These particles are subsequently used as inorganic fillers (or reinforcement) for thermally cured epoxy polymers. Epoxy-titania composites are prepared via mechanical mixing of titania particles with liquid epoxy resin and subsequently curing the mixture with an aliphatic diamine. The amount of titania particles integrated into epoxy matrix is varied between 2.5 and 10.0 wt.% to investigate the effect of sub-micron titania particles on thermal and mechanical properties of epoxy-titania composites. These composites are characterized by X-ray photoelectron (XPS) spectroscopy, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), thermogravimetric (TG), and mechanical analyses. It is found that sub-micron titania particles significantly enhance the glass transition temperature (>6.7%), thermal oxidative stability (>12.0%), tensile strength (>21.8%), and Young's modulus (>16.8%) of epoxy polymers. Epoxy-titania composites with 5.0 wt.% sub-micron titania particles perform best at elevated temperatures as well as under high stress.This paper presents the synthesis and thermal and mechanical properties of epoxy-titania composites. First, submicron titania particles are prepared via surfactant-free sol-gel method using TiCl In past few years, major research efforts have been focused on the development of high performance composite materials based on organic polymers and inorganic particles with key emphasis on developing methods to better integrate (or disperse) inorganic fillers into organic polymers. Inorganic filler particles, for example, silica , zirconiTo date, the performance of titania fillers in different polymeric matrices especially in epoxy resins has not been fully explored. There are only a few reports describing the behavior of submicron titania epoxy composites. For instance, commercially obtained submicron titania particles size: ~190 nm) have been incorporated in epoxy resin to study mechanical properties of the resulting composite materials . It was 0 nm haveOur aim is to enhance thermal and mechanical properties of epoxy polymers using titania filler and to understand the behavior of these composites at elevated temperature and under high stress. Thus, the paper presents synthesis of epoxy-titania composites via direct blending  of the as-prepared, submicron titania particles with epoxy resin monomers and subsequently curing the mixtures with a flexible diamine hardener. These composites are synthesized in the form of thin films, that is, 0.8–1.0 mm thick, and contain different ratios (wt.%) of titania particles. Thermal and mechanical properties of epoxy-titania composites are determined and reported with reference to neat epoxy polymer.−1, Dow Chemicals), poly(oxypropylene) diamine , and titanium tetrachloride  were used as precursors for preparing epoxy polymer and titania particles, respectively. All chemicals were used as received without further purification.Diglycidy lether of bisphenol A  neat epoxy polymer as a reference material for performance comparison of (b) epoxy-titania composites with different ratios (wt.%) of titania particles. The detailed procedures to prepare neat epoxy polymer, titania particles, and epoxy-titania composites are given below.Neat epoxy polymer thin films were prepared by mixing equimolar quantities of DGEBA and POPDA for 1 hour at room temperature. The mixture was subsequently poured into PTFE molds especially designed to prepare polymer films with less than 1 mm thickness. The material was degassed to remove air bubbles and then cured in an oven at 100°C for 5 hours.4  was injected through the septum, while the mixture was being stirred continuously. The reaction mixture was stirred at 60°C for half an hour. Liquid ammonia  was subsequently injected dropwise through the septum to attain a pH of 8. The mixture was aged for 6 hours at room temperature with persistent magnetic stirring. The product (white precipitates) was washed first with water (5 mL) and then with acetone (5 mL). Solid particles were recovered by centrifugation at 6000 rpm. Finally, the product was dried under vacuum at 120°C to yield 0.2 g of titania particles. These particles were subsequently used to prepare epoxy-titania composites without further heat treatment . Thermal treatment of titania particles was avoided intentionally to leave a sufficient number of hydroxyl (–OH) groups on the surface of particles for potential weak interactions with the organic matrix.To a 100 mL two-neck round bottom flask fitted with a septum, a condenser, and a magnetic bar, 40 mL of ethanol and 5 mL of water were added. TiClTo a 25 mL Erlenmeyer flask, DGEBA (0.02 mol) and the calculated amounts of titania particles were added. The mixture was stirred mechanically for 1 hour to effectively disperse titania particles. Finally, stoichiometric amount of POPDA (0.01 mol) was added to the mixture, and stirring was continued for 1 hour. The mixture was casted in PTFE molds and subsequently cured at 100°C for 5 hours. The amount of titania particles integrated into the matrix polymer was varied between 0.3 and 1.1 g to prepare epoxy-titania composites with 2.5, 5.0, 7.5, and 10.0 wt.% titania loadings. The epoxy-titania composite samples were named as ET-2.5, ET-5, ET-7.5, and ET-10 composites, respectively, conferring to the amount of titania particles (in wt.%) dispersed into epoxy polymers.α source. The XPS survey spectra were acquired in fixed analyzer transmission mode with pass energy of 150 eV.Neat epoxy polymer and epoxy-titania composites are characterized with the help of X-Ray Photoelectron Thermo VG Theta Probe spectrometer, equipped with a microspot monochromatized Al KMicroscopic characterization of thin films was carried out on JEOL JSM-5910 scanning electron microscope (SEM) equipped with tungsten filament electron emitter at accelerating voltage of 30 kV.2) atmosphere. The instrument was ramped at 10°C min−1 from −80°C to 300°C.Differential scanning calorimetry (DSC) of composite samples was performed on PerkinElmer DSC 7 instrument by heating samples in a sealed Al pan under inert  of pyrolysis for neat polymer and epoxy-titania composites.Thermogravimetric (TG) analyses were performed on PerkinElmer TGA 7 instrument. The samples were heated from room temperature to 700°C at the rate of 20°C min Program —to calcu−1 [Stress-strain curves of composite films were obtained on Testometric Universal Testing Machine M350/500. The samples for mechanical analysis were vacuum dried at 60°C for 2 hours before tensile measurements to remove adsorbed moisture, if any. The tensile tests were performed at 25°C with a constant crosshead speed of 1 mm min−1 . All meas) and oxygen (O 1s) peaks are observed around 284.8 and 531.5 eV [s) peak around 399 eV is not clear due to very low percentage of nitrogen atoms in the polymer. In contrast, the XPS survey scan of ET-10 composite demonstrates the presence of titanium (Ti 2p) peak—a doublet around 459.2 eV [s and O 1s peaks . SEM image of titania particles demonstrates that these particles have good dispersity, and they are not severely agglomerated although no surfactant or surface modifier was used.SEM images of titania particles as well as epoxy-titania composites were obtained to study the morphology, size, and shape of titania particles and their distribution in the polymeric matrix. SEM images of titania particles are shown in Tg) of neat epoxy polymer as well as epoxy-titania composites. DSC was used to study curing behavior and glass transition temperature  can freely move at this stage. However, once all monomers have reacted to form a three-dimensional polymer network, the movement of polymeric chains is considerably hindered leading to an increase in Tg after curing.Tg of epoxy polymers. At first, in comparison with neat epoxy polymer, Tg of epoxy-titania composites increases slightly at low loadings of titania particles, for example, 2.5 and 5.0 wt.%, which suggests that the polymeric matrix and the titania filler interact favorably at low filler concentration leading to a structure with improved Tg. However, as given in Tg of epoxy-titania composites is observed at higher loadings of titania particles, that is, 7.5 and 10.0 wt.%. This may be attributed to the increased distance between polymeric chains and lower crosslink density. At higher concentration of submicron titania particles, bigger particles tend to force polymeric chains to be apart and reduce the cohesive energy density [Tg of epoxy-titania composites decreases at higher loadings of titania particles.DSC scans of epoxy-titania composites with different loadings of titania particles also reveal a variable shift in  density . This ph density . It can TG analyses of epoxy-titania composites are performed to investigate thermal degradation and high temperature stability of these composites with reference to neat epoxy polymer. TG analyses of neat polymer and composite samples demonstrate a substantial increase in thermal decomposition temperatures as compared to neat epoxy polymer. Td10 and Td50 corresponding, respectively, to 10% and 50% weight loss are significantly increased at higher loadings of titania particles. It is evident that epoxy-titania composite with 7.5 wt.% titania (ET-7.5) shows the highest degradation temperatures. At 10 wt.% titania loading, Td10 and Td50 are slightly decreased, which may be attributed to the most likely microscopic phase separation between epoxy polymer and titania particles due to reduced interphase interactions. The microscopic phase separation between organic and inorganic phases cannot be avoided, though it may be delayed to a higher loading of inorganic filler by improving interphase interactions [Thermal degradation temperatures such as initial decomposition temperature (IDT) and ractions . In our Tmax, is increased as a function of titania content, and epoxy-titania composite with 10 wt.% titania (ET-10) shows the highest Tmax values as compared to neat epoxy polymer and other epoxy-titania composites. The energy of activation (Ea) for pyrolysis, as computed through Battery Program [Nonetheless, the temperature at which the rate of weight loss is maximum, referred to as  Program , also in Program . Hence, The average char yields or residues at 700°C, as measured from the respective TG traces, also increase from 8% for neat polymer to 10.5% and 12.5% for ET-5 and ET-10 composites, respectively. However, this increment in char yield is relatively smaller compared to the amount of titania incorporated into the polymer. This may be due to the presence of amorphous titania particles, which loose surface –OH groups at elevated temperature leading to a reduction in the wt.% of titania particles at 700°C. In addition, as discussed earlier, the inclusion of submicron titania particles in the polymer forces the polymeric chains to be apart thus reducing the cohesive energy density and degree of crosslinking, which may also result in a higher rate of degradation of polymeric chains thus leaving behind lower amount of organic residue compared to neat polymer.Mechanical properties of neat epoxy polymer and epoxy titania composites were determined to investigate the effect of submicron titania particles on strength and stiffness of epoxy polymer. The performance of epoxy-titania composites is decreased at relatively higher concentrations of titania particles, that is, at 7.5 and 10.0 wt.% titania. Considering the microstructure of epoxy-titania composites, this behavior can be explained by understanding the initiation and propagation of microcracks during mechanical analysis. According to pertinent literature , 29, oncμm via surfactant-free sol-gel method. The inorganic particles were subsequently dispersed into epoxy polymer matrix to prepare epoxy-titania composites with different concentrations of submicron titania particles, that is, 2.5–10.0 wt.%. The resulting epoxy-titania composites were characterized by XPS and SEM. DSC and TG results revealed significant enhancements in the glass transition (Tg) and thermal degradation temperatures of epoxy-titania composites when compared to neat epoxy polymer. Mechanical analysis of composite films also exhibited a maximum of 17% increase in Young's modulus of epoxy-titania composites with 5 wt.% titania particles (ET-5) with reference to neat epoxy polymer. Albeit the results were not promising at relatively higher loadings of submicron titania particles, thermal and mechanical properties of epoxy-titania composites were effectively improved at 2.5–7.5 wt.% titania loadings. It was observed that ET-5 composite had superior properties and generally performed best.In conclusion, the paper presented synthesis of submicron titania particles with sizes in the range of 0.7–0.8


