Osteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13  in the chondrocyte response to 14-3-3ε. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3ε-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3ε signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3ε. Direct interaction between CD13 and 14-3-3ε was then demonstrated by surface plasmon resonance. Using labeled 14-3-3ε, we also found that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3ε might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3ε–CD13 interaction could be a new therapeutic target in osteoarthritis. Summary: 14-3-3 might directly bind to CD13, which transmits a signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. Osteoarthritis is a chronic joint disease characterized by a progressive degradation of articular cartilage, synovial inflammation and remodeling of subchondral bone in response to a variety of stimuli, such as proinflammatory cytokines and mechanical loading . The catThere is now general agreement that osteoarthritis involves all structures in the affected joint, which leads to articular cartilage deterioration. Several studies have suggested that subchondral bone and articular cartilage form a functional unit, which might play a role in joint maintenance and degeneration. There is a possibility of direct signaling between the two compartments  and thatRecently, our group identified 14-3-3ε as a new soluble mediator involved in deleterious biochemical interactions between bone and cartilage . Using i14-3-3ε belongs to the family of 14-3-3 proteins, acidic proteins of 25 to 33 kDa that are highly conserved in eukaryotes . The proNo receptor for 14-3-3ε has been identified in articular cartilage. However, CD13  has recently been identified as a 14-3-3σ receptor on fibroblasts . Thus, CWe lack evidence of 14-3-3ε associated with a chondrocyte surface receptor. In the current study, we report the association of 14-3-3ε and CD13, and highlight a transmembrane signaling mechanism for 14-3-3ε-mediated MMP-3 and MMP-13 expression in chondrocytes. This interaction between 14-3-3ε and CD13, leading to a chondrocyte catabolic phenotype similar to that observed in osteoarthritis, could be a new target for treatment of osteoarthritis and could be investigated in preclinical studies.CD13 expression in articular chondrocytes was first examined in primary cultures of mouse chondrocytes. Using quantitative real-time PCR (qRT-PCR), mRNA expression of CD13 was demonstrated in murine chondrocytes, with the peak of expression at between 24 and 26 cycles (data not shown). CD13 protein expression was found in articular chondrocyte extracts by western blot analysis A and immP<0.05) and the protein level  , which demonstrates efficient gene silencing. We selected siRNA2 for the following experiments. Mouse chondrocytes were transfected with the negative control siRNA or CD13 siRNA2, then treated with 14-3-3ε for 24 h to examine MMP-3 and MMP-13 expression. Treatment with 1 µg/ml 14-3-3ε increased the mRNA and protein levels of MMP-3 by tenfold as compared with the control. The 14-3-3ε-mediated induction of MMP-3 expression and release was significantly inhibited by CD13 siRNA   with or without an aminopeptidase N inhibitor . In mouse chondrocytes, aminopeptidase N activity increased over time, to peak at 60–90 min (∼twofold increase versus control); this activity was strongly inhibited by APNi A. To tesP<0.05; P<0.05; P>0.05; To investigate the involvement of the CD13 receptor in 14-3-3ε-mediated MMP-3 and MMP-13 activity, we used an anti-CD13 blocking antibody to block only the receptor role of this protein. We pretreated primary cultures of murine and human chondrocytes with or without the anti-CD13 blocking antibody, followed by stimulation with 14-3-3ε, and compared the results in untreated and stimulated chondrocytes. In murine chondrocytes, 14-3-3ε-induced MMP-3 mRNA expression was inhibited dose dependently by anti-CD13 blocking antibody (inhibition of 64% at 1 µg/ml and 90% at 5 µg/ml), as was MMP-3 protein release (inhibition of 61% at 1 µg/ml and 87% at 5 µg/ml) P<0.05; A,B. SimiP<0.05; supplementary material Fig. S2). As observed in previous publications, MMP-13 expression in human chondrocytes was very low. Thus, the results obtained by qRT-PCR were not interpretable. Taken together, these results show that treatment with an antibody that blocks only the receptor role of CD13 greatly inhibits the 14-3-3ε-mediated mRNA and protein expression of both MMP-3 and MMP-13 in murine and human chondrocytes, which suggests that CD13 has a receptor role in transmitting the 14-3-3ε signal in chondrocytes and inducing a procatabolic phenotype.To confirm these results in human chondrocytes, cells were pretreated with a specific human anti-CD13 blocking antibody , which is widely used to block only the receptor role of human CD13 and has no effect on its enzymatic activity. Cells were then stimulated with 14-3-3ε for 24 h. 14-3-3ε-induced MMP-3 mRNA expression and protein release were significantly decreased by 43% and 59%, respectively (KD) of 2.45×10−6 M   experiments. Human recombinant CD13 was covalently immobilized on a CM5 chip. Human recombinant 14-3-3ε, a specific blocking antibody against human CD13 and an IgG1 isotype control were used as analytes. Results showed a direct interaction between recombinant CD13 and 14-3-3ε [78 resonance units (RU)], with higher binding with blocking antibody (170 RU) and no binding with IgG1 isotype control (00 RU) A. Kinetiood fit) B. These We assessed the ability of 14-3-3ε to bind to the surface of mouse chondrocytes by incubating these cells with biotin-labeled recombinant 14-3-3ε. The cells were then incubated with fluorescently labeled streptavidin and analyzed by fluorescence microscopy. Biotin–14-3-3ε bound to the chondrocyte surface A. We perin vivo , where pS is a phosphorylated Ser residue  on CD13 that could bind to 14-3-3ε, we examined the sequence and structure of CD13 (PDB ID 4FYQ) . We defiUhart and Bustos, 2013in vivo  and it i residue . Y582 wa residue C. In add residue A,B. Takein silico analysis and assess the involvement of E579FNYVW584 as the binding motif between CD13 and 14-3-3ε, mouse chondrocytes were subjected to preincubation with EFNpYVW and were then stimulated with 14-3-3ε in the presence or absence of EFNpYVW, to prevent binding to endogenous CD13. 14-3-3ε-induced MMP-3 mRNA production was dose dependently inhibited by the peptide , which might be explained by the absence of some amino acid sequences from recombinant 14-3-3ε and CD13 used in SPR analysis. Using labeled biotin–14-3-3ε, we showed that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results confirm the potential involvement of CD13 in 14-3-3ε signal transmission in chondrocytes and strongly suggest that direct interaction between 14-3-3ε and CD13 could occur in bone–cartilage communication.By SPR analysis, we found that 14-3-3ε binds directly to CD13. This technique has been found to be effective in the analysis of direct interaction between proteins. The specific interaction between 14-3-3ε and CD13 was evidenced by SPR with human recombinant CD13 covalently bound on a chip and injected human recombinant 14-3-3ε. Injection of specific blocking antibody against CD13, and IgG isotype negative and positive controls, demonstrated that the interaction between 14-3-3ε and CD13 was specific. This direct interaction was confirmed by kinetic tests, although with weaker binding affinity . However, no effect of ERK inhibitor on chondrocyte response to 14-3-3ε was found . Some published reports have demonstrated a link between 14-3-3 proteins and MAPK signaling cascades. It has recently been suggested that the stimulation of cells with 14-3-3η leads to the phosphorylation of ERK and JNK, but not p38 MAPKs, inducing mediators of inflammation and joint destruction in rheumatoid arthritis , unless stated otherwise. Fetal bovine serum (FBS) was obtained from Invitrogen . Liberase and complete protease inhibitor mixture were from Roche Diagnostics . The enhanced chemiluminescence western blot analysis kit was from Amersham Pharmacia Biotech . The immunoblot polyvinylidene difluoride membranes for western blot analysis and kaleidoscope prestained standards were from Bio-Rad . Recombinant human 14-3-3ε was from Enzo Life Sciences.Human cartilage samples were obtained from osteoarthritis patients undergoing total joint replacement surgery for osteoarthritis at Saint-Antoine Hospital . These materials are considered surgical waste and are used with informed consent and in accordance with French ethics laws . Investigations conformed to the principles outlined in the Declaration of Helsinki. The osteoarthritis diagnosis was based on clinical and radiographic evaluations according to the American College of Rheumatology criteria .3), and washed several times with phosphate-buffered saline (PBS). All enzymatic digestions were performed at 37°C, under agitation, in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/l glucose, 100 units/ml penicillin, 100 µg/ml streptomycin and 4 mM glutamine . Samples were incubated twice for 45 min each, in 25 ml Liberase (Roche Diagnostics) at 0.52 Wünsch units (UW)/ml to eliminate surrounding extracellular matrix, then incubated overnight with 25 ml Liberase at 0.13 UW/ml. The cell suspension was filtered through a 100-µm cell strainer and centrifuged for 6 min at 400 g. Chondrocytes were cultured for 8 to 10 days in 12-well plates  in DMEM containing 4.5 mg/l glucose, 100 units/ml penicillin, 100 µg/ml streptomycin and 4 mM glutamine, supplemented with 15% FBS and allowed to grow to confluence. Cells were then starved in serum-free medium containing 0.1% bovine serum albumin (BSA) for 24 h. For blocking antibody experiments, chondrocytes