D14-3-3ε contain significantly fewer germ line cells (pole cells) in their gonads, a phenotype not shared by mutants in the other 14-3-3 gene leo. We show that although D14-3-3ε is enriched within pole cells it is required in mesodermal somatic gonad precursor cells which guide pole cells in their migration through the mesoderm and coalesce with them to form the embryonic gonad. Loss of D14-3-3ε results in defective pole cell migration, reduced pole cell number. We present evidence that D14-3-3ε loss results in reduction or loss of the transcription factor Zfh-1, one of the main regulatory molecules of the pole cell migration, from the somatic gonad precursor cells.Although 14-3-3 proteins participate in multiple biological processes, isoform-specific specialized functions, as well as functional redundancy are emerging with tissue and developmental stage-specificity. Accordingly, the two 14-3-3ε proteins in Drosophila exhibit functional specificity and redundancy. Homozygotes for loss of function alleles of  The 14-3-3 proteins are small dimeric acidic proteins which are highly conserved throughout the eukaryotes in vivo specificity for 14-3-3 isotype-client interactions has been reported. For example, Cdc25C interacts with the 14-3-3ζ, ε and γ isotypes and not with the 14-3-3σ isotype 14-3-3 proteins interact with diverse cellular proteins mostly by binding to phosphorylated Serines or Threonines within particular motifs and have been associated with many cellular processes and functions leonardo, an ortholog of the mammalian 14-3-3ζ and D14-3-3εleonardo gene encodes three nearly identical protein isoforms  through alternative splicing of the primary transcript D14-3-3ε encodes a single protein in vivo. In fact, these two fly 14-3-3 proteins have been reported redundant in particular biological functions in a tissue-specific manner, but not for others Drosophila contains only two 14-3-3 genes, D14-3-3ε null mutant homozygotes is sterility An additional phenotype which characterizes Cellular movements play a crucial role in the development of multicellular organisms and can serve a variety of functions ranging from generation of different tissue layers during gastrulation to organogenesis. These cellular migrations bring into contact different cell types, which is often required for their final differentiation. The migration of primordial germ cells (PGCs) provides a model to study cellular movement and differentiation during development D14-3-3ε null mutants homozygous for the deletion ex4D14-3-3ε or the transposon insertion l(3)j2B10D14-3-3ε were reported sterile 1118w animals j2B10/+D14-3-3ε), heterozygous females laid significantly less eggs (∼18) than controls. The phenotype is a consequence of the mutations in D14-3-3ε as it is readily rescued by trangenes carrying full length D14-3-3ε cDNA under the ubiquitously expressed heat-shock promoter induced twice daily throughout development  and low (L) transgenic protein under both drivers , and obtained multiple lines capable of abrogating the endogenous D14-3-3ε . We usedD14-3-3ε mutant embryos with transgenes encoding LeoI and LeoII, but did not include LeoIII in these experiments because it is specific to adult mushroom body neurons and is more divergent from the other two D14-3-3ε mutant heterozygotes  and huckebein (hkb), engaged in normal mesoderm development per se, such as wunen (wun), necessary for pole cell migration along the basal surface of the midgut Abdominal A (abdA), Abdominal B (abdB), tinman (tin), heartless (htl), fear of intimacy (foi), trithorax (trx), trithoraxgleich (trg) και zinc-finger homeodomain-1 (zfh-1) affect normal pole cell migration and result in defective gonad formation D14-3-3ε homozygotes, namely scattered pole cells across the mesoderm and gonads devoid of, or containing few germ cells. A potential explanation for the phenotypic similarity in these loss-of-function mutants is that interaction among one or more of these proteins and D14-3-3ε is necessary for pole cell migration and/or gonad formation. In fact, an in silico search revealed that Abdominal A, Columbus, Trithorax and Zinc-finger homeodomain-1 proteins contain one or more potential 14-3-3 binding sites  and has a highly specific antibody Normal pole cell migration requires the protein products of several genes such as ng sites . Hence, D14-3-3ε mutant embryos, Zfh-1 levels were markedly reduced in all tissues where it is normally expressed and was completely absent from the mesodermal cells that constitute the somatic component of the gonad . Therefore, we provisionally conclude that D14-3-3ε regulates Zfh-1 protein stability.The absence of Zfh-1 from the presumptive mesodermally derived cells of the gonad may reflect their failure to differentiate as such, or survive altogether in  embryos . TherefoCollectively, our data demonstrate that D14-3-3ε is required within mesodermal cells which guide the pole cells during their migration and eventually participate in formation of the embryonic gonad by regulating Zfh-1 protein levels in the mesodermally derived somatic cells of the gonad.Our results demonstrate that D14-3-3ε is necessary for migration of pole cells through the caudal mesoderm to the forming gonad. Although enriched within pole cells , D14-3-314-3-3 proteins are known to bind transcription factors in a phosphorylation-dependent manner and modulate their subcellular localization and thus their activity Zfh-1 function is required for the development of the caudal visceral mesoderm, the SGPs and somatic gonadal cells per se. However, because its levels appear largely unaffected in other tissues of homozygous D14-3-3ε mutant embryos, we propose that D14-3-3ε is required for Zfh-1 entry into the nucleus and that the transcription factor is unstable in the cytoplasm in its absence. In fact, 14-3-3s are known to bind and prevent entry into the nucleus of members of the Forkhead family of transcription factors We currently do not know whether D14-3-3ε is required for Zfh-1 transcription or translation in silico results suggest that the Trx protein with eight 14-3-3 binding sites of three different consensus variants, is likely to be a bona fide 14-3-3 interacting protein, which has also been proposed to participate in germ cell migration and embryonic gonad formation Interestingly, we never observed complete loss of embryonic gonads in any of the D14-3-3ε null embryos even if they contained but a few pole cells. This is consistent with the evidence for two independent mechanisms that function in parallel and cooperatively for gonad formation. One is Zfh-1-dependent and the other requires Tin, a transcription factor without 14-3-3 binding sites , which mOocytes and embryos lacking D14-3-3ε display gross defects in anterior–posterior polarization, rendering them unviable and offers an explanation for the inability to propagate the viable homozygotes Seen in a wider context, this study focused on mechanisms of directed cellular migration. In addition to that of pole cells, such mechanisms underlie other morphogenetic movements in the embryo that give rise to musculature and nervous system, but also exhibit common features with the migration of metastatic cancer cells 2 at 21–23°C l(3)j2B10D14-3-3ε and ex4D14-3-3ε and the revertant ex5D14-3-3ε were described previously and their genetic background was normalized to our resident Cantonized 1118w. Homozygous embryos were identified from heterozygotes with the aid of the fluorescent balancer chromosome TM3GFP, , expressing ubiquitously the Green Fluorescent Protein b-Tau transgene. We generated and used the following transgenic strains: UAS-ΗD14-3-3ε, UAS-LD14-3-3ε, UAS-leoI and UAS-leoII and the RNA-interference (RNAi)-mediating UAS-IRD14-3-3ε. This transgene expresses an inverted repeat RNA from the D14-3-3ε gene. To generate UAS-IRD14-3-3ε, a 990 bp XbaI fragment of the gene containing the complete cDNA, was subcloned into pUAST 1118w background using standard methods. Of the multiple