Beta-catenin is part of a protein complex associated with adherens junctions. When allowed to accumulate to sufficient levels in its dephosphorylated form, beta-catenin serves as a transcriptional co-activator associated with a number of signaling pathways, including steroid hormone signaling pathways.4) signaling and female reproductive physiology, conditional ablation of Ctnnb1 from the endometrial mesenchymal , but not epithelial, compartment was accomplished using the Amhr2-Cre mice. Experiments were conducted to assess the ability of mutant female mice to undergo pregnancy and pseudopregnancy by or through oil-induced decidualization. The ability of uteri from mutant female mice to respond to estrogen (E2) and P4 was also determined.To investigate the role of beta-catenin in progesterone (PCtnnb1 from the mesenchymal compartment of the uterus resulted in infertility stemming, in part, from complete failure of the uterus to decidualize. E2-stimulated epithelial cell mitosis and edematization were not altered in mutant uteri indicating that the mesenchyme is capable of responding to E2. However, exposure of ovariectomized mutant female mice to a combined E2 and P4 hormone regimen consistent with early pregnancy revealed that mesenchymal beta-catenin is essential for indirectly opposing E2-induced epithelial proliferation by P4 and in some mice resulted in development of endometrial metaplasia. Lastly, beta-catenin is also required for the induced expression of genes that are known to play a fundamental role in decidualization such as Ihh, Ptch1, Gli1 and Muc1Conditional deletion of 4 and beta-catenin signaling pathways. Second, they highlight an under appreciated role for the mesenchymal compartment in indirectly mediating P4 signaling to the epithelium, a process that intimately involves mesenchymal beta-catenin. Third, the technical feasibility of deleting genes in the mesenchymal compartment of the uterus in an effort to understand decidualization and post-natal interactions with the overlying epithelium has been demonstrated. It is concluded that beta-catenin plays an integral role in selective P4-directed epithelial-mesenchymal communication in both the estrous cycling and gravid uterus.Three salient points derive from these studies. First, the findings demonstrate a mechanistic linkage between the P Then 16 hrs after the final steroid hormone injection, female mice were treated with 50 mg/kg body weight 5-bromo-2’-deoxyuridine  for 2 hours prior to euthanasia and uterine dissection. Tissues were prepared for paraffin embedding and one section (6 μm thick) from three different regions of each uterus were used to assess BrdU incorporation immunohistochemically using a BrdU staining kit  per manufacturer’s instructions. The mean percentages of BrdU positive cells in the luminal epithelium (LE) and stroma were calculated by establishing a ratio of BrdU positive cells divided by the total number of cells within the luminal epithelium or subluminal stromal compartment.To assess cell proliferation in the individual epithelial and stromal compartments, control and mutant female mice were first ovariectomized at three to five weeks of age and allowed to clear endogenous ovarian-derived sex steroids for at least one week. Female mice then received Ep < 0.05.Each experiment was independently replicated a minimum of three times with different mice in each experiment. Data in graphs represent the mean ± SEM from replicated experiments. Assignment of mice to each experiment was made randomly. Raw data were analyzed with GraphPad PRISM software (version 4.0) for simple comparisons. Mean values were considered significantly different when Our initial investigation showed that total β-catenin protein was observed throughout the implantation site on day of pregnancy (DOP) 7.5 Figure A. The hoAmhr2) gene promoter-driven Cre recombinase gene [Ctnnb1 when mated with mice harboring floxed Ctnnb1 alleles [Amhr2Cre/+;Rosa-EYFP). As anticipated, EYFP was only observed in stromal cells of the endometrium from Amhr2Cre/+;Rosa-EYFP female mice on postnatal day 13, DOP 7 and DOPP 7, while vascular endothelial  and epithelial cells were devoid of EYFP fluorescence  revealed that uteri from both Ctnnb1flox/flox control and Amhr2Cre/+; Ctnnb1d/d mice become edematous, a normal physiological response to E2. A more subtle response to E2 was observed in Amhr2Cre/+;Ctnnb1d/d uteri, but this likely stems from reduced early postnatal growth of uterine mesenchyme rather than from a lack of an E2 signaling cascade. This can be seen when comparing uteri from Ctnnb1flox/flox and Amhr2Cre/+;Ctnnb1d/d mice exposed to vehicle  in control and mutant uteri  approach and it was again determined that β-catenin-deficiency within the stromal compartment did not compromise epithelial cell proliferation , was markedly reduced in mutant uteri  of the mutant uteri , Cdkl1 (cyclin-dependent kinase-like 1), and Ldb2 (LIM domain binding 2) were shown to be down-regulated by P4 treatment in vivo[Ctnnb1flox/flox and mutant Amhr2Cre/+;Ctnnb1d/d uteri in vivo (data not shown). Conversely, S100a6 , Irg-1 (immune responsive gene-1) and Fst (follistatin), three genes shown to be up-regulated by P4[Ctnnb1flox/flox and Amhr2Cre/+;Ctnnb1d/d uteri (data not shown). Fourth, indifferent stromal cell proliferation was observed in response to a hormone regimen consistent with early pregnancy. This suggests that the proliferative stromal cell response to P4 is not dependent upon β-catenin. In sum, these findings indicate that β-catenin deficiency in the stromal compartment of Amhr2Cre/+;Ctnnb1d/d uteri results in aberrant gene expression of a specific cassette of P4-dependent genes, several of which belong to the IHH signaling cascade, but that other P4 responses are normal.Uteri from t in vivo. Based oted by P4, were eqTcf/Lef-LacZ transgenic mice [in situ. Based on this model, β-catenin activity was observed in the luminal epithelium and circular smooth muscle, an event that required the presence of an embryo. It was concluded that β-catenin was no longer active by late DOP5. However, β-catenin activity was defined by its ability to activate the Tcf/Lef-LacZ transgene, and β-catenin function was not addressed using deletional analysis . Additionally, while we and others [i.e., dephosphorylated and nuclear) β-catenin in decidualizing stromal cells, Mohamed et al. were unable to detect transcriptional activity for the TCF/LEF complex in the stromal compartment, suggesting β-catenin may regulate gene expression within the stromal compartment by a TCF/LEF-independent mechanism.Two functional studies were previously published on β-catenin in the uterus. In the first, β-catenin activity was indirectly assessed through the use of nic mice . Here, βd others  have sinet al. used the Pgr-Cre transgenic mouse line to delete β-catenin from PGR-expressing tissues, including all compartments of the uterus [2-induced morphological defects. The design, and therefore the conclusion, of our study differ to some extent from this previous report. First, while uteri from Amhr2Cre/+;Ctnnb1d/d female mice lack expression of β-catenin in the myometrial and stromal compartments, as with the Pgr-Cre model, expression of β-catenin was retained in luminal and glandular epithelia using the Amhr2Cre mouse line. Second, deletion of β-catenin in all compartments of the uterus resulted in metaplastic formation of the luminal epithelium in the intact mouse [et al., concluded that β-catenin deficiency in the epithelium was the source of the metaplastic phenotype. This conclusion is well justified in that mutations in the human Ctnnb1 gene are commonly associated with endometrial hyperplasia. Although our data does not rule out control of epithelial metaplasia by epithelial β-catenin, they indicate that, since Amhr2Cre/+;Ctnnb1d/d mutant uteri also develop metaplasia, albeit with reduced severity and incidence, the lack of β-catenin in stroma alone can dictate formation of epithelial metaplasia. As with β-catenin, deletion of APC, a component of the β-catenin signaling pathway, from the uterine stromal compartment results in a more severe phenotype where endometrial hyperplasia and carcinogenesis are observed [More recently, Jeong e uterus . Using tct mouse . Analysiobserved .2-stimulated epithelial cell mitosis. Our study also provides evidence that the stromal compartment, through activation of β-catenin, mediates as least some of the actions of P4 on the epithelium. It will now be important to delineate upstream signaling pathways that activate stromal β-catenin and to identify β-catenin target genes that are necessary for disseminating steroid hormone actions on the epithelium, in addition to its role in decidualization within the stromal compartment.Because β-catenin is connected to a multitude of cellular processes, we investigated the functional requirement of β-catenin in the stromal compartment of the endometrium for decidualization and responsiveness to steroid hormones. Our findings indicate that β-catenin is essential for early events in the terminal differentiation of uterine stromal cells. While it is well established that the stromal compartment indirectly coordinates epithelial cell proliferation through production of paracrine growth factors, deletion of stromal β-catenin did not alter ENone of the authors have competing interests.LZ completed most of the IHC and RT-PCR and some of the decidualization and histology experiments. AP completed experiments centered on progesterone and estrogen signaling, proliferative responses, receptor expression analyses, as well as some of the decidualization experiments. She also participated in drafting the manuscript. LZ assisted with IHC and animal husbandry. JT provided mice for these experiments and contributed to the experimental design and writing. JP was involved in all aspects of these studies and drafted the manuscript. All authors participated in editing and revising the manuscript. All authors read and approved the final manuscript.


