Treponema denticola inhibits neutrophil polarization and directed chemotaxis together with actin dynamics in vitro in response to the chemoattractant N-formyl-methionine-leucine-phenylanine (fMLP). Msp disorients chemotaxis through inhibition of a Rac1-dependent signaling pathway, but the upstream mechanisms are unknown. We challenged murine bone marrow neutrophils with enriched native Msp to determine the role of phospholipid modifying enzymes in chemotaxis and actin assembly downstream of fMLP-stimulation. Msp modulated cellular phosphoinositide levels through inhibition of phosphatidylinositol 3-kinase (PI3-kinase) together with activation of the lipid phosphatase, phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Impaired phosphatidylinositol-triphosphate (PIP3) levels prevented recruitment and activation of the downstream mediator Akt. Release of the actin capping proteins gelsolin and CapZ in response to fMLP was also inhibited by Msp exposure. Chemical inhibition of PTEN restored PIP3 signaling, as measured by Akt activation, Rac1 activation, actin uncapping, neutrophil polarization and chemotaxis in response to fMLP-stimulation, even in the presence of Msp. Transduction with active Rac1 also restored fMLP-mediated actin uncapping, suggesting that Msp acts at the level of PIP3 in the hierarchical feedback loop of PIP3 and Rac1 activation. Taken together, Msp alters the phosphoinositide balance in neutrophils, impairing the cell “compass”, which leads to inhibition of downstream chemotactic events.The major outer sheath protein (Msp) of  N-formyl group . Key to chemotaxis is the ability of neutrophils to polarize, with formation of a leading edge (lamellipodium) toward the chemoattractant and a trailing edge (uropod). During chemotaxis, movement of the cell is mediated by dynamic protrusion and retraction of the lamellipodium and uropod, respectively, driven by pathways that regulate remodeling of the actin cytoskeleton 3, PIP3) at the leading edge Neutrophils are key rapid-response cells of the innate immune system that are recruited to sites of infection to eradicate pathogens 2) by the lipid kinase phosphatidylinositol 3-kinase (PI3-kinase) 2 and PtdIns2, respectively Production of PIP3 is catalyzed from phosphatidylinositol 4,5-bisphosphate PtdIns P2 by theTreponema denticola is a key pathogen in the polymicrobial infection associated with chronic periodontitis T. denticola as well as in shed vesicles T. denticola's major virulence factors. It interacts with multiple host proteins and cells leading to changes in host cell homeostasis . Mice were euthanized by COT. denticola ATCC 35405 as previously described Native Msp complex was enriched from New Oral Spirochete broth cultures of C57BL/6J wild-type mice  were purchased from Charles River. Femurs and tibias were removed and cells were isolated from bone marrow by fractionation into discontinuous Percoll (Sigma) gradients . Mature neutrophils were isolated from the 80%/65% interface.Lipid phosphatase activity was measured as described previously 3 substrate for 1 hour at 37°C (Echelon). The amount of free phosphate released due to conversion by PTEN was determined using Malachite Green solution (Echelon).PTEN activity was determined using a PTEN immunoprecipitation assay. 5×102 produced was determined using a competitive ELISA assay (Echelon).PI3-kinase activity was measured using a PI3-Kinase activity ELISA: Pico (Echelon) according to the manufacturer's protocol. Briefly, 5×10Neutrophils were treated, followed by fMLP stimulation, lysed in 5X SDS sample buffer, and boiled for 10 minutes. Lysates were separated by SDS- PAGE followed by transfer to nitrocellulose and membranes were blocked with 5% skim milk/ TBS/ 0.05% Tween-20 and incubated overnight with anti-Phospho-Akt antibody . Membranes were incubated with HRP-linked secondary antibody followed by detection using the Western Lightning ECL substrate (Perkin-Elmer). Membranes were re-probed with a total Akt antibody .For immunofluorescence analysis, Msp-treated neutrophils were allowed to attach to BSA-coated coverslips followed by stimulation with fMLP. Cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100 for 15 minutes and blocked with 5% normal goat serum (NGS) for 30 minutes. Anti-Akt antibody  was diluted in 1∶50 in 5% NGS and applied for 1 hour at room temperature followed by Alex-488 goat anti- rabbit secondary antibody  for 1 hour. F-actin was labeled with Alexa-594 phalloidin  and nuclei stained with 4′,6-diamidino-2-phenylindole . Cells were examined by epifluorescence microscopy (Leica DMIRE2) and images obtained using OpenLab softwatre (Perkin Elmer).The actin capping proteins gelsolin and CapZ were analyzed as described 2PO4, 10 mM Tris pH 8.0, 5 mM imidazole), treated with benzonase , and sonicated (4×20 seconds). Samples were centrifuged and His-Tag-TAT proteins purified from supernatants using Ni-NTA superflow columns (Qiagen) according to manufacturer's protocol for denaturing conditions, except proteins were eluted from the column with 8M Urea, 100 mM NaH2PO4, 10 mM Tris pH 8.0, 0.5M imidazole. Samples were dialyzed using 15 ml Slide-a-Lyzer dialysis cassettes  overnight with 2 changes of buffer against PBS/ 5% glycerol followed by concentration with Amicon centrifugation devices .The Rac1 constitutively active  and empty TAT-HA constructs were provided by Dr. Gary Bokoch . Protein expression and purification was performed as described To determine the effect of Rac1CA or TAT-HA rescue on actin capping protein release, neutrophils were transduced with 500 nM purified TAT-protein for 10 minutes following OG permeabilization prior to Msp treatment and actin uncapping quantification as described above.Cells were treated as indicated prior to being resuspended in Hank's Balanced Salt Solution (HBSS), pH 7.4, with 1% gelatin and allowed to attach to 1% BSA coated coverslips (22×40 mm) at 37°C for 10 minutes. Coverslips were inverted onto a Zigmond chamber, and HBSS solution was added to the left chamber while fMLP was added to the right chamber. Images were taken every 20 seconds for 15 minutes using a Nikon Eclipse microscope E1000 equipped with a Hamatsu camera (model ORCA-ER). Images were analyzed using ImageJ software and chemotaxis analysis (migration and speed) performed using the manual tracking and chemotaxis tools in Image J. The number of cells polarized following 5 minute exposure to fMLP was calculated from still images obtained during the chemotaxis assay The amount of active Rac1 was determined using a PAK-PBD pulldown assay. Treated neutrophils were lysed in 2X lysis buffer ) and PAK-PBD-GST beads  were added to the lysates for 1 hour at 4°C, followed by collection. Bead samples were washed two times , followed by resuspension in 5X SDS sample buffer. Bead samples and total lysates were analyzed by SDS- PAGE and western blotting with a Rac1 antibody .P<0.05.Comparisons between two groups were performed using t-tests. Results are based on at least 3 independent experiments. Statistical significance was defined as Cellular phosphoinositide levels are tightly regulated through the actions of both PI3-kinases and phosphoinositide phosphatases. Lipid phosphatase activity was assessed in partially permeabilized neutrophils using a malachite green assay to measure dephosphorylation of a synthetic PIP3 substrate. Pretreatment of neutrophils with Msp prior to fMLP stimulation increased the release of free phosphate compared with fMLP stimulation alone, indicating activation of a lipid phosphatase . To deteAs Msp is able to inhibit Rac1 activation selectively and the polarized accumulation of PI3-kinase products in fMLP-activated neutrophils Akt is a downstream target of PI3-kinase; its recruitment and activation can be considered an indicator of PIP3 generation. PIP3 binds Akt and recruits it to the plasma membrane, where Akt is activated by phosphorylation. Immunofluorescence analysis of fMLP-stimulated PMNs revealed re-localization of Akt , green fInhibition of PTEN with the inhibitor VO-OHpic increased the PIP3 levels in neutrophils, as measured indirectly through the impact on Akt phosphorylation levels . When PIThe balance of membrane phosphoinositides in the cell regulates several functions, including actin rearrangement and subsequent locomotion. As Msp has been found to impair actin free barbed end formation in response to fMLP in neutrophils PTEN inhibition in neutrophils increased PIP3 levels , leadingSince PTEN inhibition restored ‘physiological’ PIP3 signaling in Msp-treated neutrophils, we also determined its impact on the previously recognized Msp-mediated inhibition of Rac1 activation in fMLP-stimulated cells Rac1 is the dominant mediator of actin filament uncapping downstream of the fMLP receptor Accumulation of PIP3 at the leading edge of neutrophils is crucial for the regulation of directed migration in response to fMLP through recruitment and activation of secondary mediators and local actin polymerization. Msp inhibits both neutrophil polarization and chemotaxis T. denticola Msp was reported to involve inhibition of Rac1 activation and localization de novo actin assembly at the leading edge, which ultimately would 4) inhibit functional cell polarization and chemotaxis. A major finding of this study is the crucial role of lipid phosphatases, as experimental inhibition of PTEN activity reversed all of the inhibitory effects of Msp listed above.Signaling and regulatory pathways that include plasma membrane-associated phosphoinositides, lipid kinases, and lipid phosphatases are crucial for determining the actin dynamics, polarization, and directional migration events that drive neutrophil chemotaxis. Inhibition of fMLP-stimulated neutrophil chemotaxis by We have previously shown that Msp impacts phosphoinositide production and recruitment via lipid phosphatase activity in fibroblasts, as a novel mechanism leading to actin rearrangement A recently proposed model predicts that PTEN contributes negatively to the regulation of directional sensing in neutrophils by blocking the development and maintenance of the PIP3 gradient at the leading edge It is well established that PI3-kinase and PTEN work in opposing fashion in cells, including neutrophils, to regulate phosphoinositide metabolism. PI3-kinase activity has been shown to be required for the initial polarization and chemotaxis response of neutrophils to fMLP It