transformants obtained, the line bearing an insert in the X chromosome was the most effective in silencing the endogenous gene  and “blocked” for 1 hour in BBT-250 , 10% normal goat serum (NGS). Subsequently, they were incubated with primary antibodies in 5% NGS BBT-250 as follows: chicken anti-D14-3-3ε 1∶3000, rabbit anti-vasa 1∶3000 (P. Lasko), mouse anti-GFP 1∶5000 (Molecular Probes), mouse anti-Tau 1∶3000 , and mouse anti-Zfh-1 (Z. C. Lai). Fluorescent secondary antibodies conjugated to Alexa-555, Alexa-488 and Alexa-647  were used at 1∶2000. Images from 2–3 µm optical sections were captured on a Biorad 2100 confocal microscope. The homozygous embryos were indentified based on lack of signal from the GFP-bearing balancer chromosome. Balancer chromosome homozygotes were excluded from analyses due to their abnormal appearance.Embryos were collected on agar-apple juice plates, dechorionated and fixed in 43.2 mM Hepes, 0.96 mM MgSOPole cell counts were carried out using a Biorad 2100 confocal microscope and the numbers refer to pole cell number per gonad. The results were analyzed statistically as detailed above 4, 0.48 mM EGTA, pH 6.9, 1.6% formaldehyde in 59% heptane. Subsequently, they were rinsed with 50% methanol and 50% PBT . The embryos were hydrated in PBT and incubated for 3 minutes in 40 µg/ml proteinase K, rinsed in PBT and fixed again for 25 minutes in PBT and 5% formaldehyde. They were then transferred in hybridization solution for 1–2 hours at 55°C. Finally, 40 µl of single-stranded RNA probe in complete hybridization solution was added for at least 12 hours at 55°C. The 412 retrotransposon gene RNA probe was prepared and used as previously described Embryos were collected on agar and apple juice plates, dechorionated and fixed in 43.2 mM HEPES, 0.96 mM MgSOStaged embryos or entire 2–3 day old flies as indicated were homogenized in 1× Laemli solution . The protein extracts were heated for 10 minutes at 95°C and the denatured proteins were separated by SDS-PAGE electrophoresis. The electrophoretically separated proteins were transferred onto PVDF membranes and the D14-3-3ε and β-Tubulin  proteins were detected with the respective antibodies (at 1∶2000 and 1∶500). The secondary anti-chicken and anti-mouse antibodies were used at 1∶2000 and 1∶4000 respectively. The proteins were detected by chemiluminescence (PIERCE).Figure S1IRepsilon RNAi-mediating transgene in the pUAST vector. B. D14-3-3ε abrogation by two independent transgene insetions visualized in Western blots of late embryo extracts. The two transgenic lines shown here were utilized for all experiments, with all data shown in the figures obtained with the insertion on the X chromosome (UASIRepsilonX), but verified independently with the other on the second (UASIRepsilon2).A. A map representing the salient features if the “head to head” construction utilized to generate the UAS(JPG)Click here for additional data file.Figure S2The distribution of Zfh-1 protein (green) in wild type embryos stages 8 (1), 11 (2) and 14 (3), shown with reference to the nervous system labeled with the 22c10 monoclonal antibody (blue) and the pole cells labeled with anti-Vasa (red). Zfh-1-containing cells of the gonad are indicated by arrows, whereas arrowheads point to cells expressing high levels of the protein either in the nervous system or the mesoderm.(TIF)Click here for additional data file.Figure S3Saggital sections of adult female abdomens of the indicated genotypes stained with anti-D14-3-3ε. The arrow indicates the single apparent oocyte present in the abdomen of the mutant homozygous female. Entire flies were fixed in Carnoy's fixative, paraffinized, sectioned and processed for immunohistochemistry as previously described (TIF)Click here for additional data file.Table S1Rescue of the fertility deficit of ex4D14-3-3ε (2) and J2B10D14-3-3ε homozygotes with conditional expression of heat-shock inducible full length transgenes yielding high (HhsD14-3-3ε) and low (hsD14-3-3εL) levels of the protein. Experimental conditions are as detailed on the table and the number of single crosses that yielded larvae (% Fertile) over the total number of animals crossed (# crossed) per genotype is reported.(DOC)Click here for additional data file.Table S2Results from the in silico search for 14-3-3 binding sites on proteins known to involved in pole cell migration and embryonic gonad formation . The number (# hits) of sequences on each target protein matching the indicated 14-3-3 binding motif is shown, as well as the exact sequence and its location in the protein (sequences).(DOC)Click here for additional data file.


Stationary phase containing the immobilised receptor was used to investigate the interaction between β2-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β2-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×103/M for ephedrine and (3.80±0.02) ×103/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10−4 M. Thermodynamic studies showed that the binding of the two compounds to β2-AR was a spontaneous reaction with exothermal processes. The ΔGθ, ΔHθ and ΔSθ for the interaction between ephedrine and β2-AR were −(22.33±0.04) kJ/mol, −(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were −(21.17±0.02) kJ/mol, −(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β2-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.A new oriented method using a diazonium salt reaction was developed for linking  The interactions between a protein and a drug are of growing interest due to the role in elucidating protein functions, explaining drug action mechanisms and discovering novel drug candidates Currently, a wide range of methods have been developed for investigating the drug-protein interaction, including ultrafiltration Synthesising an assay for attaching a protein to a solid matrix is a key factor for accomplishing an HPAC method. Physical absorption and random immobilised methods are commonly reported ways to synthesis an affinity stationary phase 2-adrenoceptor (β2-AR), one of the members of the GPCRs, is a crucial target of many drugs for fighting obesity and ailments of the heart and respiratory system β2-AR in vivo although they have similar structures and the same acting mechanism. One reason for this may be the different binding affinities of the two ligands to β2-AR. This work is firstly designed to establish an oriented immobilised method for fixing his-tagged fusion β2-AR on a solid matrix to synthesise an HPAC stationary phase. Further work is performed to investigate the binding behaviours of ephedrine and pseudoephedrine to β2-AR by HPAC methods.Betaβ2-AR was determined by zonal elution. The theory assumes that a known concentration of a competing agent (I) continuously passes through a column containing an immobilised ligand (L) while the injecting volume of an analyte (A) is small enough to be neglected compared with the concentration of I in the mobile phase. Assuming I and A have direct competition at a single binding site on L and the interactions have association/dissociation kinetics, Eq. (1) can be used to calculate the binding of A and I to L k' is the overall capacity factors of A ascribed to all the types of sites. In this case, the equation assumes the capacity factors of A produced by the individual sites are additive, and an independent estimate of the capacity factors due to the second type of site can be obtained, allowing for the capacity factor attributed to the first type of site 1 (Site 1) to be measured by k1' = k'-X.The binding of ephedrine and pseudoephedrine to the oriented immobilised When the column is equilibrated with an eluent at concentration [A] and [I] equal to zero, and the retention of compound A is injected as a perturbation, Eq. (2) is used to determine the binding of A to L:L and KA. For two types of binding sites, a second term is added to the k' expressions.Assuming a single type of binding site, a linear expression can be obtained by plotting 1/sqrt (k') as a function of [A] to determine mβ2-AR and ephedrine plus pseudoephedrine was described by the van't Hoff equation, listed as Eq. (3) and (4).The thermodynamic interaction between θ<0, ΔHθ>0 and ΔSθ>0, hydrophobic interaction is the main interaction force during the binding process of a drug to protein. If ΔGθ<0, ΔHθ<0 and ΔSθ>0, electrostatic interactions mainly contribute to the interaction. If ΔGθ<0, ΔHθ<0 and ΔSθ<0, Van der Waals forces or hydrogen bonds are the major forces of the binding.In these equations, R is the ideal gas constant (8.31 J/mol/K), and T is the temperature in Kelvin. According to the semi-empirical rules summarised by Ross et al. R,2S-ephedrine, 171241) and pseudoephedrine  were obtained from the National Institutes for Food and Drug Control . Fatty acid and globulin free bovine serum albumin  was obtained as lyophilized powder from Sigma-Aldrich . The Quaternary Sepharose Fast Flow pre-packed column (16×25 mm), Ni2+ Sepharose 6 Fast Flow (10×58 mm) and Sephadex G25 pre-packed column (10×100 mm) were purchased from GE Healthcare Life Sciences . Macroporous silica gel  was from Fuji Silysia Chemical Company Limited .Ephedrine  and a diode array detector . The data were collected and processed by a Chemstation 5.2 software installation.β2-AR (Refseq:MN_ 001128436) was cloned in our previous work E.coli BL21 (DE3) with an N-terminal His-tag. The transformed E. coli strain was grown overnight at 37 °C in 50 mL Luria-Bertani (LB) medium containing 100 µg/L penicillin. When the value of OD600 reached 0.4–0.6, isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 2.0 mM, and the culture was incubated at 30°C for 6.0 h. The cells containing His-tagged β2-AR were separated from the culture media by centrifugation at 6000×g for 30 min at 4.0 °C, and the supernatant was discarded. Falcon tubes containing the cell pellets were placed into a dry ice/ethanol bath for 3–5 min and were transferred to cold water for 60 s to melt the ice along the walls of the tubes. The pellet was broken free by a sterile plastic spatula and suspended in 200 mL neutral lysis buffer  in a 500-mL flask. Lysozyme (0.5 mg/mL) was added to the buffer and incubated for 2.0 h on ice with agitation to digest the cell wall. The E. Coli cells were lysed using 20 mL of a 10× Mg/Mn salt solution (100 mM MgCl2 and 10 mM MnCl2). DNA was removed by DNase (10 µg/mL) with incubation for 30 min at RT. The resulting E. coli lysate (approximately 260 mL) was placed into a dialysis bag (10 kD molecular weight cut-off) in 4 L buffer . The dialysis buffer was changed twice over a 16–24 h period. At the end of the dialysis, the cell debris was removed as a pellet by centrifugation at 10000× g for 30 min at 10 °C.The full gene of Sus scrofa β2-AR supernatant was applied to a Ni2+ Sepharose 6 Fast Flow column that had been equilibrated with dialysis buffer . The column was eluted with an increasing salt gradient of up to 500 mM imidazole in buffer A  at a speed of 1.0 mL/min. The fraction of interest was collected by eluting the column using 50% buffer B . Further purification of the collected fraction was performed on the Quaternary Sepharose Fast Flow column equilibrated with 18% buffer C . The β2-AR solution was collected by gradient elution from the column using buffer C ranging from 18%–40% with a flow rate of 5.0 mL/min. The solution was finally desalted by the Sephadex G25 column.Dialysed and centrifuged recombinant β2-AR was oriented immobilised on macroporous silica gel using a diazo coupling method  through stirring for 48 h at 60 °C. The resulting gel was filtrated by a sintered filter funnel and washed three times with methanol. The remnant methanol was removed by drying the gel in a vacuum oven at 60 °C for 12 h. The double bond in the produced gel was reduced to a single bond through reaction between the gel and 100 mL 8.6 µg/mL NaBH4 at room temperature for 12 h 2 ethanol solution  and reacting for 6 h at 90 °C. The suspension was cooled to 0 °C and alkalified with 50 mL 5% NaHCO3, followed by a sequential cleaning with saturated NaCl and distilled water 2 solution (20 mM) was added dropwise until reaching the turning point of potassium iodide starch paper changing to blue β2-AR was immobilised on the resulting gel by immediately adding the receptor  solution into the suspension and reacting for 2 h at 4 °C. Finally, the gel was washed three times with phosphate buffer .The g method . In detaβ2-AR was prepared by packing the stationary phase into a stainless steel column (2.1 mm×5.0 cm) using the slurry method at a pressure of 4.0×107 Pa. Phosphate buffer  was used as the slurry solution. The control column containing exposed silica gel and attached histidine was prepared using the same synthesis method as that of the β2-AR column.The column containing the oriented immobilised k') was calculated by detecting the retention time in triplicate for each injected sample using the equation k' = (tR−t0)/t0. When the injection solute was pseudoephedrine, Eq. (1) was used to plot the curve of 1/(k'-X) and [A], whereas Eq. (2) was used to achieve the plot between The mobile phases were prepared by adding 0 to 40 µM of ephedrine hydrochloride as the competing agent to 20 mM phosphate buffer (pH 7.4). All of the mobile phases were filtrated with 0.45 µm Millipore HV filters and degassed by ultrasonication before HPAC analysis. Working solutions of ephedrine and pseudoephedrine were prepared by dissolving the drugs in phosphate buffer  to a concentration of 1.0 mM. The detection wavelength was 220 nm with an injection of 5.0 µL for each drug. The flow rate was 0.2 mL/min for all of the analyses. Under the proposed chromatographic conditions, the void time was 1.6 min using sodium nitrite as the injection solute. The capacity factor  and (4) were used to calculate the free energy, enthalpy and entropy during the binding of the two drugs to the receptor. The semi-empirical rules by Ross et al. β2-AR.The thermodynamic investigation of ephedrine and pseudoephedrine on oriented immobilised β2-AR (Accession Number: AF000134) was obtained from Genbank. The query sequence of the target receptor was searched find out the related protein structure to be used as a template by the BLAST  program β2-AR according to the procedures illustrated by Discovery Studio 2.5 . Structural evaluation of the receptor was performed using two programs named PROCHECK and Verify 3D. Once the three dimensional model was generated, energy minimisation was carried out for the receptor before dockings. Structure of ephedrine and pseudoephedrine were constructed by DS 2.5. CDOCKER program, implemented in the software platform of DS, was used for the docking study. The pose cluster radius was set to 0.5 with a value of top hits as 10.The sequence of Sus scrofa β2-AR solution was determined by bicinchoninic acid (BCA). Using BSA as a standard, the calibration curve was plotted to assay the total protein in the solution. The linear regression equation was y = 0.0002x+0.2702, with a correlation coefficient of 0.9957. The concentration of the total protein in the β2-AR solution was (61.67±4.48) µg/mL. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the molecular weight of the protein in the desalted β2-AR solution  of the retention times on those days were 2.43%, 1.48% and 3.13%, indicating that the retention times of the drugs remained stable over the 30 days. The peak profiles were also stable over the 30 days. In conclusion, the β2-AR column had good stability for at least 30 days. The specific drugs of β2-AR give retention times approximate to the void time (β2-AR column is a consequence of the β2-AR presentation but not a nonspecific interaction with the silica gel.The stability of oid time  on the cβ2-AR column are summarised in 3x+(0.43±0.01) and a correlation coefficient of 0.9962 over the seven data points, providing a proof of one type of binding site for ephedrine binding to β2-AR. Based on which ephedrine is believed to have only one type of binding site on the β2-AR column. By dividing the intercept by the slope of 3/M (β2-AR was estimated to be (8.92±0.06) ×10−4 M.Self-competitive studies were performed where ephedrine served as both analyte and competing agent at 37 °C . In thisat 37 °C , a good e of 3/M . Throughk'-X) of pseudoephedrine and the concentrations of ephedrine, as shown in 3/M, as shown in −4 M based on the value of ephedrine calculated by self-competitive studies. Using the binding sites and the intercept of β2-AR was determined to be (3.80±0.02) ×103/M, as shown in A direct competitive study was conducted using ephedrine as a mobile phase additive and pseudoephedrine as an injection solute at 37 °C . As showβ2-AR during the interactions with the receptor. Although the association constants of the two drugs have the same order of magnitude, the exact values are varied due the high repeatability and precision of the HPAC assay, on which the spatial position of the amino groups are believed to contribute greatly to the binding of the two drugs and β2-AR. This explanation can be confirmed by the research of Vansal et al. 1S,2R-ephedrine has a maximal response relative to isoproterenol (EC50) of 72 µM, whereas 1S,2S-pseudoephedrine gives an EC50 value of 309 µM on β2-AR. Taken together, it is concluded that the varied affinity of the two drugs to β2-AR may be one of the key factors for their different pharmacological potency through the signal pathway involving β2-AR.The association constant of ephedrine is greater than that of pseudoephedrine even though the structures are similar. The difference between the structures of the two drugs is the configuration of the amino group, which produces the different spatial selectivity of the two drugs to the binding sites on β2-AR were performed at 10, 20, 30, 37 and 45 °C. Zonal elution was used to calculate the association constants of ephedrine and pseudoephedrine at those temperatures. According to eq. (3), linear relationships were found by plotting the lnKA of ephedrine and pseudoephedrine against 1000/T, with correlation coefficients of 0.9716 and 0.9771  kJ/mol and 50.94±0.31 J/mol·K. By eq. (4), the free energy during the binding was −(22.33±0.04) kJ/mol  kJ/mol , indicatθ, ΔSθ and ΔGθ for the binding of pseudoephedrine to β2-AR were −(7.48±0.56) kJ/mol, 44.13±0.01 J/mol·K and −(21.17±0.02) kJ/mol  kJ/mol . Similarβ2-AR decreased with increasing temperature, whereas the binding sites presented a positive response to an increasing temperature. When the temperature changed from 10 °C to 45 °C, the association constant of the two drugs decreased from 7.17×103/M to 5.18×103/M and from 4.93×103/M to 3.36×103/M, respectively. Meanwhile, the binding sites increased from 8.07×10−4 M to 9.48×10−4 M during the interaction, and the capacity factors of the two drugs on the β2-AR column diminished from 5.40 to 4.63 and from 6.36 to 5.60 when the concentrations were 4.0 µM. The association constants decreased 1.4 and 1.5 fold, whereas the binding sites increased 1.2 fold. At the same time, the capacity factor diminished 1.2 fold. The retention behaviour of a solute on an affinity column ascribed to the synergistic contribution of the association constant and the binding sites. For ephedrine and pseudoephedrine binding to β2-AR, the decreasing retention is mainly attributed to the diminishing association constants. The reason for this phenomenon may be related to the conformation change induced by the temperature.The association constants of ephedrine and pseudoephedrine binding to β2-AR has three types of binding sites. One is the salt bridge between the amine group of the ligand and the carboxylate side chain of Asp113 in the third hydrophobic domain. The other two types of binding interactions proceed through hydrogen bonds between the hydroxyl side chain of Ser 204 and the meta-hydroxyl group of the ligand or between the hydroxyl side chain of Ser 207 and the para-hydroxyl group of the ligand β2-AR. In this presentation, both ephedrine and pseudoephedrine gave a thermodynamic profile of ΔGθ<0, ΔHθ<0 and ΔSθ>0. This result confirmed that electrostatic interaction is the main force during the bindings. Thermodynamic studies in this work illustrated the same point to aforementioned report, which paves the way to the application of the thermodynamic method in revealing the binding mechanism of the interaction between ligands and the receptor.In the literature, it is reported that β2-AR; 1R,2S-ephedrine is the most potent. In this case, the affinity of 1R,2S-ephedrine binding to β2-AR should be stronger than the other isomers. In this work, the association constants of 1R,2S-ephedrine and 1S,2S-pseudoephedrine were calculated as (5.94±0.05)×103 M−1 and (3.80±0.02) ×103 M−1, respectively. This result is notable ascribed to the high agreement with aforementioned report. Waldeck et al 2 of about 5.95, which corresponds an dissociation constant of 11200 nM for the drug binding to β2-AR. Subsequent studies by Samama et al R,2S-ephedrine binding to wild-type β2-AR and a value of 5100±1300 nM to 2β-ARGsαS. Based on the association constant in this work, the dissociation constant for 1R,2S-ephedrine to the receptor was 168000 nM. Since the receptors used in these work are cloned with various gene sequence, it is expected that the exact values of dissociation constant for the drug differ from each other.As noted in the literature β2-AR-Fab complex) through homology searches in PDB by comparing and aligning the target sequence with that in PDB. The sequence identity was 50%. Followed by structural-based alignment between query and target receptor, C-alpha atoms were selected for the model building ascribed to the highest identity  with its own amino acid sequence (1D).One template had been identified  proved to be the exact site for the two isomers binding to the receptor. Meanwhile, no hydrogen bond was found during this interaction. It is widely known that Asp is an acidic amino acid with an isoelectric point of 2.98. In this case, Asp possesses negative charge in a solution with pH of 7.4. While in the same environment, both ephedrine and pseudoephedrine exist as positive ions. Based on these points, we concluded that the binding of the two drugs to β2-AR is driven by electrostatic interaction. This result is in good agreement with the results from thermodynamic investigation in this work and the report by Strader et al Site directed docking results showed that only one type of binding domain is engaged in the interaction between the two drugs and β2-AR . Asp 79 β2-AR onto the surface of macroporous silica gel. Compared with random immobilised assays, the current method proves to have the advantages of better maintaining the bioactive characteristics of β2-AR due to the greater amount of the immobilised protein on the gel and the increased binding sites on the surface of the receptor for recognising its ligands. The stationary phase prepared by the oriented method was also applied in exploring the interaction between β2-AR and two drugs, ephedrine and pseudoephedrine. The varied association constants of the two drugs are believed to be the key reason for their different pharmacological potency. The binding of the two drugs to β2-AR was mainly driven by the electrostatic interaction between the amino group in the structure of the drugs and the carboxylate side chain of Asp113 in the third hydrophobic domain of the receptor. The oriented method is potential in preparing stationary phases containing other receptors for HPAC to study drug-receptor interaction.A diazo coupling method was developed for oriented immobilising


