Atropine, nifedipine, tetrodotoxin, and ω-conotoxin GVIA were used to explore the agonist-induced LES contraction mechanism. Reverse transcription polymerase chain reaction and immunohistochemistry were applied to detect bombesin receptor expression. Our results indicate that GRP and DTACPN-BN, but not NMB, induced tonic contractions of the porcine LES in a dose-dependent manner, and the contractions were inhibited with selective BB2 and BB3 antagonists. The GRP-induced contraction is mainly caused by L-type Ca2+ channel-mediated Ca2+ influx. However, DTACPN-BN-induced contractions are associated with neuronal conduction. RT-PCR and immunohistochemistry revealed that BB2 and BB3 were expressed in the porcine LES. Bombesin-induced tonic contraction of the LES is mediated through BB2 and BB3. Bombesin, BB2, and BB3 agonists might have the potential to treat GERD.Gastroesophageal reflux disease (GERD) is a disorder that is related to an incompetent lower esophageal sphincter (LES). Previous studies showed that bombesin could increase LES pressure in humans and opossums. The aim of the present study was to characterize the effects of bombesin on porcine LES contraction. We used the selective agonists, neuromedin B (NMB), gastrin-releasing peptide (GRP), and [D-Tyr Gastroesophageal reflux disease (GERD) is caused by acid- or other irritant-induced injury from stomach backflow to the esophagus. Under physiological conditions, the lower esophageal sphincter (LES) is closed to prevent damage from food or acid reflux and transiently relaxes to let food pass through when swallowing occurs. The main underlying mechanism of GERD is LES incompetence, which may result from decreased LES tone or recurrent inappropriate transient LES relaxations. An incompetent LES cannot prevent acid reflux, which in turn results in damage to the esophageal mucosa, which further diminishes the LES pressure. GERD management includes lifestyle and dietary modifications, medications, which include antacids, histamine 2 receptor antagonists, and proton pump inhibitors (PPI), and surgeryBombina bombina)61), bombesin receptor subtype 2 (BB2), and bombesin receptor subtype 3 (BB3)79Bombesin is a 14-amino acid peptide and originates from the skin of the European fire-bellied toad , a BB2 agonist, gastrin-releasing peptide (GRP), and a BB3 agonist, bombesin-(6-14) (DTACPN-BN), were used to elucidate which bombesin receptor subtype or subtypes are involved in bombesin-induced LES contraction. Bombesin, GRP, and DTACPN-BN all elicited sustained and dose-dependent contraction of the isolated porcine LES sling and clasp muscle strip (The structure of the porcine LES is similar to that of the human LES. It is composed of sling muscle fibers at the greater curvature (GC) and clasp muscle fibers at the lesser curvature (LC). Bombesin receptor agonists including a BBle strip . Figure le strip . The maxle strip . The maxle strip . The maxle strip . The maxle strip . The maxle strip .2 antagonist, BN(6-14) ethylamide (DPDM-BN EA) and a BB3 antagonist, Bantag-1, were used to block GRP- and DTACPN-BN-induced porcine LES contraction, respectively. Incubation with DPDM-BN EA (1 μM) shifted the GRP concentration-response curve to the right, with three- and fourteen-fold changes at the EC50 levels for the LES sling (50 levels were 0.18 ± 0.04 μM (sling) and 42 ± 10 nM (clasp) (n = 4/group). The GRP plus 1 μM DPDM-BN EA EC50 levels were 0.58 ± 0.10 μM (sling) and 0.61 ± 0.07 μM (clasp) . These results show that DPDM-BN EA had a significant inhibitory effect on GRP-induced contraction of both the porcine LES sling and clasp muscle strips. Similarly, Bantag-1 significantly inhibited DTACPN-BN-induced contraction of the porcine LES sling and clasp muscle strips  at a concentration of 1 μM , BB2 and BB3 antagonists, respectively, before the bombesin stimulation. PD 168368 (1 μM) and Bantag-1 (1 μM) alone did not inhibit bombesin-induced contraction of both the porcine LES sling (2 antagonist DPDM-BN EA (1 μM) shifted the bombesin concentration-response curve to the right but the difference between the EC50 levels of bombesin alone and EC50 levels of bombesin plus DPDM-BN EA was not significant . In contrast, the BB2 antagonist DPDM-BN EA combining with BB3 antagonist Bantag-1 (both 1 μM) significantly shifted the bombesin concentration-response curve to the right, with ten- and sixty-fold changes at the EC50 levels for the LES sling and clasp muscle strips, respectively. Specifically, the EC50 levels of bombesin alone were 72 ± 21 nM (sling) and 10 ± 0.51 nM (clasp) (n = 4/group). The EC50 levels of bombesin plus 1 μM DPDM-BN EA combining with Bantag-1 were 0.72 ± 0.22 μM (sling) and 0.69 ± 0.14 μM (clasp), respectively . Thus, it is likely that the bombesin-induced LES contraction is mediated through BB2 and BB3.To further examine whether the bombesin-induced LES contraction is mediated through BBES sling  and clasES sling  muscle sp > 0.05, compared with GRP alone, n = 4). Similarly, atropine had no significant effect on the GRP-induced contraction . In contrast, the GRP-induced contraction of the LES sling muscle fibers was significantly inhibited by the L-type Ca2+ channel blocker, nifedipine , at a concentration of 1 μM.Sling muscle strips exhibited greater contraction forces than the clasp muscle strips. The maximal contraction forces that were induced by GRP on the sling muscle strips were one and a half times greater than the clasp muscle strips, and the maximal contraction forces induced by DTACPN-BN on the sling muscle strips were two times greater than the clasp muscle strips . Therefop < 0.05, compared with DTACPN-BN alone, n = 4).As shown in 2-specific primers, we could generate a PCR product of 487 bp  was 148 bp, as predicted. No PCR product corresponding to the NMB receptor (BB1) was detected in the cDNA that was generated from sling or clasp muscle fibers (n = 3).Using BBf 487 bp  from bot1) with a specific BB1 antibody showed negative results in both the sling and clasp muscles. However, there was positive BB1 immunostaining on the positive control tissues of Sprague Dawley rat testes (as shown in 2) was ubiquitous in the LES sling (3 were found in myenteric ganglia of the LES sling (Immunohistostaining of the neuromedin B receptor (BBES sling  and clasES sling  muscle fES sling  and clasES sling  muscles 1) as well as GRP receptors (BB2) and BB3. BB2 is widely present in the pancreas, central nervous system, and gastrointestinal tract. BB2 plays a role in many gastrointestinal functions, including gastrointestinal motility regulation, insulin stimulation, pancreatic and gastric acid secretion, colonic ion transport stimulation and the secretion of various hormones2 agonist, plays a major role in gastric emptying, small bowel transit, and gallbladder contraction2 antagonist, DPDM-BN EA, can significantly inhibit GRP-induced LES contraction, indicating that GRP causes LES contraction via BB2.Bombesin receptors are a family of G protein-coupled receptors, including the NMB receptor , which in turn induces smooth muscle contractionThe contraction of porcine LES smooth muscle fibers is modulated by multiple factors, especially autonomic neural innervations. The excitatory postganglionic vagal nerves transmit signals to nerve endings. Acetylcholine is released from the presynaptic nerve endings and stimulates postsynaptic muscarinic receptors. After stimulation of the muscarinic receptor, extracellular Ca+ channel blocker, to explore whether GRP acts on the nerve fibers or directly on the LES smooth muscle. We found that TTX did not inhibit GRP-induced contraction of the LES, suggesting that the contraction was not due to an effect of GRP on the LES neuronal fibers. Additionally, atropine, a nonselective muscarinic receptor antagonist, was applied to investigate whether GRP acts directly on the muscarinic receptor of the LES smooth muscle, resulting in contraction. The GRP-induced contraction was not inhibited by atropine, indicating that GRP does not affect the LES muscarinic receptors. In contrast, we found that nifedipine could block GRP-induced LES contractions. Nifedipine is an L-type Ca2+ channel blocker that inhibits extracellular Ca2+ influx, and LES smooth muscle contraction is generally related to an increase in the intracellular Ca2+ concentration2+ influx through the L-type Ca2+ channel was indeed a main GRP-induced contraction pathway of the porcine LES. However, the L-type calcium blocker may also decrease the intracellular calcium stores in muscle strips owing to blockade of calcium influx which is needed for calcium store refilling. Therefore, the role of intracellular calcium stores cannot be excluded completely.In the first step, we used TTX, a selective neuronal Na3 was first cloned from the guinea pig uterus3 plays a role in the regulation of energy homeostasis, glucose/insulin levels, lung development, satiety, and injury3 is present in the myenteric and submucosal ganglia in the tunica muscularis of the gastrointestinal tract and in nerve fibers between the myenteric ganglia. Additionally, with specific BB3 antibodies, it was also detected in interstitial