were treated for 15 min with SJ1D1 (1 µg/ml), a human anti-CD13 antibody (Santa Cruz Biotechnology), before stimulation with recombinant 14-3-3ε (1 µg/ml).Cartilage samples were obtained from the tibial plateau and femoral condyle of osteoarthritis patients, cut into small pieces (∼1 mmAll experiments followed protocols approved by the French or European ethics committee. Mouse articular chondrocytes were isolated by enzymatic digestion of articular cartilage from C57BL/6J mice (5–6 days old), as described previously . After 6Human cartilage samples were fixed in 4% paraformaldehyde for 72 h before paraffin embedding. Transverse sections (4 µm thick) were cut parallel to the rib axis by use of a Polycut E microtome , then mounted on slides. For labeling, slides were deparaffinized and unmasked by use of citrate buffer (pH 6) at 95°C, then blocked in a solution of TBS-TC . Human CD13 was detected with monoclonal rabbit antibody . Rabbit IgG (DAKO) was a negative control. Secondary antibody and peroxidase were added by use of the horseradish-perioxidase-conjugated LSAB+ system (DAKO). Visualization involved the DAB kit (VECTOR). Image-Pro express was used for image capture.Mouse and human chondrocytes were fixed in 4% paraformaldehyde, permeabilized (0.1% Triton X-100), then blocked in a solution of TBS-TC. Cells were exposed to anti-CD13 antibody  or an isotype control antibody . Secondary antibody and peroxidase were added by use of the horseradish perioxidase-conjugated LSAB+ system (DAKO). Visualization involved the DAB kit (VECTOR). Image-Pro express was used for image capture.Mouse CD13 knockdown was tested with two different siRNA oligonucleotide sequences: si-CD13-1, 5′-ATGGATCTTACTGAACATTAA-3′, and si-CD13-2, 5′-CCGGGTGATCCTGAGACCCTA-3′ (Qiagen). AllStars was a negative control. Mouse chondrocytes were cultured as described above. Confluent cells were removed with a mixture of collagenase P and pronase. Cells were centrifuged (1600 rpm for 6 min) and the pellet was resuspended in PBS (Sigma-Aldrich).6) were resuspended in Amaxa electroporation transfection solution with siRNA (1 µM), then placed in cell culture medium DMEM (1 g/l glucose) supplemented with penicillin, streptomycin and L-glutamine containing 10% FBS. At 48 h post-transfection, cells were starved in serum-free medium containing 0.1% BSA overnight, then stimulated with recombinant 14-3-3ε (1 µg/ml) for 24 h.Murine articular chondrocytes were transfected by use of an electroporation device (Amaxa) according to the manufacturer's instructions (Qiagen). Briefly, cells . Total RNA (0.5 µg) was reverse transcribed with use of the Omniscript RT kit (Qiagen). Relative quantification of genes involved the Light Cycler 480 Real-Time PCR Detection System (Roche Diagnostics) and GoTaq qPCR Master Mix (Promega) as described previously .For amplification of mouse cDNA, oligonucleotide primer sequences were for MMP-3, sense 5′-TGAAAATGAAGGGTCTTCCGG-3′ and anti-sense 5′-GCAGAAGCTCCATACCAGCA-3′; MMP-13, sense 5′-GATGGCACTGCTGACATCAT-3′ and anti-sense 5′-TGTAGCCTTTGGAACTGCTT-3′; CD13, sense 5′-AATCTCATCCAGGGAGTGACC-3′ and anti-sense 5′-GTGGCTGAGTTATCCGCTTT-5′; and hypoxanthine guanine phosphoribosyltransferase (HPRT), sense 5′-AGGACCTCTCGAAGTGT-3′ and anti-sense 5′-ATTCAAATCCCTGAAGTACTCAT-3′. The relative mRNA expression of MMP-3, MMP-13 and CD13 were normalized to that of HPRT, as an internal gene reference.For amplification of human cDNA, oligonucleotide primer sequences were for MMP-3, sense 5′-ATGAAAATGAAGGCTCTTCCG-3′ and anti-sense 5′-GCAGAAGCTCCATACCAGCA-3′, and human 18S, sense 5′-GCAATTATTCCCCATGAACG-3′ and anti-sense 5′-GGGACTTAATCAACGCAAGC-3′. The relative mRNA expression of MMP-3 was normalized to that of 18S, as an internal gene reference.Chondrocytes were lysed in RIPA buffer . Proteins were separated on Citerion XT 4-12% Bis-Tris gels  and transferred onto nitrocellulose membranes, which were incubated with antibodies for human or mouse CD13 (Abcam) and β-actin (Sigma Aldrich) by use of the Immun-Star Western C chemiluminescence kit (Bio-Rad). For densitometry, we used Image Gauge software version 3.0 (Fujifilm).Total mouse and human MMP-3 secretion was assayed in cell-free supernatants using an enzyme linked immunosorbent assay (ELISA) kit (R&D Systems) according to manufacturer's instructions. MMP-13 was also quantified in culture supernatants from murine chondrocytes using an ELISA kit as recommended by the manufacturer (Uscn Life Science Inc.). MMP-3 and MMP-13 concentrations were analyzed in duplicate at serial dilutions and were read against a standard curve.The activity of aminopeptidase N on cultured human and mouse chondrocytes was determined according to previously published methods . BrieflyTo test the effect of 14-3-3ε on aminopeptidase N activity, cells were incubated or not with APNi for 15 min, then stimulated with recombinant 14-3-3ε (1 µg/ml). At 24 h, cells were incubated with the substrate at 37°C for 1 h. The supernatant from each well was collected and enzyme activity was determined as described above.D) was determined by use of BIAevaluation software version 4.1. Binding and kinetics tests were first performed on empty surfaces. Data resulted from subtraction of empty-surface RU from active-surface RU.The interaction of 14-3-3ε and CD13 was measured using SPR on a Biacore 3000 instrument  in the molecular interactions facility . Human recombinant CD13  was immobilized to a CM5 sensor chip  at 11,949 resonance units (RU) by amine coupling in 10 mM sodium acetate (pH 5.0) according to the manufacturer's instructions. 14-3-3ε (10 µM), a human CD13 antibody  and an isotype antibody  were diluted in HBS-EP running buffer  , then injected over the chip at a flow rate of 5 µl/min for 5 min and dissociated for 8 min. The surface of the sensor chip was regenerated by injecting 10 mM glycine-HCl, 10 mM, pH 2.0 at a flow rate of 30 µl/min for 30 s. For kinetics study, concentrations  of 14-3-3ε were injected. Identical injections were performed in parallel over a blank surface, at 0 RU. Data were analyzed and dissociation rate constant (K3) were cultured on coverslips in a 24-well plate. After six to seven days of culture, cells were placed in culture medium supplemented with 0.1% BSA for 3 h, treated with biotin–14-3-3ε for 15 min at 4°C and washed extensively with PBS, as previously described (Recombinant 14-3-3ε (200 µg) in PBS was labeled with a 50-fold molar excess of Sulfo-NHS-LC-Biotin (Thermo Scientific) reagent, as recommended by the manufacturer. Primary murine chondrocytes (35×10escribed . For thehttp://www.phosphosite.org) (http://www.rcsb.org) under the PDB ID code 4FYQ and visualized with PyMOL Molecular Graphics System v1.5 (Schrödinger). Y582 was the unique residue inserted into an accessible and extended 20-residue-long loop. Y582 belongs to the hexamer sequence E579FNYVW584 selected for docking, to assess its capacity to accommodate into the groove of one monomer of 14-3-3ε. Chain A of the solved structure of 14-3-3ε in complex with the phosphorylated RQRpSAP ligand (PDB ID 2BR9) served as the starting coordinate. The missing atoms of K50 side-chain (lacking electronic density) were completed and the ligand was mutated into EFNYVW by use of the Build module in DiscoveryStudio, Accelrys. Two orientation modes were tested: the N-ter similar to the crystallized ligand N-ter, and the reverse C-ter. In addition, the ligands phosphorylated and not phosphorylated at Y582 were built. All these variants plus the crystal structure of chain A with RQRpSAP, as a reference, were minimized for 5000 iterations by use of the CHARMM force field  . The resce field  and the t-test to compare mean values between two groups. One-way analysis of variance (ANOVA) with Turkey Kramer multiple comparisons test was used to compare mean values using GraphPad Prism software . P<0.05 was considered statistically significant.Data are expressed as the mean±s.d. relative induction compared with the control (set to 1). Data are reported as mean±s.d. unless stated otherwise. Statistical analysis was performed with the paired Student's


Blood and Body Fluid Exposures (BFFEs) involve a complex sequence of events combining technical, human and organizational factors. Performing root cause analysis (RCA) of these events is promoted to improve safety. We conducted a RCA of a BBFE in a radiology unit using a recently developed French method called Orion.The objective was to identify how the BBFE had happened and to implement actions to prevent its reoccurrence.The starting point of the RCA was the report of a BBFE by a radiologist to occupational medicine. BBFE occurred during a non scheduled breast biopsy. Analysis was conducted in collaboration with occupational medicine and the Southwestern Centre for Healthcare Associated Infection Control. ORION comprises six steps: collecting data; rebuilding the chronology; identifying gaps; identifying contributing and influential factors; proposing actions to implement; writing the analysis report.The detailed chronology of events before, during and after the BBFE identified many gaps. The main influential factor was a sub-optimal organization during the breast biopsy: no protocol, inadequate room and time slot. Three corrective measures were retained: providing adequate safety container closer to the care procedure; providing adequate medical device to drag the carrot, reorganizing the care with an additionnal microbiopsy session close to RMI session.This first use of the ORION method to analyse a BBFE proved successful. This method seems quasi-intuitive and easier to conduct than previously described methods because it relies on a detailed chronology. It allows the implementation of BBFEs preventive measures and promotes collaborative teamwork.None declared.


