The following yeast strains were used in this study: W303-1A (MATa leu2-3, 112 ura3-1 trp1-92 AZD2281 datasheet his3-11, 15 ade2-1 can1-100) (Thomas & Rothstein, 1989), sch9Δ (W303-1A sch9Δ::URA3) and zds1Δ (W303-1A zds1Δ::repeat). Oligonucleotides used in this study for construction of strains and plasmids are described in Supporting Information, Tables S1 and S2. The sch9Δ::URA3

deletion cassette was obtained by PCR amplification of genomic DNA of strain YBY1 containing the sch9Δ::URA3 cassette with primers KSCH9-U and KSCH9-D. The deletion cassette was used to delete SCH9 gene in strain W303-1A by one-step gene replacement procedure. Strains zds1Δ::RYUR was generated using a one-step gene replacement procedure transforming W303-1A with a zds1Δ::RYUR deletion fragment, which was obtained by PCR amplification of repeat-URA3-repeat fragment on the plasmid pUC18-RYUR (Kong et al., 2007) using primers KZDS1-U and KZDS1-D. Strains zds1Δ::RYUR were spotted on plates containing 5-fluoroorotic acid to pop-out URA3 marker using the recombination of two repeat fragments VX-765 and the obtained strain zds1Δ::repeat. CZDS1 and CZDS1-D were used to confirm the deletion of ZDS1 on the genome. Plasmid YCplac22-3xHA was generated by inserting a triple copy of the HA sequence between the PstI and SphI sites and the 249-bp CYC1 terminator sequences between the SphI and HindШ sites of plasmid

YCplac22. Plasmid YEplac181-ZDS1-3xHA was generated as follows: 3317 bp of ZDS1 sequence was amplified using primers ZDS1GFP-U and ZDS1GFP-D from yeast genomic DNA. The PCR fragments were digested with SalI and NotI and inserted in the same enzyme pair-digested plasmid YCplac22-3xHA, creating an in-frame fusion between the ZDS1 ORF and 3xHA. Primers CZDS1-U and CZDS1-D were used to amplify 3861 bp

of the ZDS1 sequence from yeast genomic DNA. The PCR fragments were digested with BamHI and SalI and inserted in the same enzyme pair-digested plasmid YEplac195, creating YEplac195-ZDS1. Plasmid YCplac22-SCH9-13xMYC was generated by inserting 3052 bp of SCH9 sequence amplified from yeast genomic DNA using primers SCH9GFP-U and SCH9GFP-D between the BamHI and SphI sites and 862 bp of 13xMYC-CYC1 sequence between the SphI and HindШ sites of plasmid YCplac22. Hydroxychloroquine Plasmid YCplac22-CYC1 was generated by inserting 249 bp CYC1 terminator sequences between the SphI and HindШ sites of plasmid YCplac22. To create YEplac181-SCH9, primers SCH9GFP-U and SCH9GFP-D were used to amplify 3052 bp of SCH9 sequence from yeast genomic DNA. The PCR fragments were digested with BamHI and SphI and inserted in the same enzyme pair-digested plasmid YCplac22-CYC1. Plasmid YCplac22-YAK1-3xHA was generated as follows: 3100 bp of ZDS1 sequence were amplified using primers YAK1-U and yak1gfp-D from yeast genomic DNA. The PCR fragments were digested with PstI and SphI and inserted in the same enzyme pair-digested plasmid YCplac22-3xHA, creating an in-frame fusion between the YAK1 ORF and 3xHA.