cells of Cajal (ICC)3 in the gastrointestinal tract is not clear, and it was proposed that BB3 might be related to gastrointestinal motility regulation3 agonist, DTACPN-BN, could induce LES sling and clasp muscle fiber contractions in pigs and that the selective BB3 antagonist, Bantag-1, could significantly decrease porcine LES sling and clasp muscle fiber contractions, suggesting that DTACPN-BN causes LES contraction via BB3.BB+ channel blocker, TTX, and the neuronal Ca2+ channel blocker, CTX, were used to explore the mechanism of BB3 agonist-induced LES contraction in pigs. Our results revealed that both TTX and CTX significantly decreased BB3 agonist-induced contraction of the porcine LES sling muscle fibers. This finding implies that nerve conduction was associated with the BB3 agonist-induced LES contraction.The neuronal Na2 and BB3 transcripts in the LES sling and clasp muscles; however, BB1 transcripts were not detected. This finding is in good agreement with the results we obtained for the BB2 and BB3 agonist-induced LES sling and clasp muscle fiber contractions. Immunohistochemistry with specific BB2 and BB3 antibodies showed that BB2 was mainly expressed in the muscle, while BB3 was principally present in the ganglia. Taken together, these findings reveal that BB2 and BB3 are localized in the LES sling and clasp muscles in pigs at both the mRNA and protein levels.RT-PCR identified BB2 and BB3, but not BB1 agonists, could cause tonic LES contraction in a dose-dependent manner. Therefore, the BB2 and BB3 receptors were both involved in the bombesin-induced LES contraction. These results support the hypothesis that bombesin and its agonists are a new kind of potential drug for the enhancement of esophageal sphincter contraction in order to avoid irritant reflux. These BB2 and BB3 agonists will be helpful to treat or as adjuvant therapy for patients with proton pump inhibitor refractory GERD or those suffering from the side effects of long-term PPI use.Clinically, proton pump inhibitor refractory GERD can be non-reflux or reflux related. The reflux-related causes include residual acid reflux due to inappropriate medical treatment or poor compliance, non-acid reflux such as duodeno-gastro-esophageal (bile) reflux, hypersensitivity to physiological amounts of acid, and persistent impairment of esophageal mucosal integrity2 and BB3. This study also indicates that BB2-mediated contraction is not due to the action of nerve and muscarinic receptors, but rather is caused by the influx of extracellular Ca2+ into myocytes through L-type Ca2+ channels. In contrast, the BB3-mediated tonic LES contraction was associated with neuronal conduction. Therefore, bombesin, as well as BB2 and BB3 agonists, might have the potential to treat GERD.In conclusion, the present study showed that bombesin-induced LES contraction occurs through BB3, 122 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgCl2, 1.8 CaCl2, 11.5 glucose, pH 7.4. Additionally, the stomachs were kept on ice during transport to the laboratory, which lasted approximately 30 minutes. Carbachol, atropine, nifedipine, ω-conotoxin GVIA (CTX), Bantag-1 (BB3 antagonist), and buffer reagents were obtained from Sigma-Aldrich, MO, USA. Tetrodotoxin (TTX) was purchased from Tocris Bioscience, Bristol, UK. Bombesin, neuromedin B (NMB), gastrin-releasing peptide (GRP), and BN(6-14) ethylamide  were obtained from the American Peptide Company, CA, USA. bombesin-(6-14)  was purchased from Phoenix Pharmaceuticals, Burlingame, CA, USA. PD 168368 (BB1 antagonist) was purchased from Santa Cruz Biotechnology, Dallas, Texas, USA. These agonists and antagonists were chosen because of the specificity and high affinity for the respective bombesin receptor subtypes15Porcine stomach, including the lower esophagus, was purchased from a local slaughterhouse. The stomachs, from pigs weighing approximately 110 Kg, were placed in ice-cold Kreb-Henseleit buffer solution that had the following composition (in mM): 15.5 NaHCO2 + 5% CO2. Subsequently, the muscle strips were connected to isometric force transducers , which were connected to amplifiers and a computer recording system . The basal tension of the muscle strips was set at 1.0 g. After a 30 min equilibration period, carbachol (1 μM) was added to the organ bath, the muscle strip contractions were measured, and the carbachol was washed out. Carbachol-induced contraction served as a reference (100%) for the contractile response to bombesin or the selective agonistsThe porcine LES is often used as an animal model to study esophageal motility and GERD221 antagonist. DPDM-BN EA is a selective BB2 antagonist, and Bantag-1 is a selective BB3 antagonist. To investigate whether BB2 is involved in bombesin-induced LES contraction, the isolated sling or clasp muscle strips were treated with DPDM-BN EA (1 μM) for 6 min, followed by the addition of GRP to the organ bath in a cumulative manner3 is involved in bombesin-induced LES contraction, the isolated sling or clasp muscle strips were treated with Bantag-1 (1 μM) for 6 min, followed by the addition of DTACPN-BN to the organ bath in a cumulative mannerPD 168368 is a selective BB+ channel, muscarinic receptor, and L-type Ca2+ channel blockers, respectively. To investigate the mechanism of GRP- or DTACPN-BN-induced LES contraction in the pigs, the isolated sling muscle strips were treated with TTX , atropine , or nifedipine . GRP or DTACPN-BN was added to the muscles strips that were treated with TTX. Additionally, GRP was added to the muscle strips that were treated with either atropine or nifedipine. Furthermore, the neuronal Ca+ channel blocker, CTX (1 μM), was used to pretreat the isolated sling muscle strips, and 15 min later, DTACPN-BN was added to the organ bath2728TTX, atropine, and nifedipine are neuronal NaSling and clasp muscle fibers that were obtained from the porcine LES were stored in RNAlater solution  at 4 °C for two days to allow for tissue penetration, and then, excess liquid was removed and the tissue was stored at −80 °C until use.RNA was isolated from tissue samples with the guanidine isothiocyanate method, using the GeneJET RNA Purification Kit , according to the manufacturer’s protocol. The quality and purity of the isolated RNA were measured with an UV/Vis spectrophotometer .After RNA concentration measurements, cDNA was synthesized using the extracted RNA and the High Capacity cDNA Reverse Transcription Kit  for RT-PCR. The RNA sample preparation for reverse transcription was performed according to the manufacturer’s recommendation. A volume corresponding to 1 μg of RNA was used for the cDNA synthesis. The cDNA samples were diluted to a volume of 100 μl and prepared for subsequent PCR.1): forward 5′-GCGGACAGGTACAGAGCAAT-3′ and reverse 5′-GGCGTTTCCGTGTTTCCATC-3′; (3) gastrin-releasing peptide receptor (BB2): forward 5′-CGTGCACTGCCACATCTCTA-3′ and reverse 5′-AAACACAGCCTCTGGGATGG-3′; (4) bombesin-like receptor 3 (BB3): forward 5′-AACGCCATCCTGAAGACCTG-3′ and reverse 5′-AGAGAGCAAACAGAGCCACC-3′. The PCR products were separated by electrophoresis in a 2% agarose gel, which was then stained with ethidium bromide and visualized under UV light.Amplification of the bombesin receptor-specific sequences through PCR was carried out on a thermal cycler  with the All-in-One PCR Mix  under the following program: one cycle of 95 °C for 10 min, 35 cycles of 95 °C for 30 s, 53 °C for 50 s, and 72 °C for 30 s, and one cycle of 72 °C for 10 min. As an internal control, ß-actin was amplified. The primer sequences were the following: (1) β-actin: forward 5′-TCGGTTGGATGGAGCATCCCC-3′ and reverse 5′-GGGAAGGCAGGGACTTCCTGTAA-3′; (2) neuromedin B receptor  for BB2, or a 1:200 dilution of rabbit anti-Brs3/bombesin receptor 3 polyclonal antibody  for BB3 for 30 min at room temperature. Then, the samples were subsequently incubated with an anti-rabbit horseradish peroxidase polymer for 10 min at room temperature. The samples were then treated with a chromogen, 3,3′-diaminobenzidine tetrahydrochloride (DAB), for 10 min at room temperature and counterstained with hematoxylin for 5 min. The sling and clasp muscle sections were stained with normal rabbit IgG at equimolar concentrations as negative controls.Formalin-fixed and paraffin-embedded porcine sling and clasp muscle fibers were prepared for IHC staining. The IHC stains were performed using standard reagents and techniques on a BOND-MAX Automated Staining System (Leica Microsystems). Briefly, the deparaffinized and rehydrated sections were subjected to heat-induced antigen restoration with bond epitope retrieval solution 1 . The staining procedure involved a peroxidase block with 3% hydrogen peroxide for 5 min, incubation with a 1:300 dilution of rabbit neuromedin B receptor polyclonal antibody, LS-A825  for BB50) values. The statistical analysis of the results was performed with Student’s t-tests or one-way ANOVA followed by Tukey’s post hoc test. In all cases, the differences were considered significant when p < 0.05.These data are expressed as the mean ± standard error of the mean (SEM). GraphPad Prism 5 was used to determine the half-maximal contraction .