The exocyst is an octameric molecular complex that drives vesicle trafficking in adipocytes, a rate-limiting step in insulin-dependent glucose uptake. This study assessed the role of the exocyst complex in regulating free fatty acid (FFA) uptake by adipocytes. Upon differentiating into adipocytes, 3T3-L1 cells acquire the ability to incorporate extracellular FFAs in an insulin-dependent manner. A kinetic assay using fluoresceinated FFA (C12 dodecanoic acid) uptake allows the real-time monitoring of FFA internalization by adipocytes. The insulin-dependent uptake of C12 dodecanoic acid by 3T3-L1 adipocytes is mediated by Akt and phosphatidylinositol 3 (PI3)-kinase. Gene silencing of the exocyst components Exo70 and Sec8 significantly reduced insulin-dependent FFA uptake by adipocytes. Consistent with the roles played by Exo70 and Sec8 in FFA uptake, mCherry-tagged Exo70 and HA-tagged Sec8 partially colocalize with lipid droplets within adipocytes, suggesting their active roles in the development of lipid droplets. Tubulin polymerization was also found to regulate FFA uptake in collaboration with the exocyst complex. This study demonstrates a novel role played by the exocyst complex in the regulation of FFA uptake by adipocytes. Dietary lipids constitute approximately 40% of caloric intake in modern human diet . Free faThe exocyst is a large protein complex composed of Sec3 (Exoc1), Sec5 (Exoc2), Sec6 (Exoc3), Sec8 (Exoc4), Sec10 (Exoc5), Sec15 (Exoc6), Exo70 (Exoc7), and Exo84 (Exoc8). The exocyst complex was initially discovered in yeast as a molecular machinery that regulates the exocytosis of secretory vesicles –18. DiveIn this study, we have identified a new role for the exocyst complex in the regulation of FFA uptake by adipocytes. Our findings may shed new light on the molecular mechanism underlying FFA handling in health and diseases.2 incubator at 37°C. The adipocyte differentiation of 3T3-L1 cells was induced by changing media to DMEM, 25 mM glucose with 10% fetal bovine serum (Hyclone) containing a differentiation mix [The 3T3-L1 cells  were maintained in DMEM, 25 mM glucose (Gibco) with 10% new born calf serum  in a 5% CO-Aldrich). Three d2, media were changed to serum-free DMEM of high-glucose (25 mM) or low-glucose concentration (5.5 mM), and incubated for additional 1 hour. Cells were stimulated with 10 nM insulin for 30min in 1x assay buffer  before the assay, then the fluorescent emission from each well was measured immediately after adding QBT Fatty Acid Uptake solution [Lipid uptake assay was performed using QBT Fatty Acid Transporter Assay Kit (Molecular Devices) according to the manufacturer’s instruction . About 5solution . The unqA phosphoinositide-3-kinase (PI-3K) inhibitor (Wortmannin), MEK inhibitor (U0126), mTOR inhibitor (rapamycin), Akt1/2 kinase inhibitor (Akt1/2I), and nocodazole were obtained from Sigma-Aldrich .6 adipocytes were mixed with 100 nM RNAi duplexes and the electroporation was performed at 0.16 kV, 960 F with a Gene Pulser II (Bio-Rad). After electroporation, cells were incubated in DMEM with 10% FBS for 10 min at 37°C in 5% CO2 incubator for recovery. The sequences of stealth RNAi used were the following: Exo70: GCA GCU GGC UAA AGG UGA CUG ACU A, Exo70 control: GCA CGG UAA AUG UGG GUC AAC GCU A, Exo70 oligo #2: GCG CCA UCU UCC UAC ACA ACA ACU A, Exo70 oligo #2 control: GCG UCU AUC CUC ACA ACA AAC CCU A, Sec8: GGA GAU UGA ACA UGC CCU GGG ACU U, Sec8 control: GGA GUU CAA GUA CCC GGU AGG ACU U. The effectiveness of these Exo70 and Sec8 siRNA oligos was verified as described before [3T3-L1 adipocytes were transfected with stealth RNA interference (RNAi) duplexes (Invitrogen) using electroporation as described before . Adipocyd before .Two 6-week-old C57BL/6J male mice were purchased from The Jackson Laboratory and the inguinal fat pads were isolated for cDNA cloning of mouse Exo70. Mice were euthanized with isoflurane overdose and the euthanasia was confirmed with bilateral thoracotomy. All animal procedures were approved by University Committee on Use and Care of Animals (UCUCA) of the University of Michigan.The mouse Exo70 cDNA was obtained from mouse (C57BL/6J) inguinal adipose tissues with RT-PCR. The mouse Exo70 cDNA was cloned into pmCherry-C1 vector (Clontech). HA-tagged Sec8 expression vector was previously described and validated . The pmCFFA uptake data were analyzed with area under curves and multiple t-tests and two-way ANOVA for time-dependent FFA uptake between samples. P-value <0.05 was considered as significant.To assess the FFA uptake by adipocytes, we examined the cellular uptake of the fluorescently-labeled medium-chain FFA dodecanoic acid BODIPY-C12-FA) ,28. The -FA ,28. 7 vs. with glucose, 1.14x107, P = 0.017 at t = 3,000 s); however, under an insulin-stimulated condition, adipocyte FFA uptake did not significantly differ with and without glucose  as well as in insulin-stimulated FFA uptake   .7; insulin-stimulated condition, 2.07 x 107, P = 4.2 x 10-5 and 0.00017, compared to controls with and without insulin at t = 3,000 s). An increased dose of Wortmannin (4 μM) completely blocked the insulin-induced increase of FFA uptake . Downstream of PI3 kinase, Akt2 plays a dominant role in adipose tissue development and function [7, P = 1.6 x 10-6 compared to insulin-treated control at t = 3,000 s; P = 0.34 between Wortmannin 4 μM and Akt1/2I 10 μM). Two-way ANOVA confirmed the time-dependent (P<0.0001) and inhibitor-dependent (P<0.0001) regulation of FFA uptake. The suppressive effect of Akt inhibitor on insulin-dependent FFA uptake was found to be dose-dependent    .7 versus 2.7x107 without insulin, P = 0.03 at t = 3,000 s; 3.5x107 versus 2.9x107 under insulin treatment, P = 0.01 at t = 3,000 s)   . The int 0.0001) .The elevation of plasma FFA concentration is one of the key clinical findings observed in the patients with obesity and diabetes . The adiAkt2 plays a dominant role in insulin-dependent glucose metabolism as well as adipose tissue development ,31. InsuThe components of the exocyst complex are phosphorylated in response to insulin, EGF1, and ERK –46. The To conclude, our studies have demonstrated a novel role played by the exocyst complex in facilitating insulin-dependent FFA uptake by adipocytes. This newly identified biological pathway may help us better understand the molecular mechanism by which adipocytes regulate FFA handling in health and diseases.S1 FigIntracellular localizations of mCherry-Exo70 and lipid droplets (BODIPY 493/503) were determined by confocal microscopy. Scale = 100 μm. In each sample, 3~4 cells were analyzed. Seven samples in total were used for colocalization analysis.(TIFF)Click here for additional data file.


Protein ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs), many of which show striking preference for particular Ub linkage types. Here, we have screened for DUBs that preferentially cleave N-terminal Ub from protein substrates but do not act on Ub homopolymers. We show that members of the Ub C-terminal hydrolase (UCH) family of DUBs demonstrate this preference for N-terminal deubiquitylating activity as they are capable of cleaving N-terminal Ub from SUMO2 and Ube2W, while displaying no activity against any of the eight Ub linkage types. Surprisingly, this ability to cleave Ub from SUMO2 was 100 times more efficient for UCH-L3 when we deleted the unstructured N-terminus of SUMO2, demonstrating that UCH enzymes can cleave Ub from structured proteins. However, UCH-L3 could also cleave chemically synthesized isopeptide-linked Ub from lysine 11 (K11) of SUMO2 with similar efficiency, demonstrating that UCH DUB activity is not limited to peptide-linked Ub. These findings advance our understanding of the specificity of the UCH family of DUBs, which are strongly implicated in cancer and neurodegeneration but whose substrate preference has remained unclear. In addition, our findings suggest that the reversal of Ube2W-mediated N-terminal ubiquitylation may be one physiological role of UCH DUBs  The substrate preference of ubiquitin C-terminal hydrolase (UCH) deubiquitylating enzymes (DUBs) has been unclear. Here we report that UCH family DUBs efficiently cleave both isopeptide- and peptide-linked ubiquitin from substrates, without processing simple Ub–Ub polymers of any linkage type. Conjugation of proteins with ubiquitin (Ub) is a versatile post-translational modification that regulates a number of cellular pathways and signalling events . ModificConjugation of Ub to a substrate lysine (K) residue can be either a mono- or poly-ubiquitylation event, of which polyubiquitylation can occur via isopeptide Ub chains linked through any of seven internal K residues  [in vitro in a non-discriminatory manner [The process of Ub conjugation is regulated through the catalytic activities of deubiquitylating enzymes (DUBs), which can reverse or edit all Ub modification types to generate free monomeric Ub . The ~90y manner ,9. By coy manner . Interesy manner ,15.In vitro, purified UCH enzymes show no activity against any K-linked or linear Ub-dimers [in vitro until association with the proteasome and ASXL1 respectively, which relieve their autoinhibition and permit the hydrolysis of Ub isopeptides [The four UCH enzymes represent an unusual family of DUBs, as they are known to contain an active site cross-over loop that is thought to limit substrate accessibility –20. The b-dimers ,23, but b-dimers ,24. In ab-dimers ,26, thusb-dimers ,21. The peptides ,28.in vivo.In the present study, we set out to identify DUBs that could preferentially cleave alpha N-terminal-linked mono-Ub from SUMO2 or Ube2W. We report that members of the UCH family are capable of efficiently cleaving peptide-linked N-terminal mono-Ub, while displaying no activity against Ub dimers of any linkage type. However, UCH DUBs could also cleave isopeptide-linked Ub from lysine 11 of SUMO2, suggesting their activity is not strictly limited to peptide-linked N-terminal Ub. Thus, the reversal of N-terminal ubiquitylation may be one physiological role of UCH DUBs Escherichia coli as previously described [E. coli Rosetta (DE3) cells with a knockout in the ElaD gene (gift from Rob Layfield) which encodes a bacterial DUB [2+-nitrilotriacetate (Ni-NTA)–agarose (Qiagen) column and washed, eluted and dialysed overnight at 4°C against 50 mM Tris/HCl, 150 mM NaCl and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), pH 7.5. Purification of 6xHis-Ub-SUMO2x4ΔN11 was as above, followed by tobacco etch virus (TEV) protease cleavage to remove the 6xHis tag (1 mg of His-TEV protease per 100 mg of the fusion protein). Ub-SUMO2x4ΔN11 was purified by passing over an Ni-NTA–agarose column to remove His-TEV protease and 6xHis tag. Untagged Ub-Ube2W was purified using a Q Sepharose  ion-exchange column and eluted with an NaCl gradient (50-600 mM) and dialysed as above. Fractions were concentrated using Vivaspin centrifugal concentrator (Sartorius). Gel filtration chromatography on a HiLoad 16/60 Superdex 200 pg (for 6xHis-Ub-SUMO2x4ΔN11) or 75 pg column   was carried out as a final purification step. Ub-K11-SUMO2 and Ub dimers  were synthesized as previously described [The DUBs USP2, UCH-L1, UCH-L3, UCH-L5 and BAP1 were purified from escribed . The subrial DUB . Bacteriescribed ,31. LineIn vitro ubiquitylation of Ub-SUMO2x4ΔN11 was carried out at 37°C for 1 h as described previously [http://www.perseus-framework.org/). Ub-rhodamine-110-Gly assays were carried out as previously described [eviously . Densitoescribed .km and Vmax, each enzyme concentration was chosen so that the reaction was linear with a molar excess of the specific substrate over 60 min at 30°C (shaking at 850 rpm). Substrates were chosen according to detected activity (>10%) against the DUB. All data were plotted by SigmaPlot (version 12.5), using the enzyme kinetics tool and the following parameters: Single Substrate Study, Michaelis–Menten equation=Vmax × S/(km + S). The steady state of reactions were determined by incubating a fixed amount of enzymes  with the indicated excess of substrate.For the analysis by MALDI-TOF DUB assay, bovine serum albumin (BSA), Tris and DTT were purchased from Sigma–Aldrich. MALDI-TOF MS materials  were from Bruker Daltonics. Screening for activity and specificity of UCHL1, UCHL3, UCHL5 and BAP1 against Ub-SUMO2, Ub-Ube2W, Ub-4xSUMO2ΔN11, Ub-SUMO2Δ1-15 and Ub-K11-SUMO2 was performed as previously described . Brieflykcat/Km) were calculated to be 5.76×103 M−1·s−1 for UCH-L3 and 1.45×104 M−1·s−1 for BAP1  A are repfor BAP1 . Therefoin vitro using RNF4 and Ube2N/Ube2V1, which generates K63-linked Ub chains on the N-terminally linked Ub moiety [en bloc, but do not exert any Ub hydrolase activity towards the chains themselves.As substrates containing longer ubiquitylated chains more closely represent physiological substrates than Ub dimers, we determined how such substrates were utilized by UCH DUBs. To generate substrate containing long Ub chains, Ub-SUMO2x4ΔN11 was ubiquitylated b moiety   against Ub-Ube2W was 3.99×103 M−1·s−1 for UCH-L3, 8.86×103 M−1·s−1 for BAP1, and the highest efficiency was 1.59×104 M−1·s−1 for UCH-L5 , the product of Ube2W N-terminal auto-ubiquitylation . UCH-L3,r UCH-L5 . Therefokcat/Km) of UCHL1 and UCHL3 against Ub-SUMO2 dimers were calculated as 2.6 M−1·s−1 and 3.89×101 M−1·s−1 respectively (kcat/Km) for UCHL3 against Ub-SUMO2ΔN1-15 was 3.89×103 M−1·s−1, which is approximately 100-fold higher than the efficiency of UCH-L3 against wild type Ub-SUMO2 dimers (kcat/Km) was 2.11×103 M−1·s−1, similar to that of UCH-L3 against Ub-SUMO2ΔN1-15  ,23. It h adducts ,21. In a adducts ,28. We h in vivo –6, and tin vitro . This opkcat/Km of 5.76×103 M−1·s−1 and 3.99×103 M−1·s−1 respectively. This is several orders of magnitude lower than what has been reported for the yeast UCH enzyme YUH1 against the artificial substrate Ub-AMC (2.23×108 M−1·s−1) [3 M−1·s−1and 1×105 M−1·s−1 respectively [en bloc, completely unprocessed.Kinetic analysis revealed that UCH-L3 (and to a lesser extent UCH-L1) is capable of cleaving N-terminal peptide-linked Ub from SUMO2x4ΔN11 and Ube2W with a M−1·s−1) , but is ectively . The obsectively ,26,34. IStructural studies of UCH family enzymes either in isolation or in complex with Ub suicide probes have revealed a dynamic active site cross-over loop that has been predicted to prevent cleavage of Ub if conjugated to structured regions of protein –20,35. Tkcat/Km values of 1.59×104 M−1·s−1 and 8.86×103 M−1·s−1 respectively. Interestingly, BAP1 cleaves Ub fused to four copies of SUMO2 more efficiently than it cleaves Ub-SUMO2 dimers, and while UCH-L5 showed little activity towards Ub fusions to SUMO2, it efficiently cleaves Ub-Ube2W. This suggests that multiple factors in addition to the presence of N-terminal mono-Ub influence DUB activity. Interestingly, as UCH-L5 is a proteasome-associated DUB, one function may be to remove N-terminal Ub from substrates that are targeted to the proteasome by the Ub-fusion degradation (UFD) pathway [Our observation that UCH-L5 and BAP1 both efficiently cleave N-terminal Ub from Ub-Ube2W and Ub-SUMO2x4ΔN11 was unexpected. While individual UCH domains from UCH-L5 and BAP1 display activity towards K48 Ub dimers , full-le pathway .In summary, we have demonstrated that the UCH family members efficiently cleave isopeptide- and peptide-linked Ub from substrates but are inactive towards unbranched Ub polymers. Despite intense research into UCH family enzymes due to their links to neurodegenerative disease and cancer, the substrate preference of this DUB class have remained largely elusive. A greater understanding of the role and nature of N-terminally ubiquitinated proteins may have bearing upon our understanding of these functionally elusive proteases.


