In addition, PTENwt cells upon PTEN depletion showed mTORC2 activation. The reduced mTORC2 signaling in PTENwt cells was related to higher Rictor phosphorylation at Thr1135 residue. Phosphorylation of Rictor at Thr1135 inhibited its association with mTORC and thus there was a reduction in mTORC2 complex formation. In addition, PTENwt cells expressing mutated Rictor in which Thr1135 was substituted with alanine, showed enhanced mTORC2 formation and signaling. This enhanced mTORC2 signaling promoted inactivation of GSK3β. Thus, we established the reciprocal activation of mTORC2 and GSK3β in GBM. To the best of our knowledge, this is the first report describing role of PTEN in mTORC2 formation by promoting Rictor phosphorylation (Thr1135) in GBM. Furthermore, the drug sensitivity of mTORC2 was evaluated. A newly identified carbazole alkaloid, mahanine, showed cytotoxicity in both PTENmu and PTENwt cells. It inhibited both mTORC1/2 and AKT completely in PTENmu cells, whereas it inhibited only mTORC1 in PTENwt cells. Cytotoxity and AKT-inhibitory activity of the mTORC1/2 inhibitor was increased either by depleting PTEN or in combination with phosphatidylinositol 3 kinase inhibitors in PTENwt cells. In contrast, depletion of Rictor decreased the cytotoxicity of the mTORC1/2 inhibitor in PTENmu cells. Thus, PTEN has an important role in mTORC2 formation and also influences the effectiveness of an mTORC1/2 inhibitor in GBM.To investigate the role of PTEN (phosphatase and tensin homolog) in mammalian target of rapamycin complex 2 (mTORC2) signaling in glioblastoma multiforme (GBM), we found higher activation of mTORC2 in PTEN However, inhibition of mTORC1 induces the activation of other survival pathways and thus reduces the efficacy of rapalogs.19 Therefore, targeting mTORC2 may possibly have better therapeutic values.20 Accordingly, exploration of new improved mTORC1/2 inhibitors is of great interest.mTORC1 promotes protein translation through activation of S6K1, inhibition of 4E-BP1 and enhancement of RNA translation via S6 ribosomal protein.21 Cellular cross-talk between PTEN and mTORC1 via the phosphatidylinositol 3 kinase (PI3K)/AKT/mTOR pathway is often deregulated and enhances the malignancy.23 Although the role of mTORC1 is well characterized, the function and regulation of mTORC2 is still poorly understood.PTEN (phosphatase and tensin homolog), a tumor suppressor protein, is often inactivated in cancers.PTEN, EGFR, TP53, CDKN2A, CDK4 and LOH are several frequently mutated genes.24 PTEN mutations are frequently involved with this aggressive cancer, our initial aim was to decipher the regulation of mTORC2 with respect to PTEN wild-type (PTENwt) vs mutated conditions  is a grade IV brain tumor with higher mortality rate.  PTENmu; . This inwt cells. Furthermore, we confirmed a positive correlation between enhanced mTORC2 formation and sensitivity toward mahanine in PTEN-mutated cells. Mahanine also inhibited mTORC1 activity and may thus be considered as a potential mTORC1/2 inhibitor.Here we provide evidence for PTEN-mediated regulation of mTORC2 in GBM. We showed that PTEN mutations lead to reduced phosphorylation of Rictor at Thr1135, which in turn promotes enhanced mTORC2 formation and downstream signaling. However, higher phosphorylation of Rictor at Thr1135 leads to the reduction of mTORC2 formation in PTENmu (U87MG) and PTENwt (LN229) GBM cells and the status of PTEN was verified  in PTENwt cells , which ewt cells , demonstmu cells , confirmmu cells  and the mu cells . These fmu cells . This semu cells . As mTORmu cells .mu cells , U373MG (PTENmu) and LN18 (PTENwt). Both PTENmu cells were hyperactive in mTORC2 signaling as reflected by higher phosphorylation of mTOR (Ser2481) in contrast to PTENwt  phosphorylation compared with PTENwt  is known to inhibit mTORC2 activity as evidenced by decreased AKT Ser473 phosphorylation.wt cells . To invef Rictor . This reh PTENwt , consistwt cells with either wild-type pRK-5/Rictor (myc-tag), the T1135A mutant of pRK-5/Rictor (myc-tag) or the empty vector alone and checked the association of exogenous Rictor and endogenous mTOR by co-immunoprecipitation. Cells expressing mutated Rictor (T1135A) showed enhanced association with endogenous mTOR in comparison with wild-type Rictor/empty vector-transfected cells  may revert back because of the increased activity of S6K1. Therefore, the actual effects of PTEN were masked by the increased activity of S6K1. To reveal the definite effects of PTEN depletion upon Rictor phosphorylation, we first inhibited the S6K1 phosphorylation by disrupting active mTORC1 formation, as mTORC1 is responsible for phosphorylation of S6K1. To inhibit mTORC1 activity, we knocked down Raptor in the PTENwt cells and observed reduced phosphorylation of both S6K1 (Thr389) and mTOR (Thr2448) (wt cells triggered a complete reduction in Rictor Thr1135 phosphorylation as well as increased phosphorylation of mTOR (Thr2481). Thus, PTEN depletion coupled with S6K1 deactivation enhances mTORC2 activity through Rictor dephosphorylation (Thr1135).However, it is unusual that one phosphatase can induce hyperphosphorylation. It has been reported that S6K1 is responsible for the phosphorylation (Thr1135) of Rictor. Earlier, we found that upon PTEN depletion there was an increase in S6K1 phosphorylation (Thr389), that is, the signature of active S6K1 . AccordiThr2448) . In consThr2448) . Simulta25 It displayed a cytotoxic effect against PTENmu and PTENwt cells  was decreased only in PTENmu cells in contrast to PTENwt, where phosphorylation at this site was increased  when mTORC2 was not in significant level and mainly mTORC1 was present  positivity compared with mock-transfected cells  showed lower PI positivity, indicating lower cell death in PTENmu cells compared with PTENwt  and therefore the addition of a GSK3β-inhibitor did not exert any extra effect. In contrast, an mTORC1/2 inhibitor (mahanine) exhibited significantly enhanced cytotoxicity in PTENmu cells. This was further confirmed by demonstrating higher G0/G1 phase cell cycle arrest . This is the first evidence suggesting Thr1135 phosphorylation of Rictor is controlled by PTEN, and demonstrates PTEN as the master regulatory molecule in mTORC2 formation. In addition, we have established mahanine, carbazole alkaloid from an edible plant, as a potential mTORC1/2 inhibitor that showed cytotoxicity both in PTENmu/PTENwt cells.Mutation of PTEN is associated with high-grade GBM formation. mTORC2 is emerging as a potential drug target in PTEN-mutated cancers. The detailed mechanism of molecular regulation and cross-talk between PTEN and mTORC2 is still not clearly known. Accordingly, we investigated the role of PTEN in mTORC2 regulation and signaling in GBM. The major achievement of this study is providing evidence that PTEN36 EGFRvIII-mediated activation of mTORC2 was reversed by PTEN activity.37 A correlation between PTEN and Rictor was also reported in leukemogenesis.38 Here we showed that PTENmu cells have higher mTORC2 formation and downstream signaling than PTENwt cells as evidenced by enhanced mTOR Ser2481, AKT Ser473 and GSK3β Ser9 phosphorylations. Although a correlation between PTEN and AKT Ser473 phosphorylation was reported in lung cancer and rhabdomyosarcomas, no cross-linking molecule between these phosphatase and kinase was demonstrated.40 Here, we showed mTORC2 is a connecting molecule between PTEN and AKT.mTORC2 activity is required for generation of PTEN deletion-induced prostate cancer.34 Therefore, reduced mTORC2 activity and AKT Ser473 phosphorylation in PTENwt cells is a consequence of increased Rictor Thr1135 phosphorylation. This event was confirmed when we knocked down PTEN in PTENwt cells and observed reduced Rictor phosphorylation, and as a consequence, enhanced mTORC2 signaling. Although Rictor Thr1135 phosphorylation is responsible for decreased activity of mTORC2, reports suggest this had no effect on mTORC2 complex formation.43 However, the detailed mechanism behind reduced mTORC2 activity without hampering mTORC2 complex formation was not revealed. Our data revealed that not only was downstream signaling affected, but also there was differential mTORC2 complex formation in PTENwt and PTENmu cells. PTENwt cells lack significant mTORC2 formation, and that was reverted with PTEN knockdown. Therefore, we conclude that in the presence of PTENwt, there is enhanced Rictor Thr1135 phosphorylation, decreased mTORC2 formation, which connected to lower AKT activity, and increased GSK3β activity. The role of PTEN in regulating Thr1135 phosphorylation and mTORC2 complex formation was further demonstrated by expressing T1135A mutant Rictor in PTENwt cells. We observed significant mTORC2 complex formation and downstream signaling in the presence of PTEN when there was a mutant Rictor that cannot be phosphorylated on Thr1135. Therefore, we concluded that this may be a unique phenomenon in GBM where phospho-Rictor Thr1135 could not be recruited to mTORC2 in the presence of PTENwt.During exploration of mTORC1/2-associated molecules, we observed that Rictor is differentially phosphorylated (Thr1135) with respect to the functional existence of PTEN. Rictor phosphorylation (Thr1135) is responsible for decreased activity of mTORC2 and reduced AKT (Ser473) phosphorylation.34,43 This downstream kinase is known to be a linker molecule between mTORC1/2. Therefore, we checked mTORC1 activity and found equivalent mTORC1 formation and downstream activity as judged by comparable phosphorylation of downstream signaling molecules (S6K1 and 4E-BP1). This data reconfirmed the involvement of PTEN in regulation of mTORC2 in GBM. Therefore, S6K1 may not be solely responsible for controlling Rictor (Thr1135) phosphorylation. Simultaneous suppression of S6K1 activity and functional PTEN in PTENwt cells completely abolishes Rictor phosphorylation (Thr1135) and enhances active mTORC2 formation. However, PTEN, being a phosphatase, cannot phosphorylate a molecule directly. We assume that PTEN may be affecting some other kinases by removing inhibitory phosphorylation, and they in turn phosphorylate Rictor at the specific site. Alternatively, it may also be possible that PTEN dephosphorylates some other site of Rictor that enables the molecule to change its conformation in such a way so that the Thr1135 site is exposed for further phosphorylation by known kinases like S6K1. Experiments to distinguish between these possibilities are ongoing.Thr1135 phosphorylation of Rictor is also controlled by S6K1 via mTORC1.mu and PTENwt cells thus behaved as an mTORC1/2 inhibitor. However, in PTENwt cells, where mTORC2 was not significantly active and mTORC1 is predominant, mahanine behaves like an mTORC1 inhibitor. Here we observed an activation of the AKT pathway in PTENwt cells. Several reports indicated that mTORC1 inhibition could lead to activation of AKT by other pathways.45 This was due to release of the negative feedback loop via S6K1 leading to the activation of PI3K/AKT pathway.44 mTORC1 inhibition also leads to MAPK pathway-dependant AKT activation.45Basic understandings at the cellular and molecular level are successful when a translational or therapeutic approach works out positively. We have observed mahanine exerted cytotoxicity both in PTENwt cells in the presence of specific PI3K inhibitors. We also observed a synergistic effect when a combination of mTORC1/2 and PI3K inhibitors were used to overcome this AKT activity in PTENwt cells. These data suggest that for an effective therapeutic strategy both mTORC1/2 and PI3K inhibitors may be necessary for a beneficial effect in PTENwt GBM.This activation of AKT was reversed in PTENwt cells.Our findings demonstrated that PTEN behaved like a physiological inhibitor of mTORC2 formation. Generation of mTORC2 upon PTEN depletion provided the specific target of mahanine and hence cell death was increased in PTENmu cells where mTORC2 and AKT are active and GSK3β is inactive, treatment with GSK3β inhibitors did not add any extra benefit. In contrast, a GSK3β inhibitor exerted a significant effect on PTENwt cells where GSK3β is active.We have observed a reciprocal relationship between mTORC2 and GSK3β in GBM. Therapeutically, we also proved this reciprocal relationship. In PTEN47 Our results convincingly