Author contributions: As the find more corresponding author, MBK has had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. She supervised the study design, conduct and reporting and participated in revising the manuscript. All of the authors have seen and approved the final manuscript and have participated sufficiently in the work to take public responsibility for its content. The Canadian Co-infection cohort investigators (CTN222) are: Drs Jeff Cohen, Windsor Regional Hospital Metropolitan Campus, Windsor, ON; Brian Conway, Downtown IDC, Vancouver, BC; Curtis Cooper, Ottawa General Hospital, Ottawa, ON; Pierre Côté,

Clinique du Quartier Latin, Montreal, QC; Joseph Cox, Montreal General Hospital, Montreal, QC; John Gill, Southern Alberta CHIR-99021 research buy HIV Clinic, Calgary, AB; Mark Tyndall, Native Health Centre, Vancouver, ON; Shariq Haider, McMaster University, Hamilton, ON; Marrianne Harris, St. Paul’s Hospital, Vancouver, BC; David Hasae, Capital District Health Authority, and Dalhousie University, Halifax, NS; Julio Montaner, St. Paul’s Hospital, Vancouver, BC; Erica Moodie, McGill University, Montreal, QC; Neora Pick, Oak Tree Clinic, Vancouver, BC; Anita Rachlis, Sunnybrook Health

Sciences Centre, Toronto, ON; Roger Sandre, HAVEN Program, Sudbury, ON; Danielle Rouleau, Centre Hospitalier de l’Université de Montréal, Montréal, QC; David Wong, University Health Network, Toronto, ON; Mark Hull, BC Centre for Excellence in HIV/AIDS, Vancouver, BC; and Sharon Walmsley, Toronto General Hospital, Toronto, ON. “
“For detailed

guidance on HIV VL, resistance and genotropism testing, the reader should consult BHIVA guidelines for the routine investigation and monitoring of adult HIV-1-infected individuals 2011 [1] (http://www.bhiva.org/Monitoring.aspx). The following recommendations concern the management of patients experiencing virological failure on ART. Patient populations at the Dichloromethane dehalogenase time of virological failure will include those with no or limited HIV drug resistance through to those with three-class failure and either no or limited treatment options. For the assessment and evaluation of evidence, priority questions were agreed and outcomes were ranked (critical, important and not important) by members of the Writing Group. For patients with no or limited HIV drug resistance the following were ranked as critical outcomes: viral suppression <50 copies/mL at 48 weeks, development of resistance, discontinuation rates for clinical and laboratory adverse events. For patients with three-class failure/few therapeutic options: clinical progression, median CD4 cell count change at 48 weeks, and development of new resistance. Treatments were compared where data were available and differences in outcomes assessed.

The results highlight the importance of fungus-driven bacterial dispersal to understand the functional role of oxalotrophic bacteria and fungi in soils. VX 809 “
“Light entrainment pathways synchronize the circadian clock of almost all species of the animal and plant kingdom to the daily light dark cycle. In the Madeira cockroach Rhyparobia (Leucophaea) maderae, the circadian clock is located in the accessory medulla of the brain’s optic lobes. The clock has abundant neuropeptides with unknown

functions. Previous studies suggested that myoinhibitory peptides (MIPs), orcokinins (ORCs), and allatotropin (AT) take part in light input pathways to the circadian clock. As the sequences of AT and ORCs of R. maderae have not yet been determined, with matrix-assisted laser

desorption/ionization–time of flight mass Selleckchem Enzalutamide spectrometry, the respective Rhyparobia peptides were characterized. To search for light-like phase-shifting inputs to the circadian clock, Rhyparobia-MIP-1, Rhyparobia-AT, and Rhyparobia-ORC were injected at different circadian times, combined with locomotor activity assays. An improved, less invasive injection method was developed that allowed for the analysis of peptide effects within <2 weeks after injection. Rhyparobia-MIP-1 and Rhyparobia-AT injections resulted in dose-dependent monophasic phase response curves with maximum delays at the beginning of the subjective night, similar to light-dependent phase delays. In