Our results support the view that β-catenin signaling activity is regulated by spatial compartmentation and post-translational modifications and protein level of β-catenin alone is insufficient to count signaling activity.The stability and subcellular localization of beta-catenin, a protein that plays a major role in cell adhesion and proliferation, is tightly regulated by multiple signaling pathways. While aberrant activation of beta-catenin signaling has been implicated in cancers, the biochemical identity of transcriptionally active beta-catenin (ABC), commonly known as unphosphorylated serine 37 (S37) and threonine 41 (T41) β-catenin, remains elusive. Our current study demonstrates that ABC transcriptional activity is influenced by phosphorylation of T120 by Protein Kinase D1 (PKD1). Whereas the nuclear β-catenin from PKD1-low prostate cancer cell line C4-2 is unphosphorylated S37/T41/T120 with high transcription activity, the nuclear β-catenin from PKD1-overexpressing C4-2 cells is highly phosphorylated at T120, S37 and T41 with low transcription activity, implying that accumulation of nuclear β-catenin alone cannot be simply used as a read-out for Wnt activation. In human normal prostate tissue, the phosphorylated T120 β-catenin is mainly localized to the trans-Golgi network , and this pattern is significantly altered in prostate cancer , which is consistent with known down regulation of PKD1 in prostate cancer. These  Beta-catenin is a dual functional molecule which plays a structural role in cell-cell adhesion and a mediator for cellular signaling pathways Early studies show that β-catenin has two major pools: plasma membrane pool bound to E-cadherin which is relatively stable and cytoplasmic pool in a complex consisting of glycogen synthase kinase-3 (GSK-3), adenomatous polyposis coli (APC) and Axin that is dynamically balanced between degradation and accumulation. The degradation is ubquitin-mediated by sequential phosphorylation on S33, S37, T41 (by GSK3) and S45 (by CKI) in the absence of Wnt signaling. Recent work suggests that the N-terminal phosphorylation of β-catenin has functions beyond controlling protein degradation. β-catenin with phosphorylated S45 tends to accumulate in nucleus and spatially separates from isoform with S33/S37/T41 phosphorylation Protein Kinase D1 (PKD1), a serine/threonine kinase, has been implicated in numerous cellular functions, including cell survival, migration, differentiation, proliferation and cell-cell adhesion We showed previously that overexpression of PKD1 suppressed cell proliferation and motility Since PKD1 directly phosphorylates β-catenin at threonine-112 and 120 residues, we examined the subcellular locations of β-catenin in the presence and absence of PKD1 activity. The endogenous β-catenin localizes to plasma membrane and a portion of cytoplasm β-catenin accumulates in transGolgi Network (TGN), where it co-localizes with TGN marker P230  phosphorylation In order to confirm specificity of pT120 antibody, we first transfected HA-tagged wild type, T120I and T102I/T112R/T120I mutants of β-catenin into mouse 3T3 cell, a PKD1 positive cell line Next, we compared the pT120 β-catenin in C4-2 and C4-2/PKD1 cells. Cell lysates from C4-2 cells and C4-2/PKD1 were blotted with either a conventional β-catenin H102 antibody  or pT120 antibody . The H10To study the influence of PKD1 activity on β-catenin, we compared β-catenin subcellular distribution in C4-2 and C4-2/PKD1 cells. Subcellular fractions enriched with soluble cytosolic, nuclear or total membrane fractions (including organelle and plasma membranes) were prepared and blotted with pT120, 8E7 (for ABC), pS33/S37/T41, and H102 antibodies . In C4-2Although C4-2 cells do not express E-cadherin, C4-2/PKD1 is E-cadherin positive  due to iWe previously observed that PKD1 activity is associated with β-catenin membrane trafficking and the two proteins co-localize with multiple TGN markers in situ, we co-stained formaldehyde-fixed, paraffin embedded benign human prostate tissue using the pT120 or H102 antibody with TGN marker p230. β-catenin visualized by conventional H102 antibody typically localizes on plasma membrane as well as TGN (To study the pT120 β-catenin l as TGN . In contl as TGN . Weak stTo confirm the specificity of pT120 antibody using in immunohistochemetry (IHC), we performed peptide competition assay on pT120 antibody in IHC. The pT120 antibody staining displays a more diffuse pattern with heavy particles in cytoplasm presumably the TGN  studies were carried out. IHC was performed on serial sections of paraffinized human prostate normal and cancer samples arranged on tissue arrays to compare pT120 β-catenin to total β-catenin distribution . In normOther abnormal pT120 antibody staining patterns also found in prostate cancer samples, including strong plasma membrane  and nuclKAI1 (CD82)KAI1 actually was induced in C4-2/PKD1 cells, although the expression of β-catenin is also upregulated PKD1 suppresses cancer cell epithelial to mesenchymal transition by inhibitory phosphorylation of transcription factor Snail, a known E-cadherin repressor. Overexpression of PKD1 in C4-2 induces expression of E-cadherin and lowers tumor incidence in mice xenograft models in vivo, then the results would be interpreted that 93% prostate cancer samples have less PKD1 activity. Interestingly, reduced expression of PKD1 was found in more than 95% of human invasive breast tumors examined by antibody staining Although the critical role of Wnt signaling is demonstrated in the mutations of APC or β-catenin gene, such mutations are infrequent in prostate cancer, which counts 16% and 5%, respectively Our work reveals a new regulatory step of the dynamic equilibrium of free and bound β-catenin. The phosphorylation of T120 correlated inversely with unphosphorylated S37/T41 ABC. The distribution of pT120 β-catenin is distinct in normal and malignant prostate tissues. With malignant transformation, staining of pT120 at TGN is dramatically decreased. We propose that β-catenin T120 phosphospecific antibody can be used to study alteration in subcellular distribution of β-catenin and Wnt signaling, and possibly as biomarker in prostate and other tissues, which may facilitate risk stratification or predict disease outcomes. Further investigations on the phosphorylation relationship of T120 and S37/T41 may help to elucidate the role PKD1 in the regulation of Wnt/β-catenin signaling.109-IPSTQFDAAHPpTNVQRLAEPSQML (where pT is phosphothreonine-120) was synthesized. The peptide was injected into two rabbits to raise antisera. A corresponding nonphospho-peptide was also synthesized for antibody screening and purification. The peptide synthesis and antibody generation was performed by commercial contractor Millipore .An antigen peptide correspondent to the β-catenin T120 region, 6) were collected by scratching and washed with PBS plus a protease inhibitor cocktail and a phosphatase inhibitor cocktail . Cell pellet was resuspended in 250 microliter of a hypotonic buffer (Cytosol Buffer 1) and homogenized with a plastic pestle. After incubation 15 min on ice, cells were centrifuged at 8,000×g for 5 min. The supernatant was collected and centrifuged again at 16,000×g for 10 min and supernatant is used as soluble cytosol fraction. The initial pellet was washed with a wash buffer (Wash Buffer 2), spinned down, resuspended in 125 microliter of Nuclear Buffer 3 and homogenized. After incubating on ice for 30 min, the lysate was centrifuged at 16,000×g for 10 min. The supernatant was collected as nuclear-protein enriched fraction. The resulting pellet was resuspended in 250 microliter of Total Membrane Buffer, homogenized and incubated on ice for 30 min. The lysate was centrifuged at 16,000×g for 10 min and the supernatant was collected as membrane-protein enriched fraction. It is worth noting that the total membrane fraction generated by the kit includes plasma membrane and cellular membrane. The protein samples were separated on 10% SDS-PAGE and transferred to a polyvinylidene difluoride membranes (PVDF) using a semi-dry transfer devise (Thermo Scientific) at constant current model (0.2 A). The membrane was then incubated with TBST  plus 3% nonfat dry milk to block non-specific binding for 1 hour at room temperature. Incubation with primary antibodies (see Reagents section) was performed at 4 C for overnight. The densitometric quantification of immunoblotting was carried out with Image J software.To prepare whole cell lysates, cells cultured on monolayer were washed by phosphoate buffered saline (PBS) and dissolved in RIPA buffer For western blotting, the pT120 antiserum was used at dilution of 1∶2000. For peptide competition assay, the phospho- or nonphospho-peptides were added in the pT120 antibody solution to a final concentration of 20 nM prior to blotting. A commercial β-catenin antibody  was used for comparison at 1∶2000 dilution. The phospho-S33/S37/T41 β-catenin antibody  was used at dilution of 1∶200 for western blot. Monoclonal antibody 8E7 (Millipore) that recognizes unphosphorylated S37/T41 β-catenin was used at 1∶800 for blotting. The HA-tagged wild type β-catenin in pCS2+ mammalian expression vector, T120I and T102I/T112R/T120I mutants were described previously 4 cell/well in a 96-well plate) were transfected by 50 ng Topflash reporter, 5 ng Renilla luciferase vector and 100 ng either PKD1 expression vector or empty vector control for overnight. Luciferase activity was measured 2 days later. Relative luciferase activity (RLU) was obtained by averaging of normalized firefly luciferase activities from three samples. Normalization was performed with Renilla luciferase from Promega .Prostate cancer cell lines LNCaP  C4-2 (Urocor) and a benign prostate hyperplastic epithelial cell line BPH-1  were cultured in RPMI1640 medium plus 10% bovine fetal serum (Invitrogen). C4-2 cells that stably express PKD1-GFP (C4-2/PKD1) was previously described For immunofluorescence, cultured cells were fixed in 5% formalin in PBS for 10 minutes at room temperature. Human normal prostate tissues were deparafinized and rehydrated. The pT120 and H102 antibodies were diluted 2000-fold in PBS with 0.1% Triton X-100 and 10 mg/ml BSA. Antibody to transGolgi Network marker P230 (BD Transduction Laboratories) was used at a dilution of 1∶500. FITC and Cy3 labeled second antibodies  were used at dilution of 1∶1,000. For immunohistochemistry, the pT120 and H102 antibodies were used at 1∶1000 and 1∶100 dilutions, respectively. Human prostate tissue arrays were purchased from US Biomax. Fluorescence and IHC images were taken in an Olympus IX-51 microscope and a Nickon Eclipse 50i microscope equipped with SPOT software, respectively. The assessment of β-catenin expression in tissue specimens was done by comparison of the average staining intensity of normal samples to the staining intensities of tumor samples, and described higher or lower intensity as up regulation or down regulation respectively. To score pT120 staining in trans Golgi Network, we counted 30 normal prostate tissues for the “particle” staining pattern as described in http://www.stanford.edu/%7ernusse/pathways/targets.html. Primers used are, c-Myc, For: ACCAGCTGGAGATGGTGACCGAG; Rev: CCGAGGACGGAGAGAAGGCGCTTotal RNA was extracted with Trizol reagent (Invitrogen). Forty micrograms total RNA were used for reverse transcription in a 40 µl volume using SuperScript II kit ((Invitrogen) at 45 C for 1 hour. After RT, the reaction was diluted with TE buffer  to 200 microliter. Two microliter of the RT product was used for each PCR reaction. Thermocycle setting is 94 C for 2 minutes, 94 C for 30 seconds, 55 C for 45 seconds and 72 C for 30 seconds. PCR samples were taken at 27, 29, 31 and 33 cycles and analyzed by agarose gel electrophoresis. Wnt target genes were selected from a list in the Wnt Homepage at cyclin D1, For: TGCTGAAGGCGGAGGAGACCTGC; Rev: CAGCAGCTCCTCGGGCCGGATGGAxin2, For: TTATGCTTTGCACTACGTCCCTCCA; Rev: CGCAACATGGTCAACCCTCAGACLEF1, For: TTCAAGGACGAGGGCGATCCTCAAG; Rev: CGAGTTATTCGGGTACATAATGAID2, For: AACAGCCTGTCGGACCACAAGC; Rev: GGCTGACAATAGTGGGATGCGTCF1, For: TGGACAAGGGGGAGTCCTGC; Rev: GGTTGAGGCCAGTGGTATCGGAPDH, For:AGAAGGCTGGGGCTCATTTG; Rev: AGGGGCCATCCACAGTCCTTCFigure S1Total and pT120 β-catenin localizations in cultured prostate cell lines. (a) C4-2 cells, (b) RWPE1 cells, (c) LNCaP cells and (d) BPH-1 cells.(TIF)Click here for additional data file.Figure S2pT120 β-catenin staining in prostate tumor and adjacent normal area. A tissue core contains both tumor and normal tissues. The pT120 antibody staining reveals that pT120 β-catenin accumulates in TGN in the normal tissues . In adjacent tumor tissue, the pT120 β-catenin is more diffused in cytoplasm (upper left).(TIF)Click here for additional data file.