is clear that Msp is able to effectively inhibit phosphoinositide signaling and Rac1 activation in neutrophils. Yet, other small GTPases such as Rac2 and Cdc42 are still activated normally following fMLP stimulation Our group has recently shown in neutrophils that different Rac isoforms localize to specific membrane domains based on alterations in phospholipid membrane composition. Specifically, during chemotactic leading edge formation, accumulation of PIP3, concomitant with an increase in negative charge, recruits Rac1 to the plasma membrane Exposure of neutrophils to Msp has been reported to prevent actin assembly at free barbed ends in response to fMLP stimulation The finding that cell exposure to Msp decreased the extent of actin-uncapping in fMLP-stimulated neutrophils is contrary to our previous observations of Msp-treated fibroblasts 2 through changes in localization as well as conformational changes \ to the plasma membrane in fibroblasts PTEN can be regulated at multiple levels including transcription, post-translational as well as through changes in localization T. denticolaThough other bacterial pathogens have been reported to upset the lipid phosphatase activity of target cells, the key mechanism is often mediated by a toxic export protein injected via a transmembrane secretion system. There is no evidence of a type III or analogous secretion system in T. denticola Msp upsets the phosphoinositide balance that normally drives polarization and chemotaxis through coincident activation of PTEN and inhibition of PI3-kinase activation. The ability of Msp to diminish PIP3 levels leads to hierarchical inhibition of downstream chemotactic events, including Rac1 activation, actin filament uncapping, polarization, and directed cell migration.In summary, exposure of neutrophils to


Hsf1 and Hsf2, under normal conditions and after proteasome inhibition. We found that HSF1α is significantly more active than the β isoform after exposure to the proteasome inhibitor MG132. Furthermore, we clearly established that, while HSF2 had no transcriptional activity by itself, short β isoform of HSF2 exerts a negative role on HSF1β-dependent transactivation. To further assess the impact of HSF2β inhibition on HSF1 activity, we developed a mathematical modelling approach which revealed that the balance between each HSF isoform in the cell regulated the strength of the transcriptional response. Moreover, we found that cellular stress such as proteasome inhibition could regulate the splicing of Hsf2 mRNA. All together, our results suggest that relative amounts of each HSF1 and HSF2 isoforms quantitatively determine the cellular level of the proteotoxic stress response.Chaperone synthesis in response to proteotoxic stress is dependent on a family of transcription factors named heat shock factors (HSFs). The two main factors in this family, HSF1 and HSF2, are co-expressed in numerous tissues where they can interact and form heterotrimers in response to proteasome inhibition. HSF1 and HSF2 exhibit two alternative splicing isoforms, called α and β, which contribute to additional complexity in HSF transcriptional regulation, but remain poorly examined in the literature. In this work, we studied the transcriptional activity of HSF1 and HSF2 splicing isoforms transfected into immortalized Mouse Embryonic Fibroblasts (iMEFs) deleted for both  Proteasome is a major protein complex responsible for regulated degradation of intracellular proteins, and its activity is modified in many disorders. For example, a decrease in proteasome activity is associated with neurodegenerative diseases, whereas an increase catalytic activity is associated with cancers Moreover, both HSF1 and HSF2 exist under two different isoforms produced by alternative splicing. This increases the diversity of potential HSF homo and heterotrimers and adds more complexity to HSF regulation. Regarding HSF1, only two publications refer to the existence of two splicing isoforms (Hsf1α and Hsf1β) in mice Since the consequences of proteasome inhibition on HSF1 and HSF2 isoform expression and activity have not yet been fully characterized, we took advantage of existing cells which are deficient for both HSF1 and HSF2. Using defined transfections, we sought to determine the distinct role of each HSF1 and HSF2 isoforms independently of endogenous HSFs. Here, we show that HSF1α and HSF1β exert different transcriptional activity and we provide evidence for a specific repressor role of HSF2β. Finally, our data and analyses establish that the relative quantity of each HSF2 isoforms is regulated in a stress-dependent manner.hsf1−/− and −/−Hsf2 mice, previously provided by Dr IJ Benjamin  and described elsewhere Mixed genetic background wild type, Hsf1 and Hsf2 (−/−Hsf1.2) were obtained by intercrossing single knockout mice Immortalized Mouse Embryonic Fibroblasts wild type (WT) or deleted for both 2x-TATA-Firefly luciferase was obtained by cloning two synthetic HSEs (5′-GAAgcTTCtaGAAgcTTCtaGAAgcTTC-3′) followed by a TATA box into the pGL3 vector (Promega) at KpnI and SacI sites. The plasmid pTK-Renilla luciferase  was used as control to normalize transfection efficiency. All Hsf isoforms were cloned in pCR3.1 expression vector (Invitrogen). For Hsf1α, the coding sequence was amplified by RT-PCR from total RNA of iMEFs WT, using primers flanking the open reading frame and providing restriction site for EcoRI and NotI. Then, PCR fragment was cloned to pCR3.1 and the cloned isoform was verified by sequencing. The Hsf1β and Hsf2β cDNAs  were a gift from Dr Richard Morimoto  and they were subcloned in pCR3.1 at EcoRI site. The pβactin-HSF2α vector was a gift of Dr Kevin Sarge  and the cDNA was also subcloned in pCR3.1 at EcoRI and NotI sites.pHSEAll transfections were performed using JetPEI (Polyplus Transfection) according to the manufacturer’s instructions. Cells were harvested 24 h after transfection and 8 h of treatment with MG132 at 2.5 µM, or with an equivalent volume of dimethyl sulfoxide (DMSO) as control. For heat shock experiment, cells were maintained at 45°C during 20 min and then transferred at 37°C during 6 h for recovery. Firefly luciferase and Renilla luciferase activities were determined with Dual luciferase reporter assay (Promega).forward:CATGTCTAGTGCTGTCCAGC and reverse:5′-GAGCTCATCGACTTCTATGG-3′) were used in RT-PCR experiment. RT-PCR of the housekeeping gene Glyceraldehyde-3-phosphate deshydrogenase (Gapdh) (forward:5′-TGAAGCAGGCATCTGAGGG-3′ and reverse:5′-CGAAGGTGGAAGAGTGGGAG-3′) was used as control for the quality of extraction and retro-transcription. Densitometric analysis was made with Quantity One software (Bio-Rad).One million of WT iMEFs were plated on a 10 cm dish and treated with MG132 at 1 µM during 2 h, 4 h, 6 h, 8 h or 10 h. After 10 h of MG132 treatment, cells were allowed to recover for 1 h, 6 h or 10 h, and then harvested. Total RNAs were extracted with TRIzol reagent (Invitrogen) and 5 µg of RNAs were retro-transcribed using M-MLV RT (Invitrogen). To discriminate Hsf2α and Hsf2β, primers flanking alternative exon (forward: 5′-AGGGGAGTACAACTGCATCG-3′and reverse:5′- CAGGCGGACAAGCTTACTC-3′) forward:5′-AGTTCTGTGCAGATGAATCCCACAG-3′ and reverse:5′-GCAGATGCAGAGTTCCCATCC-3′). Experiments performed to measure Hsp70.1 transcripts used the following primers: Hsp70.1 forward: 5′-TTGTCCATGTTAAGGTTTTGTGGTATA-3′, Hsp70.1 reverse: 5′-GTTTTTTTCATTAGTTTGTAGTGATGCAA-3′. The experiments were performed at least in duplicate with one or two independent groups of embryos included in each experiment (n = 20 blastocysts). Results were normalized using 18S RNA amplification.RT-real time PCR experiments were performed on blastocysts as described in −/−Hsf1.2 iMEFs per well were seeded in a 6-well plate. Cells were transfected as described above and were harvested after 24 h of transient expression and 8 h of treatment with MG132 at 2.5 µM. To analyze the expression of endogenous HSF2, 1 million of WT cells were plated in a 10 cm dish and were treated the next day with MG132 at 1 µM, or with DMSO for 10 h. Whole cells extracts were prepared with NP-40 lysis buffer . Proteins were separated on an 8.5% or a 12% polyacrylamide gel to discriminate HSF2 isoforms and transferred to a nitrocellulose membrane (Amersham Bioscience). Anti-HSF1 (4B4) antibody (ab44819), anti-HSF2 (3E2) antibody (sc-13517) and Anti-GFP antibody (a11122) were purchased from Abcam, Santa Cruz Biotechnology and Molecular Probes, respectively.To evaluate protein expression after transfection, 200 000 Four hours of migration at 40 mA were necessary to separate HSF2α and HSF2β. Ponceau staining was performed to assess equal loading in place of classical actin control, which was excluded from the gel during the long electrophoresis.−/−Hsf1.2 iMEFs were transfected with pHSF1β-EGFP and/or with pCR3.1-HSF2α, or with pCR3.1-HSF2β. As control, cells were co-transfected with pEGFP and pCR3.1-HSF2α, or pCR3.1-HSF2β. Cells were treated with MG132 at 1 µM during 10 h. Anti-GFP antibody  was used for immunoprecipitation, carried out with Nuclear Complex Co-immunoprecipitation kit (Active Motif), according to manufacturer’s instructions.transactivation abilities of HSF1 isoforms, we first co-transfected increasing doses of expression vector coding either HSF1α or HSF1β, in −/−Hsf1.2 iMEFs, with pHSE2x-TATA-Firefly luciferase as a reporter gene. Then, cells were treated with MG132 at 2.5 µM for 8 h. As presented in transactivation and HSF1α exhibited 1.4 times higher plateau values than HSF1β. These data suggested that the α isoform was more efficient than the β one to activate HSE-dependent transcription under proteasome inhibition. Nevertheless, increasing quantities of transfected HSF1α vector was accompanied by a more pronounced basal activity (around 10 fold for 5 ng of transfected vector/1000 cells), indicating that the α isoform presents a weak stress-independent activity. To verify that the difference observed in transactivation abilities was not due to a defect in the level of expression of the transcription factors, immunoblots were performed with extracts from −/−Hsf1.2 iMEFs that had been co-transfected with an increased quantity of pCR3.1-HSF1α or pCR3.1-HSF1β, in addition to pEGFP used as a transfection efficiency control , confirming the inhibitory role of HSF2β.To verify that the inhibition observed in MG132 experiment was due to HSF2β itself and not to a side effect