a tiny droplet form on the PS film. Figure 2 Dispersion properties of FGO on PS. FE-SEM images of neat PS and the FGO/PS nanocomposites: (a) neat PS, (b) 1 wt.% FGO-OA/PS, (c) 3 wt.% FGO-OA/PS, (d) 10 wt.% FGO-OA/PS, and (e) 10 wt.% FGO-HDA/PS. TEM images of 10 wt.% (f) FGO-OA/PS, (g) FGO-DDA/PS, and (h) FGO-HDA/PS. TGA analyses were performed to investigate the thermal properties of the FGO/PS composites and pristine PS. In the thermal stabilities of FGOs (Figure 3a), the main mass loss occurred from 200°C to 500°C due to the decomposition of the alkylamine moiety [18]. The mass residues of the FGOs decreased with increased alkylamine length, from 60 wt.% for FGO-OA to 43 wt.% for FGO-DDA and 34 wt.% for FGO-HDA at 500°C.

Supernatant was then harvested from each well Flow cytometry To

Supernatant was then harvested from each well. Flow cytometry To analyze TLR9 expression on A20.IIA cells, these cells underwent intracellular staining with the Fixation/Permeabilization solution kit (BD Biosciences) and an anti-TLR9/PE mAb (BD Biosciences). Tumor burden was analyzed according to the following protocol: Fc receptors were saturated for 20 min with 10 μg/mL of anti-CD16/CD32 mAb (clone 2.4.G2), and then the cells

were incubated for 20 min with either Ro 61-8048 nmr rat IgG2a anti-CD19/APC mAb, or the corresponding isotypic mAb control (all from BD Biosciences). The living cells were defined with side scatter (SSC) and forward scatter (FSC) after autofluorescent cells were excluded. Cell phenotypes were analyzed with the LSRII cytometer and Diva software (BD Biosciences). Statistical analysis Comparisons used Student’s t-test, performed with GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Statistical significance was defined by p values less than 0.05. Results CpG-ODNs inhibit cell proliferation and induce apoptosis of malignant A20.IIA B cells in vitro TLR9 is an intracellular receptor that recognizes CpG-DNA. Cell stimulation by CpG motifs requires that they bind to TLR9. We therefore began by confirming with flow cytometry that A20.IIA B lymphoma cells express TLR9 (Figure 1A).

Figure 1 CpG inhibits cell proliferation and induces apoptotic death of A20.IIA lymphoma cells in vitro . (A) Flow cytometric analysis of TLR9 expression by A20.IIA cells after anti-TLR9 Ab staining (filled PSI-7977 mouse histogram), overlaid with isotype control

(gray line). (B) CpG inhibits the proliferation of A20.IIA cells in vitro. 104 A20.IIA cells were stimulated for 72 hours with various concentrations of CpG or control ODNs ranging from 0.0003 to 30μg/mL or with medium alone. The incorporation of the [3H] thymidine was measured by a scintillation counter. *P < 0.05; **P < 0.01. The data shown are representative of 1 of 3 experiments. (C) CpG induces apoptotic cell death of A20.IIA cell line. Cells were incubated for 72 hours with CpG or control ODNs at 3 and 30 μg/mL, or medium alone. The percentage of AnnV/PI positive cells was determined by flow cytometric analysis. ***P < 0.001. We next evaluated the Rolziracetam direct effect of CpG-ODNs on the proliferation of A20.IIA lymphoma in vitro. Based on our study of its proliferation kinetics (data not shown), tumor cells were incubated for 72 h with CpG 1826 ODNs at concentrations ranging from 0.0003 to 30 μg/mL. Cell proliferation was measured with the [3H] thymidine incorporation assay. The CpG-ODNs inhibited A20.IIA [3H] thymidine incorporation in a dose-dependent manner, whereas control ODNs had no effect on cell proliferation (Figure 1B). The maximum inhibitory effect was obtained from 0.3 to 30 μg/mL of CpG-ODNs. Based on these results, we analyzed the induction of apoptosis of A20.

Table 4 Identification of observed TRF combinations AluI a RsaI a

Table 4 Identification of observed TRF combinations AluI a RsaI a Clone libraryb RDP databasec 93 74 – Unclassified Euryarchaota 142 Out of ranged – Methanosarcina 176 74 Methanosaeta Methanosaeta 176

238/239 – Methanomicrobia 176 Out of range – Unclassified Euryarchaota 184/185 74/77 Methanosaeta Methanosaeta       Unclassified Euryarchaota       Thermoplasmatales       Methanomicrobiales       Methanosarcinales 184/185 142 – Unclassified Euryarchaota 184/185 238/239 Methanosaeta Methanosaeta       Unclassified Euryarchaota       Methanomicrobia       Methanosarcinales 184/185 259 ARC I Unclassified Euryarchaota       Thermoplasmatales       Methanomicrobiales 184/185 Out of range – Unclassified Euryarchaota       Unclassified Archaea       Methanosarcinales Out of range 74/77 – Unclassified Euryarchaota       Unclassified Archaea Out of range 238/239 – Unclassified AMN-107 clinical trial Euryarchaota     AZD1152   Methanosarcinales

Out of range 259 – Unclassified Euryarchaota a Observed TRF combinations that are not included in the table were not found in the database nor in the clone library. b Identification by comparison with predicted TRF lengths of clone library sequences. c Identification by comparison with predicted TRF lengths of RDP database sequences. d Out of range: The TRF in the database or the clone library was either shorter than 50 bases or longer than 1020 bases and would therefore not have been detected. Correlation analysis Several TRFs showed a significant correlation with process parameters (Table 5). The parameters that correlated with most TRFs were water temperature and nitrogen concentration. There were also significant correlations between several TRFs and the sludge and effluent water properties (Table 6). The parameter effluent non-settleable solids (NSS) and the concentration of carbohydrates in extracted extracellular polymeric substances (EPS) correlated with most

TRFs. No TRF showed a significant correlation with the sludge volume or shear sensitivity. Table 5 Correlations between TRF abundances and WWTP process parameters a AluI Identityb, c Observationsd Temp.e SRTf F/Mg CODh NO23-Ni AluI Farnesyltransferase 142 Methanosarcina b 2 **         AluI 176 Methanosaeta c 24     ** *   AluI 184 Methanosaeta c 33 *   *     RsaI               RsaI 142 Euryarchaeota b 3 ***       * RsaI 238 Methanosaeta c 31 * *     * RsaI 259 ARC I c 4 ***       * a The correlations are marked with asterisks corresponding to the level of statistical significance: 95% (*), 99% (**) and 99.9% (***). TRFs that are not included did not show any statistically significant correlation with any parameter. The WWTP process parameter data was taken from [22]. b Identification by comparison with the RDP database. c Identification by comparison with the clone library. d The number of times the TRF was observed. e Water temperature (°C). f Solids retention time (days). g Food to mass ratio ( g/kg*s).