In vivo imaging of androgen-mediated luciferase activity revealed several strongly expressing tissues in the male mouse as expected and also in certain female tissues. In males the testes, prostate, seminal vesicles and bone marrow all showed high AR activity. In females, strong activity was seen in the ovaries, uterus, omentum tissue and mammary glands. In both sexes AR expression and activity was also found in salivary glands, the eye (and associated glands), adipose tissue, spleen and, notably, regions of the brain. Luciferase protein expression was found in the same cell layers as androgen receptor expression. Additionally, mouse AR expression and activity correlated well with AR expression in human tissues. The anti-androgen bicalutamide reduced luciferase signal in all tissues. Our model demonstrates that androgens can act in these tissues directly via AR, rather than exclusively via androgen aromatisation to estrogens and activation of the estrogen receptor. Additionally, it visually demonstrates the fundamental importance of AR signalling outside the normal role in the reproductive organs. This model represents an important tool for physiological and developmental analysis of androgen signalling, and for characterization of known and novel androgenic or antiandrogenic compounds.Androgens, required for normal development and fertility of males and females, have vital roles in the reproductive tract, brain, cardiovascular system, smooth muscle and bone. Androgens function via the androgen receptor (AR), a ligand-dependent transcription factor. To assay and localise AR activity in vivo we generated the transgenic “ARE-Luc” mouse, expressing a luciferase reporter gene under the control of activated endogenous AR.  Androgens are responsible for masculinization of male body tissues, e.g. development of the internal and external genitalia, and in both sexes, the androgen surge at puberty drives development of secondary sexual characteristics, e.g. increased skeletal muscle bulk, voice deepening, and axillary and pubic hair growth . The priNormal male physiological development requires a controlled pattern of gene expression from fertilisation to gestation, and involves expression of the SRY transcription factor (Sex-determining Region Y) from the Y chromosome to initiate phenotypic male sexual differentiation and testis development –4. TheseIn the female, androgens are produced by the adrenal, ovarian and adipose tissues but circulate at a lower level than in the male. A key function of androgens in females is aromatisation to estrogens, but the AR is expressed in several female tissues, e.g. mammary gland, uterus, vulvar epithelium, vaginal mucosa and in ovarian follicles where it maintains follicle health during ovulation –12. In tin vivo imaging in live anaesthetized animals. Luciferase (Luc2P), being non-mammalian, gives very low background signal and has been engineered to contain degradation signals (hPEST) for rapid protein turnover, allowing real-time signal responses to changes in gene activation. Furthermore, consecutive longitudinal images can be taken using the same animal.AR function and localization may be assessed by immunohistochemistry or binding of radioactive ligands , but sucTGTTCT with a three nucleotide spacer - and therefore could not be used due to lack of discrimination [SC1.2) genes [SC1.2-ARE upstream of a minimal thymidine kinase promoter, containing a TATA box for RNA polymerase binding. We excluded adjacent endogenous DNA sequences in order to rule out binding of other transcription factors that could render the response tissue-specific, as seen for prostate specific antigen (PSA) gene promoter-reporter fusions [The consensus response element for the AR is identical to that of the closely related GR, PR and MR - a hexameric bipartite binding site, comprising of an inverted repeat of mination . However2) genes –22. Thes2) genes ,24. Our  fusions . AdditioSC1.2-ARE was used as, of several tested, it showed the greatest androgen induction and specificity for AR without activation by other transfected or endogenous steroid receptors, within the physiological range of steroid hormones  [luc2P) gene expression (+), MCF-7 cells (ERα+/PR+/AR+) and Du145 cells (AR-/GR+), and treated with the cognate ligands. LNCaP/Luc cells showed good induction of the reporter when treated with mibolerone, and dose-dependent response to a broad range of different androgens, with no significant response to estrogen (E2), progesterone (P4) or dexamethasone (Dex) , but upon transfection with an AR-expressing vector, an androgen-induced response was seen, confirming the reporter was functional . In summary, this luciferase reporter responds specifically to AR activation in a range of cell lines.Our aim was to create a reporter construct that responds specifically to active AR, with minimal crosstalk with other steroids/steroid receptors. Activity was tested initially by transient transfection into steroid receptor negative COS-1 cells alongside vectors expressing human AR, GR, ERα, and PR, and also in cells expressing one or two endogenous receptors. The al data) . The SC1pression 1A. Subspression 1B & C -ne (Dex) 1D. MCF-(2 or P4 1E. In tpression 1C & E. pression 1F. No ls.c.)) and imaged to detect bioluminescence. Male ARE-Luc mice showed significant bioluminescence/photon emission observed in body areas corresponding to the reproductive organs .The S.C. 1.2 ARE luciferase vector was used to generate transgenic ARE-Luc mice . First, we investigated AR transcriptional activity as measured by luciferase activity in adult male and female mice of approximately 6-10 weeks of age. ARE-Luc mice, along with wild type controls, were injected with luciferin substrate (150 mg/kg subcutaneous (i.p.) injection showed faster kinetics of luciferin light emission, reaching steady state maximum around 5 minutes, compared to 10 minutes for the s.c. route .Luciferase activity  was measured from lysates of several different tissues from male and female ARE-Luc mice aged 6-10 weeks. In male mice, the tissues exhibiting highest luciferase activity/mg protein content were the brain and the testes. The intestines, prostate, spleen, and eye showed a modest luciferase activity and of the tissues measured the lowest activity in males was seen in the stomach, heart and lung . Again, the lowest activity was seen in the heart and lung, as well as liver.In female mice, the strongest luciferase activity/mg protein signal was seen in the intestines (omentum included), skin, brain, bladder and spleen. Modest luciferase activity was seen in the uterus, bone, gonadal fat pads, and ovaries  – the brain signal being masked by the skull until excised. Moderate luciferase activity was also seen in the spleen, areas of the brain, bone/bone marrow and omentum attached to the intestine. Weaker but detectable AR-mediated luciferase activity was also seen in the liver, perigonadal fat deposits and specific regions of the heart .Tissues were dissected out and imaged ex vivo imaging of tissues revealed strong luciferase signal in the ovaries and the uterine horn, spleen and large tracts of the omentum attached to the intestine . Moderate activity was seen in the brain, bone and perigonadal and inguinal fat deposits.In female mice, In the brain of both sexes strong AR activity was detected in specific regions of the brain upon coronal dissection, including the cerebral cortex, thalamus and pituitary gland 3B& C.Primary ARE-Luc mouse cells were cultured from various tissues, and their steroid receptor and luciferase reporter gene expression were analysed further. Pre-adipocyte cells expressing AR, GR, ERα/β and PR  were treated with 50mg/kg/day of the antiandrogen bicalutamide, in DMSO: propylene glycol (50:50), for 48hrs. Mice were imaged at 24 and 48 hours and compared to vehicle treated controls. Bicalutamide treatment reduced luciferase expression in the gonadal region of the ARE-Luc mice at 24hrs and significantly so at 48hrs 4A& B. EEx vivo imaging of the organs confirmed that bioluminescence had dropped significantly in the intestine, ovary, and uterus  and, in rodents, probasin (PB). There is a need for models to study steroid receptor activity in physiological settings and the first mouse model allowing visualisation of endogenous steroid receptor activity was developed for the ER