related to difference in transfection efficiency, we performed a dose effect experiment . Hsf1.2−−/−Hsf1.2 iMEFs were co-transfected with expression vector coding either HSF1β-EGFP or only EGFP, in association with HSF2 isoforms. Immunoprecipitation was performed with antibodies against EGFP and blots were probed with HSF2 antibodies. Cells transfected with HSF1β-EGFP, but not HSF2, were used as control and showed no signal after immunoprecipitation, demonstrating that the HSF2 antibodies do not cross-react with HSF1β (lane 3). Interestingly, we found that both HSF2α and β isoforms interact with HSF1β (lane 4 and 5) indicating the formation of heterotrimers. Moreover, HSF2 co-immunoprecipitation required the presence of HSF1, as demonstrated by its absence in the EGFP control (lane 1 and 2).The ability of each HSF2 isoform to associate with HSF1β in response to MG132 treatment was determined by co-immunoprecipitation experiment . Hsf1.2−−/−Hsf1.2 iMEFs were transfected with HSF1β and the same amount of total HSF2, including variable HSF2β/HSF2α ratios , but considering the close structural relationship between the trimerization domains of the different HSFs, K1 and K'1 could in fact be very similar. All types of HSF trimers are supposed to bind to the DNA with the same intrinsic dissociation constant K2. Global composite constants K or K' are defined such that K = K12K2 and K' = K'12K2 (M3). There are ten different possible types of trimers, distributing over 27 trimer microstates listed below, so that 11 possible states of HSE can exist, either empty or bound by a HSF trimer. Finally, once bound to DNA, HSF trimers devoid of HSF2β promote transcription from an HSE-driven promoter with transcription rates (time−1), depending on the number of HSF1 protomers in the trimer . Let us define the following variables: x = [HSF1*], the concentration of transcriptionally active HSF1; α = [HSF2α] and β = [HSF2β]T. The probability of the different binding states of HSE can be defined as the ratio of their mass action values over the sum of all of them, written Σ.To determine how the HSF2β/HSF2α ratio impacts the transcriptional activity of HSF1β, α ratios . HSF2 prα ratios  and densα ratios . HSE-driR = K/K'. In the experiment, the total amount of HSF2 is constant but the ratio of the α and β isoforms is defined by a variable z = β/α. The fixed total concentration of HSF2 is y = [HSF2] T = α+β, so that α = y/(1+ z) and β = y z/(1+ z). The transcriptional activity (k) of the HSE driven promoter can be defined as k = Σ i Pik, where iP is the probability of presence of the HSF trimer responsible for the maximal transcription initiation rate ikTo go further, a ratio of trimerization affinity is written x+y = H). Hence, a variable can be eliminated from Eq. 1, yieldingFinally in the experiment described above, the total concentration of transfected HSF is kept constant  Both HSF1 and HSF2 exist as two main splicing isoforms, but very few studies have focused on their respective transcriptional activity. It is uneasy to address this question in cells expressing endogenous HSF1 and 2, because of the coexistence of various HSF complexes, either homo- or hetero-trimers, using potentially both existing isoforms. Therefore, we took advantage of iMEFs genetically depleted for both factors, which provide an invaluable tool to dissect the precise role of each splicing isoforms of HSF1 and HSF2.We found that HSF1α and HSF1β possessed distinct transcriptional activities, HSF1α being a more potent activator under proteasome inhibition. When HSF1 isoforms were discovered, the authors hypothesized that HSF1α should be activated at higher temperatures than HSF1β, because exon 11 encodes a putative leucine zipper increasing the length of the HR-C domain, known to stabilize HSF1 in the monomeric non-DNA binding form Regarding HSF2, previous works in yeast The absence of specific antibody for each isoform, and the technical limitations to accurately determine the ratio of isoforms in a trimer, remain a real concern to further decipher HSF2 mechanism in HSF1 inhibition. In an attempt to alternatively address this question, we have developed a mathematical modelling approach. Our model is different from those previously published on the dynamic of the eukaryotic heat shock response Expression of HSF2 isoforms is tissue-specific. For example HSF2α is the main isoform in testis, while HSF2β is dominant in brain Taken together our data demonstrate how HSF1 and HSF2 splicing isoforms contribute a new level of complexity to HSF regulation. Our results raise additional questions: how efficient is the proteotoxic response in tissues where HSF1β and HSF2β are dominant? Which evolutionary mechanisms established such complex regulation of the relative quantity of HSF isoforms? It might be critical for stressed cells to possess various ways to modulate HSF1 activity, to achieve better adaptation, and thus survival. Heat shock is an acute stress with massive protein alteration, which requires a rapid and high HSP expression. In that case, HSF1 is found in homotrimeric complex, while HSF2 become insoluble and is found in the perinuclear fraction


Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive  mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of bitter taste and as a lead for the development of broad specificity bitter blockers to improve nutrition and medical compliance.Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to β-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors,  As the primary mechanism by which animals detect and evaluate nutrients within foods and avoid ingesting toxins, the sense of taste has a significant impact on food selection, nutrition, and health. For example, the sense of taste in individuals is reported to correlate with a variety of habits including dietary preference frizzled/smoothened family of GPCRs Bitter substances are recognized by, and bind to, a family of taste receptors (TAS2Rs) that are expressed in bitter taste cells, and initiate signaling via activation of intracellular heterotrimeric G proteins p-(dipropylsulfamoyl)benzoic acid) is an FDA-approved inhibitor of the organic anion transporter Multidrug Resistance Protein 1 (MRP1) and other organic anion transporters including probenecid to facilitate dye loading. During the course of our studies of bitter taste receptor signaling, we unexpectedly discovered that probenecid inhibited the activation of the bitter taste receptor hTAS2R16 in response to its cognate ligand salicin. This activity occurred rapidly and was independent of probenecid's activity as a transport inhibitor, suggesting that probenecid interacts with the receptor rather than modulating downstream signaling processes. Consistent with its rapid inhibition, hTAS2R16 point mutations can suppress probenecid inhibition, suggesting a direct interaction with hTAS2R16 and an allosteric inhibitory mechanism in which the salicin and probenecid binding sites are distinct. Inhibition by probenecid was also observed for additional TAS2R receptors, including hTAS2R38 and hTAS2R43, but not for hTAS2R31 or for other non-gustatory GPCRs tested. In human perceptual studies, probenecid suppressed the bitter taste perception of salicin, demonstrating a correlation between the in vitro findings of probenecid inhibition and human bitter taste phenotype. The discovery of probenecid as an inhibitor of bitter taste receptors and human bitter perception offers insight into a molecular mechanism for designing modulators of human taste perception for improved food selection, nutrition, and health.Probenecid  to HEK-293T cells transiently expressing hTAS2R16 and Gα16gust44 induces an increase in intracellular calcium levels that is measured using a Ca2+-activated fluorescent dye  and hTAS2R38 (in the presence of 300 µM PTC) and calculated IC50 values of 292 µM and 211 µM respectively . To furtTo identify structural elements of the probenecid molecule that contribute to TAS2R inhibition, we tested two probenecid analogs: 4-sulfamoylbenzoic acid, comprising the core scaffold of probenecid, and ethebenecid, containing ethyl groups rather than propyl groups on the sulfamoyl group . TreatmeTo determine whether probenecid directly interacts with hTAS2R16, we screened a random mutation library of hTAS2R16 for mutations that caused a loss of inhibition by probenecid. We identified two clones containing a total of three mutations, N96T, P44T, and H113R, which were significantly insensitive to probenecid inhibition while maintaining wild-type levels of responsiveness to salicin , p<0.001in vivo. Salicin is an appropriate stimulus to confirm perceptual efficacy because it interacts principally with the receptor hTAS2R16, which has relatively few receptor polymorphisms across subjects Because multiple taste receptors, receptor alleles, and signaling pathways are involved in human taste perception, taste receptor function in cellular assays and taste perception in humans often do not correlate in vitro.We next tested for the ability of probenecid to inhibit the perceived bitterness of saccharin, a well-characterized hTAS2R31 ligand increase the sensitivity of GPCR calcium flux assays in vitro, while leaving intact the function of other bitter taste receptors and GPCRs, including hTAS2R31, CXCR4, CCR5, and βAR. Interestingly, the inhibition of multiple bitter taste receptors was also observed for GIV3727, a recently described hTAS2R antagonist The human family of TAS2Rs is comprised of at least 25 GPCRs that are highly divergent in sequence, sharing about 30–70% amino acid homology Although the interaction of probenecid with a TAS2R receptor cannot be directly measured by binding or competition assays because bitter taste ligands bind too weakly (high µM to mM EC50s), our work provides several lines of evidence that the mechanism of action for probenecid inhibition occurs by direct binding to the hTAS2R16 receptor. First, analysis of hTAS2R16 point mutations define amino acid residues involved in probenecid binding or signaling that result in decreased sensitivity to probenecid while maintaining normal responses to the ligand salicin. Second, mechanism of action studies for probenecid against the hTAS2R16 receptor demonstrate rapid kinetics for complete inhibition (within 5 minutes of probenecid treatment) and near-instantaneous action for partial (>50%) inhibition, consistent with an