contrast to Manduca sexta-AT, Rhyparobia-AT did not phase advance locomotor activity rhythms. Only injections of Rhyparobia-ORCs resulted in a biphasic light-like phase response curve. Thus, it is hypothesized that Rhyparobia-MIP-1 and -AT are candidates for relaying light-dependent delays and/or non-photic inputs to the clock, whereas Rhyparobia-ORCs Cisplatin manufacturer might be part of the light-entrainment pathways relaying phase delays and advances to the circadian clock of the Madeira cockroach. “
“Production of new neurons from stem cells is important for cognitive function, and the reduction of neurogenesis in the aging brain may contribute to the accumulation of age-related cognitive deficits. Restriction of calorie intake and prolonged treatment with rapamycin have been shown to extend the lifespan of animals and delay the onset of the age-related decline in tissue and organ function. Using a reporter line in which neural stem and progenitor cells are marked by the expression of green fluorescent protein (GFP), we examined the effect of prolonged exposure to calorie restriction (CR) or rapamycin on hippocampal neural stem and progenitor cell proliferation in aging mice. We showed that CR increased the number of dividing cells in the dentate gyrus of female mice.

5; 20 mM MgCl2; 400 mM NaCl and 50% glycerol) for ArgR5aa and ArgR149 proteins. Protein concentrations were determined using Bradford assays or using QuBit fluorimetry, and we obtained 50% purity for ArgR5aa and 30% for ArgR149. For crosslinking analysis, the proteins were also expressed as histidine-tagged

fusion proteins by cloning the coding regions of all three genes into pQE31 (Qiagen Inc.) and purifying the overexpressed proteins according the manufacturer’s Inhibitor Library in vitro instructions. The gel retardation assays were performed as described by Tian et al. (1992) and Villion & Szatmari (1998) using specific fragments labelled with digoxigenin by PCR. A 106-bp fragment containing the ARG box site was labelled with digoxygenin using 100 pmol of the following primers: ArgboxD (5′CTT GCG GAT CCG AGC TTC G) and ArgboxG (5′TTT CAG CCG AAT TCA GGG CTG) under the following conditions: 95 °C/30 s, 55 °C/30 s, 72 °C/30 s for 30 cycles, with a final extension at 72 °C/1 min. Reactions were carried out in 50-μL volumes using Natural Product Library supplier Taq DNA polymerase (Qiagen Inc.) using 120 ng of pGS38 DNA as the template. The gel shift assay was performed with 2 ng labelled DNA,

1 μg of poly-dIdC in buffer containing 20 mM Tris-HCl pH 7.5; 10 mM MgCl2; 100 mM KCl; 10 mM CaCl2; 1 mM EDTA pH 8.8; 10% glycerol; and 5 mM l-arginine. The reactions were incubated for 30 min at 37 °C before electrophoresing on a 6% polyacrylamide gel. Five millimolar and 1 mM of l-arginine were added to the gel and the 0.5 × TBE running buffer, respectively. Gels were transferred onto Hybond-N+ (Amersham Biosciences) positively charged nylon membranes and UV cross-linked. Final detections were performed using CDP-Star (NEB), according to the standard digoxygenin detection methods, and followed by exposure to a Fujifilm SuperRX X-ray

film. N-terminal 6-histidine-tagged versions of ArgR, ArgR5aa and ArgR149 were purified using Ni-NTA affinity chromatography (Qiagen Inc.) as per the manufacturer’s instructions. Purified proteins were crosslinked Casein kinase 1 with various amounts of glutaraldehyde (Fisher) in 50 mM triethanolamine pH 8, containing 150 mM KCl and 1 mM l-arginine. Cross-linked complexes were analysed by 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie Brilliant Blue. In order to obtain ArgR mutants deficient in cer site-specific recombination, we used the technique of pentapeptide scanning mutagenesis, which inserts five amino acids into random regions of a DNA sequence. We isolated a series of ArgR mutants using an in vivo site-specific recombination assay in an argR− E. coli strain (DS956) containing plasmid pCS210. Mutants were screened for their inability to delete the cer-flanked lacZ gene of pCS210, which results in a blue phenotype in X-gal-containing media. These mutants were then screened for their ability to repress the argR∷lacZ fusion in strain EC146(λAZ-7).