by Maggi and co-workers [in vitro to minimise cross talk with other steroid receptors, with specific emphasis on optimising the ARE type to exclude GR binding and activation. In a previous model, the AR activity indicator (ARAI) mouse, steroid receptor crosstalk is avoided by expressing an engineered AR, in which the native DNA-binding domain (DBD) is replaced by the Gal4-DBD, and a Gal4-responsive luciferase reporter is used [AR drives transcriptional networks in varied tissue types and is responsible for the development, maintenance and function of several tissues in the body. However, relatively few androgen-regulated genes have been well characterised, most driving functions of the prostate e.g. those encoding kallikrein enzymes . The use of a highly active luciferase (luc2P) coupled to degradation signals ensured sufficient activity for rapid and robust detection, with opportunity for rapid flux in response to changes in hormone levels. We acknowledge that a plethora of transcription factors and co-activator proteins may act in concert with the AR to fine-tune activation but no one model could reproduce this level of complexity, and therefore our model reports and assays the activity of transcriptionally active AR.The single se to GR ,26. Furtse to GR ,33,34. Tse to GR , we usedAR gene and the Hprt locus into which the luciferase reporter is inserted.Luciferase activity was seen only in those tissues expressing AR, and the reporter was silent in tissues lacking AR, indicating no constitutive activation or silencing due to positional integration effects although it is impossible to completely rule out “false negative” results due to the unlikely scenario of an as yet undetected tissue specificity of the reporter minimised by use of an artificial promoter containing a minimal ARE. Importantly, a strong reduction in luciferase signal was seen when AR was inhibited by bicalutamide – even though androgens were still circulating in the intact animal – demonstrating that the luciferase activity in these mice is indeed mediated by the AR. Although the signal in the majority of luciferase-expressing organs became almost undetectable in both sexes following anti-androgen treatment, some residual signal was seen in the most strongly positive organs e.g. the testes. This is likely due to incomplete inhibition by the competitive anti-androgen bicalutamide, as testes are known to have the highest tissue testosterone concentrations . A more Ex vivo tissue examination showed exquisitely specific localisation of luciferase activity, for example in the gonads and brain (indicating the ability of luciferin substrate to cross the blood brain barrier) in both sexes, which correlated well with AR protein expression within tissues . Within the brain, in both sexes, AR activity was highly localised to specific regions, detailed study of which will be the focus of future research. Several reports have suggested that within the brain testosterone activity is mediated by ER after aromatisation of testosterone, especially during foetal development. Certain areas of the brain are more susceptible to aromatisation and these areas adhere well to the theory, e.g. the sexually dimorphic nucleus of the preoptic area and the anteroventral periventricular nucleus [ex vivo tissue. Our model supports the hypothesis that aromatisation is not the only mechanism of androgen activity in the brain, and that AR is transcriptionally active within certain brain regions. It is not possible to state, at this stage, which androgen is mediating the effect, but future crosses of ARE-Luc mice with a 5α-reductase knockout mouse should clarify whether testosterone (aromatisable) or DHT (non-aromatisable) is responsible. nucleus . However nucleus –41. Comp nucleus . This in nucleus . We obseAnother regions showing high luciferase activity was the gastric tract, notably the surrounding intestinal omentum. The omentum represents a complex tissue that has multiple roles including fat storage ,45 and iThis study shows that the AR is indeed very active outside its well-defined role in reproductive tissues. We have observed AR expression and activity in a variety of tissues in which the role of steroid hormones are poorly understood. The retina and choroid cells of the eye showed modest AR expression and luciferase activity, as did the lacrimal glands – tissues known to express sex steroid receptors ,57. StroIn summary, we report a highly specific model for imaging endogenous AR activity in mice that overcomes previous limitations including crosstalk with other receptors, random integration, non-endogenous AR and tissue-specific expression. Our model shows that androgens have a wide range of target tissues, both in males and females, outside the most widely-studied reproductive tract. The data shown here represent a snapshot of the AR activity within adult mice (6-10 weeks), thus exclude any dramatic changes predicted during development and puberty, as well as effects of hormonal fluctuations during the female estrous cycle and less dramatic changes during aging. As well as such developmental studies, future uses of this model include evaluation and detection androgenic xenobiotics and selective androgen receptor modulators (SARMs) with the aim of producing tissue-selective antagonistic or agonistic effects in diseases such as breast and prostate cancer, polycystic ovarian syndrome and hyper- and hypogonadism .All mouse procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 under Home Office license.tk-Luc. DNA oligomers  coding for the SC1.2 ARE sequence (GGCTCTttcAGTTCT) were ligated into the Sac1/Xba1 site of p-tk-Luc to generate p-tk-Luc-ARE  promoter from the herpes simplex virus was cloned into the Sac1/Xho1 site of pGL4.18 (Promega) to generate p--Luc-ARE 1A. ThisS.C. 1.2 ARE-Luc knock-in (ARE-Luc) mouse was generated in collaboration with GenOway . The p-tk-Luc-ARE was cloned into the CBE1-HR Hypoxanthine phosphoribosyltransferase gene (Hprt) conditional targeting vector, containing the Hprt promoter and exon 1. This gene-targeting construct was introduced into male mouse embryonic stem (E14Tg2a ES) cells of the 129P2/OlaHsd strain in which the 35kb of the Hprt gene, encompassing the 5’ UTR up to intron 2, is deleted. The targeting construct then integrated into the X-chromosome by homologous recombination . Lysate (20µl) was mixed with luciferin substrate (20µl) and light emission measured using the Steadylite luciferase assay kit  in a Topcount luminometer . Light expression was then normalised to protein content as measured by Bradford Assay.2 carrier, Abbott Animal Health UK) were injected i.p. or s.c. with D-luciferin  at 150 mg/kg, 10 min before imaging. Light emission from luciferase was detected by the IVIS Imaging System 100 series (Xenogen Corporation), and overlaid as a pseudocolour image with reference scale, upon a greyscale optical image.Anaesthetized mice Click here for additional data file.Figure S2Luciferase kinetics depends on route of injection. Comparison of the time kinetics of bioluminescent signal from ARE-Luc mice injected with 150mg/kg luciferin substrate via either the i.p. or s.c. route.(TIF)Click here for additional data file.Figure S3Analysis of luciferase activity and expression in mouse ARE-Luc primary cells in culture.A, Relative mRNA expression of steroid hormone receptors in gonadal pre-adipocytes. B, Luciferase activity in gonadal adipose cells grown under conditions of hormone starvation for 72 hours and treated with 10nM hormone for 24 hours. C, Luciferase expression in various primary cell types, hormone-starved for 72 hours and treated with mibolerone (10nM) or equivalent volume vehicle (Eth) for 24 hours.(TIF)Click here for additional data file.Methods S1Further details are provided for Cell culture, Primary Cell Culture, Genomic DNA extraction and PCR, RNA extraction, RT-PCR and Southern Blotting in the Supporting Information for this paper.(DOCX)Click here for additional data file.