Hepatocellular Carcinoma (HCC) is the fifth most common cancer worldwide. β-Catenin, the central orchestrator of the canonical Wnt pathway and a known oncogene is paramount in HCC pathogenesis. Administration of phenobarbital (PB) containing water (0.05% w/v) as tumor promoter following initial injected intraperitoneal (IP) diethylnitrosamine (DEN) injection (5 µg/gm body weight) as a tumor inducer is commonly used model to study HCC in mice. Herein, nine fifteen-day male β-catenin knockout mice (KO) and fifteen wild-type littermate controls (WT) underwent DEN/PB treatment and were examined for hepatic tumorigenesis at eight months. Paradoxically, a significantly higher tumor burden was observed in KO (p<0.05). Tumors in KO were β-catenin and glutamine synthetase negative and HGF/Met, EGFR & IGFR signaling was unremarkable. A significant increase in PDGFRα and its ligand PDGF-CC leading to increased phosphotyrosine-720-PDGFRα was observed in tumor-bearing KO mice (p<0.05). Simultaneously, these livers displayed increased cell death, stellate cell activation, hepatic fibrosis and cell proliferation. Further, PDGF-CC significantly induced hepatoma cell proliferation especially following β-catenin suppression. Our studies also demonstrate that the utilized DEN/PB protocol in the WT C57BL/6 mice did not select for β-catenin gene mutations during hepatocarcinogenesis. Thus, DEN/PB enhanced HCC in mice lacking β-catenin in the liver may be due to their ineptness at regulating cell survival, leading to enhanced fibrosis and regeneration through PDGFRα activation. β-Catenin downregulation also made hepatoma cells more sensitive to receptor tyrosine kinases and thus may be exploited for therapeutics. Hepatocellular Carcinoma (HCC) is the fifth most common cancer and the third cause of cancer death worldwide CTNNB1) have been reported in 20%–40% of patients One pathway of critical importance in HCC is the Wnt/β-catenin signaling. β-Catenin is the central effector of the canonical Wnt signaling, which is a highly conserved pathway regulating critical cellular processes such as proliferation, differentiation, survival and self-renewal With this background, we hypothesized that lack of β-catenin in hepatocytes might protect against chemical-induced carcinogenesis especially in a model where HCC is conceived through tumor induction by DEN and tumor promotion through the continuous use of PB ad libitum access to PB containing drinking water for 8 months at which time mice were examined for liver tumors  were given a single dose (5 µg/gram) of DEN injection at postnatal day 14 days and two weeks later allowed r tumors . Intrigur tumors . H&E star tumors . The totr tumors .Next, we addressed the cellular mechanisms that may be the basis of enhanced tumorigenesis in KO. We identify higher numbers of TUNEL-positive hepatocytes in KO at 8 months after DEN/PB as compared to similarly treated WT, suggesting greater cell death . There wSince there have been reports from our lab and others that in response to specific injuries, there is pressure on hepatocytes that may have escaped cre-mediated deletion, our first goal was to ascertain if livers exposed to DEN/PB from KO group showed any reappearance of β-catenin especially when compared to the WT. A representative WB showed that all KO livers from DEN/PB exposed mice expressed dramatically lower levels of β-catenin as compared to WT by western blots . This waTumors in KO livers after DEN/PB are not associated with activation of traditional HCC associated receptor tyrosine kinases. Due to a paradoxical increase in DEN/PB-induced tumorigenesis in KO, we next explored possible molecular mechanisms. Both epidermal growth factor (EGF) signaling and hepatocyte growth factor (HGF) signaling are critical players in the development and exacerbation of HCC We next characterized the tumors observed in the WT and KO by immunohistochemistry for β-catenin localization. Around 31% of total tumor foci in WT mice showed nuclear β-catenin , Fig. 4.We next investigated PDGFRα signaling, which has been implicated in HCC and is involved in tumor growth, angiogenesis, and maintenance of tumor microenvironment In an additional analysis, 9 of the 55 tumors observed in the WT were concomitantly positive for both PDGFRα and nuclear β-catenin, while others were positive for either one of the two , Fig. 4.DEN/PB induced increased tumorigenesis is associated with activation of PDGFRα signaling. To further verify PDGFRα increase in the KO over WT after DEN/PB, we utilized western blots analysis. PDGFRα protein levels were higher in the KO than the WT livers  and have been described previously loxp/loxp;Alb-Cre−/−, Ctnnb1loxp/WT;Alb-Cre+/−, Ctnnb1loxp/WT;Alb-Cre−/− are referred to as wild-type or WT. These mice are in C57BL/6 background.All animal experiments were performed under the guidelines of the National Institutes of Health and the Institutional Animal Use and Care Committee at the University of Pittsburgh. The studies performed in the current report were approved by the Institutional Animal Use and Care Committee at the University of Pittsburgh. Mice with conditional deletion of β-catenin in hepatocytes with genotype- Ctnnb1ad libitum contained phenobarbital (PB) (0.05% w/v). Water containing fresh PB was prepared weekly for the duration of the studies. Mice were sacrificed at 8 months and liver collected for histology and protein analysis.Male KO (n = 9) and WT (n = 15) mice were injected intraperitoneally with DEN  at a dose of 5 µg/gram body weight at postnatal day 14 and from day 28 onwards the drinking water available Western blot Analysis: Total tissue lysates prepared in radio immuno-precipitation assay (RIPA) buffer containing 1% IgePAL CA-630, 0.5% Sodium Deoxycholate, 0.1% SDS in 1× PBS along with protease & phosphate inhibitor (1∶100) (Thermo Scientific). Proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis in 4–15% gels and then transferred to Immobilon-P membranes  in transfer buffer containing 10% methanol. Membranes were probed with primary antibodies (see below) in Tris-buffered saline with Tween-20 containing 5% nonfat milk or BSA. Horseradish peroxidase–conjugated secondary antibodies were used at 1∶50,000 dilution and signal assessed with Super Signal West Pico chemiluminescence substrate  and autoradiography. The films  were scanned to obtain integrated optic densitomery (IOD) using NIH Imager software. The average IOD for a protein was compared between the KO and WT groups and assessed for statistical significance by student t test and p<0.05 was considered significant.Antibodies. Primary antibodies used for Western blotting included: c-Met (1∶500), phospho-Met Tyr1234/1235 (1∶1000), EGFR (1∶1000), phospho-PDGFRα Tyr849 (1∶1000), phospho-IGFR (1∶1000) all from Cell Signaling Technology; β-catenin (1∶1000) from BD Biosciences; PDGFRα (1∶180), PDGFRβ (1∶500), phospho-EGFR (1∶500), phospho-PDGFRα Tyr720 (1∶200), c-myc (1∶200), PDGFA (1∶200), PDGFB (1∶200), PDGFC (1∶200), phospho-PDGFRα Tyr754 (1∶200), Glutamine Synthethase (1∶200), and GAPDH (1∶1000) all from Santa Cruz Biotechnology; phospho-PDGFRα Tyr572/574 ; Cyclin D1; alpha smooth muscle actin ; and β-actin .t test with p<0.05 considered significant.Four-micron sections of paraffin-embedded liver tissues were used for histology and immunohistochemical staining. Hematoxylin and Eosin (H&E) staining was employed to identify tumor foci. Typically four representative lobes from each mouse were included on each slide. Tumor foci were identified based on characteristic attributes such as basophilic cytoplasm and mitotic figures. For comparison, total number of microscopic nodules were counted and average numbers compared for statistical significant by student t test. P value of less than 0.05 was considered significant. For quantitative analysis of β-catenin- and PDGFRα-positivity in tumors, all microscopic foci in KO and WT livers were assessed. Any tumor foci showing cytoplasmic and/or nuclear β-catenin staining were labeled as being β-catenin-positive and any foci exhibiting cytoplasmic staining for PDGFRα as compared to surrounding non-tumor areas were labeled as being PDGFRα-positive. This enabled us to calculate percentage of β-catenin and/or PDGFRα-positive tumors in each group after DEN/PB treatment.For immunohistochemistry, antigen retrieval was achieved both by steam cooking or boiling the slides in microwave in citrate buffer for 20 minutes or 10 minutes, respectively. The sections were inactivated for endogenous peroxide, blocked and incubated with primary antibody overnight at room temperature or for one hour at room temperature, washed and incubated with appropriate biotin-conjugated secondary antibody for 30 minutes. Sections were washed, incubated with ABC reagent, washed and incubated with DAB. Sections were next counterstained with Shandon hematoxylin solution (Sigma) and cover slipped using Cytoseal XYL . For negative control, the primary antibody was omitted in the protocol. Slides were viewed under an Axioskop 40 (Zeiss) upright research microscope and digital images obtained. Collages were prepared using Adobe Photoshop CS4 software. For PCNA and TUNEL, numbers of positive cells were counted in four random 400× fields in five representative livers from each group and averages compared for significance between groups by student Hep3B cells (ATCC) were grown to 100% confluence in 10% FBS and EMEM media. Cells were trypsinized and plated in 6 well plates containing 2 ml of 10% FBS EMEM media and serum-starved overnight. Transfection was performed using pre-validated β-catenin and control siRNA (Ambion) using lipofectamine as described previously