showed the mechanism whereby PTEN mutations increase the sensitivity of cancers to mTORC1/2 inhibitors. We have established loss of PTEN induces more mTORC2 formation, and more susceptible to mTORC1/2 inhibitors. This hypothesis was further proven when we depleted Rictor from PTENmu cells, which had abundant mTORC2 formation. Depletion of Rictor leads to decreased mTORC2 formation, and therefore they behaved like PTENwt cells, with predominant mTORC1. Accordingly, mTORC1/2 inhibitors acted as only mTORC1 inhibitors and we observed a reduction in cell death. Therefore, mTORC2 is the prime factor that is responsible for differential sensitivity to mTORC1/2 inhibitors. PTEN showed its effect by inhibiting Rictor and thereafter mTORC2 kinase activity in PTENwt cells.Studies have suggested that mTORC1/C2 inhibitors have more effect on PTEN-mutated cancers.wt/PTENmu cells, however, these cells activate AKT as evidenced by the enhanced Thr308 and Ser473 phosphorylation. Phosphorylation of 4E-BP1 remained unchanged whereas S6K1 was decreased  cells and mahanine behaved like an mTORC1 inhibitor and enhanced AKT activity by the inhibition of a negative feedback loop.To correlate our observation, we found that mahanine inhibited AKT in two PTENTherefore, it is envisaged that PTEN-dependent mTORC1 and mTORC2 dynamics are not restricted only to the GBM system and could be a global phenomenon. Our findings will open up a new avenue of thought for designing modified and efficient therapy against malignancy. However, more in-depth studies are needed to uncover the molecular function of PTEN in regulation of mTORC2.All the antibodies were from Cell Signaling Technology : mTOR (cat #2983), phospho-mTOR , phospho-mTOR , Rictor (cat #9476), phospho-Rictor , Raptor (cat #2280), AKT (cat #4691), phospho-AKT , phospho-AKT , phospho-AKT , GSK-3β (cat #9315), phospho-GSK-3β , PTEN (cat #9559), β-actin (cat #4970), 4E-BP1 (cat #9644), phospho-4E-BP1 , S6K1/p70S6K (cat #9202), phospho-S6K1/phospho-p70S6K , Myc-Tag (cat #2276), horseradish peroxidase-linked anti-rabbit IgG (cat #7074), horseradish peroxidase-linked anti-mouse IgG (cat #7076). Lipofectamine LTX, plus reagent, culture medium were from Invitrogen . Antibiotic–antimycotic, propedium iodide (PI), rapamycin, GSK3β inhibitor (SB212763), PI3K inhibitor (LY294002 and wortmannin) and other chemicals were from Sigma–Aldrich . Protease and phosphatase inhibitor cocktails were from Calbiochem . Cycle Test Plus kit was from BD Bioscience . Super Signal West Pico imaging system was from Thermo Scientific .mu  and PTENwt (LN229 and LN18) human GBM cell lines  were treated with various inhibitors/drugs under identical conditions.PTENll lines  were fromu/PTENwt cells were transiently transfected with human PTEN/Rictor/Raptor targeting siRNAs . Developed bands were densitometrially analyzed by ImageJ software .25Drug/inhibitor treated or siRNA/plasmid transfected or untreated/untransfected cells were harvested and lysed and processed for western blot analysis.oC. Immunocomplex was incubated with protein A-sepharose 4B, washed with ice-cold phosphate-buffered saline, resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently identified using the respective antibodies.For detection of the mTOR complexes, cells were lysed in ice by sonication . Protein (200 μg) was incubated with anti-mTOR antibody (1:100) overnight at 4 31 Cell viability of mahanine (0–30 μM)-treated cells (1 × 104) was checked after 48 h. They were further incubated with MTT (100 μg/ml) for 3 h. Formazan crystals were dissolved in dimethylsulfoxide and optical density was taken at 550 nm.25 Cells (1 × 106) were treated with mahanine, SB216763, Wortmanin and LY294002 alone or in combination for 24 h and processed using Cell Cycle Test Plus kit.25 At least, 20 000 cells were acquired and analyzed by flow cytometry using CellQuest Pro software .49 Morphology of mahanine-treated cells were imaged by transmitted light digital inverted microscope .Mahanine was purified as described previously.t-test or Mann–Whitney U-test. Standard error bars represent the s.d. of the mean (±s.d.) and *P<0.05, denoted the significant differences between the means of the untreated and treated cells or two test groups.All the data were from at least three independent experiments and statistical analysis was performed using Graph Pad Prism 5 . The differences between the groups were analyzed by two tail Student's


DK ∼ 30 nm). Precomplexing FcεR1α with IgE potently inhibited the FcεR1α-PEAR1 interaction, and this was relieved by the anti-IgE therapeutic omalizumab. Oligomerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was also inhibited by IgE. These findings demonstrate how a protein microarray resource can be used to gain important insight into the function of platelet receptors and provide a mechanistic basis for the initiation of PEAR1 signaling in platelet aggregation.Genome-wide association studies to identify loci responsible for platelet function and cardiovascular disease susceptibility have repeatedly identified polymorphisms linked to a gene encoding platelet endothelium aggregation receptor 1 (PEAR1), an “orphan” cell surface receptor that is activated to stabilize platelet aggregates. To investigate how PEAR1 signaling is initiated, we sought to identify its extracellular ligand by creating a protein microarray representing the secretome and receptor repertoire of the human platelet. Using an avid soluble recombinant PEAR1 protein and a systematic screening assay designed to detect extracellular interactions, we identified the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) as a PEAR1 ligand. FcεR1α and PEAR1 directly interacted through their membrane-proximal Ig-like and 13th epidermal growth factor domains with a relatively strong affinity ( PEAR1 have been repeatedly linked to natural variation in response to platelet agonists in several independent studies , arrays of monomeric biotinylated “bait” proteins are screened against multimerized, enzyme-tagged, highly avid “preys”  receptor subunit α (FcεR1α) and show that multimerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was specifically inhibited by IgE.in vivo. Similarly, for multimeric protein complexes such as integrins, glycoprotein 1bαβ, and fibrinogen, care was taken to design constructs that would promote correct and active complex formation. The truncated fragments of PEAR1 and FcεR1α were designed based on their domain structures, amplified by PCR from the full-length constructs, and cloned into an expression vector containing an exogenous signal peptide and C-terminal His6 and biotinylatable peptide sequence. The 218- and 325-amino acid fragments encoding the Cε3–4 and Cε2–4 domains of the IgE constant heavy chain were amplified from plasmid pFUSE-CHIg-hE (Invivogen) and cloned into a vector containing an exogenous signal peptide and C-terminal His6 tag. IgE fragments were expressed and purified as described below. All plasmid constructs are openly available from Addgene.After compiling a list of platelet receptor proteins and classifying them into structural categories, the extent of each ectodomain was identified by careful manual examination of its structural features such as signal peptides, transmembrane regions, and glycosylphosphatidylinositol anchors; for secreted proteins, the entire protein was used. The ectodomain regions were codon-optimized for human expression, chemically synthesized with unique flanking NotI (5′) and AscI (3′) restriction sites (GeneArt AG), and subcloned into bait expression plasmids according to its structural class. Although we have previously expressed type I/glycosylphosphatidylinositol-anchored and secreted proteins for interaction screening , we desi6 tag using a bespoke supernatant loading rig and 96-well Ni2+-nitrilotriacetic acid filter plates  for 1 h and finally detected by tyramide signal amplification Alexa Fluor 555 substrate (Invitrogen) for 1 h. Between different incubation steps, slides were washed three times in PBS buffer containing 0.1% Tween 20 with gentle rocking. Arraying, incubations, and washing steps were performed at 22 °C. Positive interactions were identified and quantified by scanning slides with a ScanArray Express microarray scanner (PerkinElmer Life Sciences) at 550 nm.Normalized bait proteins were diluted in phosphate-buffered saline (PBS) supplemented with 50% glycerol, 0.02% Tween, and 0.5% bovine serum albumin (BSA) prior to printing. Bait proteins were printed on streptavidin-coated slides that also contained an inert hydrogel coating (XanTec) using a Marathon arrayer (Arrayjet) at 60% relative humidity according to the manufacturer's instructions. Printed slides were incubated for 1 h at 60% relative humidity and blocked with PBS containing 1% BSA and 10 mTo determine the effect of IgE/omalizumab and map the interacting domains of the FcεR1α-PEAR1 interaction, we used the AVEXIS method formatted on streptavidin-coated 96-well microtiter plates as described , 17. BriAll surface plasmon resonance studies were performed on a Biacore T100 instrument essentially as described . Purifie5/μl) were preincubated with either soluble recombinant purified pentameric proteins extensively dialyzed into PBS (PEAR1 (s5-PEAR1), FcεR1α (s5-FcεR1α), and control rat Cd200 all used at 10 μg/ml) or the commercially available antibodies human IgE (10 μg/ml (53 nm); Abcam), anti-PEAR1 , and omalizumab (10 μg/ml (67 nm); Xolair®, Novartis) at 37 °C as appropriate. The IgE fragments were used at an equimolar concentration of 67 nm. Platelet aggregation was triggered by collagen with the concentration adjusted to reach 40–70% platelet aggregation for each donor and measured as the percentage of change in light transmission relative to a blank (buffer without platelets) set to 100%.Venous blood was collected from normal donors following guidelines from the ethics committee of the Leuven University Hospital (number B322201111373/S53239). Platelet-rich plasma was prepared, and platelet aggregation was monitored as described . PlateleWestern blotting was performed essentially as described  using thPlatelets were stained essentially as described  using thA). In the case of multimeric protein complexes such as integrins, glycoprotein 1bαβ, and fibrinogen, care was taken to design the constructs such that they would promote correct and active complex formation; for example, secretion of integrin α chains can be dependent on the presence of the β chain . We next compiled a list of secreted and membrane proteins expressed by human platelets from 18 proteomic data sets and the literature . To increase the chances of identifying functionally relevant interactions, each protein was classified into one of six structural categories. The entire extent of the ectodomain region was then determined, and an expression construct was manually designed by pairing it with an appropriate expression vector. The final library contained 173 proteins and complexes  represented by a total of 178 plasmids . The proteins were expressed and purified as monomeric biotinylated “baits” in mammalian cells so that structurally important posttranslational modifications such as disulfide bonds and glycans were added. Similar to other secreted recombinant protein libraries that we . After purification, 126 proteins were expressed at sufficient levels for our interaction screens. As expected, Western blots of the protein library showed that the vast majority  of proteins exhibited mass heterogeneity centered on their expected size, suggesting the presence of different glycoforms . The recombinant proteins were antigenically active as assessed by demonstrating heat-labile immunoreactivity to a panel of monoclonal antibodies recognizing at least one protein from each structural category . The library of biotinylated bait proteins was serially diluted and arrayed on streptavidin-coated glass slides . Proteins immobilized on the slides retained their immunoreactivity to monoclonal antibodies known to stain native proteins at the cell surface . These plasmids and recombinant protein library represent a valuable resource for the investigation of human platelet biology particularly in regard to the role of these proteins in thrombosis.To identify an activating ligand