S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureusvirulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development.Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused  Staphylococcus aureus which is the major cause of acute bacterial skin and soft tissue infections. We conducted a high throughput screen to identify compounds that could inhibit signaling by the quorum sensing operon, agr. We found a compound that we termed savirin (S. aureusvirulence inhibitor) that could inhibit signaling by this operon. The drug helped the innate immune system in animals to clear bacteria that express this operon without affecting clearance of bacteria that do not have this operon. We addressed the mechanism of action of this compound and whether resistance or tolerance to this compound would likely develop. Our data indicate for the first time that host defense against S. aureus skin infections can be enhanced by chemical inhibition of agr-mediated quorum sensing.New approaches are needed to lessen the burden of antibiotic resistant bacterial infections. One strategy is to develop therapies that target virulence which rely on host defense elements to clear the bacteria rather than direct antimicrobial killing. Quorum sensing is a bacterial signaling mechanism that often regulates virulence in medically relevant bacterial pathogens. Therefore, drugs that inhibit quorum sensing can promote host defense by rendering the pathogenic bacteria avirulent and/or less fit for survival within the host. Our work addressed this strategy in the pathogen  Here we describe a QSI identified in a high throughput screen that selectively inhibited agr signaling in S. aureus, but not in S. epidermidis, by blocking the function of the transcriptional regulator of the operon, AgrA, preventing upregulation of the agr-regulated genes essential for skin infection. It was efficacious in murine models of agr-dependent skin infection without apparent induction of resistance or tolerance after passage in vivo. These data provide proof-of-principle that AgrA transcriptional function in S. aureus can be selectively inhibited to attenuate quorum sensing with minimal toxicity to the bacterium or induction of stress responses observed with conventional antibiotics. Thus, selective AgrA blockade could enhance agr-dependent host defense in the skin while potentially preserving the normal microbiota, limiting resistance induction, and sparing conventional antibiotics for treatment of invasive systemic infections.Approximately 90% of agr quorum sensing operon encodes two promoters agr regulated agr::P3 activation using a reporter strain where P3 drives production of GFP (ALC1743) (http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1206&loc=ea_ras). We pursued one compound where dose-response experiments using an additional reporter strain indicated that it had minimal impact on exponential phase growth during the 3 hr assay starting at a CFU of 2×107/ml and ending at ∼1×108/ml. It inhibited optimally at 5 µg ml−1 (13.5 µM)  and lipophilicity (XLogP3-3.5) meet standards for drug development The 13.5 µM) . Termed 13.5 µM) , for StaS. aureus isolates belong to one of four agr alleles depending on variations in AIP (amino acid sequence and length) and the cognate receptor, AgrC agr I alleles predominate in human disease, all four can contribute to SSTIs agr disruption should work against all agr alleles. Savirin (5 µg ml−1) inhibited agr::P3 activation in reporter strains of each agr type (in vitro and in vivo using a strain (LAC) of the epidemic methicillin-resistant USA300 clone and the predominant agr group I −1) inhibited both AIP1-induced RNAIII (agr (Δagr) and by savirin treatment  S. aureus and S. epidermidis using the online server Swissdock c of S. aureus between Tyr229, which is adjacent to a residue critical for AgrA folding agr function S. epidermidis where the key Tyr229 is a Phe and His227 is an Asn. Consistent with this, attempts to dock savirin to this site in S. epidermidis were unsuccessful, demonstrating that the DNA binding domain of AgrA is the likely target of savirin. We performed electrophoretic mobility shift assays to prove that savirin blocked the DNA binding function of AgrA. Incubation of purified AgrAc (2 µM) with the high affinity site in P2 and P3 (0.1 µM) (−1 or 13.5–432 µM) vs. vehicle inhibited this shift with an IC50 of 83 µM or 30.3 µg ml−1  suppressed constitutive luminescence without affecting viability where AIP2, an inhibitor of non-agrII AgrC signaling agr::P3 activation within the pathogen in strain LAC (1–5 µg ml−1)  shifted  µg ml−1 . To prov µg ml−1). Howeveragr virulence by microarray analysis −1. The effect of savirin vs. vehicle on AIP1 induced transcription in LAC was compared to the differences between LAC and Δagr LAC. Two hundred and five non-redundant transcripts were different and changed by greater than two fold between LAC and Δagr LAC (agr-regulated transcripts by a similar magnitude and direction including downregulation of agr secreted virulence factors (the majority of transcripts affected), transcriptional regulators, and metabolic pathways important for SSTIs agr-regulated transcripts not affected by savirin, the majority were hypothetical or involved in metabolism. In contrast, savirin affected only 5% of the non agr-regulated transcriptome  or mecA (SAUSA300 0032). Importantly, transcripts were not affected for known stress response genes and the anti-inflammatory exotoxins induced by bactericidal agents agr ablation hla, psm alpha, pvl (lukS), agrA, and agrC,  lack control of agr::P3 activation in this model causing maximal in vivo quorum sensing agr::P3 activation (AH1677) agr::P3 activation in bacteria from a lavage of the pouch as well as consequential weight loss (as a measure of morbidity) and bacterial burden in the pouch lavage and systemically in the kidney  did not affect weight loss or bacterial burden in the pouch lavage or the kidney  but not Δagr LAC would augment killing of the bacteria in vitro by macrophages. As predicted, survival of vehicle treated LAC intracellularly from 1–5 hrs was significantly greater than savirin treated LAC  vs. vehicle sequentially through the skin of ten individual mice 24 hrs after infection. We compared this to in vivo passage with sub-inhibitory concentrations of antibiotics known to induce resistance in USA300 strains, erythromycin and clindamycin, because of the genetic expression of ermC in vivo with conventional antibiotics induced resistance to killing by clindamycin  (in vitro passage every day for ten days (ndamycin  but passµg ml−1) . Equivalten days .in vivo passaged bacteria on milk agar plates where proteolysis is agr-dependent and contributes to colony growth  and neither had small non-proteolytic colonies indicative of agr dysfunction  its lack of effect on the number of Δagr bacteria 24 hr after infection, 2) its lack of effect on the number of agr+ bacteria early (day 3) at the site of skin infection, and 3) its lack of effect on macrophage or low pH killing of Δagr bacteria. Moreover, the reduction in CFU observed in our 2 models of SSTIs both at the site of infection and systemically (1.5–2.0 logs) was similar to that seen with conventional antibiotics tested in a murine surgical wound infection model In this work we have used an SMI as a tool to address many of the concerns raised about the use of quorum sensing inhibitors as therapies or adjuncts for the prevention or treatment of antibiotic resistant bacterial infections . aureus  model. WS. aureus infections involve skin and skin structures and are dependent on agr signaling in humans and animals Staphylococcus aureus). Our data are consistent with this approach and suggest that a QSI could either substitute for or be used as an adjunct to conventional antibiotics in this setting. Additionally, a QSI could be substituted for antibiotics used prophylactically to prevent wound infections until clinical signs of infection were apparent. Whether a QSI like savirin could be an adjunct with conventional antibiotics for treating systemic infections with or without a biofilm component is a matter of speculation and was not addressed by our studies. In fact, QSI's may have very different clinical utility in Gram negative and positive infections. Because even appropriate antibiotic use drives resistance S. aureus in humans agr inhibitors may have efficacy in some patients with impaired host defense systems. More experimental work is required to determine which host defense elements are essential for agr inhibitor efficacy.Because the majority of Pseudomonas aeruginosa) where QS mutants (cheaters) arise during infection by taking advantage of the metabolic effort exerted by QS enabled bacteria for survival in vivo with S. aureus infection is uncertain. Based on studies in Gram negative bacteria c, mutants resistant to savirin are most likely to be agr dysfunctional. Mutations in either agrA or agrC do arise in human infection agrA (Arg 218 to His) agr enabled bacteria during the course of infection agr mutants arise with savirin exposure, they are unlikely to have a selective advantage over wild-type bacteria. Importantly, infection with agr mutants is primarily associated with bacteremia in hospitalized patients with impaired host defense systems and not with acute skin infection in immunocompetent individuals in vivo passage in mice suggests that agr inhibitors may not drive the selection of agr mutants in skin infection. However, resistance or tolerance to agr inhibitors could arise by inducing a