Staphylococcus intermedius group’ of zoonotic pathogens, excrete two compounds in millimolar concentrations that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini. They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. In vitro studies with the N-acyl homoserine lactone (AHL) responding receptor LuxN of V. harveyi indicated that both compounds caused opposite effects on phosphorylation to those caused by AHL. This explains the quorum quenching activity. Staphylococcal strains producing yayurea A and B clearly benefit from an increased competitiveness in a mixed community.The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we report on one of the rare examples where Gram-positive bacteria, the ‘ Staphylococcus species group, which consists of closely related coagulase-positive bacterial species that play a role as zoonotic pathogens. We found that these species excrete two small compounds that inhibit both the expression of QS-controlled toxins and other QS-regulated compounds as well as growth in Gram-negative bacteria. The excreted compounds, which we named yayurea A and B, were isolated from S. delphini and structurally characterized. They represent new bacterial products, which quench the QS regulation in a wide spectrum of Gram-negative bacteria by stimulating the LuxN-mediated phosphorylation of LuxU. Furthermore, growth of yayurea A and B producing S. delphini is not suppressed by respiratory toxins when co-cultured with P. aeruginosa. This suggests that the quorum quenchers have a function in self-protection and competitiveness in natural environments shared with Gram-negatives. Here we show one of the rare cases of inter-phylum interference between firmicutes (Gram-positive) and beta-/gammaproteobacteria (Gram-negative).While studying the potential interaction of staphylococci with Gram-negative bacteria, we came across another communication system in a  V. harveyi, the QS system consists of three autoinducers and three cognate receptors functioning in parallel to channel information into a shared regulatory pathway V. harveyi produces an AHL signal termed HAI-1, 3-hydroxy-C4-homoserine lactone 54 and regulatory small RNAs In many bacteria, pathogenicity is controlled and coordinated by an inter-cellular communication process named quorum sensing (QS). QS is based on the synthesis and secretion of small hormone-like molecules termed autoinducers that bind to cognate receptors. The signal-activated receptor controls directly or indirectly expression of target genes. Since the concentration of signaling molecules in liquid culture is proportional to cell density in the culture, gene expression is coordinated in response to the bacterial population density V. harveyi, P. aeruginosa coordinates the expression of nearly 10% of its genome through three hierarchically arranged QS systems, namely Las, Rhl and Pqs Similar to QS system is a promising target for anti-virulence therapy Staphylococcus is the agr QS system In contrast to Gram-negative bacteria, many Gram-positive bacteria communicate using modified oligopeptides as signals and “two-component”-type membrane-bound sensor histidine kinases as receptors. The well-studied QS system in Staphylococcus species group, named ‘intermedius group’. This group consists of closely related mainly coagulase-positive bacterial species including S. delphini, S. intermedius, S. lutrae, S. pseudintermedius, and S. schleiferi. They are all phylogenetically related, are zoonotic pathogens, and only rarely occur in human infections. We found that these species excrete two low molecular compounds that inhibit the expression of QS-controlled toxins and other QS-regulated compounds in Gram-negative bacteria. The excreted compounds, which we named yayurea A and B, were isolated from S. delphini and structurally characterized. Yayurea A and B represent new bacterial products, and were able to quench the QS regulation in a wide spectrum of Gram-negative bacteria. Furthermore, growth of yayurea A and B producing S. delphini is not suppressed by respiratory toxins when co-cultured with P. aeruginosa. This suggests that the quorum quenchers have a function in self-protection and competitiveness in natural environments shared with Gram-negatives. Here we show an example of inter-phylum interference between Firmicutes (Gram-positive) and the Gram-negative beta- and gammaproteobacteria.While studying the potential interaction of staphylococci with Gram-negative bacteria P. aeruginosa in a co-cultivation assay, as pyocyanin production is QS controlled. We found that S. delphini DSMZ 20771 completely inhibited pyocyanin production over 24 h co-cultivation with P. aeruginosa PAO1, while Staphylococcus aureus showed no such activity  were excreted. The supernatants of S. aureus and S. epidermidis caused no effect.Our aim was to find out if some staphylococcal species are able to suppress the QS controlled phenotypes in Gram-negative bacteria. To investigate this, we tested the ability of several staphylococcal species to inhibit pyocyanin production of activity . This le did not . SterileS. delphini DSMZ 20771. Further purification revealed that the supernatant contained two compounds with different retention times (Rt) in HPLC and distinct UV spectra. We named the two compounds yayurea A and B.The QS inhibitors were isolated from the supernatant of an overnight culture of +) in ESI-MS, and showed the molecular formula, C10H13N2, in FT-ICR-MS analysis. The 1H-indole-3-ethylamine moiety was deduced from GC-EI-MS (Rt = 29.0 min). 1H-NMR spectra showed a characteristic singlet at δH = 8.55 ppm. The remaining signals were assigned to 2-(3-indoyl) ethylamine. The 13C-NMR spectrum and additional HSQC experiments displayed signals pointing to a carbonyl group at δ = 170.4 ppm, five methin groups, and two methylene groups to reveal a urea moiety. Additionally, 2D NMR experiments supported the structure of yayurea A as N-[2-(1H-indole-3-yl) ethyl]-urea (Yayurea A (indole-ethylurea) was isolated as a brownish solid, revealed an ion peak at m/z = 161 . GC-EI-MS provided a signal at Rt = 15.0 min pointing to 2-phenethylamine. The 1H-NMR again showed a set of aromatic protons and a singlet at δH = 8.54 ppm, while the 13C-NMR displayed a signal at δH = 170.4 ppm (C = O) and five aromatic and 4 aliphatic protons. In summary, the structure of N-(2-phenethyl)-urea was assigned for yayurea B . Both the synthesized and the natural compounds revealed the same chemical properties and the same QS-quenching activity in Gram-negative bacteria (data not shown).Yayurea B (phenethylurea) was obtained as a colorless solid. A preliminary molecular formula, Cayurea B . UV-Absoayurea B . In the P. aeruginosa produces various QS-controlled respiratory toxins such as pyocyanin and hydrogen cyanide S. aureusS. delphini survives better in a co-culture with P. aeruginosa than, for example, S. aureus. Indeed, co-cultivation studies with S. aureus (non-producer) or S. delphini with P. aeruginosa SH1 revealed that the viability (CFU) of S. aureus significantly decreased in the stationary growth phase, most likely due to the respiratory toxins produced by P. aeruginosa  had also no effect on viability of P. aeruginosa (Pseudomonas strains PAO1 and DSMZ 50071 showed similar results (data not shown). All in all, these results showed that production of yayurea A and B enables staphylococci to coexist with Gram-negative bacteria in a mixed community.ruginosa , while tfluenced . The addry phase . Furtherruginosa . Other tS. delphini grown in TSB. The amount of produced yayurea A and B was determined by HPLC-analysis; peak integration was correlated with standard yayurea A and B. The production of both compounds started at the transition of exponential to stationary growth phase (after approximately 4 h) and increased rapidly for the next 5 h; after 24 h little more was produced  and increased with increasing concentrations of yayurea A or B. At 250 µg/ml, prodigiosin production was inhibited at 60% (yayurea A) and 40% (yayurea B). At a concentration of 1000 µg/ml, prodigiosin production was completely inhibited by yayurea A, and to approximately 70% by yayurea B  inhibit the growth of Gram-negative bacteria. We used cholerae ; howeverV. harveyi, we performed in vitro phosphorylation assays of the corresponding signaling proteins. The full-length hybrid kinases LuxN, LuxQ, CqsS (tagged with 6 histidine residues) were heterologously expressed in an E. coli strain that lacks the F1/Fo-ATPase (to prevent ATP degradation during phosphorylation experiments), and inverted membrane vesicles prepared from this strain were directly used for the phosphorylation experiments. Then we tested the effect of yayurea A and B on the LuxN, LuxQ [in interplay with LuxP (LuxPQ)], and CqsS-mediated time-dependent phosphorylation of the HPt protein LuxU (N-(3-hydroxybutyryl)-homoserine lactone (HAI-1) (To gain insight into the molecular mechanism behind why yayurea A and B cause a decrease in bioluminescence of ein LuxU . Yayureaein LuxU . However (HAI-1) . It is kV. harveyi is exposed to these compounds. Under our test conditions, autoinducer HAI-1 was dominant in relation to yayurea A or B  the species are phylogenetically related and summarized in the ‘intermedius group’ S. schleiferi subsp. schleiferi), and oxidase negative. Interestingly, they all colonize various animals, and many represent zoonotic pathogens S. hyicus, S. chromogenes or S. muscae) inhibited QS-system in Gram-negative bacteria or produced yayurea A and B.We tested a number of staphylococcal species representatives (listed in S. delphiniS. intermediusS. pseudintermediusS. lutraeS. schleiferinuc) sequences In this study, we found that some staphylococcal species excrete two novel compounds, yayurea A and B that interfere with the QS system of diverse Gram-negative bacteria. We tested 24 staphylococcal species with respect to production of yayurea A and B by co-cultivation with Gram-negative bacteria. The results showed that only five species  spanning domains Pseudomonas, Chromobacterium, Vibrio, and Serratia).When both AI-1 and yayurea A or B were applied, HAI-1 overruled the effect of yayurea A and B Halobacillus salinus, which produces N-(2′-phenylethyl)-isobutyramide and 2,3-methyl-N-(2′-phenylethyl)-butyramide 1H-indol-3-yl)ethyl]-urea) and B (N-(2-phenethyl)-urea). Within these communities, bacteria compete with their neighbors for space and resources S. delphini was isolated from dolphin. Since both V. harveyi and S. delphini are marine bacteria and animal pathogens, it is conceivable that both bacteria share the same habitat and moreover, the same host surface. Our results demonstrated suppression of the quorum sensing regulated bioluminescence of V. harveyi during co-culture with S. delphini  was studied. Furthermore, it should be noted that HAI-1 is not constantly produced S. delphini can fully interact with LuxN from V. harveyi in the absence of any competition with HAI-1. It therefore makes sense that the zoonotic staphylococci impair the QS-system of Gram-negative bacteria for competitive reasons. The growth of these staphylococci is not impaired by P. aeruginosa because the excreted yayureas suppress the production of the QS-controlled toxins and are thereby protected from being killed by the toxins  was added, the growth of affected , but quoaffected  and 5C waffected , which iThe yayureas are potential candidates for use as anti-infectives. The only disadvantage might be the rather high concentration needed to completely quench QS; on the other hand, preliminary results suggest that they hardly have cytotoxic activities.S. delphini produces just enough yayurea A and B to almost completely silence the expression of the studied QS-regulated compounds or biofilm formation in diverse Gram-negative bacteria. In mixed cultures, the yayurea-producing staphylococci arrest Gram-negative bacteria in