This work was supported by a grant from Fondazione Cassa di Risparmio di Puglia. Conflicts of interest: The authors have no conflict of interest to declare. “
“The outcome of sudden cardiac arrest depends on the “chain of survival”1: 1 Immediate recognition of cardiac arrest and activation of the emergency response system Automated external defibrillators (AEDs) have proven valuable in out-of-hospital settings close to definitive care institutions like airports, casinos, and cruise ships—sites with a high density

of both potential victims and resuscitators.2–4 In contrast, AEDs save very few lives in residential units such as private homes or apartment complexes.5 The benefit of AEDs in remote areas without available qualified medical follow-up, such as most merchant ships, is unknown and controversial.6 The Federal Republic PD-0332991 clinical trial of Germany has decided that http://www.selleckchem.com/products/gsk1120212-jtp-74057.html AEDs with EKG display and transmission means must be present in all German-flagged merchant vessels in intermediate and long-distance trade by the end of 2012. Because there is only one German-registered

cruise ship and most ferries are exempt from this rule, nearly all AEDs will be on ships with few crew members, of whom virtually none will have any knowledge of advanced cardiac life support. Oldenburg and colleagues7 make a valuable contribution in this issue by showing that German seafarers are able to follow the AED prompts after training, but also that there is a number of other prerequisites in addition to training that must be fulfilled before the AEDs are potentially useful on board. AEDs purchased without

further instructions tend to be left unmounted and still sealed in unmarked locations. Furthermore, the authors demonstrate that selleck chemicals the available AEDs ought to be easier to operate than the ones presently in use and that one of five officers did not perform resuscitation satisfactorily by just following the AED prompts even under ideal test conditions. Immediately after training all the seafarers felt that they could perform reasonably well during a real emergency. But will they still feel confident after a few years without any practice? Like most seafaring nations, Germany demands mandatory first-aid follow-up courses for crew every 5 years. However, the American Heart Association requires recertification of both basic and advanced life support courses every 2 years, but now states that 2 years is too long an interval for skills practice and reassessment.1 Likewise, AEDs aboard will demand more frequent and thus costly re-training. Oldenburg and colleagues rightly point out that AEDs with EKG display and transmission means also have other uses on ships, but resuscitation will certainly be the main focus. Although improvement of lay rescuer education should be strongly encouraged for everyone, it is important to be realistic regarding results on ships without a doctor.

, 2004). FtsZ

polymer was collected in the pellet fraction by ultracentrifugation (Fig. 5b). In the absence of YgfX, almost all FtsZ was polymerized and collected in the pellet fraction. However, when YgfX(C)−HIS was added to the reaction mixture, FtsZ polymer formation was decreased reciprocally to the amounts of YgfX(C)−HIS added. The polymerization of FtsZ was almost completely inhibited when YgfX(C)−HIS was added to FtsZ in the 1 : 1 molar ratio. In a similar manner, the effect of PF-01367338 research buy YgfX on the ATP-dependent polymerization of MreB was analyzed. Addition of equimolar YgfX(C)−HIS almost completely inhibited MreB polymerization (Fig. 5c). These results clearly demonstrated that YgfX inhibits the GTP-dependent FtsZ polymerization, as well as ATP-dependent MreB polymerization, and that the C-terminal 87-residue cytoplasmic domain of YgfX is responsible for the inhibition of cytoskeletal polymerization. Here, we identified a novel TA system, YgfY–YgfX, on the E. coli chromosome. The toxin, YgfX,