To explore the upstream signal transduction mechanisms responsible for the imbalanced expression of glucocorticoid receptor (GR) isoforms in chronic rhinosinusitis (CRS) mucosa.An in vitro model of Glucocorticoid resistance was established by inducing nasal polyp tissue with IL-1β. Changes in the protein and mRNA expression of GRα, GRβ and the key enzymes in the p38 MAPK and JNK signal pathways were measured, respectively, before and after being induced with different doses of IL-1β and specific inhibitors of p38 MAPK, JNK, ERK, PI3K and PKC. The Glucocorticoid sensitivity was measured using in vitro Glucocorticoid binding assay. Analysis of variance (ANOVA) was used to analyze the data.P < 0.05). Either a specific inhibitor of the p38 MAPK pathway or a specific inhibitor of the JNK pathway increased the GRα/GRβ ratio and the Glucocorticoid affinity. None of the specific inhibitors of ERK, PI3K or PKC had any influence on the expression of GR isoforms.The mRNA and protein expression levels of GRα, GRβ and key enzymes of the p38 MAPK and JNK pathways increased both in time- and concentration-dependent manners in IL-1β-induced nasal polyp tissue. The expression of GRβ increased more significantly than that of GRα, and the GRα/GRβ ratio decreased in time- and concentration-dependent manners. Statistically significant differences were found in the GRα/GRβ ratio and the mRNA expression of phospho-p38 MAPK and phospho-JNK between the IL-1β-induced groups and the control groups (Our results demonstrated that the imbalanced expression of GR isoforms in nasal polyp tissue induced by IL-1β in vitro is mediated through the p38 MAPK and JNK signal pathways. Chronic rhinosinusitis (CRS) is a chronic inflammatory disease that involves the mucosa of the nasal cavity and sinus and has a 2-15% incidence rate worldwide -5. AlthoThe biological action of GC is mediated through the activation of intracellular glucocorticoid receptors (GR). There are two human GR isoforms (GRα and GRβ), and GC’s physiological actions are mainly mediated by GRα, which is far more abundant than GRβ in nasal mucosa ,7. GRβ nRecent studies -15 have 3H]-Dexamethasone was obtained from Amersham (USA). Dexamethasone was purchased from Sigma (USA). The primers and Taqman probes targeting GRα, GRβ, p38 MAPK and JNK were designed and synthesized by Applied Biosystems (USA). All other chemicals used in the study were of analytical grade and obtained from commercial sources.Primary monoclonal antibodies against GRα, GRβ, phospho-p38MAPK, phospho-JNK and β-actin, as well as a horseradish peroxidase-labeled secondary goat anti-mouse IgG antibody, were purchased from Santa Cruz Biotechnology (USA). IL-1β was purchased from PeproTech (England) and RPMI1640 culture medium was purchased from GIBCO (USA). The specific inhibitors of the p38 MAPK pathway (SB203580), ERK pathway (PD98059), PI3K pathway (LY294002), PKC pathway (Ro31-8220) and JNK pathway (SP600125) were all purchased from Calbiochem (USA).  solution containing 0.05% Tween-20. Following this, the membrane was incubated for 1.5 h at room temperature with the indicated primary antibodies (1:500). The membrane was then incubated with the corresponding horseradish peroxidase-labeled secondary goat anti-mouse IgG antibodies. Immunoreactive proteins were measured with the Enhanced Chemiluminescence (ECL) Western blot detection system . The β-actin protein served as an endogenous loading control.2O, with a total solution volume of 50 μl. The thermocycler parameters were set as: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s and 60°C for 45 s. All reactions were carried out in triplicate. The relative quantity of mRNA was determined using the comparative cycle threshold method, and values were normalized using β-actin as an endogenous control. The primers and probes employed were as follows: GRα, 5′-TGA AAA TGG GTT GGT GCTT CTA-3′ (upstream), 5′-GAC AAG AAT ACT GGA GAT TTG AGT CAA-3′ (downstream), 5′-FAM-CCT GAT GGC ACT TAG CTA TCA GAA GAC CAC AA-TAMRA-3′ (probe); GRβ, 5′-TGG CCA CCC CAA AAG GA-3′ (upstream), 5′-GAG CTC ATC CCA TGC TAA TTA TCC-3′ (downstream), 5′-FAM-AAC TAA CAT GAT TTG TGT CTA TGA AGT GC–TAMR A -3′ (probe); p38MAPK, 5′-GGC TCT GGC GCC TATG G-3′ (upstream), 5′-CCA CAC GTA ACC CCG TTT TT-3′ (downstream), 5′-FAM-TCT GTG TGT GCT GCT TTT GAC A-TAMRA-3′ (probe); JNK, 5′-TAC AGA GCA CCC GAG GTC ATC-3′ (upstream), 5′-AGA GGA TTT TGT GGC AAA CCA-3′ (downstream), 5′-FAM-TGG CAT GGG CTA CAA GGA AAA CG-TAMRA-3′ (probe); β-actin, 5′-TCC CTG GAG AAG AGC TAC GAG-3′ (upstream), 5′-GCC GTG ATC TCC TTC TGC A-3′ (downstream), 5′-FAM- CGC TCT TCC AGC CCT CCT TCC TGG-TAMRA-3′ (probe).Total RNA was reversely transcribed to cDNA using oligo (dT) 18 primers, and PCR amplifications of GRα, GRβ, p38 MAPK and JNK were performed with TaqMan universal master mix. The end products were quantified with an ABI 7900 sequence detector. Each reaction contained 25 μl 2 × Taqman PCR master, 1 μl upstream primer (10 μM), 1 μl downstream primer (10 μM), 1 μl TaqMan probe (10 μM), 5 μl cDNA and 17 μl ddH3H]dexamethasone binding assay has been used in several previous studies [3H]dexamethasone ranging between different concentrations (the range 1–50 nM), with or without a 1,000-fold excess unlabelled dexamethasone. Radioactivity was measured in liquid scintillation vials. Specific binding was calculated by subtracting nonspecific binding from the total binding. The equilibrium dissociation constant (Kd) was calculated using SCATCHARD method (by the slope of the curve in the Scatchard plot).[ studies ,20 to deP-values less than 0.05 were considered to be statistically significant.Statistical analysis was carried out using the SPSS Version 16.0 software package. Data sets for a given construct were pooled and averaged. The statistical significance of the difference between control and experimental data sets was determined by analysis of variance (ANOVA) with the least significant difference (LSD) test. Differences with IL-1β induced a decrease in the GRα/GRβ ratio in a dose-dependent manner in cultured nasal polyps in vitro.X2 = 36.031, P = 0.001; X2 = 27.533, P = 0.001).After having been cultured in the presence of 0 ng/mL, 10 ng/mL, 20 ng/mL and 30 ng/mL IL-1β for 20 h respectively, samples were harvested and GRα and GRβ protein expressions were measured. Low expression of GRα (94 kDa) and GRβ (90 kDa) was detected by using Western blot in the cultured nasal polyp tissue of the control group (0 ng/mL IL-1β), and it showed that the expression level of GRβ was lower than that of GRα .Nasal polyp tissues were cultured in the presence of 20 ng/mL IL-1β for 0 h, 10 h, 20 h and 30 h respectively. IL-1β induced the expression of GRα and GRβ in a time-dependent manner, and the expression of GRβ increased more significantly than that of GRα .0 ng/mL, 10 ng/mL, 20 ng/mL and 30 ng/mL of IL-1β were used to induce nasal polyp tissues for 20 h in vitro respectively. Both phospho-p38 MAPK (39 kDa) and phospho-JNK (46 kDa/54 kDa) protein and mRNA were extracted for quantitative measurements. A low expression of phospho-p38 MAPK and phospho-JNK was observed in the control group. IL-1β led to higher phospho-p38 MAPK and phospho-JNK expression at the protein . Treatment with 5 μM SP600125 (specific inhibitor of JNK) lead to significantly lower expression of phospho-JNK, while the expression of phospho-p38 MAPK was not found to be significantly different from that in the control group (0 μM SP600125). It was found that the expressions of GRα and GRβ also decreased, with the GRα/GRβ ratio of the 5 μM SP600125 group significantly higher than that of the control group . Further, SB203580 exhibited a much stronger inhibitive effect on GRα and GRβ expressions than SP600125 . Our results also showed that the GC affinity and sensitivity increased after the nasal polyp was incubated with SB203580  or SP600125 , but did not change when incubated with LY294002, PD98059 or Ro31-8220 .Specific inhibitors (SB203580 and SP600125) of the p38 MAPK or JNK signal pathways were separately added (at a dose of 5 μM) to the culture medium 30 min before the induction with 20 ng/mL IL-1β. The expressions of GR isoforms, phospho-p38 MAPK and phospho-JNK were measured 20 h after IL-1β induction Figure . The resA decrease in the GRα/GRβ ratio in nasal polyps is one of the important features of GC-resistant CRS. However, its etiology has remained unexplained, and there is currently no direct and effective treatment for GC-resistant CRS. In this study, IL-1β was used to induce cultured nasal polyp tissues in vitro. With the IL-1β induction, the GRα/GRβ ratio declined in both a time-dependent manner and a concentration-dependent manner, which demonstrates the successful establishment of a GRα/GRβ imbalance model in vitro. We also found that after cultured nasal polyps were induced with the same set of IL-1β concentrations, the p38 MAPK and JNK signal pathways were also activated, as the expression of key enzymes of both pathways increased in a similar concentration-dependent manner. Furthermore, the intervention with specific inhibitors of the p38 MAPK and JNK pathways resulted in an increased GRα/GRβ ratio and a recovery of GC affinity in IL-1β-induced polyps. These results suggest that the p38 MAPK and JNK signal pathways may play an important role in the IL-1β-induced GRα/GRβ imbalance in nasal polyps and may be related to GC resistance in nasal polyps. Our results also suggest that blocking these signal pathways may lead to a recovery of GC sensitivity in GC resistance nasal polyps.IL-1β is one of the important cytokines involved in the etiology of nasal mucosal inflammation. Histological studies have revealed that its expression increased in the mucosa and sinus secretions of rhinosinusitis patients, and this cytokine was confirmed to be associated with rhinosinusitis ,22. In oAlthough recent studies have confirmed that