survival response in the bacteria that leads to upregulation of efflux mechanisms. Our microarray data suggest this as a possibility but neither in vivo nor in vitro passage with savirin resulted in resistance or tolerance to agr inhibition at either the population or colony level under the conditions we used. Currently, it is impossible to predict whether these issues would arise in human infection and whether our method for chronic exposure with in vivo passage in mice actually reflects how skin bacteria would be exposed to a QSI during human infection.The potential for resistance development to QSI's has been addressed primarily in Gram negative bacteria . Human neutrophils were purchased from AllCells and the source of the neutrophils was anonymous.All animal experiments were conducted at the AAALAAC accredited Veterinary Medical Unit of the New Mexico Veteran's Affairs Health Care Service in accordance with the applicable portions of the USA Animal Welfare Act as regulated by USDA, the Eighth Edition of AIPs1-4 were synthesized by Biopeptide Co., Inc and stored in DMSO at −80°C. Savirin (3-(4-propan-2-ylphenyl) sulfonyl-1H-triazolo  quinazolin-5-one, CID#3243271) was synthesized by ChemDiv, confirmed purified by HPLC, and stored in DMSO at −80°C.S. aureus strains used in this study were as follows: USA 300 strain LAC and its agr deletion mutant as described agr I [agr::P3-gfp]) and ALC3253 (Newman [agr::P3-gfp]) as described agr I LAC [agr::P3-yfp]); AH430 (agrII 502a [agr::P3-yfp]), AH1747 (agr III MW2 [agr::P3-yfp]), AH1872 (agr IV MN TG [agr::P3-yfp]) as described agr IV clinical isolates (NRS165 and NRS166) were obtained through the Network on Antimicrobial Resistance in Staphylcoccus aureus (NARSA) supported under NIAID, NIH contract No. HHSN272200700055C. MRSA and MSSA clinical isolates were provided by Dr. Larry Massie, Pathology Service, NMVAHCS and agr typed by PCR as described Staphylococcus epidermidis strain #12228 was obtained from ATCC and a Pseudomonas aeruginosa isolate was provided by Dr. Graham Timmins, College of Pharmacy, University of New Mexico. To generate early exponential phase, non-quorum sensing bacteria, frozen stocks were cultured in trypticase soy broth (TSB) (Becton Dickinson) as described 600 in 96 well plates using a plate reader (Molecular Devices) at 37°C with shaking, reading at 30 min intervals for 16 hr. The initial cultures were sufficiently diluted such that the maximal OD600 was confirmed to be within the linear range of the plate reader (<1.25 OD600). Additionally, growth was measured in 5 ml cultures in 50 ml sterile conical tubes with shaking and the OD600 determined on 1∶2 and 1∶4 dilutions of the bacterial cultures to ensure that maximal growth was adequately detected and the OD600 of the diluted samples was under 0.8 and clearly within the linear range of the spectrophotometer.The http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1206&loc=ea_ras). Using the Hypercyt flow cytometry sampling platform 7 early exponential phase ALC1743 containing agr::P3 driving expression of GFP. After incubation for 3 hrs with 100 nM synthetic AIP1, the induced fluorescence of the bacteria was compared between vehicle controls and compounds in 0.2% DMSO. Erythrosin B generated singlet oxygen was used as a positive control to inactivate AIP1 A fluorescence-based, high throughput assay was developed to screen 24,087 compounds selected for diversity from the Molecular Libraries Small Molecule Repository of the NIH Molecular Libraries Screening Center Network (summary available at agr:: P3 reporter strains (2×107/ml TSB) were incubated (200 rpm at 37°C) in polystyrene tubes with broth, 50 nM synthetic AIP, or indicated concentrations of savirin for the indicated time. After incubation, bacteria were centrifuged , supernatants decanted, and the pellet washed with PBS/0.1% Triton X-100, fixed with 1% paraformaldehyde containing 25 mM CaCl2, sonicated, and then evaluated for fluorescence by flow cytometry . Promoter activation was measured as induction of fluorescence.Early exponential phase non-fluorescent S. aureus strains and clinical isolates were cultured as indicated in the figure legends. For S. epidermidis, overnight culture supernatant was used as a source of AIP. It was Millipore filtered and diluted 1∶2 with TSB. RNA was isolated and purified using the Qiagen RNA Protect Bacteria Reagent and RNeasy Mini Kit (Qiagen) using both mechanical and enzymatic disruption. RNA was purified with silica columns and subjected to DNase treatment to remove contaminating DNA. cDNA was generated using a high capacity cDNA RT kit with an RNAse inhibitor (Applied Biosystems) and a Bio-Rad thermocycler. Thermal cycling conditions were as follows: 10 minutes at 25°C, 120 minutes at 37°C, 5 minutes at 85°C, hold at 4°C. Quantitative PCR was performed using an ABI7500 Real-Time PCR system with Taqman Gene Expression master mix, ROX probe/quencher, and appropriate primer sequences (Applied Biosystems). Samples were assayed in triplicate. The data are represented as the fold increase of the transcript relative to 16S compared to the inoculum bacteria. The primer-probe sequences used were as follows: For S. aureus: RNAIII forward primer AATTAGCAAGTGAGTAACATTTGCTAGT, RNAIII reverse primer GATGTTGTTTACGATAGCTTACATGC, RNAIII probe FAM-AGTTAGTTTCCTTGGACTCAGT-GCTATGTATTTTTCTT-BHQ; psmα forward primer TAAG-CTTAATCGAACAATTC, psmα reverse primer CCCCTTCAAATA-AGATGTTCATATC, psmα probe FAM-AAAGACCTCCTTTGTTTGTTA-TGAAATCTTATTTACCAG-BHQ; hla forward primer ACAATTTTAGAGAGCCCAACTGAT, hla reverse primer TCCCCAATTTTGATTCACCAT, hla probe FAM-AAAAAGTAGGCTGGAAAGTGATA-BHQ; pvl-lukS forward primer CACAAAATGCCAGTGTTATCCA, pvl-lukS reverse primer TTTGCAGCGTTTTGTTTTCG, pvl-lukS probe FAM-AGGTAACTTCAATCCAGAATT-TATTGGTGTCCTATC-BHQ-2; 16S forward primer TGATCCTGGCTCAGGATGA, 16S reverse primer TTCGCTCGACTTGCATGTA, 16S probe FAM-CGCTGGCGGCGTGCCTA-BHQ; agrA forward primer CTACAAAGTTGCAGCGATGGA, agrA reverse primer TGGGCAATGAGTCTGTGAGA, agrA probe FAM-AGAAACTGCACATACACGCT-BHQ; agrC forward primer AAGATGACATGCCTGGCCTA, agrC reverse primer TGTGCACGTAAAATTTTCGCAG, agrC probe FAM- TGGTATCGAGAATCTTAAAGTACGTG-BHQ; and set7 forward primer ACGGAAAAACCAGTTCATGC, set7 reverse primer GCTTATCTTTGCCAATTAAAGCA, set7 probe FAM-CAGGTTATATCAGTTTCATTCAACCA-BHQ. For S. epidermidis: 16S forward primer TACACACCGCCCGTCACA, 16S reverse primer CTTCGACGGCTAGCTCCAAAT, 16S probe FAM-CACCCGAAGCCGGTGGAGTAACC-BHQ; and RNAIII forward primer ACTAAATCACCGATTGTAGAAATGATATCT, RNAIII reverse primer ATTTGCTTAATCTAGTCGAGTGAATGTTA, RNAIII probe FAM-ATTTGCTTAATCTAGTCGAGTGAATGTTA-BHQ.Quantitative RT-PCR was carried out for transcripts of interest relative to 16S RNA using a probe-based assay as described with minor modifications −1) or vehicle control. The cultures were washed by centrifugation and the pellet resuspended in PBS supplemented with 1% BSA. The samples were set to an OD600 of 0.4 and an aliquot was heat killed (90°C for 10 minutes) to serve as a positive control. Samples (50 µl) were mixed with 1 ml PBS/1% BSA containing propidium iodide. Membrane damage was determined by measuring bacterial fluorescence by flow cytometry (Accuri C6).Membrane integrity was measured as described using propidium iodide 2 to red in the cytosol of bacteria with higher membrane potential. The proton ionophore CCCP was used as a positive control for disrupting membrane potential. LAC was cultured with 50 nM AIP1 for 5 hr in TSB in the presence of savirin (5 µg ml−1) or vehicle control. After diluting into TSB, the bacteria were incubated with 30 µM DiOC2 in the dark for 16 min prior to analyzing by flow cytometry . Measurements from both the red and green channels were taken and data presented as a ratio of red channel divided by the green channel to reflect the shift to greater change in membrane potential.Membrane potential was measured using the BacLight Membrane Potential Kit (Molecular Probes) following the manufacturer's recommendations. Membrane potential in this assay is based on the shift between the green fluorescence of DiOCS. aureus AgrAc (residues 137–238 with an initiator methionine) deposited in the Protein Data Bank (PDB) accession number 3BS1 http://www.swissdock.ch) S. epidermidis AgrA DNA binding domain was prepared by threading the amino acid sequence (UniProt database accession number Q84FX9) onto the structural coordinates of the S. aureus protein (PDB 3BS1) using the I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). Savirin docking to S. epidermidis AgrAc was performed as described above for S. aureus AgrAc with the origin set to the Cα atom of Phe229. Structural images were generated using PyMOL .Savirin (PubChem ID SMR000016143) was docked onto the C-terminal domain of AgrA of E. coli expressing the 6X-histidine tagged C-terminal DNA binding domain of AgrA (AgrAC) from S. aureus isolate Newman was provided by Dr. Chuan He . Expression and purification of AgrAC was carried out as previously described with minor modifications C expressing E. coli were grown in Terrific broth to an OD600 of 0.6 and induced with 1 mM isopropyl β-D-1 thiogalactopyranoside overnight at room temperature. Harvested cells were flash frozen in liquid nitrogen, thawed and lysed using lysozyme and sonication. Soluble AgrAC was affinity purified using Talon Superflow Metal Affinity Resin (Clonetech) followed by gel filtration on a Superdex S200 column . Tris (2-carboxyethyl) phosphine (TCEP) at 1 mM was used as a reducing agent throughout purification. Protein was stored at −80°C in PBS, 20% glyercol, 5 mM DTT, 