was shown to inhibit cell division by interfering with the polymerization of essential Natural Product Library high throughput bacterial cytoskeletal proteins, FtsZ and MreB. Unlike another recently identified soluble E. coli toxin, YeeV, which also interacts with FtsZ and MreB, YgfX is an inner membrane protein having two TM domains. This is consistent with the previous microscopic observation of GFP-YgfX, showing that YgfX is associated with the membrane (Kitagawa et al., 2005). In this study, we also demonstrated that YgfX inhibited FtsZ and MreB polymerization through its soluble C-terminal domain. The role of the TM domains of YgfX still has to be elucidated. The localization in the inner membrane may spatially limit the YgfX activity only near the membrane. For instance, Glycogen branching enzyme Z-ring is known to be anchored to the inner membrane by ZipA (RayChaudhuri, 1999). A number of cell division proteins such as FtsW, FtsQ, FtsN, FtsL, FtsK, and FtsB also contain a TM domain(s) (Barondess et al., 1991; Dai et al., 1996; RayChaudhuri, 1999; Buddelmeijer & Beckwith, 2002; Bigot et al., 2004). Interestingly, spatially regulated inhibition of FtsZ polymerization by inner

membrane–associated MinC is responsible for the localization of Z-ring at mid-cell (Bi & Lutkenhaus, 1993). YgfX may play a similar role in temporal and spatial control of FtsZ and MreB polymerization, thus regulating cell division events in vivo. The interaction between FtsZ and YgfX was confirmed by Y2H assay. Furthermore, using Y2H assay, the region of FtsZ that is essential for the interaction with YgfX was analyzed. N-terminal 31 residues of FtsZ were not required for the interaction with YgfX. In contrast, N-terminal 31 residues are essential for the interaction with YeeV (Tan et al., 2011). This suggests that although both YeeV and YgfX target the same proteins (FtsZ and MreB) and cause equivalent morphological change, they bind distinct sites of FtsZ.

Furthermore, Chagas’ disease is becoming an GSK1120212 important health issue in the United States and Europe (Tarleton et al., 2007). During its life cycle, T. cruzi is exposed to different conditions in the insect gut, the mammalian

bloodstream and also cell cytoplasm, which required evolutionary adaptations to such environments (Brener, 1973; Kollien et al., 2001). Among them, transport processes are rapid and efficient mechanisms for supplying metabolites from parasite extracellular media, and also to regulate the first step on metabolic pathways. Trypanosomatids have a metabolism largely based on the consumption of amino acids, which constitute the main carbon and energy sources in the insect stage of the parasite life cycle (Silber et al., 2005). In T. cruzi, arginine is an essential amino acid and a key substrate for several metabolic pathways and it is obtained from the host through different transport systems or by intracellular proteolysis (Pereira et al., 1999; Canepa et al., 2004). Arginine participates in the management of cell energy through an arginine kinase (Pereira et al., 2000; Alonso et al., 2001). This enzyme, which was also found

in Trypanosoma brucei (Pereira et al., 2002b), catalyses the reversible transphosphorylation between phosphoarginine learn more and ATP, and thus phosphorylated arginine acts as an energy reservoir involved in the renewal of ATP (Pereira et al., 2002a, 2003). As phosphoarginine is completely absent in mammalian tissues, arginine kinase is a possible target for the future development of chemotherapeutic agents. Despite the relevance Tacrolimus (FK506) of amino acids in trypanosomatids, the way in which they are internalized to become available for metabolism remains relatively unexplored. In this sense, the amino acid transporters are the first cell proteins that are in contact

with solutes in the surrounding medium, and in several cases they function not only as permeases to carry the solutes into the cytoplasm but also as environmental sensors. One of the major transporter families of amino acids is AAAP (TC 2.A.18), which is largely found in plants (Young et al., 1999). In T. cruzi, members of this family were first identified by our group (Bouvier et al., 2004) and confirmed by the Tritryps genome project (Berriman et al., 2005). The T. cruzi subfamily, named TcAAAP, has >30 genes coding for proteins with lengths of 400–500 amino acids and 10–12 predicted transmembrane α-helical spanners. One interesting feature of this permease family is the absence of similar sequences in mammalian organisms; however, the presence of unidentified orthologues could not be rejected (Akerman et al., 2004). In this work we present the first functional characterization of an amino acid permease from T. cruzi. TcAAAP411 was identified as a specific arginine permease and functionally characterized in a yeast model.