a pheno- and genotypic state of low cell density, although the cells have grown to high density. The advantage for the staphylococci is twofold, on one hand, they are protected from QS-controlled toxins and on the other hand, yayurea A and B affect stationary growth of particularly those Gram-negative bacteria with prominent QS-control systems. This is one of the rare cases of inter-phylum interference between firmicutes (Gram-positive) and beta-/gammaproteobacteria (Gram-negative). A schematic presentation of the interference is shown in An interesting question is why just the ‘intermedius group’ is producing yayureas. The most likely answer is that this group shares its habitat with Gram-negative bacteria. There must be a benefit to producing yayurea A and B because they are excreted in such high amounts certainly costing energy. In addition, the yayureas show growth inhibition only at quantities that exceed the physiological concentrations. It is a clever arrangement, that Staphylococcus strains were grown in Tryptic Soy Broth  medium at 37°C, Vibrio cholerae SP27459 (O1 El Tor), Serratia marcescens, Chromobacterium subtsugae DSMZ 17043 and Pseudomonas aeruginosa were grown in lysogenic broth (LB) medium, and Vibrio harveyi DSMZ6904 in marine broth  at 28°C. The Staphylococcus strains tested in this work are listed in S. aureus (OD578 0.005) and P. aeruginosa SH1 (OD578 0.005). For the protection test, yayurea A (100 µg/ml), yayurea B (900 µg/ml), or water (control) were added to the mixed culture of S. aureus and P. aeruginosa after 3 h incubation. For co-cultivation of S. delphini with P. aeruginosa, each of the strains was inoculated with OD578 0.001, because doubling time of S. delphini was shorter than that of S. aureus. Co-cultures were aerobically grown at 37°C. For CFU determination, samples were diluted and plated on Chapman agar  and BM agar  on which P. aeruginosa grows better than the staphylococci. Colonies were counted after incubating for 24 h at 37°C; staphylococcal and pseudomonas colonies could be easily distinguished by colony shape and pigmentation.In co-culture experiments, we inoculated TSB with S. delphini . Cells were centrifuged and the supernatant was applied to an Amberilite XAD 16 resin . The column was first washed with water, then with 40% and 60% methanol and finally eluted with solvent A (80% methanol containing 5% acetic acid). Yayureas were significantly enriched in the last fraction. In the second purification step, the eluate was evaporated in vacuo, suspended in water, and further separated on an Amberilite IRC 50 cation exchange resin  after the pH was adjusted to 7.0 with 1 M NaOH. The column was washed with water, then 70% ethanol and eluted with solvent A. The eluate was concentrated in vacuo, diluted, and adjusted to a final concentration of 50 mM sodium phosphate buffer with a pH of 4.2. In the third purification step, the eluate was separated on a SP Sepharose cation exchange column  with a linear 1 M NaCl gradient in 50 mM sodium phosphate buffer on a Äkta FPLC . The two active compounds were eluted separately at 120 mM and 280 mM NaCl concentration respectively. The final purification and desalting of each peak was carried out by reversed phase preparative HPLC (RP-HPLC)  on a nucleosil 100 C-18, 8×250 mm column  with a linear water acetonitrile (containing 0.1% TFA) gradient of 0% to 60% in 25 min. Purified compounds were lyophilized and stored at −20°C. Qualitative analysis was carried out on an Agilent 1200 HPLC system  and a RP-HPLC Waters xBridge C18, 5 mm, 4.6×150 mm column. Compounds were eluted with a 15 min linear gradient of 0.1% phosphoric acid to acetonitrile at a flow rate of 1.5 ml/min. For structure elucidation, mass spectrometry was carried out on GC-MS and FT-ICR MS . NMR spectra were measured in d4-methanol and recorded on a Bruker AMX600 spectrometer , solvent was used as internal standard (δH/C 3.31/49.15 for MeOH-4d).The QS inhibitors were isolated from aerobically-grown overnight culture of S. aureus and S. delphini cells were diluted in LB medium containing 0.3% glucose to an OD578 value of 1.0 , incubated for 4 h at 37°C, then co-cultivated at 30°C with S. marcescens or P. aeruginosa; each strain was inoculated with a starting OD600 of 0.01. After 24 h of cultivation, prodigiosin of S. marcescens was extracted from the cell pellets by ethanol acidified with 4% of 1 M hydrochloric acid and then quantified by A534 nm determination. Pyocyanin in the supernatant was determined by its absorption maximum at 520 nm V. harveyi was diluted to an OD578 of 1.0 and co-cultivated with an equal amount of S. aureus or S. delphini cells in LB-MB  for 18 h. Overnight cultures of S. aureus and S. delphini cells were diluted to an OD578 of 0.1, incubated for 4 h at 37°C, then co-cultivated at 30°C with C. subtsugae, which was diluted to a final OD600 value of 2; this high OD was necessary to pronounce violacein production, whose expression is dependent on AHL at higher cell density. After 24 h of incubation, cell pellets were collected and resuspended in water. Cells were lysed by 10% sodium dodecyl sulfate and incubated for 5 min at room temperature. Violacein was quantitatively extracted from the cell by adding water-saturated butanol. The butanol phase containing the violacein was collected and determined at its absorption maximum at 585 nm.Overnight cultures of S. delphini was inoculated to a starting OD578 of 0.1 and incubated at 37°C. During the 24 h incubation, cell density was followed by OD578 and the active compounds in the supernatants were determined by RP-HPLC and phosphoric acid – acetonitrile gradient as described above. The Gram-negative representatives, E. coli, P. aeruginosa, S. marcescens, V. cholerae and V. harveyi were inoculated to OD600 of 0.1 and grown in LB for 2 h. After that time, 1 mg/ml yayurea A or B (as solution in H2O) or an equal volume of water as negative control was added. Cells were either incubated at 37°C (S. aureus or S. delphini) or 30°C . Cell density was followed for 24 h.P. aeruginosa PAO1 was diluted with LB medium to an OD600 of 0.1. The (Z-)-4-bromo-5-(bromomethylene)-2(5H)-furanone (Sigma), yayureas and tetracycline were dissolved in 10% DMSO. 190 µl diluted PAO1 was cultured in 96-well plates with 10 µl different concentrations of pure yayurea A, B, furanone, or 10% DMSO (negative control) at 30°C. After 24 h of incubation, cell density was measured at 600 nm and pyocyanin was isolated as described previously An overnight culture of S. marcescens cells were inoculated from an overnight culture to a starting OD600 of 0.1. Then 100 µl of the diluted culture was mixed with 100 µl of different amounts of the purified compounds or water in wells of 96-well microtiter plates and incubated for 24 h at 28°C. Cell density was measured at 600 nm. Prodigiosin was extracted as described above and the relative prodigiosin production was calculated as the ratio between the amount of prodigiosin at its absorption maximum at 534 nm and cell density values at 600 nm. The prodigiosin suppressing effect of yayurea A and B was determined (H2O was used as control).P. aeruginosa PAO1 or S. aureus was diluted with LB medium containing 0.3% (w/v) glucose to an OD600 of 0.1. 100 µl of the dilution was mixed with 100 µl of the yayurea solutions with the indicated concentrations, and incubated in 96-wellplates at 37°C for 24 h. To determine the biofilm formation, planktonic cells were discarded and the plates gently washed with PBS and air-dried for 30 min. The wells were stained with 200 µl of a 0.1% crystal violet solution at room temperature for 30 min. The stained biofilm was rinsed with distilled water followed by the addition of 200 µl DMSO. Absorbance of crystal violet as indicator for biofilm-forming bacteria was measured at 590 nm. The effects of the compounds were evaluated for which the sample with water was set to 100%.Biofilm formation assays were performed as described previously V. harveyi was diluted to an OD600 of 0.1 and grown with yayurea A, B, or water for 24 h at 28°C. Bioluminescence was determined in a Tecan infinite M200 plate reader . Relative luminescence was normalized by the OD600 values. The effects of the compounds were calculated by the relative luminescence, for which the sample with water was set to 100%.An overnight culture of E. coli strainTKR2000 expressing plasmids pNKN, pNKQ, and pNKS encoding wild type LuxN, LuxQ, and CqsS , respectively. Inverted membrane vesicles were prepared as described by E. coli MDAI-2 transformed with the plasmid pGEX_LuxP as described before E. coli JM109 transformed with plasmid pQE30LuxU-6His Inverted membrane vesicles were prepared using 2+-ATP, typically 100 µM [γ-32P]ATP  and 110 µM MgCl2. The reaction was stopped after 1 and 10 minutes by adding SDS Laemmli-loading buffer followed by separation of the proteins on SDS-polyacrylamide gels. Gels were dried at 80°C on filter paper, exposed to a phosphoscreen for at least 24 h, and subsequently scanned using a PhosphorImager SI . The gels presented are representative of at least three independent sets of experiments.Each QS-receptor kinase was tested as full-length membrane integrated proteins in inverted membrane vesicles. The buffer used for phosphorylation reactions was as follows: 50 mM Tris/HCl pH 8.0, 10% (v/v) glycerol, 500 mM KCl, 2 mM DTT. Each phosphorylation reaction contained equimoloar receptor concentrations , and purified LuxU at 0.1 mg/ml. For phosphorylation of LuxQ, LuxP was added at a concentration of 0.4 mg/ml. LuxP integration into LuxQ-bearing membrane vesicles was triggered by several cycles of thawing and freezing. When indicated, yayurea A or B were added to the reaction mix at a final concentration of 220 µg/ml, and HAI-1 at 20 µM. The reaction was started by adding radio labeled MgFigure S1Mass spectra of Yayurea A and B. Mass spectrometry was carried out on GC-MS and FT-ICR MS .(TIF)Click here for additional data file.Figure S2Influence of yayurea A and B on growth. Growth curve of S. marcescens, P. aeruginosa, V. harveyi and V. cholerae in BM with 1000 µg/ml yayurea A or B or equal volume of water. Arrow indicates time point (after 2 h) of the addition of compounds to the growing culture. All the measurements were made in triplicate. Bars indicate standard deviation, SD.(TIF)Click here for additional data file.Figure S3Phylogenetic tree among Staphylococcus species. The tree is based on 16S rRNA relationships according to S. intermedius, S. pseudintermedius, and S. delphini is marked in bold. Based on a combinatin of 16S rRNA and multilocus data, the group was recently complemented by the next related species S. lutrae, S. schleiferi subsp. schleiferi and S. schleiferi subsp. coagulans(TIF)Click here for additional data file.