for PEAR1, we first created a protein library that represented the cell surface receptor repertoire and secretome of the human platelet expressed as secreted recombinant proteins. We and others have previously only expressed type I/glycosylphosphatidylinositol-anchored and secreted proteins for large scale extracellular interaction screening –14, but Fig. 1 β chain , and so  β chain  . Screening the receptor microarray with the PEAR1 prey identified the high affinity IgE-binding subunit FcεR1α as a PEAR1 ligand . Because PEAR1 was presented as a bait on the microarray, to rule out any possible artifacts, we expressed the entire ectodomain of FcεR1α as a prey protein and rescreened the array. As expected, we observed that the FcεR1α prey interacted with the PEAR1 bait, showing that we could detect the same interaction but in the reciprocal bait-prey orientation .To identify an activating ligand for PEAR1 during platelet aggregation, we systematically screened the human platelet receptor microarray using the AVEXIS assay, which required expressing the entire ectodomain of PEAR1 as a recombinant pentameric, FLAG-tagged soluble “prey.” Probing the array with a control pentameric FLAG-tagged prey protein labeled the high affinity IgG receptor Fcγ2α (which directly bound the anti-FLAG antibody) and P4HB, a bait that presumably interacted with the tags on the control prey A. ScreenDK) of 27.4 ± 1.3 nm (A). The saturable binding behavior demonstrated the specificity of the interaction, which had a remarkably high affinity when compared with similar receptor-ligand interactions, which are usually in the micromolar range when measured using the same approach (B). By using the AVEXIS assay and a series of structure-guided truncations in both PEAR1 and FcεR1α, we showed that the minimal FcεR1α binding unit on PEAR1 was contained solely within the 45 amino acids comprising the 13th epidermal growth factor domain (A and C). Similarly, the PEAR1 binding site was located within the membrane-proximal Ig-like domain of the FcεR1α ectodomain, the same domain bound by IgE (B and C), raising the possibility that IgE may prevent PEAR1 binding when complexed with its receptor. Indeed, IgE, when bound to FcεR1α, could potently inhibit the FcεR1α-PEAR1 interaction at low concentrations, consistent with the high affinity of IgE for FcεR1α (D). To show that this inhibitory effect was due to blocking of the FcεR1α-PEAR1 interaction and not steric interference with the large (190-kDa) IgE molecule, we demonstrated that smaller subfragments of the IgE heavy chain that bound FcεR1α also blocked the FcεR1α-PEAR1 interaction (E). To demonstrate the specificity of this effect, we showed that addition of an anti-IgE monoclonal antibody that prevents IgE binding to FcεR1α  could relieve the inhibitory effect of IgE on the FcεR1α-PEAR1 interaction (F).To validate and quantify the interaction, we used surface plasmon resonance and observed clear saturable binding between PEAR1 and FcεR1α with an equilibrium binding constant . Unlike PEAR1 (A). The specificity of this effect was demonstrated by precomplexing the FcεR1α pentamers with IgE to completely block FcεR1α-mediated PEAR1 signaling, suggesting the possibility that IgE could act as an endogenous plasma-borne restrictive regulator of thrombus formation (B). PEAR1 oligomers modestly inhibited platelet aggregation (A) and did not trigger known FcεR1α signaling effectors such as the phosphorylation of PLCγ (data not shown), suggesting that they competed with membrane-tethered PEAR1 for FcεR1α binding. Finally, oligomerized FcεR1α, but not a control, was able to trigger PEAR1 phosphorylation with similar potency to an activating anti-PEAR1 antibody (C) and led to clear AKT phosphorylation, a known mediator of PEAR1 signaling .To investigate the role of the FcεR1α-PEAR1 interaction in platelet activation, we confirmed cell surface expression of PEAR1 , 9 and Fke PEAR1 , we did ke PEAR1 A. The spormation B. PEAR1 regation A and didantibody C and ledignaling  , of any  that is currently licensed for the treatment of severe persistent allergic asthma . It is a


Division site selection presents a fundamental challenge to all organisms. Bacterial cells are small and the chromosome (nucleoid) often fills most of the cell volume. Thus, in order to maximise fitness and avoid damaging the genetic material, cell division must be tightly co-ordinated with chromosome replication and segregation. To achieve this, bacteria employ a number of different mechanisms to regulate division site selection. One such mechanism, termed nucleoid occlusion, allows the nucleoid to protect itself by acting as a template for nucleoid occlusion factors, which prevent Z-ring assembly over the DNA. These factors are sequence-specific DNA-binding proteins that exploit the precise organisation of the nucleoid, allowing them to act as both spatial and temporal regulators of bacterial cell division. The identification of proteins responsible for this process has provided a molecular understanding of nucleoid occlusion but it has also prompted the realisation that substantial levels of redundancy exist between the diverse systems that bacteria employ to ensure that division occurs in the right place, at the right time. Current Opinion in Microbiology 2014, 22:94–101Growth and development: prokaryotesThis review comes from a themed issue on Frédéric BoccardEdited by Issue and the EditorialFor a complete overview see the Available online 17th October 2014http://dx.doi.org/10.1016/j.mib.2014.09.020http://creativecommons.org/licenses/by/3.0/).1369-5274/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license . During replication the newly synthesised sister chromosomes rapidly segregate ‘origin-first’ in opposite directions, before the replication forks rendezvous and terminate in the terminus region . Once chromosome replication and segregation are complete the cell is ready to divide. In Bacteria this normally occurs by binary fission and in almost all species this is initiated by the assembly of the tubulin homologue FtsZ into a ring-like structure (‘Z-ring’) at the nascent division site  can itself act as a cell cycle ‘checkpoint’ and prevent division until the replicated sister chromosomes have segregated—a process termed nucleoid occlusion , 17, 18.nucleoid occlusion proteins were identified. Noc in B. subtilis .••.B. subs (SBSs) a [25, 26B. subtilis [E. coli [er regions  and the observation of large ribbons of FtsZ-SlmA-SBS by electron microscopy Tonthat et al.  proposed [et al.  presente in vivo , 27•. Mo in vivo .E. coli, Klebsiella pneumoniae and Vibrio cholerae have revealed that SlmA binds the SBS as a dimer of dimers [c.a. 4–8 dimers of SlmA per SBS. If multiple dimers of SlmA are also present in vivo this could allow nucleation of FtsZ filaments within non-productive structures, and remain compatible with the observation that only one FtsZ binding site per dimer is required for activity. Despite this possibility, such a mechanism now seems unlikely. Du and Lutkenhaus [in vitro under all conditions previously tested. Crucially, the identification of FtsZ mutants that are insensitive to this activity even though they can still interact with SlmA-SBS demonstrates unambiguously that SlmA plays an active role in disassembling FtsZ polymers  and forms large nucleoprotein complexes at the NBSs [Understanding the mechanism by which Noc acts has proved more challenging. In the NBSs ••. It lothe NBSs ••. An eathe NBSs , 30. RecB. subtilis poses additional challenges  was recently shown to promote the redistribution of Z-rings from mid-cell to the cell poles  of E. coli [Ter MD at mid-cell so that it can be processed by FtsK [et al. [Ter appears to leave mid-cell just prior to constriction, might this process also communicate the completion of chromosome segregation? [Another possibility is that the activity or organisation of the nucleoid may itself play a role in restricting Z-ring assembly . One cla E. coli  is recru E. coli •. This c by FtsK , 49, 50. [et al. , suggest [et al. . Interesegation? •.Streptococcus pneumoniae this process may play a more prominent role, especially given the small volume of these cells and the observation that the Z-ring appears to assemble on top of unsegregated nucleoids [A third distinct possibility is that DNA-translocases present in the division machinery such as FtsK/SpoIIIE proteins might clear the DNA from beneath the closing septum , 53. In ucleoids , 54, 55.ucleoids , 35•.B. subtilis min noc background the frequency of Z-ring assembly was both dramatically reduced and subject to a significant delay. Nevertheless, in a small population of cells there was a substantial preference for Z-ring assembly at mid-cell, independently of either the Min system or the nucleoid [In contrast to the well-studied systems that negatively regulate division site selection, it has been proposed that additional mechanisms might exist that act positively, for example, by contributing an essential division component or by counteracting an inhibitor , 56. Rodnucleoid •, which nucleoid •. CurrenStreptomyces spp., which are multi-nucleate filamentous bacteria that produce long chains of spores, FtsZ initially assembles extended ‘spiral’ structures on top of the nucleoids during sporulation, before forming a ‘ladder’ of Z-rings between the segregated nucleoids [et al. [Schizosaccharomyces pombe, whereby the nucleus restricts the localisation of Mid1p, which acts positively to position the division site [Positioning at midcell of FtsZ) was found to positively regulate Z-ring positioning in Myxococcus xanthus, although it probably acts indirectly since a direct interaction with FtsZ could not be established [pomZ cells never occurred over the nucleoids, indicating that M. xanthus probably contains a nucleoid occlusion system [In ucleoids . Remarkaucleoids ••. But h [et al. •• highliion site , 60. Morablished . Interesn system .Caulobacter crescentus lacks obvious homologues of MinCD or nucleoid occlusion factors, an alternative mechanism has been identified that combines aspects of both systems. MipZ (mid-cell positioning of FtsZ), a divergent ParA/MinD family ATPase, is essential for the correct placement of the division site [Corynebacterineae (see for [Corynebacterium glutamicum PldP, an orphan ParA-like division protein, localises over the nucleoid and at the nascent division site [C. glutamicum [Mycobacterium smegmatis, but whether it acts directly remains unclear [Although ion site ••. It foion site ••. Strikion site . Consequion site , 63. Nevion site . Interes(see for  in-depthion site . Since nutamicum , 67. Sim unclear .B. subtilis and E. coli, Noc and SlmA also play integral roles in helping to specify the spatial and temporal organisation of division. Noc may also play a role in restricting division to one plane in S. aureus. Nonetheless, it is clear that in some bacteria other factors may act to protect the DNA at later stages of division, for example, DNA translocases. One important area of future research will be how nucleoid occlusion varies with growth rate, particularly since during slow growth, in E. coli at least, the chromosomes appear to segregate as cell division initiates [V. cholerae that have more than one chromosome? As the range of organisms studied and the development of novel genetic and cell-biology tools continues to rapidly expand, we anticipate new insights into how diverse organisms tackle this fundamental problem.The identification of Noc and SlmA provided a molecular basis for nucleoid occlusion and considerable progress has been made in the last decade in understanding the mechanisms by which these factors act. While the primary role of nucleoid occlusion is almost certainly to prevent catastrophic guillotining of the genetic material, in nitiates . Anothernitiates  or in banitiates , 72. Sim• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