1 mM TCEP and 1 mM MgCl2. Electrophoretic mobility shift assays (EMSA) using purified AgrAC (2 µM) were performed as described agr P2 and P3 C, DNA probe, vehicle and/or savirin (5–160 µg ml−1 or 13.5–432 µM) were loaded in Tris-acetate-EDTA (TAE) buffer containing 10 mM dithiothreitol. Assays including the 16 bp probe were run with 10% native polyacrylamide gels.lux reporter strain, AH3048, was generated by transforming S. aureus Δagr strain ROJ48 agrA gene cloned into plasmid pEPSA5, which placed transcription of agrA under the control of the xylose-inducible Tx5 promoter. To construct plasmid pCM63, the agrA gene was PCR amplified from AH1263 genomic DNA using primers CML609 (5′-GTTGTTGAATTCCCATAAGGATGTGAATG-3′) and CLM610 (5′-GTTGTTTCTAGACTTATTATATTTTTTTAACGTTTCTCACCG-3′), the PCR product was digested with EcoRI and XbaI, and ligated into similarly digested pEPSA5. Preliminary experiments demonstrated that light production by AH3048 increased in a xylose dose-dependent fashion, without impacting growth, up to a xylose concentration of 0.25%. For testing the impact of savirin on light production, AH3048 cultures were not induced with xylose because the constitutive level of agrA transcription from pCM63 was sufficient for luminescence induction. An overnight culture of AH3048 grown in TSB with 10 µg ml−1 chloramphenicol (for plasmid maintenance) was used to inoculate (at 1∶500 dilution) TSB containing antibiotic in 96-well microtiter plates (Costar 3603) at 200 µl per well. A 2-fold serial dilution series of savirin (0.4–6.3 µM or 0.29–2.33 µg ml−1) was used and the concentrations were tested in quadruplicate. Microtiter plates were incubated at 37°C with shaking (1000 rpm) in a Stuart SI505 incubator  with a humidified chamber. Luminescence and OD600 readings were recorded at 30 min increments using a Tecan Systems  Infinite M200 plate reader. Maximal light production occurred after 6 hrs of growth. As a specificity control, a 2-fold dilution series (0.5 nM to 1000 nM) of AIP-2  was tested in quadruplicate, as well as 12 control wells containing vehicle (DMSO). As positive controls, two compounds demonstrated by others to inhibit AgrAc in EMSA assays, diflunisal and 4-phenoxyphenol (Sigma) An AgrA-dependent agr mutant in the presence or absence of savirin (5 µg ml−1), the bacteria were grown for 5 hr in TSB with 50 nM AIP1 or an equivalent amount of DMSO as the vehicle control and processed for microarray analysis as described agr vehicle vs. Δagr savirin, and LAC vehicle vs. Δagr vehicle, n = 3. The bacterial RNA was purified as described http://www.ncbi.nlm.nih.gov/projects/geo/) under the accession number GSE52978. All microarray data are MIAME compliant.To compare the transcript levels of LAC and the Δ600 after bacterial strains were grown overnight in TSB in the presence or absence of savirin (5 µg ml−1). The assay was performed using rabbit erythrocyte lysis as described 50.Alpha hemolysin activity was measured in 0.45 µm filtered cultured supernatant standardized by ODagr group I clinical isolate #32 generated with either savirin (5 ug ml−1) or DMSO vehicle control was 0.45 µm filtered, stored at −80°C, and thawed on ice. Human neutrophils (AllCells) were washed twice in saline to remove EDTA, suspended in RPMI with 10 mM HEPES and 1% HSA, and assessed for viability by Trypan blue staining (>97%). The experiment was run in triplicate and each tube contained 3×106 neutrophils in 100 µl RPMI to which was added 100 µl of either RPMI, TSB diluted 1∶5 or 1∶10 in RPMI, or treated supernatants diluted 1∶5 or 1∶10 in RPMI. PBS with 0.1% Triton-X100 (100 µl) was used for 100% lysis. Tubes were incubated at 37°C in a 5% CO2 incubator for 1 and 2 hours. At each time, the tubes were centrifuged at 13,000 rpm, at 4°C, for 5 minutes. Cell free supernatant (100 µl) was transferred to a micotiter plate and immediately processed for LDH according to the Cytotoxicity Detection Kit (Roche). A blank was created for each plate with 10%TSB in RPMI. The data are depicted as the percentage of total lysis after correction for LDH release stimulated by media alone.The ability of secreted toxins to lyse human neutrophils was determined by LDH release. Overnight supernatant from MRSA in vivo experiments, savirin was solubilized at 1 mg ml−1 in 0.5% hydroxypropyl methylcellulose (Sigma) in endotoxin-free sterile water made 3.0 mM in NaOH with cell culture tested 1 N NaOH (Sigma), and put through a 0.22 µM filter (Millex-GV). The vehicle control was the HPMC used to solubilize the savirin. Sample sizes were determined by preliminary experiments to determine the number of mice required to observe significance. Dermonecrosis model: SKH1 hairless immunocompetent mice  were obtained from Charles Rivers . At Day 0, early exponential phase bacteria (4×107) washed in sterile normal saline were injected concurrently with savirin (5 µg) or vehicle in 50 µl subcutaneously into the flank using a 3/10 cc insulin syringe with a 28 ½ gauge needle (Becton Dickinson). For delayed delivery, 10 µg savirin was administered 24 and 48 hr after infection in 50 µl. The animals were divided into two groups to have equivalent mean abscess sizes prior to administering drug or vehicle. Abscess area  and ulcer area  were measured with calipers as described 2) was calculated from the equation (π/2)[(length of the abscess)×(width of the abscess)]. The flat ulcer area (mm2) was calculated from the equation (length of the ulcer)×(width of the ulcer) or alternatively from digital images using Adobe Photoshop standardized to a micrometer with equivalent results. On Day 3 or Day 7, the mice were euthanized using isoflurane inhalation. The abscess/ulcer area was excised (1.5 cm2) and the spleens removed. Tissues and spleens were placed in 1 ml of HBSS/0.1% HSA in a bead-beating tube containing sterile 2.3 mm beads (Biospec) and were processed for bacterial CFU by homogenizing the spleens in a bead beater, diluting all samples 1∶10 in 1 ml PBS/0.1%Triton, sonicating, and plating serial dilutions on blood agar as described Airpouch model: age matched −/−Nox2 male mice (Jackson Labs) or C57BL/6 male mice (Charles Rivers) were infected with either 2×107 early exponential phase non-fluorescent AH1677 bacteria (−/−Nox2) or 5×107 LAC Δagr (C57BL/6) into an air pouch generated by the injection of 5 ml of air subcutaneously as described For all agr LAC (2×107/ml TSB) were incubated for 5 hr at 37°C with shaking (200 rpm) in the presence of savirin (5 µg ml−1) or vehicle control. Bacteria were opsonized (1×108/ml) with rabbit IgG anti-Staphylcoccus aureus (Accurate Antibody YVS6881) (100 µg/ml) in phenol red-free Dulbecco's Modified Eagle Media, DMEM, containing 4.5 g/L D-glucose/2%Hepes+1% FCS). The experiment was performed in triplicate. Murine macrophage RAW264 cells (5×106) in 250 µl of DMEM+2% FCS were combined with 5×106 opsonized bacteria in 250 µl of DMEM+1% FCS (MOI 1∶1) in sterile polystyrene 12×75 mm tubes, centrifuged briefly to initiate contact, and incubated for 1 hr at 37°C in 5% CO2. The infected cells were treated with lysostaphin (Sigma) (2 µg/ml for 15 min) to kill extracellular bacteria and then washed and suspended in fresh media. Half of the samples were incubated for an additional 4 hrs. To determine the intracellular CFU at 1 and 5 hr, the relevant cells were centrifuged, suspended in PBS/0.1% Triton-X-100 and sonicated to disrupt cells and dilutions plated on blood agar. The cell line was tested for Mycoplasma sp. contamination by PCR (Life Technologies).Early exponential phase LAC+50 nM synthetic AIP1 or Δagr LAC  were incubated for 5 hr at 37°C with shaking (200 rpm) in the presence of savirin (5 µg ml−1) or vehicle control. Bacteria were centrifuged, washed, resuspended in DMEM/2%Hepes acidified with either HCl to pH 2.5 or 10 µg ml−1 linoleic acid (Sigma) and incubated for the indicated times. Dilutions were plated on sheep blood agar to determine the residual viability.Early exponential phase LAC+50 nM synthetic AIP1 or Δ7) were injected simultaneously with erythromycin (Sigma) (16 µg) and clindamycin (Sigma) (0.12 µg) or savirin (5 µg) into the flank of a SKH1 mouse as described above. At 24 hr, weight loss was recorded and the mouse was euthanized using isoflurane inhalation. The abscess area was measured, excised, and placed in 1 ml of HBSS/0.1% HSA in a bead-beating tube and processed as described above. Serial dilutions of the bacteria from the abscess were plated on blood agar plates and were incubated at 37°C for 24 hr. Colonies were lifted from the blood agar plates and sonicated in saline until a desired OD600 was reached. These bacteria were injected simultaneously with erythromycin (16 µg) and clindamycin (0.12 µg) or savirin (5 µg) into the flank of the next SKH1 mouse. The procedure was repeated through ten mice total for each treatment. After the tenth mouse in each group was euthanized, the bacteria taken from the plate were concentrated and stored in TSB/10% glycerol at −80°C for subsequent analysis.The induction of resistance to clindamycin in LAC served as a positive control and is based on the presence of ermC (SAUSA300 pUSA 030007) which confers erythromycin and clindamycin resistance 6 ml−1) were incubated in 5 ml TSB with the addition of erythromycin (16 µg ml−1) and clindamycin (0.12 µg ml−1) or savirin (5 µg ml−1) at 37°C with shaking (200 rpm). After 24 hr, the bacteria were washed by centrifugation and diluted into 5 ml fresh TSB and new addition of drug or savirin was added to the respective tubes for a total of ten days. The passaged bacteria exposed