The pervasive use of cell phones impacts many people–both cell phone users and bystanders exposed to conversations. This study examined the effects of overhearing a one-sided (cell phone) conversation versus a two-sided conversation on attention and memory. In our realistic design, participants were led to believe they were participating in a study examining the relationship between anagrams and reading comprehension. While the participant was completing an anagram task, the researcher left the room and participants overheard a scripted conversation, either two confederates talking with each other or one confederate talking on a cell phone. Upon the researcher’s return, the participant took a recognition memory task with words from the conversation, and completed a questionnaire measuring the distracting nature of the conversation. Participants who overheard the one-sided conversation rated the conversation as significantly higher in distractibility than those who overheard the two-sided conversation. Also, participants in the one-sided condition scored higher on the recognition task. In particular they were more confident and accurate in their responses to words from the conversation than participants in the two-sided condition. However, participants’ scores on the anagram task were not significantly different between conditions. As in real world situations, individual participants could pay varying amounts of attention to the conversation since they were not explicitly instructed to ignore it. Even though the conversation was irrelevant to the anagram task and contained less words and noise, one-sided conversations still impacted participants’ self-reported distractibility and memory, thus showing people are more attentive to cell phone conversations than two-sided conversations. Cell phone conversations may be a common source of distraction causing negative consequences in workplace environments and other public places. Much of the research has used a simulated driving task to examine these effects and has found that drivers using cell phones are slower to change speed or direction While most research on cell phones has focused on drivers, more recent research has shown pedestrians are also affected by their cell phone usage. Pedestrians talking on cell phones have impaired visual attention while crossing the street Cell phone use can clearly have a negative impact on the user’s cognition particularly when the user is driving or crossing streets on foot. A question worth considering is whether there is any impact on bystanders who overhear cell phone conversations, since there are possibly millions of bystanders to the millions of cell phone conversations that take place yearly. Surveys have shown that 82% of respondents are annoyed by others’ cell phone use in public The only study thus far to examine the cognitive effects of one-sided conversations on bystanders is a study by Emberson, Lupyan, Goldstein, and Spivey Our study tests the cognitive effects of one-sided conversation and serves as a bridge between research done in a well-controlled laboratory setting and a completely naturalistic setting. We were able, with the help of confederates, to have a realistic situation in a controlled environment. In contrast to Emberson et al. In our experimental paradigm, participants were led to believe they were participating in a study examining the relationship between anagrams and reading comprehension. While the participant was completing an anagram task, the researcher left the room under the pretext of needing to make more copies. The participant overheard confederate(s) engaged in either a one-sided or two-sided conversation while the researcher was out of the room. After the researcher returned, she explained that the purpose of the study was to see how a conversation affected a person. The participant took a recognition test, assessing their memory for words from the conversation, and completed a questionnaire regarding the distractibility of the conversation. Based on abundant experimental and survey research showing the distracting nature of cell phones, we predicted one-sided conversations would be more distracting than two-sided conversations because of the unpredictable nature of a one-sided conversation. It was hypothesized that participants exposed to the one-sided conversation would be more distracted by it and would thus make more mistakes on the anagram task, but perform better on the memory task.This study and consent process received approval from the University of San Diego’s Institutional Review Board. Participants provided their written consent to participate in this study. All participants were 18 years of age or older.Participants were 164 undergraduate students taking an Introduction to Psychology class at the University of San Diego. Fifteen participants guessed the purpose of the study and their data was not analyzed. Of the remaining 149 participants, 110 were female and 39 were male, with a mean age of 18.48 years . The ethnicities of the 149 participants were: 117 Caucasian, 15 Asian-American, 14 Hispanic, and 2 African American, and 1 Pacific Islander. Participants were recruited from the Psychology Department’s participant pool. Introductory Psychology students were required to either earn five research participation credits or perform alternative assignments. This study counted for two credits.The demographics contained questions about age, gender, ethnicity, and primary spoken language. It also contained four questions about reading interests and word puzzles to maintain the perception that the study was about the relationship between anagram performance and reading comprehension.easy anagrams, where first and last letters were in the correct position so that only middle letters are scrambled , and hard anagrams, where all of the letters were scrambled (e.g. suohe = house). This procedure was based on the methodology of Foley, Foley, Wilder, and Rusch Anagrams were used as an attention measure since the participant attempted to complete them while the conversation was taking place. Two types of scrambled words were used: The conversation was a scripted conversation that lasted approximately seven minutes and covered three topics: a birthday party for dad, shopping for furniture, and meeting a date at the shopping mall. A one-sided conversation was conducted by a confederate talking on the cell phone; whereas two-sided conversations were carried out by two confederates speaking to each other. Confederates read the conversation off of a computer screen during the study. Each speaker said 506 words. Words that were tested on the recognition test only came from the conversation uttered by the speaker seated next to the participant. In both the one-sided and two-sided conversations, the confederate sitting next to the participant read from the same role in the script. None of the words on the recognition tests were repeated by the second speaker so that the participant only heard the recognition words one time, even if he/she overheard a two-sided conversation.Participants answered the following three questions to determine if they knew the purpose of the study: “What do you think the purpose of this study is?”, “Do you have any questions about this study?” and “Do you think there is any other purpose to the study? If yes, please describe fully.”A recognition test was used to assess memory. This test took place approximately four minutes after the conversation ended. It consisted of seventy words from five categories. Participants were tested on their ability to discriminate (1) actual words from the conversation, (2) related words , but not part of the conversation, and (3) other categories with varying relatedness to the conversation topic. Relatedness to the conversation topic was operationally defined as the number of words from that category that appeared in the conversation. For example, no words from the ‘beach’ category occurred during the conversation. In contrast, 10 words, such as “potatoes,” that fit into the food category appeared in the conversation. There were also six conversation words in the ‘school’ category and zero conversation words in the ‘household items’ category. None of these conversation food words appeared in the recognition test; for example “potatoes” did not appear in the recognition test, but a new word “salad” did. Mistakes were tracked to see if participants were more likely to make false alarms for food words as opposed to beach words. This procedure was meant to assess not only how many conversation words filter into the participant’s memory, but also what type of content was ‘picked up’ by the participant. During the recognition test, one word was shown on the computer screen at a time, and participants were asked to determine whether each word was part of the conversation they had just heard. To respond, the participant was asked to choose their confidence level on a scale of 1–6, from “definitely not” to “definitely yes.” Responses included: definitely not, probably not, maybe not, maybe yes, probably yes, and definitely yes.strongly disagree to strongly agree.A seven item self-report questionnaire was administered on the distracting nature of the scripted conversation. The questionnaire was adapted from Monk et al. At the end of the study, participants were asked about their thoughts regarding the study (“what were you thinking/feeling/considering doing?”), whether they were able to look at all the anagrams, and whether they were able to hear both sides of the one-sided conversation. They were also asked whether they had heard talk on campus about this particular study and were asked again about the purpose of the study.Subjects were randomly assigned to one of three conditions:two-sided conversation – participant heard both sides of a conversation taking place between two confederates.one-sided/cell phone conversation – participant heard one side of a cell phone conversation taking place between a confederate and the researcher who was not in the room.one-sided conversation with silent confederate – participant heard one side of a cell phone conversation, but there was also a second, silent, confederate present. This condition was used to determine if any differences between condition 1 and 2 were due to the different number of people in the room. It was thought that if the silent confederate appeared to ignore the conversation, the participant might also, making the results more similar to a two-sided conversation.Participants were led to believe they were participating in a study examining the relationship between the ability to unscramble anagrams and reading comprehension. Trained confederates also appeared to be participating in the same study for research credits. For the two-sided conversation (Condition 1) and the one-sided conversation with silent confederate (Condition 3), two confederates showed up for the study; while one confederate showed up for the one-sided conversation (Condition 2). One participant was tested at a time.The researcher indicated where each participant should sit. The room in which the experiment took place had three desks adjacent to each other, each with its own desktop computer. The desks were separated by a two-inch wide partial partition that extended approximately three feet from the wall and was five and a half feet high; the partition extended seven inches past the edge of the desks. The participant sat closest to the door and the confederates sat in the seats next to the participant, separated by the partition. The participant and the confederates always sat in the same seats so that the participant sat next to the confederate saying the half of the conversation that all participants would hear. This seating arrangement minimized differences between the volume of the words overheard that would be later tested on in the recognition task.After seating the participant and confederate(s) and administering the informed consent, the researcher described the study as investigating the relationship between anagrams and reading comprehension. The researcher explained that the results of the anagram task would be used to determine which group each participant would be placed in for the reading comprehension portion of the study, thus imparting some consequences on the participant’s anagram performance. The researcher gave the participant a copy of the anagrams to unscramble. The researcher then pretended to realize that the other anagram copies were bad copies in which half the anagrams were not printed. The researcher showed the participant and confederate(s) the bad copies, apologized, and explained that he/she needed to leave the room to make more copies of the anagrams for the other subject(s) (the confederate(s)). The researcher instructed the participant to go ahead and complete the anagrams since they were only part of the ‘pre-experiment’, and that the