the decreased GRα/GRβ ratio is one of the important features in GC-resistant CRS, the signal transduction mechanism responsible has not yet been fully identified, meaning that further research is needed. Before now, it has been suggested that p38 MAPK and JNK are involved in the signal transduction of GR. This study examined the activation of the p38 MAPK and JNK signal pathways in the IL-1β-induced samples, and found that the expression of activated key enzymes (phospho-p38 MAPK or phospho-JNK) increased in the same manner as the GRβ/GRα ratio. The above results suggest that the p38 MAPK and JNK signal pathways may be associated with the change of GRα/GRβ ratio. Furthermore, the ratio of GRα/GRβ and GC affinity were observed to increase after the p38 MAPK or JNK pathways were blocked by specific inhibitors, SB203580 and SP600125, respectively, indicating that the p38 MAPK and JNK signal pathways are indeed involved in regulating the ratio of GR isoforms and GC sensitivity in IL-1β-induced nasal polyp tissues in vitro. In order to further exclude the possibility of SB203580 or SP600125 directly affecting GR isoform expression, we performed a negative control experiment (samples not induced with IL-1β). Neither SB203580 nor SP600125 led to changs in GR isoform expression in this negative control experiment. The above results show that the synthesis of GR isoforms may be regulated through p38 MAPK and JNK signal pathways in the IL-1β induced nasal polyps. We believe that the change in the GRα/GRβ ratio may cause the insensitivity of nasal polyps to GC. Blocking the above signal pathways helped to increase the GRα/GRβ ratio, which may improve GC sensitivity in nasal polyps. Our results are consistent with recent studies reported by other researchers on the signal pathways involved with GR expression and GC sensitivity in brain tissue .Although our study showed that both the p38 MAPK and JNK signal pathways were involved in the regulation of GR isoform expression in IL-1β-induced nasal polyps in vitro, more research needs to be carried out in order to determine how these two pathways regulate this process. In this study, we found that both SB203580 and SP600125 can influence the expression of GR isoforms, but blocking the p38 MAPK signal pathway with SB203580 cannot change the expression of a key enzyme in the JNK pathway, and vice versa. The above evidence indicates that the two signal pathways involved in regulating GR expression operate in parallel, and there was no significant interaction observed. Our study also found that SB203580 has a more significant effect on GR isoform expression and GC affinity than SP600125, suggesting that the p38 MAPK may be a more important pathway than the JNK pathway.In contrast to the results of our own experiments described above, a recent study by Zijlstra and colleagues  reportedIn summary, our study showed that the p38 MAPK and JNK signal pathways are involved in regulating the expression of GR isoforms in IL-1β-induced nasal polyps in vitro, as their activation resulted in a decrease of GRα/GRβ ratio. Further, our results suggest that these two pathways are involved in the etiology of GC-resistant CRS. Our findings also provide deeper insights into the pathogenesis of GC resistance in CRS and provide further clues as to which pathways should be targeted in developing a more precise treatment for GC-resistant CRS. However, the precise upstream signal mechanisms involved in GR regulation are very complex. In this study, only five signal pathways were investigated. A complete elucidation of the pathological mechanisms of GC-resistant CRS requires further thorough study on all other related pathways.


In particular, the PTP scaffold was conjugated at the C2 position with the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole, a group believed to confer potency and selectivity toward the human (h) A2B adenosine receptor (AR) to the xanthine ligand 8-(1-(3-(trifluoromethyl)benzyl)-1H-pyrazol-4-yl)-1,3-dimethyl-1H-purine-2,6-dione (CVT 6975). Interestingly, the synthesized compounds turned out to be inactive at the hA2B AR but they displayed affinity at the hA3 AR in the nanomolar range. The best compound of the series (6) shows both high affinity (hA3 AR Ki = 11 nM) and selectivity  at the hA3 AR. To better rationalize these results, a molecular docking study on the four AR subtypes was performed for all the synthesized compounds. In addition, CTV 6975 and two close analogues have been subjected to the same molecular docking protocol to investigate the role of the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole on the binding at the four ARs.A new series of pyrazolo[4,3- ES-MS: (MH+) 518.2. Anal. Calcd. for C25H18N9OF3 (MW 517.47): C, 58.03; H, 3.51; N, 24.36. Found: C, 57.92; H, 3.44; N, 24.18.N-{8-Methyl-2-[1-(3-trifluoromethyl-benzyl)-1H-pyrazol-4-yl]-8H-pyrazolotriazolopyrimidin-5-yl}-phenyl-acetamide (6): Yield 85%, white solid; mp 190°C (EtOAc-light petroleum); IR (KBr): 3240–2990, 1675, 1605, 1580, 1525 cm-1; 1H NMR (CDCl3) δ: 3.96 ; 4.27 ; 5.20 ; 7.03–7.37 ; 7.82 ; 7.94 ; 7.97 ; 8.82 . ES-MS: (MH+) 518.2. Anal. Calcd. for C26H20N9OF3 (MW 531.49): C, 58.76; H, 3.79; N, 23.72. Found: C, 58.93; H, 3.65; N, 23.88.2-(4-Methoxy-phenyl)-N-{8-Methyl-2-[1-(3-trifluoromethyl-benzyl)-1H-pyrazol-4-yl]-8H-pyrazolo triazolopyrimidin-5-yl}-phenyl-acetamide (7): Yield 77%, yellow solid; mp 178°C (EtOAc-light petroleum); IR (KBr): 3245–2975, 1680, 1615, 1570, 1515 cm-1; 1H NMR (CDCl3) δ: 3.97 ; 4.18 ; 4.25 ; 5.21 ; 7.03–7.37 ; 7.57 ; 7.83 ; 7.96 ; 7.98 ; 8.78 . ES-MS: (MH+) 562.2. Anal. Calcd. for C27H22N9O2F3 (MW 561.52): C, 57.75; H, 3.95; N, 22.45. Found: C, 57.53; H, 3.86; N, 22.33.1 and hA2A AR; 50 mM Tris/HCl, 10 mM MgCl2, 1 mM EDTA, pH 8.25 for hA3 AR), frozen in liquid nitrogen and stored at -80°C. For the measurement of adenylyl cyclase activity only one high speed centrifugation of the homogenate was used. The resulting crude membrane pellet was resuspended in 50 mM Tris/HCl, pH 7.4 and immediately used for the cyclase assay.All pharmacological methods followed the procedures as described earlier. –36 In br1 ARs 1 nM [3H]CCPA was used (KD = 0.61 nM), whereas 10 nM [3H]NECA were used for A2A (KD = 20.1 nM). The high-affinity agonist [3H]HEMADO (KD = 1.1 nM) was used for A3 AR binding at a concentration of 1 nM. Non specific binding of [3H]CCPA was determined in the presence of 1 mM theophylline, in the case of [3H]NECA and [3H]HEMADO, 100 μM R-PIA was used. Ki-values from competition experiments were calculated with the program SCTFIT. ATP for 20 min in the incubation mixture as described without EDTA and NaCl. CCPA ([3H]2-Chloro-N6-cyclopentyladenosine) (hA1 AR), [3H]NECA ([3H]5'-N-ethylcarboxamidoadenosine) (hA2A AR) and [3H]HEMADO ([3H]2-(1-Hexynyl)-N-methyladenosine) (hA3 ARs) were used as radioligands in binding assays. Inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells expressing hA2B receptors was determined as a measurement of affinity of compounds at the hA2B AR (Newly synthesized compounds ( hA2B AR . 33–35]–353–7) w2B AR. On the contrary they have shown an unexpected high affinity, with some in the nanomolar range, towards the hA3 AR subtype. In 3 selective agonist [3H]HEMADO  from single experiments for compounds 6 and 4, respectively, are reported. The affinity at the hA3 AR seems to be more related to the substitution at the N5 position than to the substituent present at the C2 position. In fact, the N5-unsubstituted derivative 3, proved to be almost inactive at all four ARs, while, the introduction of a substituent at the N5 position  induced a recovery of affinity at the hA3 AR with good levels of selectivity versus the other receptor subtypes. In particular, the introduction of an arylcarbamoyl moiety, which has been demonstrated optimal substituent for the hA3 AR, triazolopyrimidines bearing a 1-(3-trifluoromethyl-benzyl)1H-pyrazol-4-yl moiety at the C2 position in order to explore the effect on affinity and selectivity profiles at the four ARs. In particular, the 1-(3-trifluoromethyl-benzyl)1H-pyrazol-4-yl group when attached at the 8 position of xanthine derivatives, such as CVT-6975 (1), is known to confer high affinity for the hA2B AR. Curiously, the synthesized compounds were inactive at the hA2B AR but demonstrated activity as potent and selective antagonist of the hA3 AR. A molecular docking study was performed in order to rationalize the influence of a 1-(3-trifluoromethyl-benzyl)-1H-pyrazole branch. We observed that only the hA3 AR has the topological features to accommodate this new series in agreement with experimental data. Interestingly, the ability to host such a big moiety can be ascribed to an accessory binding pocket present in the hA3 AR, which is formed by specific unconserved residues. Two possible binding modes were proposed in which the substituents in C2 and N5 are reciprocally inverted. Further studies will be needed to validate which one is predominant or if they may co-exist. Although derivatives more potent at the hA3 AR, and with a better drug-like properties than compound 6, have been already reported in literature, our derivative shows high selectivity for hA3 AR over the other ARs subtypes .We have presented a novel series of pyrazoloHEMADO. Representative curves  from single experiments with Ki values of 17 and 39 nM for compounds 6 and 4, respectively, are reported.Both compounds show high affinity binding to hA(PDF)Click here for additional data file.S2 Fig3 shows in a radioligand competition assay with the A1 selective radioligand [3H]CCPA a Ki value of 764 nM. The curve shows total binding to hA1 ARs from a representative single experiment.Compound (PDF)Click here for additional data file.S3 Fig3 shows in a radioligand competition assay with the nonselective radioligand [3H]NECA a Ki value of 6820 nM. The curve shows total binding to hA2A ARs from a