The scale bar represents substitutions per site. NJ trees were constructed using mega 4.1 with 1000 bootstrap replications and Kimura 2-parameter as

a model KU-60019 research buy of nucleotide substitution. All the termites from 14 populations representing two genera, and two families (seven Odontotermes horni, five Odontotermes spp. and two C. heimi colonies), screened initially for the presence of Wolbachia, using PCR assays with the wsp gene were found to be positive. At least one MLST gene was sequenced for all the 14 colonies (Table 1). Repeated failures to PCR amplify Wolbachia genes (MLST and 16S rRNA gene) resulted in incomplete profiles. Sequence typing was performed using the Wolbachia MLST database (http://pubmlst.org/Wolbachia/), which resulted in the assignment of full sequence type (ST) to four strains (Table 1). All of the alleles and allelic profiles characterized for the termite strains were new to the MLST database, except for B supergroup C. heimi Wolbachia (Table 1). Alleles were shared among some of the strains, showing their relatedness, but at the same time showing a distinct difference for other strains. The divergence among the strains accounted for 591 variable AZD2014 datasheet sites (VI) out of 2079 sites (28.43%), with a concatenated alignment of all five Wolbachia MLST genes (Table 2). The gene hcpA showed the highest

nucleotide divergence with 154 variable sites out of 444 (34.38%), followed by fbpA with 128 variable sites out of 429 (29.83%) (Table 2). The average Ka/Ks per gene was found to be ≪1 (the average Ka/Ks across genes is 0.0933), which indicated strain evolution mainly by synonymous substitutions. This is compatible with a scenario of a strong purifying selection. Recombination

events were not indicated in either single gene or concatenated MLST dataset alignments (P<0.05) by MaxChi analyses. Phylogenetic reconstructions for all genes by both Bayesian inference and neighbor-joining methods showed similar results, and therefore only Bayesian phylogenetic trees were included. Phylogenetic reconstructions based on the concatenated alignment Florfenicol of hcpA, gatB, coxA, ftsZ and fbpA genes indicated a strong clustering of termite Wolbachia from this study with Wolbachia from bed bug Cimex lectularis and form a sister clade within F supergroup with different species of scorpion genus Opistophthalmus (Fig. 1). Two major clusters were observed for termite Wolbachia in the present study (MCT, W5, G29 and RA) (T1, T3, T21 and THYD) (Fig. 1). On the basis of single-gene analyses, all the strains consistently belonged to the same supergroup. All Odontotermes spp. harbored F supergroup Wolbachia, with the exception of a population from O. horni (T2). Two C. heimi colonies harbored two different supergroup Wolbachia. One of these (TERMITE3) belonged to supergroup F, whereas the other (TLR) belonged to supergroup B (Table 1, Fig. 2).

The scale bar represents substitutions per site. NJ trees were constructed using mega 4.1 with 1000 bootstrap replications and Kimura 2-parameter as

a model LDK378 supplier of nucleotide substitution. All the termites from 14 populations representing two genera, and two families (seven Odontotermes horni, five Odontotermes spp. and two C. heimi colonies), screened initially for the presence of Wolbachia, using PCR assays with the wsp gene were found to be positive. At least one MLST gene was sequenced for all the 14 colonies (Table 1). Repeated failures to PCR amplify Wolbachia genes (MLST and 16S rRNA gene) resulted in incomplete profiles. Sequence typing was performed using the Wolbachia MLST database (http://pubmlst.org/Wolbachia/), which resulted in the assignment of full sequence type (ST) to four strains (Table 1). All of the alleles and allelic profiles characterized for the termite strains were new to the MLST database, except for B supergroup C. heimi Wolbachia (Table 1). Alleles were shared among some of the strains, showing their relatedness, but at the same time showing a distinct difference for other strains. The divergence among the strains accounted for 591 variable http://www.selleckchem.com/products/pci-32765.html sites (VI) out of 2079 sites (28.43%), with a concatenated alignment of all five Wolbachia MLST genes (Table 2). The gene hcpA showed the highest