Pin site infections are a common complication of external fixation that places a significant burden on the patient and healthcare system. Such infections increase the number of clinic visits required during a patient’s course of treatment, can result in the need for additional treatment including antibiotics and surgery, and most importantly can compromise patient outcomes should osteomyelitis or instability result from pin loosening or need for pin or complete construct removal. Factors that may influence the development of pin site infections include patient-specific risk factors, surgical technique, pin design characteristics, use of prophylactic antibiotics, and the post-operative pin care protocol including cleansing, dressing changes, and showering. Despite numerous studies that work to derive evidence-based recommendations for prevention of pin site infections, substantial controversy exists in regard to the optimal protocol. This review comprehensively evaluates the current literature to provide an overview of factors that may influence the incidence of pin site infections in patients undergoing treatment with external fixators, and concludes with a description of the preferred surgical and post-operative pin site protocols employed by the senior authors (ATF and SRR). Pin site infections are common orthopaedic problems that may arise from percutaneous pins or wires –62. ThesMost pin site infections are treatable with improved wound care and a short course of oral antibiotics . Deep tiThere is no accepted definition of pin site infection universally . CompariPin site reaction: Represents normal/physiologic changes in skin colour, skin warmth, and pin site drainage and resolves within 72 h.Pin site colonization: Includes Erythema, warmth, drainage, possible pain, and positive culture.Pin site infection: Includes all of the above, possibly with the addition of pus, pin loosening, or increased microbial growth on cultures. Recognizing that these definitions can be tedious a pin site infection may be defined, for practical purposes, as the presence of any classic signs or symptoms of infection around a pin or wire that requires treatment with antibiotics, pin removal, or debridement . Once deNot all patients are susceptible to pin site infections , 50, 57 p < 0.01) [External fixator parameters affect the risk of pin site infections. Increased duration of pin fixation was associated with a higher rate of pin site infection in a cohort of 27 patients with 178 total pin site infections [ < 0.01) . Skin te < 0.01) . Pin insAlthough pin site infections are complications, severe problems may follow that could compromise treatment goals and increase patient morbidity , 36, 39.p = 0.003) [In the presence of a pin site infection, the risk of intramedullary infection is increased from 6 to 70 % in patients undergoing conversion from external to internal fixation (= 0.003) . These o= 0.003) . In LATN= 0.003) . With th= 0.003) . Recentl= 0.003) .Although pin infections are a common complication of external fixation, there is no consensus regarding the optimal measures that should be used to prevent them. The variations in surgeon and nursing preference are, in part, due to limitations and gaps within the current literature. Specific limitations, outlined in Table Attention has been placed on the development of materials and specialized coatings of pins that could potentially reduce rates of infections. Examples include titanium pins and hydroxyapatite (HA) coatings. Titanium is thought to improve the metal–skin interface by inciting a smaller inflammatory response than stainless steel. HA enhances osseointegration of the pin, decreases motion at the interface, and lowers the loosening rate which would, otherwise, be a major contributor to infection.p > 0.05 for all outcomes). It was concluded that the additional cost of titanium alloy pins was not warranted given that pin site complications were not different compared with stainless steel pins.In a randomized controlled trial of 80 patients (320 pins) with unstable distal radius fractures treated with small AO external fixators, patients had either titanium alloy or stainless steel pins of identical geometry. After an average of 44 days in the external fixator, there were no statistically significant differences in the total pin site infection rate , pin loosening, or need for premature fixator removal (Staphylococcus epidermidis to stainless steel screws was significantly lower in the presence of HA coating.In a randomized study of 19 patients (76 pins) undergoing hemicallotasis in the treatment of medial compartment knee osteoarthritis, Magyar et al.  demonstrp < 0.001).Pommer et al.  conducteIn the context of a systematic review where the qualifying studies are evaluated for possibilities of confounding or bias, the conclusion was although HA pin coating reduced rates of loosening, there was insufficient evidence to determine whether this brought benefits to rates of deep infection, malunion, or the need for pin removal secondary to infection .p = 0.16), although the XCaliber group had significantly more pain at rest, pain during activity and greater paracetamol use. The authors concluded that the thread design of the standard pin was adequate in this clinical setting.Pin geometry and thread design are parameters that has been studied in the context of reducing pin site infection. W-Dahl and Toksvig-Larsen  randomizAlternative pin coatings, including silver and gold, have been used experimentally to reduce infection. These have been shown to prevent bacterial adhesion or growth, but clinical use is not widespread possibly due to the associated expense and need for further clinical testing.Surgical technique is quoted anecdotally as a means of lowering the incidence of pin infection. The goal is to prevent injury to the bone and soft tissues and subsequent bacterial colonization of necrotic tissue. Intra-operative precautions such as protecting soft tissues with drill sleeves, using sharp drill bits, avoiding thermal necrosis when using power drills, and preventing ischaemic necrosis of skin by implanting wires or pins without excessive skin tension are all measures thought to reduce pin site infections , 37, 42.Many investigators have attempted to determine the efficacy of different cleansing solutions in reducing the rate of pin site infection. This section will focus on the few randomized studies that aimed to determine the effect of using different cleansing solutions as the main variable.® chlorhexidine-impregnated discs changed weekly. The study was powered to detect a 5 % difference in infection rate, but after a mean follow-up of 5.9 weeks, the study failed to reveal any significant differences in pin site erythema, drainage, cellulitis, antibiotic use, pin removal rate, and pin loosening. It was concluded that sterile dry dressings changed weekly are an adequate and inexpensive choice; the study limitations include unblinded evaluation of infection and dressing changes done by the treating surgeon and of uncertain patient compliance. Henry [Egol et al.  publishee. Henry  led a pre. Henry  completee. Henry , inadeque. Henry , and inae. Henry .Lethaby et al.  publishep < 0.0001), more frequent presence of S. aureus , greater use of antibiotic treatment , and greater use of pain medications at the time of pin extraction (p = 0.03). However, there was no significant difference in infection rates, rates of pin loosening, or incidence of higher grade infections on the Checketts–Otterburns scale. Although this study demonstrates that a chlorhexidine cleansing solution may lead to lower rates of pin site bacterial colonization and decreased use of antibiotics and pain medications as compared with saline, it is limited in that the pin site infection rate was not shown to differ. A more recent cohort study suggested that chlorhexidine cleansing solution leads to decreased pin site infection rates. However, surgical techniques and other aspects of pin site care differed between the two study groups confounding the interpretation of the results [W-Dahl and Toksvig-Larsen conducted a prospective cohort study of 49 patients (196 pins) undergoing tibial osteotomy and external fixation for knee deformity correction in which pin site infection rates were compared at 1, 6, and 10 weeks post-operatively with two different cleansing solutions: (1) chlorhexidine (2 mg/ml) and (2) normal saline (0.9 % sodium chloride) cleansing solutions . It was  results .In summary, it is unclear whether cleansing pin sites is necessary to reduce the risk of infection. The ideal pin site cleansing solution is yet to be identified, but there is some evidence that chlorhexidine may be useful to decrease pin site colonization, antibiotic use, and pain.The optimal frequency for pin site care is unclear. One study found that the frequency of pin site cleaning is a factor that could potentially affect pin site infection rates . Fifty pDoes the type of pin site dressing influence infection rate? Although many investigators have looked into this, a conclusion on the efficacy of certain dressings alone is not possible because the study groups differ in other aspects of pin site care or surgical technique. Seven randomized trials were identified that were designed to specifically evaluate the efficacy of various dressings in reducing pin site infection rate , 60, 61.®) in adults with unstable displaced distal radius fractures. Grant et al. [In the remaining four randomized trials, no dressing type was shown to be superior , 18, 37.t et al.  studied t et al. .While many orthopaedic surgeons have guidance on when patients may shower or bathe post-operatively with the intent of reducing the risk of pin site infection, there is little direction from the literature. One case series suggested that showering after post-operative day (POD) 5 is not only acceptable, but could be used successfully as the only means of pin site care . The autThere are a small number of studies that seek to determine when surgical wounds may be exposed to showering. A cohort study of 192 patients undergoing thoracolumbar spinal operations showed no difference in wound infection rates with early showering  versus a control group  . These pS. aureus, or distribution of infection grade at weeks 1, 6, and 10 post-operatively. Therefore, it was concluded that a single preoperative dose of prophylactic antibiotics is adequate in this clinical setting.Oral antibiotics are often prescribed for prophylaxis against pin site infection, but evidence-based guidelines are lacking . To addrA study by Magyar et al.  supportep = 0.003). Therefore, the authors concluded that the local antibiotic administration reduced pin site infection rates, likely by reducing local bacterial flora.In addition to oral prophylactic antibiotics, local administration of antibiotics has been hypothesized to reduce rates of pin site infection . A totalp = 0.019). However, there was no difference in infection rate between the titanium control and tantalum groups (p = 0.230).Chou et al.  reportedIn addition to the factors discussed, there are other aspects of pin site care or operative technique that may affect infection rates. There are no controlled studies that address whether to use sterile or non-sterile technique when administering pin site care. There is an uncertainty over the effects of pin site massage to relieve skin tension, whether dressings are advantageous (versus no dressing) and whether to remove pin site eschar.Over 200 new external fixators are placed annually at the senior authors’ institution; the management of pin sites and pin site infections is an essential part of daily clinical practice. The prevention of infection protocol includes the following: intravenous antibiotics are given prior to skin incision and continued for 24 post-operatively; no tourniquet is used; and pins are inserted after pre-drilling with sharp drills using sleeves to protect soft tissues. Electrocautery is avoided at pin sites to prevent tissue necrosis. HA-coated tapered pins with cortical threads are used exclusively. For most applications, 6.0-mm-diameter pins are used; however, 4.5-mm pins are used in adult foot and forearm cases and for paediatric patients. Pin diameter is ensured to be <33 % of the bone diameter. Pin site care starts on POD 2. Pin care is done once daily by cleaning each pin site with a new sterile cotton-tipped applicator that has been soaked in a solution of 1:1 hydrogen peroxide and normal saline. Dry sterile gauze is wrapped around each pin site. Daily showering is encouraged on POD 4 where the patients