effect on an upstream signaling component, such as the receptor itself. The effect of probenecid is also observed in the presence of inhibitors against MRP transporters  The identification of point mutations at residues 44 (P44T) and 96 (N96T) of hTAS2R16 that significantly suppress the ability of probenecid to inhibit salicin activity help to define probenecid's mechanism of action on the receptor. Both mutations affect probenecid activity without affecting salicin activity, suggesting an allosteric mechanism with distinct sites on the receptor for salicin and probenecid. This is in contrast to GIV3727, where mutations in hTAS2R43 and hTAS2R46 that confer resistance to inhibition alter both the specificity and activity of agonist compounds, suggesting an overlapping binding site As suggested by structural studies of class A GPCRs The intracellular location of the probenecid binding site suggests that probenecid may potentially act by uncoupling G proteins from the receptor. If so, probenecid may be a useful reagent for understanding signal transduction for TAS2R receptors. Additional mutational analysis to further define the binding sites for probenecid and salicin on hTAS2R16 will be important for a complete understanding of the molecular mechanism of probenecid inhibition and may provide insight for the rational design of effective bitter blockers.It is interesting to note that well-known polymorphisms in several TAS2Rs are found in the first intracellular loop, near the location of the P44 mutation in hTAS2R16 that confers probenecid resistance. For example, P49 of hTAS2R38 is located in the first intracellular loop and is part of the well-known PAV (taster) haplotype that confers sensitivity of individuals to PTC n-propyl groups, is consistent with the binding of probenecid to a site on the intracellular face of the receptor, such as one that blocks binding of a G protein to the receptor.Our studies using probenecid analogs suggest that inhibitor hydrophobicity is important for the pharmacological activity of probenecid. In particular, the propyl groups of probenecid may provide a better fit for the size and/or hydrophobicity of the putative probenecid-binding site on hTAS2R16. The ability of probenecid to cross the plasma membrane in vitro provides a mechanistic explanation for its ability to inhibit human bitter taste perception of salicin in vivo. Future perceptual testing with a variety of diverse bitter compounds will help to determine whether the inhibitory effect of probenecid in vivo includes additional TAS2Rs.Finally, the ability of probenecid to completely inhibit the cellular response to salicin Salicin, probenecid, 6-n-propylthiouracil (PROP), 4-sulfamoylbenzoic acid, N,N-diethyl-4-sulfamoylbenzoic acid (ethebenecid), chloroquine diphosphate, saccharin sodium salt hydrate, aloin, quinine HCl, indomethacin, and isoproterenol were purchased from Sigma . Probenecid (Sigma P-8761) was dissolved at 500 mM in 1 N NaOH and titrated to pH 7.0. Phenyl-β-D-glucoside was purchased from TCI . SDF-1α and RANTES were purchased from Peprotech , and phenylthiourea (PTC) was purchased from Alfa Aesar .α16 chimera containing the last 44 amino acids of rat gustducin (Gα16gust44) was generated by overlap PCR. All constructs were sequence verified.hTAS2R16 (N172/H222 variant) HEK-293T cells were transfected with hTAS2R expression constructs using Lipofectamine 2000 (Invitrogen) in poly-lysine coated, black 384-well plates with clear bottoms (Costar) and incubated for 22 hours at 37°C. Growth media was removed and cells were washed twice with HBSS containing 20 mM HEPES, then loaded with a calcium indicator dye in HBSS containing 20 mM HEPES  with or without 1 mM probenecid. Cells were incubated at 37°C for 1 hour in the presence of both dye and probenecid, then moved to a Flexstation II-384 (Molecular Devices) set for 32°C. After a 15-minute temperature equilibration (without washout), indicated compounds were injected (at t = ∼25 seconds) and fluorescence was measured for 100 to 180 seconds, reading every 3 seconds. Data sets were analyzed and represented as % over baseline signal using Prism 5.0 software . For Schild plots, replicates of raw calcium flux values were expressed as % over baseline signal. The mean value at 36 seconds  for each concentration of TAS2R ligand in the presence of the indicated concentration of probenecid was plotted against the log of ligand concentration. Data points were fit using non-linear regression in GraphPad Prism.Subjects for perceptual studies were recruited and tested with a protocol approved by the Office of Regulatory Affairs at the University of Pennsylvania. Written consent was obtained on a Regulatory Affairs-approved consent form. For the saccharin stimulus and a parallel salicin stimulus, subjects were given 2.3 mM lactisole in mixture with the stimuli, to inhibit the sweet taste of saccharin. Subjects were presented with the stimuli, either 30 ml of 10 mM salicin, 250 mM saccharin plus 2.3 mM lactisole, or 10 mM salicin plus 2.3 mM lactisole in 40 ml medicine cups (Baxter), and were asked to rate the bitterness intensity on a general labeled magnitude scale (gLMS) that ranged from “Barely Detectable” to “Strongest Imaginable” along a computerized vertical 96 mm scale with magnitude labels spaced semi-logarithmically. Subjects immersed the tongue (anterior 2 cm) into the stimulus solution for five seconds while sealing their lips around their tongue to control the area of stimulation. They were then given a series of five cups containing 10 ml of either 10 mM probenecid (dissolved in 1 N NaOH and pH adjusted to 7.4 using 1 M HCl) or a control stimulus (8.1 µM quinine HCl) and asked to rinse with each cup for two minutes and expectorate after each rinse. After rinsing, subjects were again presented with the same stimulus tasted before treatment and asked to rate the bitterness intensity from the anterior tongue. All stimuli were tested blindly with both rinses in a 2×2 design. 8.1 µM quinine HCl was selected as the control rinse to match the bitterness intensity of 10 mM probenecid based on pilot testing with ten subjects to control for bitter taste cross adaptation effects of the inhibitor.Figure S1Analysis of hTAS2R16 H113R single mutant for probenecid sensitivity. HEK-293T cells were transfected with hTAS2R16 variant H113R followed by challenge with 3 mM salicin in the presence or absence of probenecid . The H113R mutant demonstrated wild type levels of sensitivity to probenecid and salicin. The light gray trace represents inhibition of wild type hTAS2R16 in this experiment and is shown for comparison.(TIFF)Click here for additional data file.


Serines 452/453 and 456/457 were phosphorylated upon phosphatase blockade by calyculin A, or in response to IL-1 or translational stressors such as anisomycin and sorbitol. Deletion or phospho-mimetic mutations of aa 452–457 of TAB1 retain TAB1 and p38 MAPK in the cytoplasm. The TAB1 mutant lacking aa 452–457 decreases TAB1-dependent phosphorylation of p38 MAPK. It also enhances TAB1-dependent CCL5 secretion in response to IL-1 and increases activity of a post-transcriptional reporter gene, which contains the CCL5 3′ untranslated region. These data suggest a complex role of aa 452–457 of TAB1 in controlling p38 MAPK activity and subcellular localization and implicate these residues in TAK1- or p38 MAPK-dependent post-transcriptional control of gene expression.TAB1 was defined as a regulatory subunit of the protein kinase TAK1, which functions upstream in the pathways activated by interleukin (IL)-1, tumor necrosis factor (TNF), toll-like receptors (TLRs) and stressors. However, TAB1 also functions in the p38 MAPK pathway downstream of TAK1. We identified amino acids (aa) 452/453 and 456/457 of TAB1 as novel sites phosphorylated by TAK1 as well as by p38 MAPK in intact cells as well as  A complex interplay of protein kinases and their phosphorylated substrates regulates major aspects of immune and stress responses TAK1 activation is tightly controlled by reversible phosphorylations, non-degradative ubiquitination and by protein:protein interactions. The latter include interactions with TAK1-binding proteins (TAB) 1–3 which all have been shown to participate in TAK1 activation. Hence, TAB1–3 can be viewed as crucial regulatory subunits of the active TAK1 kinase complex The TAB1 subunit is also present in TAK1/TAB2-polyubiquinated immunoprecipitated protein complexes after IL-1 stimulation In addition to TAK1, TAB1 interacts with p38 MAPK and activates its autophosphorylation by an allosteric mechanism. TAB1-mediated p38 MAPK autoactivation occurs independent from all three p38 MAPK-activating kinases  but accounts for only a small portion of overall p38 MAPK activity in a cell-and stimulus-dependent manner As illustrated in the upper panel of By mass spectrometry and by phospho-site specific antibodies, TAB1 was shown to be phosphorylated at S423, T431 and S438 by ERK1, p38 MAPK or JNK In particular, the physiological role of TAB1 is still enigmatic. While ablation in mice or RNAi-mediated suppression of TAB1 has no effect on IL-1-, TNF-, or TLR-induced activation of NF-κB, JNK, or p38 MAPK signaling pathways, suppression of TAK1 abolishes these signals In previous experiments aimed at investigating the effects of activated p38 MAPK or TAK1 on TAB1 we have described three different posttranslationally modified forms of TAB1 that can be distinguished based on mobility shifts upon SDS-PAGE In addition to the already described S423, T431 and S438 residues, the C-terminal part of TAB1 contains several conserved serine/threonine residues which may also be subject to regulatory phosphorylations . As exemIn contrast, deletion of six serines comprising aa 452–457 (TAB1ΔS) abolished TAB1 form 1 in response to both, overexpressed MKK62E/p38 MAPK , lane 7 In order to identify which of the six serines were phosphorylated, we generated phospho-specific antibodies against di-phosphorylated peptides containing either SS452/453 or SS456/457. A careful mutational analysis revealed the specificity of these antibodies for their respective residues . As showin vitro with a kinetic similar to that of the already known p38 MAPK site S438 physiological settings . Howeverin vitro kinase activity of immunoprecipitated TAK1:TAB1 complexes . Therefore, we tested if TAB1 was able to affect phosphorylation of p38 MAPK