4 in 1975 to 3.2 in 2012 and the total morbidity increased from 229 to 2092.[4] Small Molecule Compound Library The incidence of endometrial cancer is

likely to continue to increase based on these recent trends. Discovering the causes of the increase and establishment of prophylactic measures and new therapeutic strategies requires an improved understanding of the carcinogenic mechanisms of endometrial cancer. Environmental factors, including estrogen, an abnormal mismatch repair (MMR) system, genetic abnormalities, and aberrant methylation of DNA and microRNA, are currently proposed as major mechanisms of carcinogenesis in endometrial cancer. Endometrial cancer is defined as type I or II based on clinicopathological properties. Type I endometrial cancer more commonly develops in

premenopausal or perimenopausal women and occurs in an estrogen-dependent manner via atypical endometrial hyperplasia. The tumor is positive for the estrogen receptor and progesterone receptor, shows well-differentiated endometrioid adenocarcinoma, has a lower frequency of lymph node metastasis, shows little muscular invasion, and often has a relatively favorable prognosis. In contrast, type II endometrial cancer Galunisertib purchase tends to develop in postmenopausal women in an estrogen-independent manner, and is thought to be due to de novo carcinogenesis that develops directly from the normal endometrium, rather than via endometrial hyperplasia or undiagnosed precancerous lesions. The tissue type is specific, including extremely poorly differentiated endometrioid adenocarcinoma Progesterone and serous adenocarcinoma, and the prognosis is often poor. This review focuses on the mechanisms of carcinogenesis in endometrial cancer that have recently emerged. Estrogen is a steroid hormone that promotes the development of female

genitalia, including the endometrium, vagina, vulva and mammary gland. Estrogen passes through the cell membrane and binds to estrogen receptor (ER) in the cytoplasm. ER forms dimers and regulates gene expression via estrogen response elements in promoter regions of target genes. ER has ligand- and DNA-binding domains, and ligand-independent activation function (AF)-1 and ligand-dependent AF-2 transcriptional activation domains.[5] The balance of transcriptional activation domains varies among tissues, with dominance of AF-2 in mammary gland cells and AF-1 in endometrial cells.[6, 7] Miyamoto et al.[8] suggested that mismatch repair (MMR) deficiency was the most important abnormality in early-stage endometrial cancer, and examined the correlation between MMR and estrogen. Expression of hMLH1 and hMSH2, which are important MMR proteins, was examined by immunostaining and showed a strong positive correlation with blood estrogen. MMR activity in endometrial epithelial cells in vitro also showed a dose-dependent increase with higher estrogen levels.

Microscope test and ink test were performed to identify a typical scabies Small molecule library case. Complete blood cell counting showed white blood cells (WBC) 3.73 × 109/L, neutrophil 1.49 × 109/L, red blood cell 2.81 × 1012/L, hemoglobin 69 g/L, hematocrit 17.5% and platelets 75 × 109/L. A doctor performed bone marrow biopsy and this demonstrated hyperplasia anemia. A thyroid

function test displayed the free thyroxine (free T4) to be 4.15 ng (normal range: 0.89–1.80 ng/dL), free triodothyronine (free T3) to be 3.48 pg/mL (normal range: 2.30–4.20 pg/mL), thyroid stimulating hormone (TSH) < 0.01 IU/mL (normal range: 0.35–5.50 IU/mL) and TSH receptor antibody (TRAb) at 36% (normal: 0–9%). ESR was 140 mm/h, and RF was positive with a titer of 1 : 320. Erythrocyte direct antiglobulin (Coombs) test for autoimmune hemolytic anemia, Ham test for paroxysmal nocturnal hemoglobinuria and purified protein derivative (PPD) skin test were negative. Anti-nuclear antibody (ANA), anti-ds-DNA antibody, and anti-elutable nuclear antigen antibodies were negative. A chest computed tomography (CT) scan was normal. The patient was diagnosed with Felty's syndrome (FS), Graves disease and scabies. Treatment was started with sulfur