representative single experiment.Compound (PDF)Click here for additional data file.S4 Fig6 are reported superposed to the coordinates of compound 36 to reveal the similarity in the accommodation of the common 1-(3-Trifluoromethyl-benzyl)-1H-pyrazole residue. The coordinates of compound 36 in B are obtained from a secondary docking solution. (C) Per residue electrostatic interaction energy map and per residue hydrophobic interaction score map. The maps are calculated for a selected pose of compound 6 inside the hA3 AR binding site. Electrostatic energy values are expressed in kcal mol–1, whereas hydrophobic scores are expressed in arbitrary hydrophobic units.The hypothetical binding modes (A and B respectively indicated) of compound (TIF)Click here for additional data file.S5 Fig1, hA2A, hA2B and hA3 ARs are reported in panels A, B, C and D respectively. In panel D, the profiles of the two predominant binding modes for hA3 AR, A (red) and B (blue), are showed. In Panel E the location of residues Met86, Ser181, Ser247 and Asn250 (in cyan) and Phe168, Trp243, Leu246 and Tyr265 (in green) in the A3 AR and the corresponding residues in the others AR subtypes is indicated by the ball representation of alpha Carbon atoms.The contributes to electrostatic and hydrophobic energy interactions for hA(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Advanced castration resistant prostate cancer (CRPC) is often characterized by an increase of C-terminally truncated, constitutively active androgen receptor (AR) variants. Due to the absence of a ligand binding domain located in the AR-C-terminus, these receptor variants  are unable to respond to all classical forms of endocrine treatments like surgical/chemical castration and/or application of anti-androgens.in vivo was demonstrated using a chick chorioallantoic membrane xenograft assay.In this study we tested the effects of the naturally occurring stilbene resveratrol (RSV) and (E)-4--N, N-dimethylaniline, a fluorinated dialkylaminostilbene (FIDAS) on AR- and ARΔLBD in prostate cancer cells. The ability of the compounds to modulate transcriptional activity of AR and the ARΔLBD-variant Q640X was shown by reporter gene assays. Expression of endogenous AR and ARΔLBD mRNA and protein levels were determined by qRT-PCR and Western Blot. Nuclear translocation of AR-molecules was analyzed by fluorescence microscopy. AR and ARΔLBD/Q640X homo-/heterodimer formation was assessed by mammalian two hybrid assays. Biological activity of both compounds in vivo.The stilbenes RSV and FIDAS were able to significantly diminish AR and Q640X-signalling. Successful inhibition of the Q640X suggests that RSV and FIDAS are not interfering with the AR-ligand binding domain like all currently available anti-hormonal drugs. Repression of AR and Q640X-signalling by RSV and FIDAS in prostate cancer cells was caused by an inhibition of the AR and/or Q640X-dimerization. Although systemic bioavailability of both stilbenes is very low, both compounds were also able to downregulate tumor growth and AR-signalling RSV and FIDAS are able to inhibit the dimerization of AR and ARΔLBD molecules suggesting that stilbenes might serve as lead compounds for a novel generation of AR-inhibitors. Therefore, treatments for patients suffering from locally advanced or metastatic prostate cancer (PC) include the reduction of circulating androgens by surgical or chemical castration as well as the blockade of the androgen receptor (AR) with anti-androgens. Unfortunately the effects of endocrine therapies are only transitory. Although the majority of patients initially responds to first line androgen deprivation therapy (ADT), PC-cells almost invariably acquire the ability to grow and survive in the presence of sub-physiological levels of circulating androgens. This state of the disease, whose onset is characterized by rising titres of serum prostate specific antigen (PSA), is known as castration resistant prostate cancer (CRPC). Although CRPC is resistant to standard endocrine therapies, it is not completely refractory to further hormonal manipulation and AR-signalling still continues to play a pivotal role in the progression of CRPC. The proposed mechanisms that underlie AR function in this clinical setting are quite varied: These include intratumoral androgen synthesis, increased AR-expression and/or stability, deregulation of AR cofactors, aberrant kinase pathway activation as well as AR-mutations broadening AR-ligand specificity in vitro and in vivoin vitro and in vivo. The present study shows for the first time that the modulation of AR- as well as ARΔLBD-signalling by synthetic and naturally occurring stilbenes is caused by an inhibition of AR- or ARΔLBD-dimerization.With the emergence of constitutively active ARΔLBD in late stage CRPC Renilla reniformis luciferase reporter plasmid (pRL-TK) used for the control of transfection efficiency was purchased from Promega . 3,4',5-trans-trihydroxystilbene or resveratrol (RSV) and its synthetic analog (E)-4--N,N-dimethylaniline, a cell permeable fluorinated N, N-dialkylaminostilbene (FIDAS-3) pSG5-AR, encoding a wild type full length AR (919 amino acids (aa), termed thereafter AR) was supplied by Dr. Z. Culig . The expression plasmid pAR-t1EosFP coding for a green fluorescent EosFP-AR-fusion protein was a generous gift from Dr. F. Oswald . The expression plasmids pCruz-ARQ640X and pEGFP-ARQ640X coding for the c-terminally truncated AR-mutant Q640X (aa 1–640) were provided by Dr. J. Céraline . The PSA-reporter plasmid pPSA-61luc was a generous gift of Dr. J. Trapmann ; the pARE(2x)-luc reporter plasmid pLC0548, created by H. Lebedur was provided by Dr. A. Allera . AR-negative PC-3 and the AR-positive LNCaP and 22Rv1 cells were purchased from the American Type Culture Collection . The CRPC cell line LNCaP C4-2 originally described by Wu et al. PC-3-cells were seeded in 24-well plates and grown in the absence of DHT for 24 hours. Subsequently, cells were transfected with pAR-t1EosFP and pEGFP-ARQ640X, respectively. After 24 hours pAR-t1EosFP transfected cells were treated with ethanol (solvent control) or 5 nM DHT for 2 hours in presence/absence of RSV (100 µM) or FIDAS (50 µM). pEGFP-ARQ640X transfected cells expressing constitutively active GFP-tagged Q640X did not need an androgenic stimulus and were treated with RSV and FIDAS only. Subsequently, nuclear/cytoplasmic fluorescence was determined in the cells using fluorescence microscopy.Prostate cancer cells were lysed in a buffer containing 10 mM Tris/HCl pH 7.6, 5 mM EDTA, 50 mM NaCl, 50 mM NaF and 1% Triton X100 (v/v). Cell nuclei and debris were removed by centrifugation , and the supernatant was tested for protein concentration using the BCA-method . Cell extracts were electrophoresed through a 10% SDS-PAGE gel  and electroblotted onto a nitrocellulose or PVDF . AR protein was detected by monoclonal mouse antibody AR441 , detection of β-actin with mouse monoclonal Anti-β-Actin  served as loading control. Immunoreactive bands were localized using horseradish peroxidase-labeld goat anti-mouse antibody . Immunoreactive complexes were visualized by direct detection of chemoluminescene using the Fusion FX7 Imaging and Gel documentation system and FUSION-CAPT software .in vitro toxicicity  and different reporter gene constructs -luc) using Polyfect . pRL-tk-LUC was co-transfected as an internal control for transfection efficiency. 24 hours after transfection, cells were treated with/without 5 nM DHT in the presence/absence of the stilbenes RSV (50 and 100 µM) and FIDAS (50 µM). After 24 hours reportergene activity was measured using the Dual-Luciferase Reporter Assay  according to the manufactures instructions. Within this time frame RSV as well as FIDAS did not exhibit a significant city see .Renilla reniformis luciferase for transfection control. The pACT-vector contains the herpes simplex VP16 activation domain upstream of a multiple cloning region. The genetic information coding for the interactive proteins of interest  were subsequently cloned into the pBIND and pACT–vectors to generate fusion proteins with the DNA-binding domain of GAL4 and the activation domain of VP16. The pGAL4 and pVP16 fusion constructs  were transfected along with pG4.31-luc which encodes a GAL4-dependent firefly luciferase reporter. Dimerization of two chimeric fusion proteins like AR-VP16/ACT and AR-GAL4/BIND (AR homodimer), Q640X-VP16/ACT and Q640X-GAL4/BIND (Q640X homodimer) or AR-VP16/ACT and Q640X-GAL4/BIND (AR/Q640X-heterodimer) promoted the activation of the M2H reporter pG4.31-luc with subsequent expression of firefly luciferase. Amounts of firefly luciferase and Renilla luciferase were subsequently detected using the Dual-Luciferase Reporter Assay System.Dimerization of AR and/or Q640X was analyzed using the CheckMate Mammalian Two-Hybrid (M2H) System  according to the manufactures instructions. The CheckMate M2H contains 2 expression vectors termed pBIND and pACT. The pBIND vector expresses a yeast GAL4 DNA-binding domain upstream of a multiple cloning region and a SV40-controlled Total RNA was isolated from 22RV1, LNCaP and LNCaP C4.2 cells using the RNeasy Mini kit  according to the manufactures instructions. The mRNA levels of full length AR and the AR splice variant AR-V7 were determined by the relative quantification method (ΔΔCt) using glucose 6-phosphate dehydrogenase (G6PD) as housekeeping gene. The quantification was performed with TaqMan probes along with the QuantiFast Multiplex RT-PCR + R Kit  on a ViiA 7 Real-Time PCR . Primers were designed to span exon/exon boundaries in order to avoid signals from residual genomic copies of the gene. Primers and MGB-probes  assay as an animal substitute model In order to analyze the effects of FIDAS Data are reported as means ± standard deviation. Analysis was performed with Student's T-test (two tailed for independent samples) with p<0.05 