in intact cells. Both, TAB1 and p38 MAPK were co-expressed and the phosphorylation state of p38 MAPK was investigated by immunoblot analysis of whole cell lysates . This effect is not changed by co-expressed wild type MYC-TAB1 (rd column). However, co-expression of TAB1ΔS or TAB1 4SE but not of a TAB1 4SA mutant resulted in an almost complete cytosolic re-localization of both, p38 MAPK and TAB1 (th to 6th column). These results suggested that the serine cluster or its phosphorylation regulate cytosolic retention of TAB1 and of p38 MAPK.To further reveal the role of aa 452–457 in p38 MAPK signaling, we analyzed the effects of deletion (TAB1ΔS), and phosphomimetic (TAB1 4SE) or phosphorylation-deficient (TAB1 4SA) mutants on both, p38 MAPK and TAB1 localization. In agreement with Collectively, the results shown in In order to assign additional biological functions to aa 452–457 of TAB1 we used stably reconstituted TAB1-deficient Mefs infected with retroviral expression vectors containing either untagged  or MYC-tCCL5 expression. As TAB1-deficient Mefs were difficult to transfect transiently we analyzed A549 cells which strongly upregulate CCL5 protein and mRNA in response to IL-1 in a TAK1-dependet manner . This would suggest that the protein:protein interaction is either very labile or indirect. However, TAB1 promoted p38 MAPK phosphorylation primarily at T180 and the gain-of-function mutant of p38 MAPK Y182F promoted TAB1 phosphorylation at aa 452–457 adding further evidence for a mutual control of both proteins .CCL5 mRNA stability  CCL5 3′UTR and pSGG-luciferase-EMPTY , cloned into pCR-Blunt II-TOPO and then subcloned into the EcoRI and XhoI sites of pCS2-MT. A ClaI fragment of pCS2-MT-MYC(6)-TAB1 was then cloned into the ClaI site of pM5XNeo to generate pM5XNeo-MYC-TAB1. pM5XNeo-TAB1 was generated by excising the MYC-tags by EcoRI digestion and religation. Recombinant retroviruses and infections of Mef cells were done exactly as described in For retroviral expression, a cDNA encoding TAB1 was amplified from pCDNA3.1-HA-TAB1 Calcium phosphate transfections and reporter gene assays were performed as described 5 in 9,4 mm wells. 24 h later cells were transfected in duplicates with 2.5 µg plasmid DNA containing 0.4 µg pSV40-ß-galactosidase + 0.1 µg pSGG-luciferase- CCL5 3′UTR or pSGG-luciferase-EMPTY in combination with 2 µg empty vector control or pCS2MT-MYC-TAB1wt or pCS2MT-MYC-TAB1ΔS) and 3 µl PLUS reagent and 3.75 µl lipofectamine in DMEM per well. After 6 h at 37°C, 5% CO2, the medium was complemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin. The next day, cells were harvested and splitted into two halves one of which was used to extract mRNA and the other was lysed for determination of luciferase activity or equal expression of the TAB1 constructs.Lipofectamine transfections of A549 cells were performed according to the manufacturer's instructions . Specifically, A549 cells were seeded at 5–6×10CCL5 (Hs00171085_m1), ß-actin (Hs99999903_m1) and luciferase .RT-qPCR was performed as described 3VO4, 5 mM sodium pyrophosphate, 0,1% ß-mercaptoethanol and a Roche protease inhibitor mix). HEK293IL-1R cells . p38 MAPK was immunoprecipitated from 1.5 mg cell extract protein in , 1% Triton X-100, 1 mM Na3VO4, 2 mM DTT, 50 mM NaF) using rabbit polyclonal anti p38 MAPK antibodies in vitro kinase assays.For immunoprecipitation of p38 MAPK , cells win vitro kinase assay or were splitted into two halves one of which was boiled for 5 min in 2× Roti-Load (Roth) before loading on 8% SDS-PAGE. The other half was redissolved in 10 µl washing buffer for in vitro kinase assay.For immunoprecipitation of TAK1-TAB1  or GFP-T3VO4, 1 mM PMSF, Roche protease inhibitor mix, 1 mM DTT, 1 µM microcystin), sonified 3×20 seconds on ice and centrifuged at 100.000×g for 20 min at 4°C. 1.5 mg of the supernatant was used for IP with 1.5 µg of anti TAB1 (H-300) or IgG (sc-2027) antibodies coupled for 1 h to 15 µl of Trueblot anti-mouse Ig immunoprecipitation beads . After 2 h, beads were washed three times in the lysis buffer including 450 mM NaCl. Proteins were eluted by boiling in 2× Roti-Load before loading on SDS-PAGE.For immunoprecipitation of endogenous TAB1  cells wein vitro kinase assay to measure TAK1 kinase activity using recombinant HIS(6)-MKK6 and γ[32P]ATP as substrates was performed as described Immunoprecipitation of the endogenous TAK1-TAB1-TAB2 complex  using TAin vitro kinase assays were carried out at 37°C for 30 min with agitation in 30 µl of reaction buffer containing 10 µl H2O including approximately 1 µg of recombinant GST or HIS-tagged protein, 10 µl of redissolved protein G Sepharose beads containing kinases immunoprecipitated from cell extracts and 10 µl . For the experiments shown in 32P]ATP were added to the reaction buffer and phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography.Unless stated otherwise, HEK293IL-1R cells were cultured in 9,4 mm wells. After 24 h of transfection, cells were seeded for 24 h in μ-slides VI (Ibidi). After washing, cells were fixed with 4% paraformaldehyde in Hank's BSS (PAA Laboratories) for 5 min, blocked with 10% normal donkey serum (Dianova) for 30 min and incubated with primary and secondary antibodies diluted in Hank's BSS containing 0.1% Saponin (Sigma) for 2 h at room temperature. The primary antibodies were anti-TAB1 , anti-FLAG M2 , anti-MYC 9E10  and anti-P(SS452/3)-TAB1 (1∶100). Cy3-congugated  and FITC-conjugated  secondary antibodies were used. For controls, primary antibodies were omitted. Nuclei were stained with Hoechst 33342 (Invitrogen). Fluorescence imaging was performed on a Leica DM IRE2 fluorescence microscope. Captured images were analysed with the Leica FW4000 Fluorescence Workstation software.Immunoblotting was performed essentially as described The mouse cytokine array panel A kit (ARY006) and the mouse CCL5 duoset (DY478) were used according to the manufacturer's instructions .2 flask of cells were transfected with 10 µg of expression vectors for HA-TAK1, MYC-TAB1, p38 MAPK plus MKK62E (5 µg each) and 20 µg of pDest26-HIS(6)-Ubiquitin or pDest26-HIS(6). Total amount of DNA was adjusted to 50 µg by adding pCDNA3.1. After 24 h cells were lysed in 1 ml of lysis buffer . Lysates were sonicated to shear DNA and cleared by centrifugation for 5 min at 15.000×g. 3 mg of lysate protein were incubated with 50 µl of Ni2+- NTA agarose for 3 h at room temperature. Beads were collected by centrifugation, washed twice in lysis buffer, twice in a buffer containing 1 volumes of lysis buffer and three volumes of buffer TI , and twice with buffer TI alone. Bound proteins were eluted for 5 min at 95°C in SDS PAGE sample buffer. 120 µg of proteins from the initial lysate (input) were precipitated by adding one volume of icecold TCA (10%). The pellet was collected at 15.000×g for 15 min at 4°C, washed once in two volumes of icecold EtOH (100%), dried in a speed vac and resuspended in 100 µl of SDS PAGE sample buffer at 95°C for 5 min. Bound and input protein samples were separated by 8%SDS-PAGE including 4.5% glycerol and analysed by western blotting.Ubiquitination of TAB1 and TAK1 was determined as described in Figure S1Evidence for new phosphorylation sites in TAB1 in addition to S423, T431 and S438. A) HEK293IL-1R cells were transiently transfected with expression vectors for HA-TAB1 wild type, or versions in which S423/T431/S438 (STS) were mutated to alanine as indicated alone or in combination with GFP-YopP, FLAG-p38 MAPK plus MKK62E or HA-TAK1. B) A similar experiment as in A) was performed using a TAB1 mutant in which S425 was mutated to alanine (HA-TAB1 S425A). 24 h later, cells were lysed followed by immunoblotting (IB) to detect HA-TAK1, FLAG-p38 MAPK or HA-TAB1 using the indicated antibodies. Black arrowheads indicate the three forms of TAB1 (numbered 1–3) with different mobility on SDS-PAGE as previously described by us (TIF)Click here for additional data file.Figure S2Validation of phospho-specific antibodies recognizing phosphorylation of SS452/453 and SS456/457 of TAB1. HEK293IL-1R cells were transiently transfected with expression vectors for FLAG-p38 MAPK plus MKK62E (A) or HA-TAK1  in the indicated combinations with wild type HA-TAB1 (wt) or versions in which serines 452, 453, 456, 457 were point mutated to alanine  or to glutamic acid (C). 24 h later, cells were lysed and expression of FLAG-p38 MAPK or HA-TAK1 and expression or phosphorylation of HA-TAB1 was analyzed by immunoblotting (IB) using the indicated antibodies. Black arrowheads indicate forms of TAB1 with different mobility as described in the legend of (TIF)Click here for additional data file.Figure S3Amino acids 452–457 play only a marginal role in TAK1 activation and in ubiquitination of the TAK1-TAB1 complex. A) HEK293IL-1R cells were transiently transfected with GFP-TAK1 and the indicated TAB1 expression vectors. 24 h later, cells were lysed, TAK1 was immunoprecipitated using GFP antibodies and its kinase activity was determined in vitro using recombinant HIS(6)-MKK6 and 32P-ATP as substrates. Reaction mixtures were separated on SDS-PAGE and phosphorylated MKK6 was detected by autoradiography (left panel) as described previously in vitro kinase assays as described in A). Lane 1 indicates a sample in which the cell extract was omitted in the kinase reaction to control for autophosphorylation of bacterially expressed HIS(6)-MKK6. D) HEK293IL-1R cells were transiently transfected with empty vector or an ubiquitin expression vector (HIS-Ubiquitin) and combinations of epitope-tagged expression vectors for TAK1, TAB1, p38 MAPK and MKK62E as indicated. 24 h after transfection, cells were lysed in denaturing buffer. Ubiquitinated proteins were purified on Ni2+-NTA agarose. Lysates and affinity purified proteins were analyzed by immunoblotting using the indicated antibodies. Details are described in the methods section and in (TIF)Click here for additional data file.Figure S4TAK1-dependent activation of the CCL5 gene in A549 cells. A459 lung epithelial carcinoma cells were treated for 30 min with the TAK1 inhibitor 5Z-7-oxozeaenol (1 µM) followed by IL-1α (10 ng/ml) for 6 h, IL-1 alone or were left untreated. Thereafter, CCL5 secretion in the supernatant (A) or CCL5 mRNA expression (B) were determined by specific ELISA and RT-qPCR, respectively. Shown are mean values +/− s.e.m. form 3 independent experiments.(TIF)Click here for additional data file.