ointment, leflunomide 20 mg, propylthiouracil 50 mg and celecoxib (Celebrex) 200 mg daily. After CP-868596 price 7 days of therapy, the scabies symptom was significantly improved. The manifestations of RA and Graves disease were partly relieved. However, the enlarged spleen was still palpable about 5 cm below the left costal margins. White blood cells and neutrophils were reduced from 3.6 × 109/L and 1.45 × 109/L, to 2.7 × 109/L and 1.17 × 109/L, respectively 2 weeks after the admission, as showed in Figure 1. Two weeks after the admission, oral 10 mg prednisone daily find more was added to the patient’s regimen. There was a dramatic increase in the WBC and neutrophil counts, and normal counts were restored within 1 week after the treatment, as displayed in Figure 1. Dramatically, the enlarged spleen contracted to normal size. FS is

characterized by the triad of RA, neutropenia and splenomegaly. We present here a rare case of a 36-year-old woman with FS, hyperthyroidism and scabies. In the present case, the patient presented with skin infection in neutropenic settings. Neutropenia and splenomegaly with elevated ESR, elevated levels of serum C-reactive protein and RF, joint deformities, joint pain, X-ray manifestations in the hands and anemia of chronic disease pointed toward FS. Enlargement in size of the thyroid gland and serum test of thyroid function and specific antibodies indicated the diagnosis of hyperthyroidism.[1] Treatment was commenced with disease-modifying anti-rheumatic drugs (DMARDs), including leflunomide and Celebrex for RA, sulfur ointment for scabies and propylthiouracil for hyperthyroidism. The scabies was cured, and the symptoms of RA and hyperthyroidism were largely relieved after 1 week of treatment.

The mean cell survival was calculated from three independent experiments. Sensitivity to metronidazole was also

evaluated using E-test strips on BHISA according to the manufacturer’s instructions (AB Biodisk, Solna, Sweden). Bacteroides fragilis 638R and recQ mutant cells were grown on BHISA before colonies were scraped off and resuspended in phosphate-buffered saline (PBS). Aliquots of these suspensions were fixed in glutaraldehyde (2% v/v in PBS) and prepared for transmission electron microscopy (TEM) (Simpson et al., 2006). Ultrathin sections were viewed using a JEOL 1200EX II TEM. Further cell samples were either Gram stained or the DNA Androgen Receptor antagonist and membranes were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg mL−1) and FM4-64 (1 mM), respectively. Fluorescence microscopy was performed at × 1000 magnification using a Zeiss Axiovert 200 microscope and photographed using a Zeiss Axiocam. Pictures were analysed using axiovision 4.6. To visualize DNA strand breaks, genomic DNA was extracted and the presence of double- and single-strand breaks was analysed by neutral and alkaline agarose gel electrophoresis, respectively (Abratt et al., 1990; Dachs et al., 1995). Analysis of the B. fragilis 638R genome sequence revealed the presence of three putative RecQ genes, identified by ORF numbers BF638R_3282 (Q1), BF638R_3781 (Q2) and BF638R_3932 (Q3). These ORFs encoded deduced proteins of 607

amino acids (aa), Adenosine 634 aa and 726 aa in length, respectively. These Doxorubicin cell line B. fragilis 638R loci corresponded to BF3249, BF3706 and BF3892, respectively, from strain NCTC 9343. Q1 was most similar to E. coli RecQ (43.7% aa identity), while Q2 and Q3 showed 39.6% and 38.9% aa identity to it, respectively. The presence of multiple RecQ homologues in prokaryotes had not been described before this study on B. fragilis,