This paper reviews the move from virtual reality exposure-based therapy to augmented reality exposure-based therapy (ARET). Unlike virtual reality (VR), which entails a complete virtual environment (VE), augmented reality (AR) limits itself to producing certain virtual elements to then merge them into the view of the physical world. Although, the general public may only have become aware of AR in the last few years, AR type applications have been around since beginning of the twentieth century. Since, then, technological developments have enabled an ever increasing level of seamless integration of virtual and physical elements into one view. Like VR, AR allows the exposure to stimuli which, due to various reasons, may not be suitable for real-life scenarios. As such, AR has proven itself to be a medium through which individuals suffering from specific phobia can be exposed “safely” to the object(s) of their fear, without the costs associated with programing complete VEs. Thus, ARET can offer an efficacious alternative to some less advantageous exposure-based therapies. Above and beyond presenting what has been accomplished in ARET, this paper covers some less well-known aspects of the history of AR, raises some ARET related issues, and proposes potential avenues to be followed. These include the type of measures to be used to qualify the user’s experience in an augmented reality environment, the exclusion of certain AR-type functionalities from the definition of AR, as well as the potential use of ARET to treat non-small animal phobias, such as social phobia. According to Moore’s law, the number of transistors on integrated circuits doubles approximately every 2 years Moore, . This grThe objective of this paper is twofold. First, it reviews the move from virtual reality (VR) systems to augmented reality (AR) systems in the treatment of phobias. Second, it highlights four issues relating to AR: (a) qualifying an AR experience necessitates a set of AR specific instruments [not necessarily those used to qualify a virtual environment (VE) experience]; (b) historically, AR applications have been around a long time before the term “AR” was assigned to the concept; (c) presently, certain AR-type functionalities are excluded from the definition of AR; and (d) the use of augmented reality exposure-based therapy (ARET) has advantages over virtual reality exposure-based therapy (VRET), but these advantages could be exploited beyond the treatment of small animal phobia.in imago and in vivo exposure-based therapies, are presented. Next, VRET, the “direct ancestor” to ARET, is introduced; its documented successes, as well some of as its advantages over traditional exposure-based methods are presented. After this overview, the focus shifts toward AR, including how it distinguishes itself from VR, some of its advantages over VR, and what criteria must be met to consider a functionality as AR. At this point, the instruments presently used to measure an AR user’s experience are discussed, and some concepts to be measured in AR are proposed. The next section addresses the history of AR. This is accomplished in two parts. While the first covers, some of the major events that occurred after the coining of the phrase “augmented reality,” the second addresses the period going back to the roots of AR, a time-frame less covered by previous publications. From this historic account will emerge an issue relating to the definition of AR: the present one leaves certain AR-type functionalities nameless. A solution will be proposed to close this semantic gap. Next, the AR enabling technologies, and some of the technical challenges faced by the developers are put forward. A variety of AR applications are listed. In particular, publications pertaining to the use of ARET are reviewed; these eight studies either test the efficacy of ARET protocols, compare ARET protocols to other types of exposure-based protocols, compare ARET technologies, or simply quantify users’ experiences in an ARET environment. Finally, the last discussion point addresses the limited use of ARET in the treatment of phobias, other than small animal phobias. One of the plausible reasons behind this self-imposed restriction, a possible way to break free of it, as well as the potentially resulting opportunity to expand ARET to the treatment of social phobia are discussed. To close, a conclusion reiterates the major points of the paper.To this aim, the article first addresses some of the evolutions that have led to the use of AR in the treatment of specific phobias. To establish the framework of AR, it is useful to distinguish it from VR. Thus, the paper presents some definitions relating to the technology , some of the concepts commonly used to quantify and qualify a user’s experience of virtual stimuli , as well as some of the non-mental health applications of VR. After having covered these basics, the focus shifts toward mental health. More specifically, the implications of suffering from a phobia and two of the possible  treatments, The exact definition of the word “virtuality” depends on the context of its use. However, in the domain of VEs, Theodore Nelson’s definition is pertinent; he defines the virtuality of a thing as the “seeming” of that thing . It is comprised of visual stimuli projected on a surface  and, generally, acoustic stimuli produced by an electronic device . Further to these, a VE may also expose the user to haptic (contact), olfactory, or even gustatory stimuli . However, each of these techniques has major drawbacks: while a patient may be unwilling to face the actual threat in vivo, it might prove too difficult for a patient to mentally visualize the anxiety inducing threat. In fact, it has been reported that when patients find out that the therapy entails facing the threat, about 25% of them either refuse the therapy or terminate it  is not “transported” to a different location, and consequently, there is no immersion In an ARE, measures of realism  and reality (level to which the user experiences the hybrid environment as authentic) are still pertinent. However, this may not be the case for presence. If Heeter’s  conceptuOn the other hand, a measure addressing the alignment of real and virtual elements could contribute to an overall assessment of the quality of a user’s experience in an ARE; this could be in the form of a measure of co-existence between the virtual and the non-virtual elements. Further co-existence measures could assist in qualifying the experience of an ARE. These could include co-existence measures between the user and virtual elements, as well as between the user and non-virtual elements .The term “augmented reality” was introduced in 1990 by Tom Caudell while working on Boeing’s Computer Services’ Adaptive Neural Systems Research and Development project in Seattle  gunsight. The concept behind the gunsight was published by Grubb : “the siThe difficulty of categorizing this type of application as AR persists for the follow-on systems. As it was known that the trajectory of the bullets was influenced by the shooting aircraft’s flight parameters, the newer generation of gunsights started to take these into account when displaying the aiming reticle. This type of application does, to a certain extent, take real world parameters into consideration, but it still does not align real and virtual objects. Thus, it does not quite meet Azuma and colleagues’ definition of AR.The first operational system that did meet all three of Azuma and colleagues’ premises of AR seems to have been the AI Mk VIII Projector System (earlier variants had been successfully tested but never entered service). As the name suggests, the radar picture of the AI Mk VIII radar picture was projected onto the pilot’s windscreen, thereby superimposing the virtual cue onto the real world position of the target aircraft , the helmet mounted sight (HMS), and the helmet mounted display (HMD). The latter, invented in the 1960s by Professor Ivan Sutherland and his graduate student Bob Sproull, can be considered as one of the major technological breakthrough that furthered the development of AR . As AR functionalities may co-exist with such AR-like functionalities , it could be useful to find a term that describes functionalities that don’t register real and virtual objects with each other. Using Azuma and colleagues’ widely accepted definition of AR as an anchor, the authors of the present paper propose the term “non-registered augmented reality” (NRAR) to describe functionalities that: (1) combine real and virtual objects in a real environment; and (2) run interactively, and in real time; but (3) don’t register  real and virtual objects with each other.While the exact configurations of AR systems vary, their common elements include: (a) a means of providing a geospatial datum to the synthetic elements; (b) a surface to project the environment to the user; (c) sufficient processing power to generate the 3-D synthetic elements and merge them with