nucleotide divergence with 154 variable sites out of 444 (34.38%), followed by fbpA with 128 variable sites out of 429 (29.83%) (Table 2). The average Ka/Ks per gene was found to be ≪1 (the average Ka/Ks across genes is 0.0933), which indicated strain evolution mainly by synonymous substitutions. This is compatible with a scenario of a strong purifying selection. Recombination

events were not indicated in either single gene or concatenated MLST dataset alignments (P<0.05) by MaxChi analyses. Phylogenetic reconstructions for all genes by both Bayesian inference and neighbor-joining methods showed similar results, and therefore only Bayesian phylogenetic trees were included. Phylogenetic reconstructions based on the concatenated alignment else of hcpA, gatB, coxA, ftsZ and fbpA genes indicated a strong clustering of termite Wolbachia from this study with Wolbachia from bed bug Cimex lectularis and form a sister clade within F supergroup with different species of scorpion genus Opistophthalmus (Fig. 1). Two major clusters were observed for termite Wolbachia in the present study (MCT, W5, G29 and RA) (T1, T3, T21 and THYD) (Fig. 1). On the basis of single-gene analyses, all the strains consistently belonged to the same supergroup. All Odontotermes spp. harbored F supergroup Wolbachia, with the exception of a population from O. horni (T2). Two C. heimi colonies harbored two different supergroup Wolbachia. One of these (TERMITE3) belonged to supergroup F, whereas the other (TLR) belonged to supergroup B (Table 1, Fig. 2).

3%) had their first permanent molars and incisors evaluated using the European Academy of Paediatric Dentistry evaluation criteria for MIH. Mothers completed a medical history questionnaire-based interview performed in the schools by a trained examiner. Results.  For children with MIH, 6% reported no relevant medical history; the remaining 94% reported various medical conditions putatively associated with MIH compared with 70% for the non-affected group. Post-natal medical conditions (33.3%) were most frequently reported. When

data were split into the possible risk effect groups, maternal psychological stress PLX3397 mouse (OR, 3.24), frequent exposure to ultrasonic scans during the last gestational trimester (OR, 2.51) and birth order as a fourth sibling or later (OR, 3.17 and 5.73, respectively) were previously unreported significant risk factors and postulated as contributing to, or causing the defect. Conclusions.  Children with MIH had experienced a greater number of medical conditions than their unaffected peers with no single health Carfilzomib event identified as a risk factor. “
“Children in Gaza Strip suffer from a high prevalence of dental fluorosis. To estimate and compare total daily fluoride (F) intake (TDFI) and investigate the relative contributions of different sources of F to TDFI, in 3- to 4-year-old

children in Gaza Strip, exposed to low (<0.7 mg/litre), moderate (0.7–1.2 mg/litre) or high (>1.2 mg/litre) F concentrations

in tap water. A 3-day food diary and samples of tap water, drinks, foods, toothpastes and toothbrushing expectorate were collected from 216 children receiving low (n = 81), moderate (n = 72) or high (n = 63) F concentrations in tap water. F concentration of samples was analysed using an F-ion-selective electrode. TDFI from all sources was estimated. Data were analysed by anova and Tukey’s test. The mean (±SD) F concentration in low, moderate and high F tap waters was 0.21(±0.15), 0.91(±0.13) and 1.71(±0.35) mg/litre, respectively. Mean (±SD) TDFI was 0.02(±0.01), 0.04(±0.01) and 0.05(±0.03) mg/kg bw/day, respectively (P < 0.0001). Foods made the largest contribution (63.9%) to TDFI. Total daily fluoride (F) intake increased as F concentration Farnesyltransferase in tap water increased. Foods were the primary source of F. Programmes for monitoring fluoride expose should consider the fluoride concentration of water used for food preparation and local dietary behaviours. “
“International Journal of Paediatric Dentistry 2013; 23: 188–196 Objective.  The study investigated the influence of exposure to anti-asthmatic medications and of various factors on the caries prevalence in children in Slovenia. Methods.  The study population consisted of children aged 2- to 17 years (n = 220) under treatment for asthma, who had used anti-asthmatic medications for at least 1 year; 220 controls were matched for age.