to erythromycin and clindamycin either in vivo or in vitro and non-passaged bacteria (1×106 in 5 ml TSB) were incubated overnight (18 hr) with erythromycin (16 µg ml−1) and increasing doses of clindamycin (0.06–1.0 µg ml−1) and plated to determine viability. Either in vivo or in vitro passaged bacteria exposed to savirin and non-passaged bacteria (2×107 ml−1 TSB) were evaluated for the ability to respond to savirin by incubation in 1 ml cultures with 50 nM synthetic AIP1 in the presence of additional savirin (1–5 µg ml−1) for 2 hr at 37°C with shaking (200 rpm). Expression of RNAIII was determined by qRT-PCR as described above.early exponential phase LAC  (A) at 3 hr of incubation compared to (B) log CFU at 3 hr from a starting CFU of 2×107/ml. Mean ± SEM, n = 5. ***p<0.001 by two-tailed Student's t-test.Effect of savirin on (TIF)Click here for additional data file.Figure S2agr::P3 promoter activation in all 4 agr alleles. Effect of 5 µg ml−1 savirin vs vehicle on agr::P3 promoter activation in an agr I strain (AH1677), an agr II strain (AH430), an agr III strain (AH1747), and an agr IV strain (AH1872) after incubation for 14 hr. Data are represented as mean fluorescence units of total S. aureus ± SEM, n = 3. *** p<0.001 by two-tailed Student's t-test.Savirin inhibits (TIF)Click here for additional data file.Figure S3S. aureus and S. epidermidis in bulk cultures. (A) S. aureus and (B) S. epidermidis were incubated with savirin (5 µg ml−1) (red line) vs. vehicle (blue line) in 5 ml cultures with shaking for the indicated times. The cultures were diluted 1∶2 and 1∶4 before reading at OD600 to ensure that the readings were within the linear range of the spectrophotometer. Mean ± SEM, n = triplicates of a representative experiment. ***p<0.001 **p<0.01, *p<0.05 by two-tailed Student's t-test.Effect of savirin on growth of (TIF)Click here for additional data file.Figure S4A) Membrane integrity measured as propidium iodide uptake by LAC agr+ cultured overnight with savirin (5 µg ml−1) vs. vehicle control. Heat killed LAC was a positive control for the assay. Mean ± SEM, n = 3. (B) Membrane potential measured as a shift in fluorescence (DiOC2) of LAC plus 50 nM AIP1 cultured for 5 hr with savirin (5 µg ml−1) vs. vehicle control. CCCP-treated LAC was a positive control for the assay and demonstrated collapse of membrane potential. Mean ± SEM., n = 3. ***p<0.001 **p<0.01, *p<0.05 by two-tailed Student's t-test.Savirin does not affect membrane potential or membrane integrity. ((TIF)Click here for additional data file.Figure S5agrA reporter activation in an agr null strain expressing a plasmid for agrA where agr::P3 drives luminescence, AH3048, after 6 hr of growth. AIP2 as an inhibitor of non-agrII AgrC signaling was used as a specificity control. Viability is represented as OD600. Data are represented as the mean ± SEM of quadruplicates of a representative experiment.Effect of diflunisal and 4-phenoxyphenol on AgrA promoter activation. Effect of increasing concentrations of diflunisal and 4-phenoxyphenol (1.56–100 µM) vs vehicle on (TIF)Click here for additional data file.Figure S6agr-dependent virulence factor production in clinical isolates. Effect of savirin (5 µg ml−1) vs vehicle on overnight alpha-hemolysin production (measured as HA50) by current clinical isolates that represent MRSA and MSSA from multiple different sites of infection and of agr alleles I–III.Savirin inhibits (TIF)Click here for additional data file.Figure S7agr+ vs Δagr LAC for clearance from the skin. Hairless SKH1 mice were infected with 4×107 bacteria and the abscess CFU determined at the indicated times. Data are represented as mean ± SEM, n = 4 mice per group. *p<0.05, **p<0.01 by two-tailed Mann-Whitney U test.Comparison of USA300 LAC (TIF)Click here for additional data file.Table S1Changes in the USA300 LAC transcriptome by microarray.(DOCX)Click here for additional data file.Table S2agr+ and Δagr.Transcripts upregulated by savirin in LAC (DOC)Click here for additional data file.


In chronic kidney disease, quality of life is adversely affected. We hypothesized that biomarkers of renal function could also be determinants of subjective well-being (SWB) in Swedish elderly subjects. SWB was assessed by the Psychological General Well-Being index (PGWB index) in two study groups: Active seniors (AS) consisted of community-dwelling elderly Swedes leading an active life (2 (AS vs DGM). In the AS cohort cystatin C was negatively related to PGWB index in women , and the covariates age and BMI did not improve the models. The renal biomarkers were unrelated to the PGWB index in the DGM cohort. Cystatin C in the AS cohort was adversely related to the PGWB subdimensions anxiety, depressed mood, positive well-being, and vitality in women, but in men only to depressed mood . In the DGM cohort, depressed mood in men was also significantly related to cystatin C (P = 0.050), but not in women.There were no significant differences in cystatin C and eGFR values between the two cohorts: cystatin C medians 0.88 vs 0.86 mg/L and eGFR 73 vs 80 mL/min/1.73 mRenal function even within the normal range, measured by serum cystatin C concentration, has significant and sex specific associations with subjective well-being and its subdimensions in healthy elderly subjects. Maintenance of good renal function in aging may be of importance in maintaining a high subjective well-being. While it is well known that in more pronounced renal dysfunction , quality of life (QoL) is adversely affected [During our whole life span, factors that influence health and functioning are accumulated; these factors include genetics, environment, and individual life experience and exposures . Moderataffected –10 thereaffected . In partS-creatinine and S-cystatin C are commonly used as biomarkers to calculate eGFR. Among the disadvantages of C-creatinine as a GRF marker are its dependence on muscle mass, age, race and sex of the subjects , tubularWe hypothesized that a possible negative effect of kidney function on SWB could contribute substantially to the total variance in SWB in an elderly population even in the absence of CKD. The aim of this study was thus to look for possible associations between established biomarkers of renal function  and subjective well-being in a sample of 689 Swedish elderly subjects.Active Seniors (AS) cohort was recruited during 2003 and 2004 by a multi-phase sampling procedure aimed at an elderly retired population living in various communities in Central Sweden. The locations for the recruitment were selected to represent a broad range of socioeconomic levels and included rural as well as urban and suburban areas. The sample consisted of 389 senior citizens and was recruited from several retired persons’ organisations, which implicates that they are independent and socially active. Being retired, living independently in their own homes in addition to participation in such organisations were the sole inclusion criteria, not pre-set health criteria. We have designated them as ‘Active Seniors’ in contrast to elderly persons that do not engage themselves in such social activity. All were Caucasians, most of them born in the 1920’s and 1930’s, mean age at sampling was 74 ± 5 years for both sexes and the sex ratio M/F was 127/262. Based upon the cystatin C values in different age groups   and the Senior subjects with subjective or objective cognitive complaints (DGM cohort) were recruited from an incident case study at the University Hospital, Örebro Sweden [DSM-IV criteria ICD-10 criteria was used to divide dementia patients into different diagnostic categories. Mixed dementia (F 00.2 according to ICD-10) was diagnosed in cases of coexistence of AD and a history of vascular risk factors e.g. transitory ischemic attack(s). atrial fibrillation, hypertension, diabetes mellitus, Hachinski ischemic score ≥ 6 and CT scan showing ischemic changes. Probable AD was diagnosed in accordance with the NINCDS-ADRDA criteria. Of the 73 subjects classified as ND, 39 were MCI. Everyone in the study group completed the PGWB questionnaire without assistance during their visit at the Memory unit. Based upon the cystatin C values in different age groups  approved the study. Height and weight  were measured and BMI (kg/m2) was calculated. The resting systolic and diastolic blood pressure was measured using an automatic oscillometric method . The equipment has been validated.http://www.mapi-trust.fr, was used to measure subjective well-being or distress [The Psychological General Well-Being (PGWB) index, accessible from MAPI Research institute, distress . It consBlood samples were taken with the subjects in the supine position, by venipuncture using vacuum tubes with gel, Becton Dickison. Serum was obtained after clotting for 30 to 60 minutes at room temperature and centrifuging at room temperature for 10 minutes at 2000 g. All samples were stored at -80°C. The serum samples were analysed on a Hitachi 911 multianalyser, Roche, Mannheim, FRG. Creatinine was analysed using a enzymatic method, Crea plus, from Roche/Boehringer Mannheim FRG, and the calibrator was IDMS standardised. Albumin, IgG, IgA and IgM were analysed by immunoturbidimetry using reagents from DAKO, Copenhagen, Denmark and cystatin C by immunoparticle turbidimetry from Gentian AS, Moss, Norway. LDL and HDL cholesterol were measured by direct, homogeneous assays based on detergent treatment of the serum. N-geneous™ HDL-c and N-geneous™ LDL reagents, respectively, from Genzyme Corporation, Cambridge, MA, USA.Estimated glomerular filtration rate (eGFR) was calculated