The human vasculature includes a vast network of microcapillaries networking the body and is a major target for non-carcinogenic effects of space radiation in the body. The brain microvascular system is crucial to healthy functioning of the brain and its pathology is not only a primary event in a range of neurodegenerative diseases but also an important influencing factor in many others. The vasculature is maintained by angiogenesis regenerating vessels as they are needed, this is particularly relevant if the blood–brain barrier is damaged by agents such as space radiation, thereby creating the need for angiogenic regeneration. The resulting lack of vasculature due to the inhibition of re-growth of vessels can, in turn, lead to a negative feedback loop and further pathologies.Using three-dimensional human vessel cultures with human umbilical vein and brain microvascular endothelial cells, we have developed assays that determine at what stage angiogenesis is inhibited by ionizing radiation. The relative biological effect of high linear energy transfer (LET) 1 GeV Fe ions compared with low LET 1 GeV protons is only one for developing vessels but greater than four for mature vessels. This action of low LET protons on developing vessels was surprisingly effective (50% inhibition with 40 cGy exposure) and together with the effect of high LET ions may represent a significant hazard in the space environment.The morphology of developing vessels 5 days after exposure showed significant differences that suggest distinct mechanisms of inhibition. Cells exposed to protons failed to make connections with other cells. Conversely, cells exposed to Fe ions extended cellular processes and made connections to other cells but did not develop a central lumen. The microtubule and actin cytoskeletons showed differences indicating that motility at the extending tips of endothelial cells is inhibited by protons but not Fe ions. Actin-rich protrusive structures that contain bundled and dynamic microtubules showed a 65% decrease when exposed to high-energy protons but not with the same dose of high-energy Fe ions. Since protein kinase C (PKC) has long been known to stimulate angiogenesis, we hypothesized that rescue of the capillary phenotype after proton exposure would be possible by stimulating PKC before irradiation. One-day-old vessel cultures were treated with 30 and 60 nM phorbol ester (PMA) 15 min before irradiation. Stimulation of PKC restored capillary formation in proton-treated cultures but not in Fe ion-treated cultures. More specifically, stimulation of PKC by PMA was able to restore the tip motility that was inhibited by low LET ions [Further studies with various charged particles showed that low LET ion particles (Proton and He ions) with an LET lower or equal to 1 keV/μm inhibit vasculogenesis in the same way as protons. Higher LET charged particles  with an LET ≥8 keV/μm inhibit vasculogenesis in the same way as Fe ions.In conclusion, we have shown that low and high LET ions inhibit the formation of brain capillaries by different mechanisms. For low LET ions, inhibition involves regulation of PKC-dependent motile tips leading to a failure of cellular processes to migrate through the matrix and meet up with other processes. For high LET ions, the cells fail to complete angiogenesis by not migrating and forming tubular structures. This complexity of response opens up possibilities of greater control over angiogenesis and the resulting pathologies during coincident exposure or therapy. For exposure in space, knowledge of these mechanisms will enable more precise risk assessment and mitigation strategies. For radiotherapy, treatment could be manipulated to utilize the radiation effectively.


An ability to sense pathogens by a number of specialized cell types including the dendritic cells plays a central role in host's defenses. Activation of these cells through the stimulation of the pathogen-recognition receptors induces the production of a number of cytokines including Type I interferons (IFNs) that mediate the diverse mechanisms of innate immunity. Type I IFNs interact with the Type I IFN receptor, composed of IFNAR1 and IFNAR2 chains, to mount the host defense responses. However, at the same time, Type I IFNs elicit potent anti-proliferative and pro-apoptotic effects that could be detrimental for IFN-producing cells. Here, we report that the activation of p38 kinase in response to pathogen-recognition receptors stimulation results in a series of phosphorylation events within the IFNAR1 chain of the Type I IFN receptor. This phosphorylation promotes IFNAR1 ubiquitination and accelerates the proteolytic turnover of this receptor leading to an attenuation of Type I IFN signaling and the protection of activated dendritic cells from the cytotoxic effects of autocrine or paracrine Type I IFN. In this paper we discuss a potential role of this mechanism in regulating the processes of innate immunity. We have previously observed and reported that the activation of unfolded protein responses during infection with some viruses such as vesicular stomatitis virus and hepatitis C virus compromises the cellular responses to the cytokines that belong to family of Type I interferons (IFNα/β). These effects apparently rely on the activation of a specific protein kinase termed PERK that transduces signals from unfolded proteins to accelerate phosphorylation-dependent degradation of the IFNα/β receptor. Here we found that the same pathway can be PERK-independent and can be stimulated by signaling induced in the specialized cells (e.g. dendritic cells that produce IFNα/β), which recognize pathogens (e.g. viruses). This work delineates a mechanism by which the degradation of IFNα/β receptor is accelerated. Our studies also highlight the importance of this mechanism for limiting the magnitude and the duration of IFNα/β signaling and maintaining the survival of IFN-producing dendritic cells. Cytokines that belong to the family of Type I interferons , play an important role in innate immunity Type I IFN mediates its effects through the stimulation of the Type I IFN receptor (which consists of IFNAR1 and IFNAR2 chains) and the subsequent activation of the Janus kinases TYK2 and JAK1, phosphorylation of STAT1/2 proteins, and ensuing trans-activation of a plethora of interferon-stimulated genes. The products of these genes directly suppress the spread of some pathogens  and cooperate with inflammatory cytokines in DCs to promote antigen presentation linking innate and adaptive immunity  within IFNAR1's degron All responses to IFNα/β require the presence of the Type I IFN receptor at the cell surface We have recently discovered an alternative (TYK2 activity-independent) signaling pathway that leads to S535 phosphorylation and to the subsequent ubiquitination and downregulation of IFNAR1 in the absence of a ligand. As a result, IFNAR1 is being removed from the surface of those cells, which have not been yet exposed to IFNα/β Here we report that the DNA-containing herpes simplex virus (HSV) also induced the ligand/TYK2-independent phosphorylation and downregulation of IFNAR1. Remarkably, these effects required neither viral protein synthesis nor PERK activity but relied on the activation of the PRR signaling pathways that center around the activation of the p38 protein kinase. This kinase was required for the phosphorylation of the IFNAR1 priming site leading to an ensuing phosphorylation of the IFNAR1 degron on Ser535/526, acceleration of IFNAR1 degradation, and attenuation of IFNα/β signaling. These events appear to be important for the protection of DCs from autocrine/paracrine Type I IFN.RNA-containing viruses (HCV and VSV) can downregulate IFNAR1 in human KR-2 cells that harbor a catalytically inactive TYK2 and that are deficient in IFNα-stimulated Ser535 phosphorylation of IFNAR1 and the degradation of this receptor chain Ifnar1 allele was replaced with a mutant allele that lacks Ser526  that did not induce changes in the status of IFNAR1 (data not shown) until later periods of the infection (24–30 h post infection) which were chosen for the maximal expression of viral proteins to induce UPR. Under these conditions, HCV and VSV required PERK activity to promote IFNAR1 phosphorylation and degradation PERK (PERKfl/fl) where PERK is acutely excised upon transduction with a retrovirus encoding the Cre recombinase We further tested the requirement of PERK by using MEFs from mice harboring a conditional knockout allele of 7 to 3 pfu. Remarkably, the treatment of KR-2 cells with a high dose of either active or inactive HSV sufficed for inducing the priming phosphorylation of IFNAR1 within 60–90 minutes (We next tested a possibility that a requirement for prolonged infection and ensuing HSV replication needed to observe the effects of low doses of HSV (MOI 0.1) might be foregone if more viruses are used initially. To this end, we compared the effects of HSV (at MOI 5.0) that was either sham treated or irradiated with UV for inactivation. The latter procedure decreased the titer of this viral preparation from 7×10 minutes . Further minutes  indicatiS532A mutant . To determine the role of IFNAR1 proteolytic turnover in this process we used a standard approach of blocking the protein synthesis by treating the cells with cycloheximide. Under these conditions, the treatment of cells with inactivated HSV markedly accelerated the rate of degradation of either endogenous IFNAR1  or exogeA mutant . TogetheOne possibility is that such a signaling pathway could be initiated by the recognition of pathogen patterns within inactivated HSV. HSV was reported to activate Toll like receptors (TLR), including TLR9, via viral genomic DNA To test this possibility, we aimed to determine whether other known inducers of PRR signaling were capable of stimulating priming phosphorylation of IFNAR1. To this end, we switched from KR-2 fibrosarcoma cells to the types of cells that actually function to present foreign antigens, and, accordingly, express numerous types of pathogen recognition receptors. The treatment of human monocytic U937 cells with inducers of TLR9 (CpG) or TLR4  led to a robust phosphorylation of IFNAR1 on its priming site . FurtherSignaling pathways triggered by the activation of PRR are known to induce a number of important regulatory kinases such as Jun N-terminal kinases (JNK), IκB kinases (IKK), stress-activated p38 protein kinases, and mitogen-activated Erk kinases  was assessed by immunoblotting using a phosho-S532-specific antibody as in . Under tubstrate . Finallyubstrate . This acubstrate . Given tubstrate , these rConsistent with the importance of priming phosphorylation in the ligand-independent pathway, the treatment of U937 cells with LPS activated p38 kinase and also stimulated the phosphorylation of S535 within the degron of IFNAR1 . This phWe then investigated the effects of PRR signaling on the degradation of IFNAR1. Consistent with previous reports in other cell lines We next sought to investigate whether the acceleration of IFNAR1 degradation by PRR signaling may attenuate the cellular responses to Type I IFN. We focused on the activation of STAT1 (assessed by its tyrosine phosphorylation) in mouse bone marrow macrophages that robustly responded to exogenous murine IFNβ, and where such a response could be readily abolished if neutralizing antibodies against IFNα and IFNβ were added to the reaction , it