Conversely, a randomized placebo-controlled trial of

pneumococcal polysaccharide and conjugate vaccines showed no virological differences between adult groups on or off HAART [16], and an observational study of diphtheria/tetanus/acellular pertussis (DTaP) immunization of 2–9-year-olds receiving HAART also reported no effect on HIV viral load [17]. Whether there are long-term consequences of repeated bursts of HIV viraemia post-vaccination is unknown [18] and there is currently no evidence that vaccination adversely affects the pace of HIV disease progression [19]. HIV-positive children are at greater risk of vaccine-preventable infections than other children, yet vaccination coverage in this DAPT nmr group is suboptimal Nutlin-3a cell line in populations across Europe [20-22]. Reasons for this may include physician uncertainty regarding the safety or appropriateness of vaccinating such children, deferral at times of intercurrent illness, or concerns that ‘intervention fatigue’ in patients may have adverse effects on HAART adherence. National and international societies recommend vaccination of HIV-positive children with some modification of routine schedules; for example, recommendations are available from the World

Health Organization (WHO)/United Nations Children’s Fund (UNICEF), the American Academy of Pediatrics and the British HIV Association (BHIVA) [5, 23-25]. Variation among these guidelines, compounded by differences among national schedules, may serve to reduce vaccine coverage in this vulnerable patient group. The development of uniform schedules for all HIV-positive children

enough living in European countries would be greatly beneficial, especially as new and more effective vaccines become available which potentially confer more benefits for HIV-infected children than for other children. However, achieving uniformity in guidelines is challenging given the inherent variation of the clinical, immunological and virological status of the cohort across Europe and within individual nations. Furthermore, increasing numbers of HIV-infected children living in Europe originate from developing countries and have incomplete or unknown vaccination status, unrelated to their immunological status or whether they are receiving HAART [26]. Recommendations need to accommodate the different requirements of (a) newly diagnosed children, whether immunocompetent or already immunocompromised; (b) those on HAART, whether complete or incomplete responders; (c) partially immunized or nonimmunized children within these groups; and (d) children during time periods when they fall below thresholds for effective or safe immunization. Yet European guidelines must also aim to minimize deviation from existing routine schedules, lest they generate confusion and further reduce vaccine uptake.

Ocular input to Vc/C1 units by bright

light or hypertonic saline was markedly reduced by PH disinhibition and reversed completely by local Vc/C1 application of SB334867. OxA applied to the Vc/C1 surface mimicked the effects of PH disinhibition in a dose-dependent manner. OxA-induced inhibition was prevented by co-application of SB334867, but not by the orexin-2 receptor antagonist TCS Ox2 29. PH disinhibition and local OxA application also reduced the high threshold convergent cutaneous receptive field area of ocular units, suggesting widespread effects on somatic input to Vc/C1 ocular units. Vc/C1 application of OxA or SB334867 alone did not affect the background selleck screening library discharge of ocular units and suggested that the PH–OxA influence on ocular unit activity was not tonically active. Vc/C1 application of OxA or SB334867 alone also did not alter mean arterial pressure, whereas PH disinhibition evoked prompt and sustained increases. These results suggest that stimulus-evoked increases in PH outflow acts through OxA and orexin-1 receptors to alter the encoding properties of trigeminal brainstem neurons responsive to input from the Selleckchem MS 275 ocular surface and