researcher would pick up the participant’s anagram answer sheet upon the researcher’s return. After the researcher left the room, the confederate either received a phone call from the researcher or engaged in a conversation with the other confederate. For one-sided conversations, the cell phone was placed out of view on the desk and was answered after the first vibration. During the one-sided conversations, the confederate sat next to the participant and read from the script. For the two-sided conversations, only the confederate sitting next to the participant said the words that were later included in the recognition task.Upon the researcher’s return, the participant was given the manipulation check in which they were asked to write down what they thought was the purpose of the study. The confederates were then excused and the participant was given a surprise recognition memory test on the computer using MediaLab (Empirisoft). Finally, the participant was asked a series of questions regarding the distracting nature of the conversation  on a Likert scale of 1–7, and were interviewed about their thoughts regarding the study. The participant was then debriefed, asked to maintain confidentiality regarding the study, and thanked for their time.Participants’ data were not analyzed if the participant guessed the purpose of the study (n = 15). This left 60 participants who overheard the two-sided conversation, 56 who overheard the one-sided conversation, and 33 participants who overheard the one-sided conversation with a silent confederate. A three-way analysis of variance (ANOVA) showed that the one-sided and one-sided with silent confederate conditions were not significantly different from each other on either the attention or memory tests (see Recognition test and d’) so these two conditions were combined, leaving 60 participants who overheard the two-sided conversation, and 89 who overheard the one-sided conversation. A Chi-square test comparing participants in the three conditions revealed that participants in the three conditions were not significantly different in regards to gender, ethnicity, and primary spoken language  or age . A seconA Cronbach’s alpha was used to determine whether the seven statements of the distractibility scale were measuring the same concept. The distractibility scale had a Cronbach’s alpha of.752. The item measuring the believability of the conversation had a corrected item-total correlation of.072. Since the believability was not related to the distractibility of the conversation, it was dropped from further distractibility analysis. Believability was significantly different between groups  = 5.102; p = .025. However, further analyses revealed that believability was not related to performance on either the anagram or recognition task, or to any performance differences between the one-sided and two-sided conditions. The Cronbach’s alpha for the remaining six questions on the distractibility scale was.796.An overall distractibility score was calculated for each participant by summing each participant’s responses to the remaining six items of the distractibility scale. An independent sample t-test indicated participants exposed to the one-sided conversation reported more distraction  than those exposed to the two-sided conversation  = 4.915, p<.001). A comparison between conditions indicated that the distractibility scale did not meet Levene’s test of equality of variances (F = 5.566 and p = .020) and therefore, we adjusted the degrees of freedom from 147 to 102.682.A multivariate analysis of variance (MANOVA) compared the responses between conditions to the individual questions on the distractibility scale . ParticiThe number of anagrams that were solved correctly and incorrectly was tallied. Incorrect anagrams included responses in which participants crossed out or erased their answers as well as wrong answers. Paired-samples t-test revealed that participants correctly solved more easy anagrams  than hard anagrams  = 26.150, p<.001; d' = Z(hit rate) − Z. Hit rate = hits/(hits+misses). False alarm rate = false alarms/. In cases where the response rate was 0, the response was converted to 1/(2N) where N = the number of trials For the recognition task, participants’ responses were measured in terms of d’, a formula that takes into account each participant’s ability to correctly discriminate stimuli amongst novel distractors A three way ANOVA comparison of the d’ for the on-sided condition versus the two-sided condition and one-sided condition with silent confederate revealed a main effect for conversation type.  = 5.487, p = .005). Post hoc tests revealed that one-sided condition vs. one-sided with silent confederate condition were not significantly different from each other, p = .521. However, the one-sided condition was significantly different from the two-sided condition, p = .002, and the one-sided condition with silent confederate was also significantly different from the two sided condition, p = .038. Since the one-sided condition and the one-sided condition with silent confederate were not significantly different from each other but both of these groups were significantly different from the two-sided group, we collapsed the data for the one-sided group with the one-sided with silent confederate group.There was a significant effect of conversation type on d’ . An indeThe confidence levels for different types of responses were compared. Participants were more confident in their correct rejections  than their misses  = 2.993; p = .003; The accuracy for each type of confidence rating was also calculated: 1) definitely (definitely yes and definitely not), 2) probably (probably yes and probably not), and 3) maybe (maybe yes and maybe not). A multiWe hypothesized that the self-report questionnaire, anagram task, and recognition task would reveal that participants who overheard the one-sided conversation would be more distracted than participants who overheard the two-sided conversation. As predicted, participants exposed to the one-sided conversation did report being more distracted by the conversation than those who overheard the two-sided conversation. In addition, those who overheard the one-sided conversation had a more accurate performance on the recognition task, and were more confident and accurate in their responses to words from the conversation. Although participants completed more easy than hard anagrams, and females performed better on the anagram task than males, there were no significant differences between the different conversation conditions on the anagram task.Several components of the distractibility questionnaire were significantly different between the two conditions. Participants who overheard the one-sided conversation rated the conversation as more noticeable, and distracting. They were also more surprised that the conversation took place than participants in the two-sided condition. Participants who overheard the one-sided conversation were also more likely to rate the content and volume of the conversation as annoying than those who overheard the two-sided conversation. These results are in agreement with those of Monk et al. The annoyance that participants who overheard the one-sided conversation felt is consistent with surveys that have shown people are annoyed by other’s cell phone use in public In regards to results of the anagram task, females have been shown to perform better than males on anagrams tasks In this study, participants who overheard the one-sided conversation performed better on the recognition memory task than those who overheard the two-sided conversation, indicating that they remembered more words that were said in the conversation. The one-sided condition’s better memory for the conversation occurred despite an experimental design in which participants were not asked to focus on the stimuli of interest. In memory studies, the researcher typically knows that the participants attended to the stimuli; for example, the participant is asked to study the stimuli or there is only one set of stimuli e.g., . Even inMany studies have examined performance regarding unattended stimuli e.g., . The resThere were also significant differences between conditions in the accuracy of high confidence responses on the recognition test. Participants in the one-sided condition were more likely to be correct when they said that words were “probably” part of (or not part of) the conversation. When participants were unsure about a word’s presence in the conversation and chose “maybe”, there were no significant differences between groups. Hoffman et al. The main strength of the current study is its realistic design. Participants were led to believe that the conversation was not part of the study. Similar to cell phone conversations in natural settings, the conversations in our study had an element of surprise and bystanders to the conversation decided themselves whether or not to attend to the conversation. When people are distracted by a conversation while working on a task, they are not usually warned a conversation will take place. Moreover, if participants had been asked to ignore the conversation, it might lessen the generalizability of the study. The current study is also unique in several other regards. First, the participants heard the conversation only one time as people would in naturalistic settings. Second, participants were tested on two cognitive abilities, attention to the anagram task during the conversation and memory for the conversation after it ended.A possible limitation of this study may be the difference in number of words overheard in the two types of conversations. Compared to the two-sided conversation, those who overheard the one-sided conversation heard only half of the words. It’s possible that participants who overheard the one-sided conversation performed better on the recognition task because of experimental confounds. Participants in the one-sided condition heard less noise and had less information to remember, and possibly the confederates spoke louder during the one-sided conversation than the two-sided conversation. We did not control for the difference in sound and word count which may have contributed to the differences in distraction. Nevertheless, in Emberson et al. Although the volume for each conversation type was not recorded, attempts were made to ensure that each participant was tested under similar conditions. For example, in order to reduce between-subject volume differences, only the confederate sitting next to the participant said the words that were tested on the recognition task. While participants in the one-sided conversation were more likely to rate the volume as annoying, the average rating for the one-sided conversation was 4.35 on a scale of 1 to 7, midway between a score of 4, neither distracting nor not distracting and 5, slightly distracting. Thus it seems that the volume of the one-sided conversation was only mildly annoying. It’s possible that participants rated the volume of the one-sided conversation as more annoying even though there were no volume differences between the conversations. Prior research has shown that participants can rate the volume of one-sided conversations as more annoying even when volume is controlled Although, we did not record volume levels for the conversations described in this study, we have completed a follow-up study in which volume was recorded for 54 participants . The conversation in the follow-up study was not identical to the one described in this paper, but was centered on the same list of words and was thus tested with the same recognition test. Although, there were no significant differences in volume between the two groups , the d’ for the 1 one-sided conversation was significantly greater than the d’ for the two sided conversation (p = .028). Since both studies were conducted using the same procedure in the same room, and d’ was significantly different despite equal volume levels in the follow-up study, we have reason to think volume probably played a negligible role in this study.It is likely that cell phone conversations would impact a bystander’s cognition. Monk and colleagues This study is the first to have observed cognitive effects of cell phone conversations on bystanders in a realistic context. Participants rated the cell phone conversation as more distracting and were more likely to remember content from the conversation, even though they were working on another task and were unaware that the conversation was part of the study. These results have implications for workplace environments, transportation hubs and other public areas. Future studies should explore how attention and cognitive effects of cell phone use vary as a function of conversation volume and content. Additionally, it will be important to determine what types of tasks are subject to performance impairments by overheard cell phone conversations.