remove the dressings, allowing water to rinse the frame and use of an antibacterial liquid soap. The leg and frame are patted dry with a clean towel. Patients are allowed to swim in a chlorinated pool after 4 weeks. Most patients are allowed weight bearing as tolerated. Over time, some patients develop a non-infectious dermatitis for which we have learned that further dilution of the hydrogen peroxide solution is helpful. For example, instructions would be changed to use 1 part hydrogen peroxide and 3 parts normal saline.When a patient presents with a pin site reaction, they are encouraged to start twice daily pin care for that pin, reduce activity and weight bearing, and start oral antibiotics if these measures fail or if symptoms worsen. If a patient presents with a pin site infection, they are started on oral antibiotics and instructed to start twice daily pin site care for that pin. A reduction of weight-bearing activity in conjunction with elevation of the limb (if swelling is present) is recommended. If the infection does not resolve completely, pin cultures are taken and a second antibiotic is added empirically. Culture-specific antibiotics are then started when the results become available. For persistent infections, radiographs are obtained and evaluated for pin loosening. If half-pin loosening is not clear from the radiographs, the pin is then disconnected from the frame and tested. Loose pins are removed immediately in the office. Persistently infected tensioned wires are also removed in the clinic. When vital pins or wires are removed, the patient is brought to the operating room for frame modification urgently. At that time, the pin site is debrided and if there is accompanying cellulitis, the patient admitted for IV antibiotics and elevation. Patients who have had deep infections  are monitored for signs of osteomyelitis. Typically, removal of the foreign body  is enough to cure the infection. When in doubt, an MRI  is obtained to evaluate the need for repeat debridement. After treatment with the external fixator is complete and at the time of frame removal, all pin sites are debrided. Since HA pins have been used routinely, loose pins are very rare at the time of frame removal and most pins are still fixed securely in the bone.Pin site infections remain a common clinical problem in patients treated with external fixators. Future research in this area holds promise in elucidating additional effective preventative measures. Given the limitations of the current literature, well-designed clinical trials will be instrumental in assisting clinicians choose optimal pin site care regimens, pin designs, and operative techniques when working with patients with percutaneous orthopaedic pins and wires. Techniques which reduce intramedullary nail contamination for patients previously treated with external fixation can minimize deep infection, including with the LATN technique  or by avS. aureus, in contrast to the 100 % infection rate in the uncoated pins (p < 0.01). Although the extraction torque of the coated pins decreased over the 14-day study period, it was superior to the uncoated pins. These results suggest that this coating may be a viable option to reduce the risk of infection while maintaining a stable interface with bone. In an in vitro study by Chen et al. [S. aureus and S. epidermidis adhesion in comparison with plain titanium pins without increased cytotoxicity to precursor osteoblast cells. The prevention measures tested in the Chou [Continued focus on materials development and novel methods of prophylactic antibiotic administration may provide insights that can be tested further in human clinical trials. Specifically, efforts are being made to produce an improved pin coating that has antimicrobial properties. This would have implications for external fixation but would revolutionize internal fixation for use over a site of previous pin use and for routine use as a prophylactic measure against infection. The findings of Chou et al.  are intrn et al. , a silvethe Chou , DeJong the Chou , and Chethe Chou  studies,Puckett et al.  have takThere is no consensus on the precise definition of a pin site infection, but this frequent complication of external fixation is a cause of considerable cost and patient morbidity. Patients with advanced age, multiple medical comorbidities, a prolonged duration of treatment in the external fixator, and those undergoing active correction are at an increased risk of pin site infections which may lead to additional complications such as osteomyelitis, pin loosening, loss of alignment, or premature removal of the external fixator.The literature is limited with regard to prevention of pin site infection. A small number of studies have been published that guide the orthopaedic surgeon to choosing strategies to reduce the risk of pin site infections: hydroxyapatite pin coatings improve osseointegration, extraction torque, and pin site infection during bone transport or tibial lengthening; meticulous operative technique is an important factor—soft tissue disruption and thermal damage should be minimized when inserting pins or wires—and care should be taken to reduce the skin tension at pin or wire sites.There is no strong evidence to guide choice of dressing type, cleansing regimen, or other aspects of pin site care. There is suggestion that chlorhexidine may be superior to saline as a pin site cleansing solution and that daily cleansing with saline is not superior to weekly cleansing. With regard to pin site dressings, there is suggestion that polyhexamethylene biguanide dressings, and possibly silver sulphadiazine dressings, may reduce pin track infection rates. However, there are several other trials showing the effect of dressing type to be negative and the question remains as to whether post-operative pin site dressings are important. With regard to pin site care, commencement of dressing changes on POD 2–3 may be convenient, as the drainage associated with pin site reaction normally decreases by this time. Clinicians should use personal judgement and experience until better evidence is available and, especially in the light of weak evidence, should consider the cost–benefit ratio of any pin site care regimen used.The optimal regimen and time course are yet to be determined for prophylactic antibiotics. There is some evidence that 3 days of prophylactic antibiotics is not superior to one preoperative dose in the setting of elective tibial osteotomy and external fixation for knee deformity. In patients undergoing treatment with opening-wedge osteotomy by hemicallotasis for osteoarthritis of the knee, 2 weeks of prophylactic antibiotics was determined to be optimal, although the study was limited by a lack of statistical analysis. As for local prophylactic antimicrobials, there is some clinical and laboratory evidence that administering them reduces the incidence of pin site infections, although this method is not a standard of practice currently. Until more evidence is available, the choice of prophylactic antibiotic regimen should be guided by the clinician’s experience for the particular orthopaedic operation, by patient comorbidities and a past history of infection.As many questions remain over how to reduce the risk of pin site infections effectively in patients treated with percutaneous orthopaedic pins and wires, an important measure that can be taken at this point in time is to teach patients how to recognize the signs and symptoms of pin site infection promptly so they may seek treatment as soon as possible. Specifically, pain at a pin site may precede infection at which point oral antibiotic treatment may be initiated .Surgeons and nursing staff should adopt a uniform pin care protocol that works for their patients and that can be taught to everyone involved in that patient’s care. Using a consistent protocol will help to ensure that the patient is not getting different information from different members of the healthcare team, a common problem that can lead to confusion and loss of confidence. Providing patients with a handout describing the pin site care protocol is an effective way to communicate to home nursing and family members that are involved in the pin site care. Audits of the protocol with a review of the latest studies on pin infection and prevention will allow for updating the protocol and delivering high-quality care.


Actinomucor elegans T3 was investigated. Flavourzyme hydrolysate showed the highest level of degree of hydrolysis (DH) (45.87%) followed by A. elegans T3 proteases hydrolysate (37.84%) and alcalase (30.55%). The A. elegans T3 proteases was observed to be more effective in generating small peptides with ACE-inhibitory activity. The 3 kDa membrane permeate of A. elegans T3 proteases hydrolysate showed the highest ACE-inhibitory activity with an IC50 of 0.79 mg/mL. After chromatographic separation by Sephadex G-15 gel filtration and reverse phase-high performance liquid chromatography, the potent fraction was subjected to MALDI/TOF-TOF MS/MS for identification. A novel ACE-inhibitory peptide (VQY) was identified exhibiting an IC50 of 9.8 μM. The inhibitory kinetics investigation by Lineweaver-Burk plots demonstrated that the peptide acts as a competitive ACE inhibitor. The razor clam hydrolysate obtained by A. elegans T3 proteases could serve as a source of functional peptides with ACE-inhibitory activity for physiological benefits.Angiotensin I-converting enzyme (ACE) inhibitory activity of razor clam hydrolysates produced using five proteases, namely, pepsin, trypsin, alcalase, flavourzyme and proteases from  C-terminal residues (His-Leu) by the action of ACE, thus resulting in vasoconstriction, ultimately leading to the increase in blood pressure ) as the independent variable and with the reciprocal of HA production (1/[V]) as the dependent variable [The inhibition kinetics of the peptide on ACE was investigated using HHL as a substrate. Lineweaver-Burk plot was used to determine the type of inhibition of the peptide. The ACE reactions were carried out at various substrate concentrations  in the absence and presence of two different concentrations of the peptide (2 and 5 μg/mL). Linear interpolation was plotted with the reciprocal of HHL concentration . The statistics analysis was carried out using SPSS 20.0 . Differences among treatments were determined by one way ANOVA. The A. elegans T3 proteases was found to be the most efficient in producing small peptides with the best ACE-inhibitory activity. A novel potent ACE-inhibitory peptide, VQY, with the IC50 value of 9.8 μM, was purified from the hydrolysate by a series of chromatographic separations and identified by MALDI/TOF-TOF MS/MS. Lineweaver-Burk plots revealed that the peptide exhibits strong competitive inhibition activity against ACE. This is the first report of ACE-inhibitory peptides derived from enzymatic hydrolysates of razor clam. It is highly recommended that the ACE-inhibitory peptides from razor clam hydrolysates be employed in the development of nutraceuticals and pharmaceuticals for the treatment of hypertension.The present study revealed that enzyme hydrolysates of razor clam have good potential for the production of ACE-inhibitory peptides. Among the proteases tested in this trial,


This study investigates the function of 5-HT type 3 (5-HT3) receptors using a computational approach. Antagonists of the 5-HT3 receptor are currently one of the most effective therapeutic agents in treatment of chemotherapy-induced nausea, vomiting, and irritable bowel syndrome. Several experimental studies have shown the effect of pharmacological agents such as 9-tetrahydrocannabinol (THC), the psychoactive component of Cannabis, Cannabidiol (CBD), a non-psychoactive ingredient of Cannabis plant, Menthol, Propofol, and etc. on the functional human 5-HT3 receptors expressed in Xenopus oocyte as well as rat nodose ganglion neurons ,2. 5-HT Experimental inhibition of 5-HT3 shows similar trend in computational binding energies to the lower binding site (THR 361).Docking calculations provide explanation of molecular basis of difference in inhibition by menthol like compounds. Similar binding energies for THC and CBD corresponds to their similar inhibition of membrane currents measured in experiment.