deep tissues of the eye. “
“Visual sequential search might use a peripheral spatial ranking of the scene to put the next target of the sequence in the correct order. This strategy, indeed, might enhance the discriminative capacity of the human peripheral vision and spare neural resources associated with foveation. However, it is not known how exactly the peripheral vision sustains sequential search and whether the sparing of neural resources has a cost in terms of performance. To elucidate these issues, we compared strategy and performance during an alpha-numeric sequential task where peripheral vision was modulated in three different conditions: normal, blurred, or obscured. If spatial ranking is applied to increase the peripheral discrimination, its use as a strategy in visual sequencing should differ according to the degree of discriminative

information that can be obtained from the periphery. Moreover, if this strategy spares neural resources without impairing the performance, its use should be associated with better performance. We found that spatial ranking was applied when peripheral vision was fully available, reducing the number Megestrol Acetate and time of explorative fixations. When the periphery was obscured, explorative fixations were numerous and sparse; when the periphery was blurred, explorative fixations were longer and often located close to the items. Performance was significantly improved by this strategy. Our results demonstrated that spatial ranking is an efficient strategy adopted by the brain in visual sequencing to highlight peripheral detection and discrimination; it reduces the neural cost by avoiding unnecessary foveations, and promotes sequential search by facilitating the onset of a new saccade.

The incubation temperature was set to 37 °C. As positive controls for cell surface exposure, strains JG137 and JG138, producing OmcAstrep and MtrCstrep, were chosen; as a control for OM integrity under the incubation conditions, the periplasmic c-type cytochrome MtrA containing an N-terminal strep-tag (MtrAstrep) was Copanlisib mouse produced in an S. oneidensisΔmtrA background (JG50) (Schuetz et al., 2009). Cells were grown anaerobically overnight in minimal media with fumarate as an electron acceptor. At an OD578 nm of ∼0.2, 0.1 mM arabinose

was added to induce OM cytochrome and MtrA/MtrB production. After 4 h of production, cells were harvested and washed twice with mineral media without fumarate and lactate and then resuspended in HEPES buffer (100 mM, pH 7.5) containing 50 μM MgCl2 to obtain a final OD578 nm between 3 and 5. All further measurements were performed in independent duplicates in an anaerobic glove box. Specific reduction rates were obtained by normalization to the protein content of the cell suspension. Fifty microliters of the cell suspension was pipetted in a well of a microtiter plate. The assay was started through the addition of 150 μL of a solution containing 10 mM lactate and 10 mM ferric citrate. At different time points (0–30 min), the reaction was stopped by

the addition of 100 μL 3 M HCl. The Fe2+ concentration of the samples was determined using the ferrozine reagent (Viollier et al., 2000). The MFC setup used in this study features an anode I-BET-762 clinical trial and cathode chamber with a working volume of 8 mL each, separated by a Nafion-117 membrane (Quintech, Göppingen; Kloke et al., 2010). A saturated calomel reference electrode (SCE) was Thiamine-diphosphate kinase separated from the anode compartment by another Nafion membrane. Electrodes were made of graphite felt cubes (Alpha Aesar, Karlsruhe) connected to platinum wires (0.1 mm; Chempur, Karlsruhe). The anode compartment was filled with 5.5 mL mineral media containing 50 mM lactate and 0.1 mM arabinose. Five hundred microliters of a cell suspension with an OD578 nm of 4 was added to start the experiment. All MFC

experiments were performed in duplicate and conducted at a constant temperature of 30 °C. The whole setup was connected to a potentiostat (Pine Instruments, Grove City). The standard measurement protocol consisted of two phases: after a conditioning period with a constant current flux over 5 h (0.3 μA cm−3), MFC cultures were subjected to a continuous increase in current density at a rate of 1.1 μA cm−3 h−1 over 45 h (current sweep phase). The anode compartment was continuously flushed with nitrogen gas to maintain anoxic conditions. Additional terminal electron acceptors were not added. A markerless multideletion mutant in all annotated OM cytochromes of S. oneidensis was constructed to generate a strain platform that allows for analysis of OM cytochrome activity without the potential detection of redundant activities from similar proteins.