could be noticeably increased by incubating these cells with the IFNAR1-neutralizing antibody . Similar data were obtained when Annexin V-negative CD11c-expressing cells were analyzed  are distinguished with an advanced ability to recognize a variety of pathogenic patterns and, upon this activation, synthesize and secrete Type I IFNs and other cytokines that participate in shaping the immune responses As indicated by our data . Vectors for bacterial expression of GST-IFNAR1 and mammalian expression of human and murine Flag-IFNAR1 were described previously 2 or sham treated for the same time period.VSV  and HSV-1  were propagated in HeLa cells. These cells were also used to determine the viral titers in serially diluted stocks using methylcellulose method. In experiments requiring inactivated virus, virus suspension was placed in Petri dishes and exposed either to UV-C light (254 nm) for 5 min to achieve the total dose of 1500 J/mHuman monocytic U937 and HeLa cells were from ATTC. Replication-deficient lentiviral particles encoding shRNA against GFP (shCON), PERK, p38, or the empty virus control were prepared via co-transfecting 293T cells with three other helper vectors as described previously This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania (protocol # 800992). Every effort was made to minimize animal suffering. IFNAR1-null mice were kindly provided by D.E. Zhang (UCSD). Bone marrow-derived macrophages (BMM) and bone marrow-derived dendritic cells (BMDC) were produced as described previously These assays were carried out as described previously 2, 1 mM NaF, 1 mM NaVO3) and ATP (1 mM) at 30°C for 30 min. Samples were then separated by 10% SDS-PAGE and analyzed by immunoblotting with phospho-specific antibodies.Kinase assays were carried out as described previously Figure S1PCR analysis of DNA from tails of back-crossed chimeric Ifnar1S526A mice of the indicated gender. Presence of the SA alleles in 3 founders is indicated by a PCR product that migrates slower due to a remaining loxP site.(TIF)Click here for additional data file.Figure S2BMDC from wild type mice or IFNAR1 S526A mutant mice were untreated or treated with neutralizing anti-IFNAR1 antibody  or normal mouse IgG (mIgG) and activated by LPS (2 µg/mL for 48 h). Percent of CD11c-positive and Annexin V-negative viable DCs is depicted as an average of three independent experiments (±S.D.).(TIF)Click here for additional data file.


The enteric nervous system contains excitatory and inhibitory neurons which control contraction and relaxation of smooth muscle cells and gastrointestinal (GI) motor activity. Nitric oxide (NO) plays an important role as a non-adrenergic non-cholinergic inhibitory neurotransmitter in the enteric nervous system, which activates the NO-GC/cGMP/PKG signalling pathway and thus relaxation of the smooth musclulature in the GI tract. Interstitial cells of Cajal (ICC) act as pacemaker cells in the GI tract by generating slow waves of depolarisation to induce rhythmic smooth muscle contractions. In addition, our previous work established a surprising role of ICCs in excitatory and inhibitory nitrergic neurotransmission. The aim of the present study was to investigate molecular and cellular mechanisms, which mediate regeneration of intestinal slow-waves and inhibitory nitrergic neurotransmission upon acute damage of the ICC network.CreERT2c-Kit knock-in allele at the endogenous c-Kit locus. This tamoxifen inducible mouse model enables genetic manipulation and depletion of ICC as well as regeneration studies at defined time points during development and in adults in vivo. To investigate the role of ICC in transducing the nitrergic inhibitory signal, we deleted cGMP-dependent protein kinase I (Prkg1), the central mediator of the non-adrenergic, non-cholinergic neurotransmission in ICC using floxed Prkg1 animals. Furthermore we crossed CreERT2/c-Kit+ mice with conditional DTA/LSL-R26+ animals, which carry a latent diphtheria toxin A (DTA) expression cassette to deplete the ICC network. Using these models, we subsequently investigated molecular and cellular mechanisms which mediate regeneration of slow-waves and GI motility over time.To evaluate the role of ICC in excitatory and NO-dependent inhibitory neurotransmission after acute damage, we generated a Prkg1 in ~40% of all ICC abolished specifically the NO-dependent component of the inhibitory junction potential in colonic circular smooth muscle cells. This resulted in a significantly disturbed GI motility with a profound increase in total GI transit time, as seen in animals with a disruption of the ICC network due to expression of DTA. Interestingly, GI motility, slow-wave activity and enteric neurotransmission recovered completely within 5 weeks. However, we found no overt recovery of ICC cell number, reexpression of Prkg1 or proliferation of precursor cells.Deletion of in vivo genetic evidence for a surprising plasticity of ICC which restores normal gut function after damage of the ICC network.Our results suggest that adaptive mechanisms of the remaining ICC restore pacemaker activity and enteric neurotransmission. Therefore, we provide first


Nucella lapillus generally has thicker shells at sites sheltered from wave action, where crabs are abundant and pose a high risk of predation, than at exposed sites where crabs are rare. We studied two populations showing the opposite trend. We reciprocally transplanted snails between field sites and measured shell length, width and lip thickness of those recaptured 12 months later. Snails transplanted to the sheltered site grew larger than sheltered-site residents, which in turn grew larger than transplants to the exposed site. Relative shell-lip thickness was greater in residents at the exposed site than at the sheltered site. Transplants from shelter to exposure developed relatively thicker shells than their controls and relatively thinner shells from exposure to shelter. Progeny of the two populations were reared for 12 months in a common garden experiment presenting effluent from crabs feeding on broken conspecifics as the treatment and fresh sea-water as the control. The crab-effluent treatment decreased foraging activity, concomitantly reducing cumulative somatic growth and reproductive output. Juveniles receiving crab-effluent grew slower in shell length while developing relatively thicker shell lips than controls, the level of response being similar between lineages. F2 progeny of the exposed-site lineage showed similar trends to the F1s; sheltered-site F2s were too few for statistical analysis. At sexual maturity, shell-lip thickness was greater in snails receiving crab-effluent than in controls, indicating plasticity, but was also greater in the exposed-site than in the sheltered-site lineage, indicating heritable variation, probably in degree of sexual thickening of the shell lip. Results corroborate hypotheses that ‘defensive’ shell thickening is a passive consequence of starvation and that heritable and plastic control of defensive shell morphology act synergistically. Shell thickening of juveniles was similar between lineages, contrary to hypotheses predicting differential strengths of plasticity in populations from low- or high-risk habitats.The intertidal snail  It is widely reported that certain rocky shore gastropods develop thicker-walled shells at sites sheltered from wave action, where crabs are often abundant and pose a high risk of predation, than at exposed sites where crabs tend to be scarce e.g. . Shell-wNucella lapillus (L.) that contravene the general trend by having thicker shells at a site exposed to heavy wave action and free of crabs than at a more sheltered site infested with crabs. In contrast to shell thickness, shell shape in the two populations follows the normal trend for N. lapillus in which shells at exposed sites have relatively shorter spires and larger, wider apertures than shells at sheltered sites.Accordingly, we combined a reciprocal transplant experiment in the field with common garden experiments deploying laboratory-hatched progeny to examine plastic and heritable components of variation in shell morphology in two populations of The reciprocal transplant and common garden experiments were designed to examine the roles of inheritance and plasticity on adaptive shell morphology and to yield samples for studying associated gene expression. Because of the large volume of data, effects of reciprocal transplantation and common garden conditions lacking wave action or crab-effluent on shell shape are presented elsewhere N. lapillus were obtained from two sites in North Wales, U.K.  had grown to a shell length of 8–12 mm (August 2008), they were colour-coded with a waterproof pen according to lineage (exposed or sheltered).Source populations of Procedural details are given in Pascoal et al. −1with permanent aeration supplied from air-diffusion stones. Ambient temperature fluctuated seasonally between 8–16°C. Twenty-five F1s of sheltered site ancestry and 90 of exposed site ancestry were allocated per tank . After allowing snails to acclimatize for 1 wk, two tanks were left unchanged as controls and two were supplied with water-borne olfactory cues assumed to be perceived by N. lapillus as risk of crab predation Carcinus maenas, carapace width 8–12 cm, collected from the Menai Strait, were placed in the 25-litre cistern supplying the treatment-tanks and fed cracked adult N. lapillus. Crabs that died were replaced within 48 h. Tanks were spatially transposed at monthly intervals to avoid incidental position effects. Barnacles attached to stones were renewed as needed to maintain an unlimited supply of food for the N. lapillus in each tank.A ‘control garden’ and a ‘treatment garden’, each with two replicates, were established in four 25-litre aquaria. The control garden presented an artificial environment lacking most natural characteristics including the effects of wave exposure typical of the exposed field site and of crab predation characterizing the sheltered field site. The treatment garden presented an olfactory cue signaling risk of crab predation. Seawater was supplied via two constant-head cisterns at a rate of 3 ml sAt 6 months and again at 12 months from the beginning of the experiment, snails were photographed and their shell-lip thickness measured as above. Shell length and width were measured using tpsDig and MODICOS software, as above.At 12 months, tissue and shell mass were measured (±0.0001 g) after breaking the shell, extracting the tissue and drying for 24 h at 80°C. This procedure could be applied only to snails of exposed-site ancestry, as destructive sampling for a parallel study had depleted the sheltered-site lineage.1 generation fortuitously laid eggs in all tanks. Hatchlings (F2s) were grown for 10 months, then genetically assigned to exposed- or sheltered-site ancestry  and comparison of adjusted means was based on models lacking the interaction term.Mean shell-lip thickness, shell mass and tissue mass were adjusted to appropriate covariates by ANCOVA of log10-transformed data.Shape of the shell and of the external and internal rims of the aperture was quantified by using tpsRelwto generate relative warps. After confirming their independence of centroid size, scores on the first three relative warps were subjected to MANOVA (SPSS 11.3). To aid interpretation of relative warp analysis, width adjusted to length was compared among treatments