although it is well documented in higher eukaryotes. For example, the human genome encodes five RecQ proteins (BLM, WRN, RecQL1, RecQL4, RecQL5), while Arabidopsis thaliana encodes seven RecQ homologues (Hartung & Puchta, 2006). Our analysis revealed that the annotated genomes from 14 members of the genus Bacteroides encoded multiple putative RecQ homologues (Fig. 1a; Table S2). The significance of multiple RecQ homologues in Bacteroides is unclear. All the B. fragilis 638R RecQ homologues contained two of the signature amino acid domains representative of all known RecQ helicases, namely a helicase domain (with the essential DEXX motif [X representing alanine (A), histidine (H), serine (S) or aspartate (D) residues]) as well as a helicase C-terminal domain (Fig. 1b). The helicase domain from all three homologues further contained the conserved regions 0 to VI (Bennett & Keck, 2004). The BF638R_3932 (Q3) homologue contained an incomplete HRDC domain, whereas the RecQ coded by BF638R_3781 (Q2) was unusual as it completely lacked an HRDC domain.

Six reference lines were measured on the study cast: D + E space, arch width, arch length, intercanine width, intercanine length, and arch perimeter. For each participant, the D + E space of the contralateral intact primary molar served as a control. A paired t-test was used to compare the cast measurements between initial examination and 12-month follow-up. A t-test was used to compare D + E space changes with those of the control group. Results.  The D + E space of the extraction side after 12 months was significantly smaller than that of the control side (P < 0.05) and the initial D + E space (P < 0.05). A significantly

greater arch perimeter, intercanine width, and intercanine length were found after 12 months compared with the initial parameters. No significant differences were found, however, in arch width or arch length between the initial examination OSI-906 mouse and the 12-month follow-up examination (P > 0.05). Conclusions.  The 12-month space changes in the maxillary dental arch after premature loss of a primary maxillary first molar consist mainly of distal drift of the primary canine toward the extraction site. Mesial movement of permanent molars or tilting of the primary molars did not occur. An increased arch dimension was found especially in the anterior segment (intercanine width and length). There is no need for the use of space maintainers from the results in this study

in cases of premature loss of a primary first molar. “
“International Journal of Paediatric Dentistry 2010; 20: 347–352

Aim.  To investigate the prevalence of dental www.selleckchem.com/products/PLX-4032.html fluorosis in children who had participated in an oral health programme between the ages 2–5 years, including fluoride tablets from the age of 2 years. Design.  The study group consisted of 135 10- to 11-year-old children who had participated in the programme, including parent education, tooth-brushing instruction and prescribed fluoride tablets Urocanase (0.25 mg NaF) (2–3 years: 1 tablet/day; 3–5 years: 2 tablets/day). The prevalence of dental fluorosis in the study group was compared with that in a nonintervention reference group consisting of 129 children of the same ages. The analysis was based on photos of the permanent maxillary front teeth using the Thylstrup & Fejerskov (TF) Index. Results.  No statistically significant difference in prevalence of dental fluorosis was seen between the two groups. Forty-three percent of the children in the study group and 38% in the reference group had fluorosis, the majority of a mild nature (TF-score 1). None had a TF score above 2. The pattern was the same after correction for parent reported intake of tablets at 3 and 5 years of age. Conclusion.  Introduction of fluoride tablets at the age of 2 years did not result in increased prevalence of dental fluorosis. “
“International Journal of Paediatric Dentistry 2012; 22: 92–99 Background.

, 1999). This opens the possibility for easier heterologous Selleckchem Gefitinib production of mycobacterial glycoproteins in the nonpathogenic and fast-growing streptomycetes. However, it has not been formally proven that glycosylation of mycobacterial proteins

is carried out by the same yeast-like protein mannosylation system in streptomycetes. Here, we show that the Apa protein is expressed and glycosylated by S. coelicolor, a strain that is taxonomically very close to S. lividans, but has the advantage of a well-developed system for genetic manipulation. Using a series of constructed null mutants, we demonstrate that Ppm and Pmt activities are essential for Apa glycosylation. We also show that Lnt1, the homologue of the D1 or Lnt domain of M. tuberculosis