using the equation which include both creatinine and cystatin C from the Chronic Kidney Disease Epidemiology collaboration. This equation includes both creatinine and cystatin C as well as age, sex and race . In the All variables were checked for normality of distribution and collinearity before analysis. Continuous variables with a skewed distribution were transformed using natural logarithms before regression analyses were made. The Mann-Whitney rank sum test was used to compare the PGWB sum and PGWB sub-dimension between gender in the two cohorts, median and interquartile range for descriptive statistics of these variables.A forward multiple regression was used to determine the degree to which variance in PGWB index was explained by age, BMI, gender and, cystatin C or creatinin. To examine which of the subdimensions contributed to the variance in PGWB index ANCOVA analyses were done with age, BMI, and biomarkers of kidney function as covariates. All multivariate regression models were performed after stratification, since we have found profound differences between SWB in the AS and DGM cohorts .Statistical analyses were performed using SPSS version 15.0 , and PGWB Statistical significance were considered with a probability value <0.05.In the AS study group, 4 subjects failed to complete the PGWB questionnaire and responses from 12 subjects failed to answer one or two questions randomly distributed among the 22 questions. The missing answers were imputed using the manual from MAPI research institute.2 was similar, 22%, in both the AS and DGM cohorts. A cystatin C concentration >1.1 mg/mL was found in 16% of subjects in the AS cohort and 22% in the DGM cohort.Baseline characteristics of the two study groups are shown in Table There were group differences in SWB, assessed by the PGWB instrument, between the two cohorts, see Table P = 0.06). Just as in women, addition of age and BMI as covariates did not change this pattern; cystatin C retained its β value around the same numerical level (-10.7), again showing borderline significance (P = 0.07).In the AS cohort, cystatin C was significantly and negatively related to PGWB index in women, explaining ≈ 5% of the variance in SWB, as seen in Table P = 0.02) but explained only ≈ 2% of its variance. Adding the covariates age and BMI increased the explanatory power of the model to ≈ 6%. In men, creatinine was unrelated to PGWB index, and adding age and BMI as covariates did not improve the insignificant R2 . In the DGM cohort too, cystatin C in men was significantly and adversely correlated with depressed mood (P = 0.050), although the variance explained was minor (≈ 1%). In the AS cohort, there was also an adverse relation between positive well-being and cystatin C (P = 0.03), explaining ≈ 2.5% of that subdimension.In men, in the AS cohort there was a significantly adverse relation between cystatin C and depressed mood, which explained ≈ 6% of its variance , two cohorts of Swedish elderly subjects with a low prevalence of chronic kidney disease (CKD) as judged from their serum cystatin C concentrations.There were marked gender differences in the adverse relation between cystatin C and the subdimensions of SWB. In women in the AS cohort all subdimensions except self control were adversely related to cystatin C, whereas in the DGM cohort cystatin C was unrelated to any SWB subdimension. , or on hemodialysis, with QoL as outcome variable, not SWB. Health Related Quality of Life (HRQoL) has been found to be reduced in proportion to the severity of CKD , 23. OurIn the Health, Aging and Composition study, cystatin C was not associated with depression at baseline but participants having cystatin C higher than 1.25 mg/L had an increased risk to develop diagnosed depression . In the SWB includes a person’s evaluation of his or hers life, both positive and negative feelings as well as perceptions of one’s situation. In our model cystatin C explained 6% of the SWB variance which is regarded as clinically relevant. SWB has been associated with longevity –28. ChidSWB differs between the sexes , 32 as aThere are some limitations in our study. SWB is a function of many variables such as socioeconomic status, marital status, physical health, life expectations –37, and Renal function even within the normal range, assessed by serum cystatin C concentration, has significant and sex specific associations with SWB and its subdimensions. Maintenance of good renal function in aging may therefore be of importance not only in preventing somatic deterioration in old age but also in maintaining a good subjective well-being.


Posterior vertebral column resection (PVCR) is an effective alternative for treating rigid and severe spinal deformities. Accurate placement of pedicle screws, especially apically, is crucial. As morphologic evaluations of thoracic pedicles have not provided objective criteria, we propose a thoracic pedicle classification for treating rigid and severe spinal deformities.et al.  using a morphological method.A consecutive series of 56 patients with severe and rigid spinal deformities who underwent PVCR at a single institution were reviewed retrospectively. Altogether, 1098 screws were inserted into thoracic pedicles at T2-T12. Based on the inner cortical width of the thoracic pedicles, the patients were divided into four groups: group 1 (0–1.0 mm), group 2 (1.1–2.0 mm), group 3 (2.1–3.0 mm), group 4 (≥3.1 mm). The proportion of screws accurately inserted in thoracic pedicles for each group was calculated. Statistical analysis was also performed regarding types of thoracic pedicles classified by Lenke P < 0.008) except groups 3 and 4 (P > 0.008), which were then combined. The accuracies for the three new groups were 35.05%, 65.34%, and 88.32%, respectively, with statistically significant differences between the groups (P < 0.017). Rates of screws inserted in thoracic pedicles classified by Lenke et al.  were 82.31%, 83.40%, 80.00%, and 30.28% for types A, B, C, and D, respectively. There was no statistically significant difference (P > 0.008) between these types except between type D and the other three types (P < 0.008).There were statistically significant differences in the rates of screws inserted in thoracic pedicles between the groups  contains supplementary material, which is available to authorized users. The combined use of posterior vertebral column resection (PVCR) and pedicle instrumentation has allowed greater deformity correction and overall spinal balance than are achieved with conventional approaches. In recent years, reports on the clinical use of this technique have gradually increased. In particular, PVCR may be the best procedure for correcting severe (Cobb angle >100°), angular, and rigid (flexibility <10%) spinal deformities with significant decompensation in the coronal or sagittal plane. Studies have indicated, however, that severe spinal deformities are often accompanied by congenital or secondary pedicle deformities that might be associated with a high risk of malpositioned pedicle screws and vascular or neurological complications [When PVCR is performed to treat severe spinal deformities, the most crucial step is to insert the screw within the pedicle. Factors affecting the accuracy of thoracic pedicle screw insertion have been well studied. However, most studies focused on the morphology of the pedicles –5, whichThe present study included a consecutive series of 56 patients with severe and rigid spinal deformities undergoing PVCR at a single institution between October 2004 and July 2010. A total of 1098 thoracic pedicles were studied, statistically analysed, and classified to demonstrate the clinical significance of treating severe and rigid spinal deformities with PVCR.A consecutive series of 56 patients with severe and rigid spinal deformities who underwent PVCR at a single institution between October 2004 and July 2010 were included in the study. The Scientific Committee of the Second Affiliated Hospital of Kunming Medical University, Yunnan Province, China gave academic, institutional, and ethical approval of the research protocol and procedures. All participants were provided with full information about the study’s procedures and aims and gave informed consent to participate in the study, according to the principles of medical ethics of the World Health Organization and the Helsinki Declaration. All patients gave informed consent for their radiological images and information to be published. There were 20 male and 36 female patients with an average age of 21.5 years (range 10–48 years) at the time of the surgeries. The patients had a main curve with average Cobb angles of 104.6° ± 35.9° and 94.8° ± 27.7° in the coronal and sagittal planes, respectively. One surgeon performed all of the operations. The screws were inserted with the free-hand technique using a 2-mm hand drill. A total of 1098 thoracic pedicle screws were inserted at T2-T12. The average follow-up was 48 months.The patients underwent standing anteroposterior and lateral radiography before and after the surgery. The Cobb angle of the curve was measured to evaluate spinal deformities in the coronal and sagittal planes. All thoracic pedicles to be instrumented were scanned preoperatively using Philips 256 computed tomography (CT) equipment . Volume rendering (VR) and multiplanar reconstruction (MPR) were also performed. The inner cortical width of thoracic pedicles was measured horizontally at the narrowest point of each thoracic pedicle. Postoperatively, axial CT imaging and reconstruction of the thoracic pedicles