Haliclona viscosa. The aim of this study was to test Haliscosamine in vitro and in vivo for its antifungal activity against Fusarium oxysporum f.sp. melonis causing fusarium wilt of melon.Marine sponges are a potential source of new molecules with diverse biological activities. We have previously isolated a sphingosine derivative, (9Z)-2-amino-docos-9-ene-1,3,13,14-tetraol  from the Moroccan sea sponge in vitro test showed that haliscosamine has a similar effect as DESOGERME SP VEGETAUX®. In addition, in vivo showed a significant effect against Fusarium oxysporum f.sp. melonis. Taking to gather, our results suggest that haliscosamine constitutes a potential candidate against Fusarium oxysporum f.sp. melonis and the possibility to use in phytopathology.Overall,  Thus, the suppression of this pathogen is considered urgent and a big challenge for this type of agriculture. Indeed, preventive treatment using chemical pesticides is the only way to fight these fungi. However, chemical pesticides sprayed into the air or discharged into the soil can be harmful to the environment and to humans. Biological antifungal may be an alternative. Furthermore, Marine sponges are a potential source of new biological compounds with diverse biological activities -2-amino-docos-9- ene-1,3,13,14-tetraol) . This result indicates that the inhibition of Fusarium was a concentration-dependent manner of Haliscosamine  show highly significant (P < 1‰) factor treatment (intergroup variation). Comparison of means by Duncan's test helped highlight homogeneous groups at the 5%. Haliscosamine 1% has an effect similar to DESOGERME SP 2% on the inhibition of Haliclona sponges are known for their high chemical various secondary metabolites with interesting biological activities    to see the possibility of its use as a biopesticide.Haliscosamine isolated from the Moroccan sponge, Fusarium oxysporum f.sp. melonis, Fom 20474 CECT (Coleccion Espanola de Cultivos Tipo) was used in this study -2-amino-docos-9- ene-1,3,13,14-tetraol) and it is active against human pathogenic yeasts, Candida albicans, Candida tropicalis and Cryptococcus neoformans. Haliscosamine used in this study, was isolated as described previously , used in this study as a positive control, is an algaecide, fungicide and bactericide product used in Morocco both to remove algae, fungi and bacteria in irrigation systems and also to disinfect soil. It consists of 20 g/L of polyhexamethyle bioguanidine hydrochlorique and 50 g/L of N-alkyl dimethyl benzyl ammonium chloride  and then dried and placed on previously inoculated PDA medium. Plates were first kept at 4°C for at least two hours to allow the diffusion of chemicals, and then incubated at 28°C. Inhibition was scored by the absence of any contact between the discs and fungi after 48 h of incubation then inhibition zones were measured. Standard disks of the DESOGERME SP VEGETAUX® (20 μl/disc), served as the positive antifungal controls. All the assays were carried out in triplicate.This test uses Potato Dextrose Agar (PDA) as medium [Difco]. Conidial suspension was prepared from a 5-dold fungal culture  and adjusted with Malassez’s cellule in sterile water in order to obtain a final concentration of 10To determine whether the haliscosamine has fungistatic (temporary inhibition) or fungicide (permanent inhibition) effect on FOM, agar cylinder was cut out from inhibition zone and placed on the PDA medium and revival of their growth was observed. The fungicidal effect was where there was no growth after additional nine days of incubation at 25°C; whereas, a fungistatic effect was where temporary inhibition of mycelial growth occurred  were planted in 20-cm-diameter pots containing 2.5 L of sterile substrate [Plantaflor PROFI TYP3]. The haliscosamine was dissolved in DMSO and solutions of 1% and 2% were prepared in the irrigation water. Then, the pots were irrigated for two days by each of these solutions. On the third day, a 7-d-old FOM culture grown in potato dextrose broth (PDB) was added to the pots containing plants. Pathogen inoculum which consisted of a mixture of conidia and chlamydospores, was added to the potting mix at a rate of 1000 propagules/g of substrate : All plants of NCI were infested with Negative control Uninfested (NCU): No plants of CNU has infected or treated.Two negative controls (without any prior treatment) were used:One-way analysis of variance (ANOVA) was used to highlight the effect of treatment on the development of the plant pathogen. Averages of infected plants of different treatments were compared by Duncan test. P-value <0.05 was considered as a significant difference. Statistical analysis of data was performed using the SPSS software package 10.0 (SPSS Inc. USA).


One proposed mechanism for assembly of secreted heteropolysaccharides by many Gram positive bacteria relies on the coordinated action of a polymerization complex through reversible phosphorylation events. The role of the tyrosine protein kinase transmembrane modulator is, however, not well understood.wzb, wzd and wze genes from Lactobacillus rhamnosus ATCC 9595 and RW-9595 M contain motifs also found in corresponding proteins CpsB, CpsC and CpsD from Streptococcus pneumoniae D39 (serotype 2). Use of an anti-phosphotyrosine antibody demonstrated that both Wzd and Wze can be found in tyrosine phosphorylated form. When tyrosine 266 was mutated to phenylalanine, WzdY266F showed slightly less phosphorylated protein than those produced by using eight other tyrosine mutated Wzd genes, when expressed along with Wze and Wzb in Lactococcus lactis subsp. cremoris MG1363. In order to demonstrate the importance of ATP for the interactions among these proteins, native and fusion Wzb, Wzd and Wze proteins were expressed and purified from Escherichia coli cultures. The modulator protein, Wzd, binds with the phosphotyrosine kinase Wze, irrespective of its phosphorylation status. However, Wze attained a higher phosphorylation level after interacting with phosphorylated Wzd in the presence of 10 mM ATP. This highly phosphorylated Wze did not remain in close association with phosphorylated Wzd.The protein sequences deduced from the Lactobacillus rhamnosus thus carries out tyrosine phosphorylation of Wzd in addition to auto- and trans- phosphorylation of the kinase itself.The Wze tyrosine kinase protein of The online version of this article (doi:10.1186/s12866-015-0371-2) contains supplementary material, which is available to authorized users. Lactobacillus rhamnosus RW-9595 M stimulates interleukin, tumor necrosis factor and interferon gamma in mouse splenocytes  and RW-9595 M [GenBank: AY659979]  . For sturocedure .Table 2L. lactis subsp. cremoris MG1363 was used as host strain for the Wzy-dependent polymerization complex from L. rhamnosus RW-9595 M. A total of ten plasmids were constructed for expressing Wze and Wzb in conjunction with the wild type or nine mutated versions of the wzd gene in L. lactis subsp. cremoris MG1363 . Each Δwzd gene was amplified and then cloned into pMG36EB  and the resulting recombinant plasmids were separately transformed in L. lactis using the previously-published electroporation procedure [rocedure . The insE. coli culture was diluted 100-fold with fresh LB broth supplemented with ampicillin, which was incubated at 37°C with shaking until A600 reached 0.5 to 0.6 . Induction was initiated by adding IPTG to 1 mM and incubation continued for 3 h with shaking at 37°C. Cells were centrifuged at 18000 X g for 20 min at 4°C and the pellet was suspended in 15 ml STE buffer  containing 200 μg/ml lysozyme. After 10 min incubation on ice, the cells were sonicated  and then centrifuged at 18000 X g for 20 min at 4°C [2 and 20 mM β-mercaptoethanol) containing 10% sarcosyl and incubated overnight at 4°C [6-Wzd was added to 1 ml Ni2+ nitrilotriacetic acid (Ni2+-NTA) agarose resin (Qiagen) with 20 mM imidazole. Batch binding was carried out overnight (18 h) with gentle stirring at 4°C. For GST-Wze, glutathione sepharose 4B  was utilized for purification. When needed to ensure complete dephosphorylation, a 0.3 ml aliquot of resin-bound His6-Wzd was treated with 600 U YOP  at 30°C for 3 h, then inactivated by heat (65°C for 1 h). YOP-treated and untreated aliquots (0.3 ml) were then transferred to columns and washed 3 times with PBS containing 20 mM imidazole.An overnight n at 4°C . The celt at 4°C . The resg 15 min, then the pellet was washed three times with PBS containing 5% Triton X-100 and 2 M urea, followed by two washes with PBS. The washed pellet was suspended in 5 ml PBS containing 2% sarcosyl then proteins were precipitated by adding 7.5 ml acetone followed by centrifugation for 30 min at 18000 X g. Sarcosyl was eliminated by two washes with 2.5 ml 70% ethanol then the pellet was suspended in 1 ml PBS containing 5 mM MgCl2 and 1 mM DTT.For proteins without any tag (Wzb and Wze), the cell pellet from 100 ml of induced culture was suspended in 15 mL PBS (pH 7.3) then incubated for 20 minutes at 25°C with 200 μg/ml lysozyme. Lysis was completed by two passages on a French Press  followed by centrifugation at 18000 X L. lactis subsp. cremoris cells grown to 5 × 108 CFU/ml (A600 of 0.65) in GM17 was centrifuged at 20000 X g for 10 min at 4°C. The pellet was suspended in 1 ml TE buffer (10 mM Tris (pH 8.0) and 0.1 mM EDTA) and mechanically disrupted in a mini beadbeater  with 1 g of 0.1 mm dia. glass beads  by two 30 s treatments with 1 min cooling on ice between two treatments [A volume of 15 ml of eatments . Twenty in vitro protein interaction was carried out by His-pull down assay. Either 1 ml of purified Wze or 10 ml of diluted cell lysate of GST-Wze with 20 mM imidazole were added to an aliquot (300 μl) of the resin-conjugated His6-Wzd and incubated overnight (18 h) with stirring at 4°C. In duplicate samples, 10 mM ATP (Sigma) was added before the overnight incubation. The protein mixtures were then transferred to a column and washed with PBS containing 20 mM