considered as significant.Various natural and synthetic stilbenes were recently shown to exhibit strong chemopreventive and anti-proliferative effects in experimental PC Effects of FIDAS on AR-signalling were analyzed using an AR-specific reporter gene assay. Full AR activity was achieved following stimulation of the cells with 5 nM DHT (stilbene untreated controls set at 100%). In LNCaP and 22Rv1 cells treated with FIDAS (50 µM) transcriptional activity was diminished to 35% and 18%, respectively. RSV-controls at the same concentration reduced AR transcriptional activity of DHT-stimulated LNCaP and 22Rv1 cells to 47% and 39%, respectively. When treated with the maximal concentration of 100 µM RSV transcriptional activity of the AR was reduced to 33% in LNCaP and 9% in 22Rv1 cells.Several studies suggest that the predominant mechanism enabling RSV to inhibit AR-signalling is through a down regulation of AR-mRNA The ability of the stilbenes RSV and FIDAS to modulate basal endogenous AR or ARΔLBD mRNA-levels were studied by qRT-PCR. As endogenous ARΔLBD we analyzed the expression of the splicing variant AR-V7 , a prototypical ARΔLBD In agreement with previous findings In LNCaP and LNCaP C4-2 cells FIDAS also induced a dose dependent decrease of AR and AR-V7 mRNA . The FIDThe ability of high RSV concentrations to increase AR mRNA levels in LNCaP and the general inability of FIDAS to reduce AR and AR-V7 mRNA levels in 22Rv1, although the compounds diminished AR-signalling in both cell lines, prompted us to analyze the effects of RSV and FIDAS on AR-protein levels in LNCaP and 22Rv1 cells. This approach is furthermore supported by a report of Harada et al. showing that RSV represses AR target gene expression, at least partially, by regulating AR-degradation As seen in In consequence, in order to analyze the direct effects of FIDAS and RSV on AR and ARΔLBD-signalling without interference from changes in AR mRNA levels, the following experiments were performed in PC-3 cells transiently transfected with AR and/or Q640X100 µM RSV: AR-activity 16% and Q640X-activity 46% - 50 µM FIDAS: AR-activity 10% and Q640X-activity 23%). The fact that both compounds were able to inhibit the LBD-lacking Q640X clearly shows that RSV like FIDAS does not attenuate AR-function through binding to the AR-ligand binding domain.In order to analyze the effects of RSV and FIDAS on ARΔLBD-signalling, AR-negative PC-3 were co-transfected with the constitutively active ARΔLBD-variant Q640X and an AR-dependent reportergene pARE(2x)-luc. Co-transfected pRL-tk-LUC served as transfection control. In parallel, experiments were performed with full length AR in presence/absence of DHT. Both RSV and its fluorinated counterpart FIDAS were able to significantly down-regulate the transcriptional activities of AR  and Q640v567es was shown to form transcriptionally active heterodimers In the absence of androgens, co-expression of a full length AR with an ARΔLBD like Q640X or ARBased on our results that AR-expression and AR-stability cannot fully account for RSV or FIDAS-mediated inhibition of AR-signalling, we analyzed the effects of both stilbenes on the nuclear translocation of AR and Q640X. Previous studies analyzing the effects of RSV on the nuclear translocation of AR remained controversial In order to generate genomic signals the AR has to dimerize with another AR-molecule in the nucleus The M2H is a powerful tool to identify protein-protein interactions in living cells. In contrast to conventional M2H procedures that are mainly using isolated functional domains of proteins to analyze protein-protein interaction, our experimental setup is based on fully active transcription factors fused to a DNA-binding GAL4-binding domain or a VP16-activation domain . Upon dimerization the chimeric GAL4/BIND and VP16/ACT receptor fusion proteins are able to promote the activation of the M2H reporter controlled by a GAL4-responsive promoter. However, a M2H-approach for the analysis of dimerization processes using fully active transcription factors like AR or Q640X requires extensive control experiments (see supporting information). In contrast to the nucleosolic formation of AR-dimers following androgenic stimuli in vivo showed that ID/MyoD-dimerization remained unaffected by RSV and FIDAS in the M2H, the latter suggesting that the effects of stilbenes on AR- and Q640X-dimerization are specific (data not shown).As can be seen in in vitro test concentrations) were administered intravenously (CAM-vein) allowing the systemic spread of the substances in the chick embryo-CAM system in vivo. The downregulation of proliferation as reflected by KI67 was more pronounced in the AR-positive LNCaP than in the AR-negative PC-3 . Additionally AR-activity was inhibited by both stilbenes as depicted by a down regulation of PSA in the tumor xenografts . Our results show that even low systemic concentrations following intravenous injection are sufficient to impair proliferation as well as AR-signallingThe poor aqueous solubility of RSV and RSV-derivates like FIDAS Although the majority of patients initially respond well to ADT, PC almost invariably recurs in a form that is no longer susceptible to classical endocrine therapies. This state of the disease, whose onset is characterized by rising titres of serum PSA at castrate levels of circulating androgens, is known as CRPC. Interestingly, the AR still plays a central role at this stage of the disease. The mechanisms allowing CRPC cells to activate the AR despite subphysiological levels of circulating androgens include intratumoral androgen synthesis (steroidogenic pathway), deregulation of steroid receptor cofactors and/or increase of intracellular AR-levels (hypersensitive pathway), point mutations broadening AR-ligand specificity (promiscuous pathway) and AR activation through tyrosine kinase-dependent mechanisms (outlaw pathway) in vitro, they are unable to activate the full panel of AR-dependent genes in vivo both, the AR and ARΔLBD are expressed in CRPC cells, it was suggested that ARΔLBD- receptors must act in concert with full length AR to activate AR-dependent genes in CRPC v567es and Q640X form heterodimers in vitro, the functional relationships between the AR and ARΔLBD remain largely unknown The AR is a ligand binding transcription factor of the steroid hormone receptor superfamily. In the absence of androgens, the AR is predominantly located in the cytoplasm. Upon androgen binding the AR-protein translocates into the nucleus where it dimerizes with another AR-molecule. The AR-dimer subsequently binds to androgen response elements present in the cis-regulatory regions of target genes. Early functional studies showed that a deletion of the AR-LBD situated in the C-terminus of the AR, results in constitutive activation of the receptor molecule To date various studies focused on the isolation of small molecule inhibitors, able to destabilize AR-molecules Resveratrol (RSV), a natural stilbene found in grapes and wine is characterized by a broad range of anticancer, antioxidant, anti-inflammatory and cardioprotective activities in vivo  FIDAS was selected because of its relatively long half life Ex3dup) Ex3dup and AR-V7 has been shown so far Several studies suggested that a RSV-induced decrease of AR-mRNA is the predominant mechanism responsible for the anti-androgenic effects of RSV in vivo AR and ARΔLBD are concomitantly expressed it was hypothesized that under androgen deprived conditions, AR and ARΔLBD must form transcriptionally active AR/ARΔLBD-heterodimers in order to activate AR-dependent genes In order to analyze the effects of FIDAS and RSV on AR/ARΔLBD-signalling, experiments were performed in PC-3 cells, transiently transfected either with AR and/or Q640X, an ARΔLBD recently isolated from a metastatic castration resistant tumor The decrease of AR and Q640X-signalling following stilbene treatment was not due to an inhibition of nuclear translocation as shown by fluorescence microscopy of green fluorescent AR or Q640X fusion proteins transfected into PC-3 cells. Although these findings differ from a study analyzing AR nuclear translocation in LNCaP cells In order to generate genomic signals the AR and Q640X must form receptor dimers in the nucleus in vitro experiments. Using a modified chorioallantoic membrane xenograft model we proved that even low systemic concentrations RSV and FIDAS were able to inhibit the proliferation of PC cells. Interestingly, the inhibition of proliferation was the most pronounced in AR-positive cells and paralleled by a down regulation of PSA in the tumor xenografts. Although numerous clinical data are available on the safety of RSV as a dietary supplement , the effects of a long term treatment with high doses of RSV remain to be elucidated. Moreover, the pharmacokinetics, bioavailability, and toxicity of synthetic RSV-derivatives like FIDAS, generated to improve the low bioavailabilty of natural stilbenes, need to be determined.Unfortunately, the concentrations of FIDAS and RSV, necessary to inhibit AR-dimerization are relatively high. Results from pharmacokinetic studies indicate that circulating stilbenes are rapidly metabolized in the body and raise doubts on the physiological relevance of the concentrations typically used for The effects of stilbenes on the AR and androgen-responsive genes are complex. However, gene expression data suggest that the down-regulation of androgen-responsive genes is not solely due to decreased levels of the AR Figure S1Representative control experiments for the M2H. PC-3 cells were transiently transfected with pAR-VP16/ACT (pAR-ACT), pAR-GAL4/BIND (pAR-BIND), pQ640X-VP16/ACT (pQ640X-ACT), pQ640X-GAL4/BIND (pQ640X-BIND) or the empty vectors pACT and p-BIND as shown in (TIF)Click here for additional data file.Table S1Effects of RSV and FIDAS on the cell viability in vitro.(DOC)Click here for additional data file.