Ppm, is dispensable for glycosylation of the Apa protein and of the bacteriophage φC31 receptor and that, in contrast to mycobacteria, the homologous Lnt1 of S. coelicolor does not interact with the Ppm protein. Given the phylogenetic relationship between mycobacteria and streptomycetes, we also explored the functionality of M. tuberculosis Ppm and Pmt in S. coelicolor, as this might provide a way for production of mycobacterial glycoproteins by introducing a cognate glycosylation system in a heterologous host; we show that Ppm, but not Pmt, is functional when heterologously expressed. Escherichia coli strains Dabrafenib ic50 were grown in 2XYT medium (Sambrook & Russell, 2001). Growth of Streptomyces mycelium, preparation of spores, transformation with polyethylene glycol, conjugations, and phage propagation were carried out according to Kieser et al. (2000). For protein expression experiments, S. coelicolor was grown in LB broth containing 34% sucrose to obtain dispersed mycelial growth (Lara et al., 2004). Unmarked Aurora Kinase deletion mutants were obtained by the PCR targeting procedure of Datsenko & Wanner (2000) on relevant cosmids carrying the cloned regions of interest

of the S. coelicolor chromosome (Redenbach et al., 1996), followed by recombination of the mutations into the chromosome as described by Gust et al. (2004). All mutants were verified by PCR and sequencing to confirm replacement of the relevant gene with the 81-bp in-frame ‘scar’ sequence (Gust et al., 2004). The cosmids used were St6D7A, StE87, and 2StG2, which carry the cloned ppm, pmt, and lnt1 genes, respectively. Table 1 lists the strains, plasmids, and bacteriophage used in this study, while Supporting information, Table S1 lists the oligonucleotides used. Plasmid construction and purification were carried out according to Sambrook & Russell (2001). DNA amplification was carried out using PfuUltra DNA polymerase AD and site-directed mutagenesis using the QuikChange kit (both from Agilent Technologies). A detailed description of plasmid construction is provided in Data S1.

This led us to develop an assay based on the fact that the transcription factor, NsrR, responds specifically and with very high sensitivity to NO located in the cytoplasm rather than outside the cytoplasmic membrane (Bodenmiller & Spiro, 2006; Tucker et al., 2008). The assay was used to compare the effects on NsrR-dependent transcription of mutations INCB024360 datasheet in genes for enzymes implicated in NO production, as well as the effectiveness of externally added NO and nitrite as sources of cytoplasmic NO. Strains of E. coli K-12 and plasmids used in this study are listed

in Table 1. The nsrR::kan mutation was transferred by P1 transduction from E. coli strain JOEY 60 to RK4353 to construct strain JCB 5222. The strain to be tested was transformed with the Phcp::lacZ fusion plasmid, pNF383, (Filenko et al., 2007). A plasmid with a synthetic promoter with a consensus FNR-binding site linked to lacZ that is repressed by FNR was used in control experiments designed to Belnacasan datasheet distinguish between NO-induced damage to FNR and NO-induced derepression of Phcp (Williams

et al., 1998). Purified transformants were grown in minimal salts medium (MS) supplemented with 5% (v/v) LB, 0.4% (v/v) glycerol, 20 mM trimethylamine-N-oxide, 20 mM sodium fumarate and 35 μg mL−1 tetracycline. Cultures were started with 2% inocula that had been grown overnight at 37 °C with aeration in 5 mL LB in 25 mL conical flasks. Multiple anaerobic cultures were incubated statically at 37 °C in test tubes filled with 15 mL of medium. Once the optical density at 650 nm had reached 0.2 or above, one culture was left as an unsupplemented control; other cultures were supplemented