imidazole. In duplicate wash solutions, either 200 μM or 10 mM ATP was added. When necessary, elution of bound proteins was carried out with 1 M imidazole.The 6 or GST was carried out with primary antibodies using a 1:1000 dilution of mouse monoclonal IgG2ak anti-histidine tagged antibody (Millipore) or mouse monoclonal IgG anti-GST tag antibody (Millipore), respectively. All membranes were incubated for 1 h with 1:20000 dilution of secondary antibody (goat anti mouse IgG) coupled with horseradish peroxidase (HRP) (Millipore). Supersignal substrate (Thermo scientific) was used for HRP detection and visualised by exposure to Amersham Hyperfilm ECL (GE).Proteins separated by SDS-PAGE were transferred by semi-dry electroblotting onto polyvinylidene difluoride membranes in triplicate to detect proteins with three different antibodies. Phosphotyrosine protein detection was carried out with primary antibodies: the membrane blots were incubated overnight at 4°C using a 1:1000 dilution of mouse monoclonal IgG anti-phosphotyrosine 4G10 antibody (Millipore). Detection of tagged protein with HisNo human subjects, human material, or human data were used in this study.L. rhamnosus, three proteins may play a role in the control of EPS production, namely Wzb (phosphatase), Wzd (tyrosine protein kinase transmembrane modulator) and Wze (phosphotyrosine kinase). Details of comparative sequence analysis of Wzb, Wzd and Wze can be found in the Additional file L. rhamnosus strain RW-9595 M were transferred to L. lactis subsp. cremoris MG1363 in order to discover the phosphorylation state of the three proteins in vivo in a Gram-positive expression system, as well as to demonstrate the importance of individual tyrosine residues to total Wzd phosphorylation and to the phosphorylation state of Wze. In order to examine protein interactions in vitro and their effect on phosphorylation, the three genes wzb, wzd and wze were expressed in E. coli to obtain tagged and untagged proteins.Polymerization must be strictly controlled with respect to precursor availability and the energy necessary to form glycosidic bonds as well as to transport the units out of the cell, and finally to attach the polymers to the cell in the form of capsular material or to release them. Significant advancement has been made in understanding some interactions among components of the biosynthetic complex, but further work is needed to elucidate how these interactions promote the assembly of polysaccharides on the cell surface. In wzd genes changing a different tyrosine codon to phenylalanine were expressed individually in concert with wze and wzb from L. rhamnosus RW-9595 M in L. lactis subsp. cremoris MG1363 carrying one of the nine constructed plasmids (Table L. lactis subsp. cremoris MG1363 with or without plasmid pMG36CT were probed with the anti-phosphotyrosine antibody (data not shown). All nine separate tyrosine mutated Wzd proteins (33 kDa) were phosphorylated  or C41(DE3) carrying pQEWzd  or BL21(DE3) carrying pGSTWze  was recognized by the anti-phosphotyrosine antibody, which shows tyrosine phosphorylation  and C41(DE3) carrying the vector plasmids did not show any tyrosine phosphorylated proteins, with or without added ATP (data not shown). In the absence of added ATP, the His and C41(E. coli C41(DE3) pGSTWze  pGSTWze (data not shown) for the same amount of protein. The level of phosphorylated GST-Wze protein did not differ in the presence or absence of 10 mM ATP. However, the untagged native Wze protein was not phosphorylated when produced by E. coli strains NM522 or BL21(DE3), whether or not ATP was present.The presence of GST-Wze was confirmed by anti-GST antibody and, as a control, GST expressed alone was not detected by the phosphotyrosine antibody (data not shown). The anti-phosphotyrosine antibody did detect phosphorylated GST-Wze in cell lysates of oli C41DE pGSTWze E. coli host strains tested, the tyrosine phosphorylation of His6-Wzd depended on the strain of E. coli used for expression. The initial level of phosphorylation of these two proteins did not change when ATP was added. The cause of the difference between strain BL21(DE3) and its derivative mutant C41(DE3) has been attributed to a lower amount of T7 RNA polymerase in strain C41(DE3), which reduces transcription levels of cloned genes, actually leading to a more stable over-expression of recombinant proteins by strain C41(DE3) [6-Wzd may conceivably be carried out by a PTK of E. coli BL21(DE3), as was observed in studies of other proteins [E. coli host strains used. UDP-glucose dehydrogenase YwqF from B. subtilis was phosphorylated by PTK Wzc from E. coli, showing that Gram-negative PTK is capable of phosphorylating proteins from Gram-positive species. Wze possesses a tyrosine in a similar position as the tyrosine 569 implicated in autophosphorylation of Wzc (PTK) in E. coli [E. coli carries out tyrosine phosphorylation. Although serine phosphorylation of GST has been demonstrated previously [While GST-Wze was slightly phosphorylated in all C41(DE3) . Phosphoproteins . In our  E. coli . The aut E. coli . Thus, ieviously , no tyroeviously . To our 6-Wzd. Wzb was not retained by His6-Wzd after washing the resin (data not shown), suggesting that no stable interaction was present between these two proteins. After incubating resin-bound His6-WzdC41 and Wze together, the resin was divided into two fractions for washing either with or without ATP. Wze was retained by His6-Wzd using the same washing conditions as with Wzb , so they could be tested for affinity to resin-bound His2+ resin in the absence of His6-Wzd (data not shown). A cell lysate of E. coli C41(DE3) pGSTWze was passed through a column containing purified His6-Wzd expressed by either E. coli BL21(DE3) (His6-WzdBL21) or C41(DE3) (His6-WzdC41) bound to the Ni2+ resin  in S. thermophilus CNRZ1066 [L. rhamnosus could be involved in the production of EPS as proposed for EpsC. Indeed, several mutations occurring in the CpsC transmembrane modulator of a number of S. pneumoniae strains lead to defective cell wall attachment of CPS [L. rhamnosus Wzd sequence, equivalent tyrosine residues can be found in similar positions (Y33 and Y77)  is located in the C-terminal cytoplasmic domain. The wzd, wze and wzb genes from L. rhamnosus RW-9595 M were thus expressed in L. lactis subsp. cremoris MG1363 in order to verify which tyrosine residues could be involved in Wzd phosphorylation and whether Wze is then phosphorylated in vivo. While each of the nine separate tyrosine mutations led to Wzd phosphorylation, mutation of the C-terminal tyrosine (Tyr266) showed slightly weaker Wzd phosphorylation than most of the other eight mutations. This suggests that more than one tyrosine residue participates in Wzd phosphorylation, although Tyr266 appears to be slightly more important. In all cases, Wze was phosphorylated, although the phosphorylation levels of Wze were slightly lower when Wzd was mutated in positions Y33 (N-terminus), Y141 or Y266 (C-terminus). A previous study observed that the C-terminal cytoplasmic domain of CapA (transmembrane modulator) was essential for phosphorylation of CapB (kinase) in Staphylococcus aureus [et al. [Wzd from s aureus . Tocilj  [et al.  proposedS. pneumoniae D39, when the cps2C (transmembrane modulator) was deleted, no capsule was detected, but low molecular weight products could be observed [cps2C, while some mutants were able to maintain wild type levels of total CPS production, including the Y82C mutation [epsA was deleted in L. lactis [S. pneumoniae strain D39 contains 5 tyrosine residues while Cps2C from strain Rx1-19 F contains only 3, all located within the first 100 N-terminal residues, as does the modulator of S. thermophilus and S. aureus. No tyrosine residue appears conserved among all 13 putative PCP2b sequences compared from staphylococci, streptococci, lactococci and lactobacilli, although the tyrosine positioned right after the first transmembrane domain is found in 12 out of the 13 sequences aligned. This indicates that the precise function of PCP2b could differ among species. The lactobacilli PCP2b sequences contain a supplementary stretch of 46 amino acids including 3 to 5 tyrosine residues that are not found in sequences from streptococci, lactococci and staphylococci. Wzd (L. rhamnosus), EpsA (L. lactis subsp. lactis or cremoris) and EpsB (L. johnsonii) have a total of 9, 7 and 6 tyrosine residues, respectively. The genera Lactobacillus and Lactococcus have common conserved tyrosines  in their modulator protein sequences. These conserved tyrosines, especially the tyrosine located near the C-terminal transmembrane region, may be associated with polymer extension and release instead of attachment. An equivalent C-terminal tyrosine 266 is absent from the CpsC sequences of S. pneumoniae, and S. thermophilus as well as the CapA sequence of S. aureus. Future work will need to focus on how these differences impact the production of capsular polysaccharides versus the production of released high molecular weight EPS.In . lactis . Cps2C fOur study shows that the activity of Wzd is also modulated through tyrosine phosphorylation of more than one tyrosine residue, allowing the phosphorylation of Wze. This new information suggests that further modification of the model for the control of EPS elongation can be proposed Figure . The nonL. rhamnosus ATCC 9595 and RW-9595 M produce exopolysaccharides through a polymerase-dependent mechanism. Future experiments will focus on the role of the Wzb phosphatase in complex formation or dissociation, the relationship between the Wzd/Wze complex formation and EPS polymerization as well as the interaction of other proteins with this complex, including interactions of the polymerization complex with the proteins involved in synthesizing and transporting the repeat units. This approach will contribute to determining their function in modulating the biosynthesis of exopolysaccharides by L. rhamnosus.