with a thickness of 0.8 mm was performed parallel to the axis of the inserted screw. A senior doctor evaluated the accuracy of the thoracic pedicle screws’ placement. Penetration of the internal and external cortical walls of the thoracic pedicles was measured in millimetres and classified into three degrees: 0 (no penetration); 1 ; 2 . The accuracy of thoracic pedicle screw placement was confirmed using the following criteria: (1) Pedicle screw trajectory during the surgery was accomplished with a 2-mm hand drill. A 1-mm self-made pedicle trajectory probe was used to confirm that the five walls of the channel were intact. (2) Postoperative CT scanning of the instrumented thoracic pedicles was performed to confirm that the axis of each screw was completely within the pedicle. Degree 0 and 1 penetration of the thoracic pedicle indicated accurate placement of the screw. (3) No complications relevant to thoracic pedicle screws occurred postoperatively, such as nerve root irritation or spinal cord complications.2 test was used for comparison of the accuracy of thoracic pedicle screws between the groups. Statistical analysis was performed using SPSS software . Statistical significance was defined as P < 0.05 for overall comparisons and as P < 0.008 for comparisons between the four groups.The inner cortical width of the thoracic pedicles was measured in millimeters, and the pedicles were then classified into four groups based on their inner cortical width: group 1 (0–1 mm), group 2 (1.1–2.0 mm), group 3 (2.1–3.0 mm), group 4 (≥3.1 mm). The placement accuracy of the thoracic pedicle screws in the groups was calculated. The χ2 test was again used to compare the accuracy of thoracic pedicle screw placement in these groups. Statistical analysis was performed using SPSS. Statistical significance was defined as P < 0.05 for overall comparisons and as P < 0.017 for comparisons between the three new groups. The accuracy of thoracic pedicle screw placement was also calculated based on the morphological classification of pedicles [2 test was used for comparison of the accuracy of thoracic pedicle screw placement among different morphological types. Statistical analysis was performed using SPSS. Statistical significance was defined as P < 0.05 for overall comparisons and as P < 0.008 for comparisons between different types.The four groups were then classified into three new groups: new group 1 (0–1 mm), new group 2 (1.1–2.0 mm), new group 3 (≥2.1 mm). The χpedicles , and theP < 0.008) between all of the groups except between groups 3 and 4 (P > 0.008)  were accurately instrumented with pedicle screws. Among the original four pedicle groups, 68 screws were accurately inserted into 194 thoracic pedicles in group 1 (0–1.0 mm inner thoracic pedicle width), 115 screws in 176 thoracic pedicles in group 2 (1.1–2.0 mm), 304 screws in 352 thoracic pedicles in group 3 (2.1–3.0 mm), and 339 screws in 376 thoracic pedicles in group 4 (≥3.0 mm). The accuracies of thoracic pedicle screw placement were 35.05%, 65.34%, 86.36%, and 90.16% in groups 1, 2, 3, and 4, respectively. Statistical analysis was performed to compare the accuracy of thoracic pedicle screw placement among these groups. The differences were statistically significant (P < 0.017) , 115 screws in 176 thoracic pedicles in new group 2 (1.1–2.0 mm), 643 screws in 728 thoracic pedicles in new group 3 (2.1–3.0 mm). The accuracies of thoracic pedicle screw placement were 35.05%, 65.34%, and 88.32% in new groups 1, 2, and 3, respectively. The accuracy was then compared among these new groups and the difference was found to be statistically significant among all of the new groups , except between type D and the other three types (P < 0.008) , etc.. These treatments to correct and fix spinal deformities have been well accepted by spine surgeons. However, for severe, rigid, and angular spinal deformities, it is not easy to obtain desirable correction results using these approaches.In recent years, some authors have reported the clinical use of posterior VCR (PVCR) for correcting spinal deformities in patients with spinal scoliosis or kyphosis , 8. In pMany factors have an impact on the accuracy and safety of thoracic pedicle screw placement. The rate of thoracic pedicle screw malpositioning during correction of spinal deformities is as high as 3.0–44.2%, and the rate complications related to pedicle screws is 0.9% –12. For The present study included a consecutive series of 56 patients with severe and rigid spinal deformities who underwent PVCR at a single institution between October 2004 and July 2010. A total of 1098 thoracic pedicles instrumented at T2-T12 were reviewed. We have proposed criteria for establishing a thoracic pedicle classification based on the inner cortical width of thoracic pedicles as determined on CT scans, and believe these criteria can be of clinical significance.When the accuracy of pedicle screw placement is evaluated, routine radiography is of little help to the surgeon to determine directly whether the pedicle has been penetrated. The accuracy of routine radiography for evaluating pedicle screw placement varies significantly, ranging from 10% to 83% . RoutineSome studies indicated that the gaps between thoracic pedicles of the upper and middle thoracic vertebrae and dural sac were small, whereas the gap between the pedicles of the lower thoracic vertebrae and dural sac are as large as 2 mm . TherefoWhether the inner or outer cortical width of thoracic pedicles should be measured when studying the morphology of thoracic pedicles has been debated , 17–19. et al.[Along with the pedicle classification method of Lenke et al., previouet al.[et al.[P < 0.008). The statistical data of the current study and our clinical experiences indicated that, among many other factors affecting the accuracy and safety of thoracic pedicle screws, the angulations of the spinal deformities in the coronal plane and the rotation of the vertebral body only indirectly affected the accuracy and safety of thoracic pedicle screw placement by increasing the difficulty of the surgeries. The only crucial factors that had a direct impact on the accuracy and safety of thoracic pedicle screw placement and safety were (1) whether the pedicle had a channel and (2) the inner cortical width of the thoracic pedicles.The study by Liljenqvist et al. noted thl.[et al., there wTherefore, we propose the following thoracic pedicle classification based on the inner cortical width of the thoracic pedicle on CT scans: type I thoracic pedicle, which has no channel and an inner cortical width of 0–1 mm; type II thoracic pedicle, which has a channel. Type II thoracic pedicles are then subclassified: type IIa, which has an inner cortical width of 1.1–2.0 mm, and type IIb, which has an inner cortical width of ≥2.1 mm. As the thoracic pedicle classification criteria have been quantified and the morphology of the thoracic pedicles is not taken into account, the consistency and reliance of this new thoracic pedicle classification method is an improvement. Comparisons on the accuracy of thoracic pedicle screws between the two classification methods showed that thoracic pedicle classification determined by the inner cortical width of the pedicle on CT images can help surgeons predict whether a screw could be accurately inserted into the thoracic pedicle—which was of clinical significance.Regarding the distribution of different types of thoracic pedicle, types I, IIa, and IIb thoracic pedicles accounted for 17.67%, 16.03%, and 66.30%, respectively, of the total thoracic pedicles studied. The accuracies of pedicle screw placement in types I, IIa, and IIb thoracic pedicles were 35.05%, 65.34%, and 88.32%, respectively. Evaluation of thoracic pedicles prior to or during PVCR procedures is of significant importance. Type IIb pedicles of the thoracic vertebrae are suitable for screw insertion, and screw placement can be attempted in type IIa pedicles. In contrast, the accuracy of thoracic pedicle screw placement in type I pedicles is extremely low, particularly when the thoracic pedicles are adjacent to apical vertebrae or are above or below vertebrae that are to be resected. In these cases, PVCR should be selected cautiously or completely abandoned.PVCR is an effective approach for treating severe spinal deformities. Whether the screw can be inserted into a morphologically abnormal pedicle is crucial for accurate pedicle screw placement. In the present study, we noted that the inner cortical width of the thoracic pedicles was the sole decisive factor for predicting the accuracy of thoracic pedicle screw placement. On that basis, we propose quantification classification criteria for thoracic pedicles that could help surgeons predict whether a screw could be inserted into a thoracic pedicle during PVCR, thus guiding the instrumentation of thoracic pedicles with the free-hand technique.The inner cortical width of thoracic pedicles was the sole factor crucial for predicting the accuracy of thoracic pedicle screw placement. We proposed a quantification classification method of thoracic pedicles based on determining the inner cortical width of pedicles using CT: Type I thoracic pedicle has no channel and an inner cortical width of the thoracic pedicle of 0–1 mm. Type II thoracic pedicle has a channel, which is subclassified into type IIa  and type IIb . The proposed thoracic pedicle classification method can help surgeons predict whether the screw can be inserted into a thoracic pedicle, thus guiding accurate thoracic pedicle instrumentation when PVCR is performed.