as stated in the text with 2.5 or 10 mM sodium nitrite, 20 mM sodium nitrate, or 5–20 μM nitric oxide saturated water (NOSW) prepared as described by Vine & Cole (2011). Nitric oxide saturated water was added repeatedly at 30 min intervals under the surface of the culture using a sterile syringe and needle to avoid exposure to oxygen. A magnetic stirrer was used very briefly to ensure that the NOSW was distributed evenly throughout the culture, Dichloromethane dehalogenase but to avoid aeration. Cultures were incubated statically at 37 °C. Bacteria were grown in MS supplemented where indicated with 10% LB, 0.4% glycerol, 20 mM TMAO, 20 mM sodium fumarate and 2.5 mM sodium nitrite or 20 mM sodium nitrate. All cultures were started with inocula that had been grown at 37 °C with aeration in 2 mL LB in a test tube for at least 2 h. Anaerobic cultures were incubated statically overnight at 30 °C 250 mL flasks filled with 250 mL of medium. The optical density at 650 nm was monitored until it had reached 0.6–0.8, then the bacteria were collected by centrifugation (8000 g, 2 min, 4 °C). The bacteria were resuspended in 10 mL phosphate buffer and were homogenized. The washed bacteria were collected by centrifugation (3000 g, 3 min), then resuspended in 0.5–1 mL phosphate buffer to give an optical density at 650 nm of 70–90.

This led us to develop an assay based on the fact that the transcription factor, NsrR, responds specifically and with very high sensitivity to NO located in the cytoplasm rather than outside the cytoplasmic membrane (Bodenmiller & Spiro, 2006; Tucker et al., 2008). The assay was used to compare the effects on NsrR-dependent transcription of mutations TSA HDAC ic50 in genes for enzymes implicated in NO production, as well as the effectiveness of externally added NO and nitrite as sources of cytoplasmic NO. Strains of E. coli K-12 and plasmids used in this study are listed

in Table 1. The nsrR::kan mutation was transferred by P1 transduction from E. coli strain JOEY 60 to RK4353 to construct strain JCB 5222. The strain to be tested was transformed with the Phcp::lacZ fusion plasmid, pNF383, (Filenko et al., 2007). A plasmid with a synthetic promoter with a consensus FNR-binding site linked to lacZ that is repressed by FNR was used in control experiments designed to Roxadustat ic50 distinguish between NO-induced damage to FNR and NO-induced derepression of Phcp (Williams

et al., 1998). Purified transformants were grown in minimal salts medium (MS) supplemented with 5% (v/v) LB, 0.4% (v/v) glycerol, 20 mM trimethylamine-N-oxide, 20 mM sodium fumarate and 35 μg mL−1 tetracycline. Cultures were started with 2% inocula that had been grown overnight at 37 °C with aeration in 5 mL LB in 25 mL conical flasks. Multiple anaerobic cultures were incubated statically at 37 °C in test tubes filled with 15 mL of medium. Once the optical density at 650 nm had reached 0.2 or above, one culture was left as an unsupplemented control; other cultures were supplemented

as stated in the text with 2.5 or 10 mM sodium nitrite, 20 mM sodium nitrate, or 5–20 μM nitric oxide saturated water (NOSW) prepared as described by Vine & Cole (2011). Nitric oxide saturated water was added repeatedly at 30 min intervals under the surface of the culture using a sterile syringe and needle to avoid exposure to oxygen. A magnetic stirrer was used very briefly to ensure that the NOSW was distributed evenly throughout the culture, Selleck Bortezomib but to avoid aeration. Cultures were incubated statically at 37 °C. Bacteria were grown in MS supplemented where indicated with 10% LB, 0.4% glycerol, 20 mM TMAO, 20 mM sodium fumarate and 2.5 mM sodium nitrite or 20 mM sodium nitrate. All cultures were started with inocula that had been grown at 37 °C with aeration in 2 mL LB in a test tube for at least 2 h. Anaerobic cultures were incubated statically overnight at 30 °C 250 mL flasks filled with 250 mL of medium. The optical density at 650 nm was monitored until it had reached 0.6–0.8, then the bacteria were collected by centrifugation (8000 g, 2 min, 4 °C). The bacteria were resuspended in 10 mL phosphate buffer and were homogenized. The washed bacteria were collected by centrifugation (3000 g, 3 min), then resuspended in 0.5–1 mL phosphate buffer to give an optical density at 650 nm of 70–90.