Through distinct protein complexes it regulates different levels of available cellular energy substrates required for cell growth. One of the important functions of the complex is to maintain available amino acid pool by regulating protein translation. Dysregulation of mTOR pathway leads to aberrant protein translation which manifests into various pathological states. Our review focuses on the role mTOR signaling plays in protein translation and its physiological role. It also throws some light on available data that show translation dysregulation as a cause of pathological complexities like cancer and the available drugs that target the pathway for cancer treatment. The regulation of translation is crucial for controlling cell growth and proliferation while translation dysregulation results in aberrant growth and tumorigenicity . Transla Streptomyces hygroscopicus that was isolated from a soil sample of Rapa Nui islands in the 1970s . Ra. RamTORC in vivo . Th. Th85]. S101 is necessary for phosphorylation of S65 and S112 has been shown to affect binding to eIF4E although it does not affect phosphorylation at other sites. The kinase responsible for the phosphorylation of these two serine sites has not been identified. S83 is conserved in all three 4E-BPs but does not seem to control translation initiation. The exact function and regulation of each phosphorylation site and which phosphorylations are dependent on one another are still not very well understood .Besides the eIF4E binding motif, there are two other key regulatory motifs in 4E-BP1, a RAIP motif, named after its sequence Arg-Ala-Ile-Pro at the N-terminus, and an mTOR signaling (TOS) motif at the C-terminus  90, 91], 9190, 9 in vitro [ in vitro by mTORC1 but these sites are considered rapamycin insensitive in cells [ in vivo; moreover some studies suggest that another unidentified 4E-BP1 kinase might exist [Although mTORC1 has been implicated in the regulation of 4E-BP1 phosphorylation there are various conflicting reports. For instance, rapamycin treatment leads to loss of phosphorylation at S65 and T70 on 4E-BP1 while mTORC1 has a modest effect on the phosphorylation of these sitesin vitro . On the in cells . It is pht exist . In diffht exist . Whetherht exist .There are some major discrepancies regarding the identity of phosphorylation sites required for the release of 4E-BP1 from eIF4E. T37 and T46 phosphorylations have been reported to have either little effect on eIF4E binding , 87 or t in vivo can be significantly different from its effect in cultured cells [Rapamycin resistant phosphorylation of 4E-BP1 has been reported in regenerating rat livers where rapamycin inhibits activation of S6K1 in response to partial hepatectomy, but 4E-BP1 phosphorylation remains uninhibited, suggesting that the effect of rapamycin on 4E-BP1 functioned cells . Rapamyced cells . Severaled cells . Torin ied cells . The acted cells , 107. 4Eed cells . One meced cells  while aned cells .Various growth-inhibitory conditions and physiological stresses also lead to shutdown of mTOR signaling and 4E-BP1 dephosphorylation, which indicates that this may be a common response of cells to unfavorable conditions .mTORC1 upregulates protein synthesis by various other mechanisms. Maf1 which is a Pol III repressor is inhibited by mTORC1 phosphorylation and thus induces 5S rRNA and tRNA transcription , 113. Thγ (PPAR-γ) is the main regulator of adipogenesis which is also activated by mTORC1 [α (HIF1α) which is a positive regulator of many glycolytic genes [Proliferating cells also require lipids in addition to protein to synthesize plasma membranes and other macromolecules and mTORC1 controls this synthesis of lipids  via the y mTORC1 , 121. mTic genes , 122.mTORC1 is also an important negative regulator of autophagy, a eukaryotic homeostatic process in which various cytoplasmic components such as damaged organelles and intracellular pathogens are degraded inside lysosomes . mTOR inThe mTOR pathway is related to tumorigenesis because of its vital role in cell growth, proliferation, and metabolism. The aberrant activation of mTOR pathway either by loss of tumor suppressors or activation of oncogenes promotes tumor growth in various malignant cell lines. The upstream and downstream elements of the mTOR pathway are dysregulated in different human cancers. The overexpression of different growth factor receptors like IGFR (insulin like growth factor receptor) and HER2  and mutations in the PI3K can lead to activation of AKT and mTOR pathways . This mT in vivo [ in vivo [The downstream effectors of mTORC1, 4E-BP1, eIF4E, and S6K1 are also associated with various malignancies. eIF4E is an oncogene as it is overexpressed in many human cancers with poor prognosis and its overexpression results in transformation of cells in vivo , 134. eI in vivo . Dysregu in vivo , 137 whe in vivo , 139. Th in vivo . S6K1 is in vivo  and its  in vivo .mTORC2 also plays a role in cancer since the mTORC2 subunit RICTOR is also overexpressed in multiple cancer types  and its Lipid synthesis is increased in proliferating cancer cells and PI3K/AKT mediated high glycolytic rates produce ATP and the other building blocks required for lipid synthesis . The mTOActivated mTOR pathway is also related to various familial cancer syndromes. The loss of tumor suppressor LKB1 (liver kinase B1), which is a key kinase for activating AMPK , resultsmTOR inhibitors can be broadly grouped into two classes: the allosteric inhibitors of mTORC1  and the mTOR kinase inhibitors.Rapamycin was the first mTOR inhibitor but despite its antitumor activity in preclinical models it was not successful as an anticancer drug. Subsequently several analogs of rapamycin, now called rapalogs, with better solubility and pharmacokinetic properties were synthesized such as temsirolimus, everolimus, and deforolimus. Like rapamycin these rapalogs form a complex with intracellular receptor FKBP12 which binds to mTOR and inhibits mTORC1 downstream signaling. In phase II and III clinical trials, everolimus and temsirolimus were effective in treating RCC , neuroendocrine tumors, and Mantle cell lymphoma and both have been approved by FDA for treatment of RCC . However In vitro, AZD8055 effectively inhibits cell proliferation and induces autophagy and, in vivo, AZD8055 inhibits tumor growth [In order to overcome the limitations of rapalogs, ATP-competitive mTOR kinase inhibitors were developed that directly target the mTOR catalytic site and inhibit phosphorylation of mTORC1 substrates S6K1 and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and mTORC2 . These ir growth . In addir growth . Signifir growth . Treatmer growth .The drawback with mTOR kinase inhibitors as with rapalogs is that the mTORC1 feedback loop can be relieved which leads to activation of PI3K or MAP kinase signaling . Thus duAnother mechanism that explains the resistance to mTOR kinase inhibitors is that cancer cells downregulate the expression of 4E-BPs which leads to an increase in the eIF4E/4E-BP1 ratio, and it is this change in eIF4E/4E-BP1 stoichiometry that limits the sensitivity of cancer cells to catalytic site TOR inhibitors suggesting that the eIF4E/4E-BP1 ratio might act as a predictive marker for treatments using catalytic TOR inhibitors .Dual PI3K/mTOR inhibitors were developed because of the above concerns over mTOR inhibitors. This was made possible because of the high homology that is shared by the kinase domains of PI3K and mTOR . These mmTOR pathway plays a key role in nutrient homeostasis that regulates cellular growth and proliferation. mTOR regulates protein translation through effector molecules S6K1 and 4E-BP1. Dysregulation of the pathway is complicated by cross-talk between mTOR and other signalling pathways like AKT and PI3 kinase. Though mTOR pathway dysregulation manifests into various pathological states, it is not the only candidate responsible for the effect. Further downstream signalling is very complex that is understood by the fact that therapeutic regimens that target only mTOR are not very effective to treat cancer. Dual inhibitors that target both mTOR and PI3 kinase have shown promise in combating the disease.


Rhinella.Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act  as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity, Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Bufotenine was isolated from In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action.This work do not present or propose bufotenine as a drug for the treatment of rabies due to the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule, may lead to the choice or development of a tryptamine analogue presenting potential clinical use.The online version of this article (doi:10.1186/1678-9199-20-45) contains supplementary material, which is available to authorized users. Fractions were manually collected and submitted to mass spectrometry (MS and MS2 ESI-IT-TOF) analyses. The fraction containing bufotenine was then purified using a C18 column , with a linear gradient of B over A 13% to 15% in 15 minutes, at a constant flow rate of 1.1 mL.min-1, at 4°C, monitored by UV absorbance at 214 nm. Bufotenine peak was manually collected, dried and submitted to 1H-NMR spectroscopic analyses and/or biologic assays.The collection and housing of Anadenanthera colubrina seeds, which contain up to 2% of the seed dry weight in bufotenine. The seeds were obtained from the legitimate supplier Arbocenter Comércio de Sementes Ltda, Birigui, São Paulo (batch 0019), Brazil. The extraction of bufotenine was performed as described by Stromberg  method, according to Takeuchi et al.  and Mosmet al. , with moin vitro, being necessary some kind of additional test to monitor cell infection, such as tests with antibodies bound to fluorescent substances [Rabies virus does not cause cytopathic effects in cells cultured bstances . For thiet al. [4 cells/well) were deposited on 96-well tissue culture microtiter plates with 50 μL of each bufotenine dose (from 0.5 to 4 mg/mL) and 50 μL of PV virus previously diluted at 1:100 (IDCC100). As negative inhibition control, only 50 μL of MEM-10 was added to the cells and as positive inhibition control ketamine was used , according to Lockhart et al. [2 atmosphere, the medium was removed by suction from all wells and the microplates were cooled on ice. The cells were fixed by adding acetone 80% in water (kept at –20°C). After 15 minutes, the plates were emptied by inversion and dried at 37°C according to Smith et al. [et al. [This test was based on simplified fluorescent inhibition microtest (SFIMT), acording to Favoretto et al. , with mot et al. . After a [et al. . The sta [et al. . After oet al. [et al. [4 cells/well) were deposited on 96-well tissue culture microtiter plates containing different bufotenine doses , and 50 μL of PV virus previously diluted at thirty-fold the FFD50 value. As negative inhibition control, only 50 μL of MEM-10 was added to the cells and as positive inhibition control ketamine was used . After a 20-hour period of incubation at 37°C under a humidified 5% CO2 atmosphere, the medium was removed by suction from all wells. The procedures of cells fixation and staining were the same described in “Fluorescence inhibition test” section. The microtiter plates reading was quantitatively performed in inverted fluorescence microscope , where each field with fluorescence were counted, being 0 the minimum (positive control) and 18 the maximum (negative control) number of infected fields. The IC50 was defined as the inhibition concentration of bufotenine that reduced the number of fields with fluorescent foci to 50% when compared with the negative control.This test was based on rapid fluorescent focus inhibition test (RFFIT) acording to Smith et al. , adapted [et al. , with moprior to the addition of the virus and, secondly, to test a possible “treatment effect”, bufotenine was added in different times , after incubation of cells and PV virus. All the other procedures were the same as described in “Fluorescent focus inhibition test” section.The time course effect of bufotenine was examined on PV virus with two minor modifications of the fluorescent focus inhibition test. First, to test a possible “protector effect”, bufotenine was added in different times , 50) and inhibition (IC50) concentrations were calculated from concentration-effect curves after linear regression analysis. The results represent the mean ± standard error of the mean values of two different experiments (triplicates).The 50% cytotoxic  when compared to the negative (cells + MEM-10) and positive controls (cells + DMSO 20%) .The 3.9 mg/mL dose of Fluorescence inhibition testBufotenine was able to inhibit the infection significantly, showing dose-response effect when tested at a concentration of 0.5 to 3 mg/mL Figure .Figure Fluorescent focus inhibition test-1. It was also possible to observe the dose-response effect. Moreover, there was a time course dependency of the effect regarding the moment of PV virus administration, being bufotenine most effective when added along with the cells and PV virus  mg.mL-1  and cutaneous wound healing in rats, among others [There are recent described effects of hydroalcoholic extracts of different parts of g others –37. Howeg others –40.Among the possible described mechanisms of rabies virus penetration known (or postulated), the bufotenine inhibition of rabies virus infection observed in this study appears to be related to the competition to the cells’ receptors, since it could only be observed when simultaneous administration of bufotenine and PV virus was performed Figure . Howevernd generation active molecules, since rabies is still a disease with no cure prognosis and it kills, every year, thousands of people around the world [Numerous authors have described that there are high-affinity receptors at host cells specific for rabies virus, which include AChR – and/or associated molecules – that act as receptors. Although it was not possible to unequivocally demonstrate that the inhibitory effect of bufotenine was mediated by its binding to the acetylcholine receptor, the results of this work open important perspectives in the study of the mechanism of action of bufotenine, as well as the elucidation of the mechanisms of the virus pathogenicity. Altogether, our data may aid in the development of 2he world . FurtherThis work do not present or propose bufotenine as a drug for the treatment of rabies for several reasons, including the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule may lead to the choice or development of a tryptamine analogue presenting potential clinical use. These tryptophan derived alkaloids are very interesting building blocks in organic chemistry syntheses . Our nexRhinella jimi specimens were performed under license number 15964-1 from the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA).The collection and housing of 2 fragmentation profile of (A) m/z 205 (bufotenine) and (B) m/z 219  molecules. Note the common m/z 160 ion. (DOCX 333 KB)Additional file 1: Figure S1: ESI-IT-TOF MSAdditional file 2: Figure S2: Figure showing (A) C18-RP-HPLC profile of the separation of (B) bufotenine and (C) 5HTQ, at 4°C. (DOCX 131 KB)1H-NMR spectrum of bufotenine purified from R. jimi skin secretion. (DOCX 113 KB)Additional file 3: Figure S3: Interpretation and annotation of A. colubrine seeds, as represented by (B) the RP-HPLC profile. (DOCX 134 KB)Additional file 4: Figure S4: Figure showing (A) ESI-IT-TOF MS profile of bufotenine purified from Additional file 5: Figure S5: Cytotoxicity evaluation of bufotenine, as assayed at the most effective antiviral dose. (DOCX 35 KB)Additional file 6: Figure S6: Percentage of viable cells after treatment with different bufotenine concentrations compared with negative (cells + MEM-10) and positive (DMSO 20%) controls. (DOCX 46 KB)