How do the holidays – and the possible New Year’s resolutions that follow – influence a household’s purchase patterns of healthier foods versus less healthy foods? This has important implications for both holiday food shopping and post-holiday shopping.207 households were recruited to participate in a randomized-controlled trial conducted at two regional-grocery chain locations in upstate New York. Item-level transaction records were tracked over a seven-month period (July 2010 to March 2011). The cooperating grocer’s proprietary nutrient-rating system was used to designate “healthy,” and “less healthy” items. Calorie data were extracted from online nutritional databases. Expenditures and calories purchased for the holiday period (Thanksgiving-New Year’s), and the post-holiday period (New Year’s-March), were compared to baseline (July-Thanksgiving) amounts.p<0.001), with 75% of additional expenditures accounted for by less-healthy items. Consistent with what one would expect from New Year’s resolutions, sales of healthy foods increased 29.4% ($13.24/week) after the holiday season compared to baseline, and 18.9% ($9.26/week) compared to the holiday period. Unfortunately, sales of less-healthy foods remained at holiday levels ($72.85/week holiday period vs. $72.52/week post-holiday). Calories purchased each week increased 9.3%  after the New Year compared to the holiday period, and increased 20.2%  compared to baseline.During the holiday season, household food expenditures increased 15% compared to baseline ($105.74 to $121.83; Despite resolutions to eat more healthfully after New Year’s, consumers may adjust to a new “status quo” of increased less-healthy food purchasing during the holidays, and dubiously fulfill their New Year’s resolutions by spending more on healthy foods. Encouraging consumers to substitute healthy items for less-healthy items may be one way for practitioners and public health officials to help consumers fulfill New Year’s resolutions, and reverse holiday weight gain. How do the holidays – and the possible New Year’s resolutions that follow – influence a household’s purchase patterns of healthier vs. less-healthy foods? Holiday weight gain has been largely documented, but less understood is the potentially compensating cycle of food purchasing that might start much earlier in the season and run much later. Food purchases dictate the total calories available in the home and over-purchasing can have a long-term impact on weight gain that would not be reflected in short-term studies of holiday weight gain. During the holiday season, weight gains of 0.37 kg to 0.93 kg do occur, and may not be reversed after the holidays are over It is widely assumed that people increase junk-food purchasing and consumption during the holidays and there are several behavioral factors that may explain why individuals are caught overindulging. Longer eating durations, eating with others, easy access to foods, and increased portion sizes are all prevalent during the holidays, and have all been associated with increased intake After the holidays, New Year’s resolutions to reach the “ideal weight” are often quickly abandoned as individuals return to previous habits Participants provided written informed consent before purchase-monitoring began, and the study was approved by the Cornell University Institutional Review Board.This study was conducted as part of a larger seven-month study on purchasing patterns of healthy and relatively less healthy foods. In order to designate healthy and relatively less healthy foods, the research team identified a grocer in the Northeastern United States that had developed a proprietary health rating system for their products. This system, know as Guiding Stars, utilizes a scoring algorithm to categorize foods and beverages into one of four groups–zero, one, two, and three stars–with three stars being the most nutritious Participants for the study were recruited during the months of June and July 2010 in three stores located in a Western New York city with 62,235 residents. Individuals were notified of the study through face-to-face public intercept, emails, word of mouth, and flyers posted in the grocery stores. Shoppers who did more than 75% of the household shopping were targeted so the study would capture a more representative picture of purchasing for an entire household. In addition, shoppers completed a demographic survey assessing income, age, education level, employment status, family size, and marital status.As an incentive for participation, individuals received a 10% discount on purchases of rated items (including zero starred items), which was loaded onto a Bank of America debit card at the end of each week. Each participant received a specific ID card, which was scanned during every transaction. The grocer separated these transactions and sent the data to the research team. With this data, the research team determined the participant’s reimbursement and loaded the reimbursement onto the debit card once a week. This 10% discount lasted during the baseline data collection period from July 17 through September 6.Over the course of 37 weeks, from July 17, 2010 to March 12, 2011, daily-itemized transaction level data were collected for 207 participating households. Each household had a specific identification code, which the grocer used to separate transaction records. These records were then sent to the research team via secure electronic transmission. Data in the transaction records included the health rating of each item purchased, price, product descriptions, total quantity of each item purchased, and total expenditures. With this data, groupings of products by health-rating, “healthy” or “less-healthy,” could be constructed.Additionally, the grocer utilized a category code to classify its approximately 20,000 products. This category code contained nearly 1000 groups into which specific items were classified. In order to obtain nutrition information, the highest frequency item purchased within these product groups was used to extract calorie, fat, and sugar information from online nutrition databases and manufacturer websites. These data were then merged to the transaction data for analysis.In each household, data were aggregated each week, Sunday through Saturday, such that one outcome measure of interest is weekly household food expenditures. Since we utilized the participating grocer’s proprietary rating system we identified healthy foods as receiving at least one star, and relatively less healthy foods as receiving no stars. We then separated expenditure data into purchases of healthy and relatively less healthy foods.The second outcome measure utilized in analysis was weekly per-serving calorie aggregates. This measure was generated by generating a calorie per-serving value for each item purchased and summing these per-serving calories by week for each household. We used a per-serving measure since this is the information available on food labels. In addition, this measure standardizes caloric amounts for all food items making comparisons much more transparent.In the analysis, we used ordinary least squares regression to estimate the difference of holiday and post-holiday purchasing when compared to a baseline level. We refer to the period lasting from July 17 to November 13, 2010 as the baseline period, the period lasting from November 14, 2010 through January 1, 2011 as the holiday season, and the period lasting from January 2 to March 12, 2011 as the post-holiday season. In the regression analyses we controlled for treatment condition received in a larger study conducted concurrently, age, BMI, income, education, and number of children in the household.p<0.001) on food and beverage items with $12.11 (p<0.001) spent on less healthful foods and $3.98 (p<0.05) spent on healthier items during the holiday season. About 75% of additional expenditures were dedicated to less healthful food items.Average household weekly (Sunday through Saturday) expenditures shown in p<0.001), a 23.7% jump in weekly expenditures compared to the baseline period. Households spent about the same on the less-healthy items in the post-holiday season as they did during the holiday season . However, when compared to baseline levels, households increased expenditures on the more nutritious foods by an estimated $13.24 (p<0.001) after the holidays. This more than triples the $3.98 increase in expenditures seen in the holiday season (p<0.001).In the post-holiday period, households continued the upward trend in expenditures and spent an additional $25.01 per week (p = 0.006). Yet in the New Year these gluttonous foods accounted for 55.7% of expenditures, a decrease of 1.4 percentage points from baseline (p = 0.057). Since expenditures on the more calorically-dense foods remained flat, this drop in expenditure shares reflects the increase in expenditures on the healthier items in the post-holiday period. While additional expenditures on the more nutritious items in the post-holiday period nearly tripled additional expenditures in the holiday period, households were actually spending $0.53 more per item on these foods in the post-holiday period than the holiday period. Thus in the New Year, households tended to seek out the more expensive versions of the healthier items instead of seeking the best value.Interestingly, the mix of foods purchased during the holiday season shifted in favor of the less healthy foods as the share of expenditures dedicated to these foods increased from 57.1% during the baseline period to 59.3% during the holiday period (p<0.0.003) during the holiday period relative to the baseline period, and nearly 91% of this increase was due to additional purchases of the more calorically-dense foods. For the healthier foods, the increase in total weekly per-serving calories purchased was not significant at conventional levels suggesting a strong focus on the more fattening foods.Given these purchasing patterns, were households stocking up with more calories during the holiday and post-holiday periods ,4? Totalp<0.001) relative to baseline, more than doubling the 440 calorie increase evident in the holiday season. Of this 890 calorie increase, 560 calories were attributed to the calorically-dense foods while healthier items made up the additional 330 calories, suggesting a greater focus on healthier items post-New Year’s Day. Yet, since expenditures on the less-healthy foods remained constant, households may have been purchasing less-healthy foods with higher calorie content.Even more intriguing is that contrary to well-intentioned New Year’s resolutions, additional weekly per-serving calories purchased increased to 890 in the post-holiday period (p<0.453) in the holiday period to 330 (p<0.001) in the post-holiday period, a 725% (p<0.001) jump. This increase in healthy-item purchasing from the holiday period to the post-holiday period accounts for over half of the additional 450 (440 to 890) calories purchased in the post-holiday period compared to the holiday period. In other words, additional weekly per-serving calories for each healthy item purchased increased slightly by 10 calories in the post-holiday period, indicating that households were purchasing more expensive and somewhat more calorically dense “healthier” items.When we compared additional weekly per-serving calories purchased of healthy foods from the holiday and post-holiday seasons, we found that total weekly per-serving calories purchased jumped from 40 (As might be expected, during the holiday season individuals spent more money on food and purchased more weekly per-serving calories than during the summer/fall. Our findings agree with previous research which has also shown increased calorie intake during the holiday season due to consumption of more energy dense foods as well as purchasing for larger groups While expenditures on the healthier starred foods increased by more than nine times, and total weekly per-serving calories purchased from starred foods rose by 725% in the post-holiday period, households maintained the level of non-starred item purchasing they had adopted during the holiday period. Consequently, if households resolved to eat more healthfully in the New Year, they may have fulfilled this desire not by decreasing purchases of less-healthy items or total weekly per-serving calories, but by buying fewer, but more expensive and slightly more calorically-dense healthy items. Because holiday food purchases are generally driven by tradition versus health concerns, it is possible that participants remained in a state of willful ignorance surrounding the nutrition content of their holiday purchases The finding that after New Year’s purchasing of healthy items increased and relatively less-healthy items remained the same as during the holiday period suggests that even though many people make a New Year’s resolution to eat healthier or lose weight, consumers are making purchasing decisions that only partly support these goals. The sustained increased purchasing seen after New Year’s may be due to the status-quo bias, which suggests that even when people recognize that making a change would be best for them, they still continue to follow their behavioral scripts Because the study did not track a full year of food purchasing, it is impossible to see when purchasing might return to pre-holiday levels. One hypothesis is that as winter ends, and people start gearing up for summer and “bikini season,” food purchasing decreases and establishes the pre-holiday baseline observed in our study from July to November. Although little work has been done elucidating the seasonality of weight gain/loss, a study of advertising in popular women’s magazines suggested that dieting themes were dominant during the summer months In addition to adjusting to a new purchasing status quo, increased purchasing of healthier items, and sustained elevated purchasing of non-starred relatively less healthy items in the post-holiday period may indicate that people were experiencing the impact of a “health halo.” Previous research has illustrated that low-fat claims on foods led to greater consumption of those foods by reducing consumption guilt and increasing perceived serving size Limitations to the study include the fact that the study tracked purchasing for a household, and not actual food consumption. However, previous research has indicated that purchasing records such as receipts provide an accurate record of actual consumption Strengths of the study include its long duration, which allowed examination not only of holiday purchasing, but also of pre- and post-holiday purchasing. Although the study was not able to track all of spring/summer purchasing, it still gives a very interesting picture of food purchasing expenditures over ¾ of the calendar year. The study is also the first to elucidate purchasing differences between “healthy,” and “less-healthy” items over the holiday period, providing interesting insight into how the six-week holiday season impacts not just overall purchasing, but specific categories of items. Results suggest that future research could focus on ways to help consumers decrease their purchasing of less nutrient-dense items once the holiday season is over.Small yearly weight gains of one to two pounds may be a large contributor to the high rate of overweight and obesity in America Several strategies may be useful in controlling the increased food purchasing seen both during the holidays and after the holidays. Pre-determined shopping lists may deter impulsive purchasing at the grocery store, which tends to result in purchases of less nutritious foods


Non-ketotic hyperglycemia is an unusual and rare cause of hemichorea-hemiballismus. Correction of the hyperglycemia usually results in total resolution of the signs and symptoms. We present the case and medical imaging findings of a 66-year-old female who presented with steadily worsening choreiform and ballistic movements of the right upper and lower extremities over a 2-week period. Her serum glucose was greater than 600 mg/dL, and no ketones were present. CT scan and MR demonstrated left basal ganglia abnormalities suggesting hyperglycemia-related hemichorea-hemiballismus syndrome. Restoration of euglycemia led to eventual resolution of all symptoms. Knowledge of this disorder is paramount so as to rule out other causes of intracranial pathology. Hyperglycemia is a rare cause of movement disorders. We present the case and medical imaging findings of a 66-year-old female who presented with worsening choreiform and ballistic movements secondary to non-ketotic hyperglycemia that subsequently improved with insulin treatment.A 66-year-old female presented to the emergency department with steadily worsening purposeless involuntary movements of the upper and lower right extremities over the prior 2 weeks. Two days prior, the patient had visited the emergency department and was found to have a serum glucose greater than 600 mg/dL and no ketonemia. Prior to this she had no known history of diabetes. She briefly received insulin and intravenous fluids but was sent home on metformin and sitagliptin when she declined to use insulin at home. She then returned with worsening involuntary movements.Physical examination was remarkable for choreiform and ballistic movements of the right upper and lower extremities. These movements did not lessen with sleep. Laboratory evaluation again showed severe hyperglycemia without evidence of ketonemia. CT scan and MR with diffusion-weighted imaging showed abnormalities in the left basal ganglia suggesting possible hyperglycemia-related hemichorea-hemiballismus syndrome with no evidence of stroke , 2. AggrAlthough uncommon, hemichorea-hemiballismus syndrome due to non-ketotic hyperglycemia can be the first presenting sign of diabetes mellitus or can occur after many years of poor glycemic control . Women aResolution of symptoms usually occurs rapidly with restoration of euglycemia, although in some instances, similar to our patient, the symptoms may persist for some time . Since t