by ANCOVA of logNo specific permits were required for the described field studies, which took place on shores with public right-of-way and did not involve endangered or protected species. The location is not privately-owned or protected in any way.Statistical comparison of mean log(shell-lip thickness) adjusted to the geometric mean of shell length and width, hereafter referred to as shell size, between transplants and controls within lineages was invalidated by disparate covariate values ; other cUnadjusted mean log(shell-lip thickness) was similar between controls and transplants within each lineage .Within 12h of exposure to crab-effluent, snails tended to crawl up the sides of the tanks and were less frequently seen than controls among stones bearing the food supply of barnacles. Snails receiving crab-effluent produced fewer egg capsules than controls , but there was no difference in the size of egg capsules produced  or in the number of eggs per capsule .1 generation) mean shell length was ranked higher in controls than in snails receiving crab-effluent . At 12 months from the beginning of the experiment, mean shell length was not significantly different among categories , whereas in the F2 generation (exposed-site only) mean shell length was greater for control than for treatment snails .At 6 months from the beginning of the experiment  adjusted to log(tissue mass) was greater in snails exposed to crab-effluent than in controls, but within those categories there was no difference between generations , Table 11 snails , Table 1At 6 months from the beginning of the experiment, log(shell-lip thickness) adjusted to shell size was greater in snails receiving crab-effluent than in controls, but within those categories there was no difference between lineages , Table 2Shape of the shell Mean scores on RW3 differed between treatment and control snails of exposed-site ancestry, all other comparisons being statistically non-significant . Although the marked increase in shell mass per unit tissue mass of snails experiencing crab-effluent is consistent with proposals that increased shell thickness is an actively induced defensive response to risk of crab predation Nucella lamellosa.Relative shell-lip thickness of juveniles at 6 months was markedly greater in snails receiving crab-effluent than in controls. Such an early ontogenetic response will be especially effective in promote fitness because vulnerability to crab predation falls most heavily upon juveniles whose shells have not yet reached a size-refuge from predation At 12 months, the difference in relative shell-lip thickness between snails receiving crab-effluent and controls had become much greater in the exposed-site than in the sheltered-site lineage. Experimentally induced changes in shell shape were subtle and statistically significant only for exposed-site snails. Snails exposed to crab-effluent had slightly wider shells than controls, with narrower aperture internal rims. Narrowing of the internal rim of the aperture, with little change in the external rim, reflects thickening of the rim itself. Thickening in response to crab-effluent, however, may be confounded with thickening due to sexual maturation In conclusion, our common garden experiment corroborates previous studies demonstrating a negative influence of crab-effluent on prey foraging activity, tissue growth and shell growth, and a corresponding positive influence on shell thickness. Our results are consistent with, but do not critically test, the hypothesis that shell thickening is a secondary consequence of starvation rather than a direct response to predation risk. Further support of the starvation hypothesis, however, may be adduced from the slower growth and development of thicker shells by snails transplanted from the sheltered to the exposed field-site where feeding opportunities are more frequently constrained by weather than at the sheltered site.Littorina saxatilisNucella lamellosaN. lapillus, at least during the vulnerable juvenile phase. Induction of defensive phenotype might be expected to be stronger in populations from habitats where risk of predation is high when integrated over time, but which varies unpredictably in the shorter term N. lapillus from a sheltered than from an exposed site, situated within 14 km of our sites. Development of secondary sexual characters such thickening of the shell rim further complicates interpretation. Moreover, even congeners may show contrasted morphological changes. Whereas N. lapillus develops a narrower aperture correlated with an elongated spire N. lamellosa develops greater globosity in response to crab-effluent Whereas greater plasticity has been reported in


Objective: Early studies of plastic surgery patient triage using telemedicine are descriptive and deal with feasibility rather than accuracy. The inpatient study arm compares on-site wound-evaluation accuracy with remotely viewed digital images. The outpatient arm prospectively compares on-site and remote diagnosis, management, and outcomes in a busy, urban, reconstructive-surgery clinic. The concurrent 6 patient case studies illustrate significant systems improvement by using remote consultation. Methods: A total of 43 inpatients and 100 consecutive outpatients were evaluated by on-site and remote surgeons as performed in previous arms with digital-camera and store and forward technology. Consent was obtained from all patients participating. Agreements regarding diagnosis  and management  were calculated. Results: In the first study arm, on-site and remote agreement (46%-86% for wound description and 65%-81% for management) generally matched agreement among on-site surgeons (68%-100% and 84%-89%). Moreover, when on-site agreement was low (68% for edema), agreement between on-site and remote surgeons was also low (57%). Remote evaluation was least sensitive detecting wound drainage (46%). On-site surgeons opted for more treatment, often prescribing antibiotics and admitting the patient. The second teleconsult arm provides further evidence of accuracy, overall agreement of 32%, sensitivity 48.55%, specificity 96.92%, positive predictive value 49.26%, negative predictive value 96.83%, and P < .001 regarding diagnosis . Patient transfer, postoperative monitoring, and outcomes via electronic image transfer, as well as cost-benefit analysis of this clinic-based study, are presented. Conclusions: eConsultation renders similar outcomes to standard, on-site examination in a selected group of plastic surgery patients. Remote evaluation may assist triage decisions, thereby decreasing emergency room throughput time and office-visit frequency, supplementing satellite facility consultation by plastic surgeons, and providing real-time postoperative assessments, thereby improving quality and reducing costs. Telemedicine is in broad use in radiology and cardiology, where the electronic transmission and initial evaluation of radiographs and electrocardiographic tracings improve the efficiency of clinical care.In 1998, Stoloff et alGiven the reliance on photography for surgical outcome evaluation and achieving reproducible and valid results in research, Galdino et alWe seek to develop an understanding of the accuracy of remote temporal and physical digital interpretation. It is our observation that the feasibility of patient triage for most plastic and reconstructive surgery consultations is less dependent on the quality of photographs than it is on the ability to process and remotely interpret such images. Standardization in the emergency room (ER) is less practical when triaging surgical patients. Because of the scarcity of studies examining nonstandardized photography with low-quality digital imaging, we set out to determine the validity in plastic surgery e-consultation and ER triage. For the purpose of this manuscript, eConsultation refers to remote interpretation and management utilizing store and forward digital images.In the initial study arm, 43 wounds in 37 inpatients were photographed with a Canon A80 camera (resolution 4.0 megapixels). On-site evaluation consisted of bedside examination by 2 physicians at the time of digital imaging and transmission. Remote evaluation consisted of 2 physicians examining digital images remotely at the time of transmission and 2 physicians examining digital images remotely at later time. A questionnaire was recorded by all physicians, and agreements regarding wound description and wound management were calculated among on-site surgeons and remote surgeons and between on-site and remote surgeons.In the second arm, 100 patients were photographed at an urban outpatient plastic surgery clinic with a Fujifilm FinePix A330 digital camera (resolution 2.0 megapixels) without standardized photography. A medical student, untrained in medical photography, took the pictures. On-site evaluation consisted of bedside examination by a surgeon at the time of digital image capture and transmission and remote evaluation consisted of digital image consultation via store and forward technology at a later date. The remote surgeon was given no information in addition to the picture. An injury questionnaire was recorded and agreements regarding diagnosis were calculated between on-site and remote surgeons with SAS statistical software program version 9.2. A series of 5 clinical cases is presented in which patient transfer decisions, home-based postoperative monitoring, and overall outcomes were improved and costs reduced via electronic image transfer. These studies were approved by the institutional review board.P < .001, regarding diagnosis .Inpatient examination by on-site surgeons agreed 68% to 100% for wound description and 84% to 89% for wound management , showed 68% to 100% agreement among on-site surgeons for wound description and 84% to 89% agreement for wound management. A similar study in vascular surgery revealed comparable results among on-site surgeons (64%-85% for wound description and 63%-91% for wound management).,17A review of the trauma and burn literature reveals wound evaluation studies using high-quality digital images. Galdino et al14P < .001 with an overall sensitivity 48.55%, specificity 96.92%, PPV 49.26%, and NPV 96.83%  and nonstandardization of photographs showed an overall agreement of 32% with Therefore, the original hypothesis that digital image quality and standardization of photos do not affect diagnosis and management cannot be rejected. Table P < .001, overall sensitivity 48.55%, specificity 96.92%, PPV 49.26%, NPV 96.83%; category analysis: P < .001, agreement 26.67% to 41.18%, sensitivity 36.36% to 50%, specificity 92.22% to 96.92%, PPV 41.46% to 63.64%, NPV 90.86% to 99.49%; and 24 choice analyses: P value, sensitivity, specificity, NPV, and PPV (Table ER physicians treating surgical patients who require plastic surgery consultation can use store and forward technology. Halstead et alForty-three inpatients and 10 consecutive outpatients were examined on site.Medium- and low-quality digital images were obtained without standardization and evaluated using store and forward technology. Accuracy and concordance rates were high. Variations in concordance were comparable to on-site variability. Five cases demonstrate systems improvement using eConsultation. The authors conclude that eConsultation is an accurate, cost-saving, time-saving technique in the evaluation and management of select plastic surgery patients.