the pointing device’s input; and (d) adequate graphics power to animate the scene on the display . However, as AR requires that only a few virtual elements be designed, the cost of producing the environment is reduced. Furthermore, unlike VR, AR does not “extract” the user from the real world  results concerning a comparative study between IVET and ARET , a projection-based AR system for therapy for small-animal phobia . While it may be difficult for cyberpsychology laboratories working without significant technical support to implement alternative tracking technologies, it could be constructive for those who do benefit from such support to experiment ARET protocols using alternative tracking methods ; such developments may unlock ARET’s access to many potentially useful treatment protocols. One of these may be the treatment of social phobia. Presently, some of the VEs destined to provide support in the treatment of social phobia rely on public speaking tasks. Depending on the target population, the environment often consists of public speaking rooms such as auditoriums, conference rooms, and classrooms. In this type of situation, one advantage of ARET is that the exposure could take place in the actual places the patient encounters his difficulties . Thus, assuming that the required tracking technology is developed, AR could provide such The purpose of this paper was to review the move from VRET to ARET. Unlike VR, which entails a complete VE, AR limits itself to producing certain virtual elements to then merge them into the view of the physical world. Although the general public may only have become aware of AR in the last few years, AR type applications have been around since beginning of the twentieth century. Since then, technological developments have enabled an ever increasing level of seamless integration of virtual and physical elements into one view. Like VR, AR allows the exposure to stimuli which, due to various reasons, may not be suitable for real-life scenarios. As such, AR has proven itself to be a medium through which individuals suffering from specific phobia can be exposed “safely” to the object(s) of their fear, without the costs associated with programing complete VEs. Thus, ARET can offer an efficacious alternative to some less advantageous exposure-based therapies. Above and beyond presenting what has been accomplished in ARET, this paper also raised some AR related issues, and proposes potential avenues to be followed. These include the definition of an AR related term, the type of measures to be used to qualify the experience of ARE users, as well as the development of alternative geospatial referencing systems, which themselves, may open the door to other ARET applications, such as the treatment of social phobia. Overall, it may be said that the use of ARET, although promising, is still in its infancy but that, given a continued cooperation between clinical and technical teams, ARET has the potential of going well beyond the treatment of small animal phobia.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The toxicity of doxorubicin, leading to an irreversible heart failure, limits its use as chemotherapeutic agent. The beneficial effects of early administration of β-blocker were reported in patients with heart failure due to doxorubicin, suggesting an important role of β-adrenoceptors (β-ARs). This study aimed to identify a putative target (β-AR and/or its effectors) at the early phase of a chronic doxorubicin-induced cardiomyopathy (Dox-CM) in a rat model.−1) and validated by echocardiography and left ventricle (LV) catheterization. The β-AR protein expressions in LV were evaluated by western-blot at days 35 (d35) and 70 (d70) after the first doxorubicin injection. Ex vivo cardiac contractility  was evaluated on isolated heart in response to specific β-AR stimulations at d35.Dox-CM was induced by six doxorubicin injections (cumulative dose: 15 mg.kg3-AR expression was only decreased at d70 (-37±8%). At d35, β1-AR expression was decreased by 68±6%, but ex vivo β1-AR function was preserved due to, at least in part, an increased adenylyl cyclase response assessed by forskolin. β2-AR expression was increased both at d35 (+58±22%) and d70 (+174±35%), with an increase of ex vivo β2-AR response at d35. Inhibition of Gi protein with pertussis toxin did not affect β2-AR response in Dox-CM hearts, suggesting a decoupling of β2-AR to Gi protein.At d35, Dox-CM hearts were characterized by mild LV systolic and diastolic dysfunctions, which were exacerbated at d70. In Dox-CM hearts, β1/β2-AR imbalance in early Dox-CM and reveals the important role that β2-AR/Gi coupling could play in this pathology. Our results suggest that β2-AR could be an interesting target at early stage of Dox-CM.This study highlights the β Anthracyclines, like doxorubicin (Dox), epirubicin and daunorubicin, are among the most effective drugs used in chemotherapy for cancer patients. Since the late 60s, Dox is frequently used against a variety of cancers including Hodgkin’s lymphoma 1- and β2-adrenoceptor (β-AR) subtypes in the pathogenesis of Dox-cardiotoxicity 3-AR subtype et al., demonstrated that left ventricular (LV) diameters remained constant and diastolic function was better preserved after Dox-treatment in patients receiving carvedilol, compared to placebo et al., demonstrated that metoprolol, a β-blocker without antioxidative properties, failed to give cardioprotection in lymphoma-treated doxorubicin patients As in different HF etiologies, Dox-CM is characterized by an alteration of adrenergic system 2-AR expression was increased from 35 days after the first Dox-injection, this effect was maintained until 70 days after the first Dox-injection, resulting in an increase of β2-AR-induced contractility. In addition, β1-AR function was preserved, in spite of decreased β1-AR protein expression. This discrepancy could be explained by an increase of adenylyl cyclase (AC) expression and/or activity as illustrated by an increased forskolin-induced response in Dox-CM rats.The aim of the present study was to identify a putative target (β-AR and/or its effectors) involved at the early phase of a chronic Dox-CM in a rat model. This study demonstrated for the first time that βAll experiments were performed in accordance with the 1996 Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health. The protocol was approved by the Direction Départementale de la Protection des Populations (agreement number C-44 015) and all efforts were made to minimize suffering.ad libitum.One hundred and fifty eight male Sprague-Dawley rats (225–250 g) were purchased from Janvier  and were housed under standard conditions of room temperature, humidity (40–60%) and 12 h light/dark cycle. Food and water were available −1) over a period of two weeks, with a total cumulative dose of 15 mg.kg−1 body weight (Dox-CM: n = 100). Age-matched rats injected with saline were used as controls (Ctrl: n = 58). Rats were then bred in animal housing until three weeks after the last injection (day 35 (d35); Ctrl: n = 46, Dox-CM: n = 76) or until eight weeks after the last injection (day 70 (d70); Ctrl: n = 12, Dox-CM: n = 24). At d35, twenty Dox-CM rats and twenty Ctrl rats were randomly selected to be hemodynamically explored by echocardiography-Doppler. Then either rats were hemodynamically assessed by LV catheterization, or rat hearts were removed either to test ex vivo cardiac contractile function or to perform biochemical studies. At d70, rats were used to perform echocardiography-Doppler and biochemical studies.Doxorubicin  was administered intraperitoneally in six equal injections  equipped with a 10 MHz sectorial probe. Rats were anaesthetized with a gas-mixture of 1% isoflurane  in OAll images were digitally stored on hard disks for off-line analysis . Measurements were made on five cardiac cycles and averaged for each data value. The following parameters were determined as recommended by the American Society of Echocardiography −1 O2 flow to limit hemodynamic repercussion. Body temperature was monitored by rectal probe and maintained constant (37°C) by warming-blanket. The right carotid artery was isolated, ligated at the proximal part and the pressure catheter was inserted in. Signals were recorded using an Analogic/Digital converter , stored and displayed on a computer by the IOX1.5.7 Software System (EMKA Technologies). Data were analysed using Datanalyst software (EMKA Technologies). The following parameters were obtained: LV end diastolic pressure (LVEDP), LV contraction and relaxation velocities , the index of LV relaxation constant (Tau).LV catheterization was performed, at d35, by a 2F microtip pressure catheter . Anaesthesia maintenance on spontaneously breathing rats was performed with an inhalational anaesthesia system for small animal . Isoflurane was delivered through a nose mask at a concentration of 2% volume and 1 L.minThe animals were thereafter sacrificed by injection of a pentobarbital overdose.1-AR, β2-AR, β3-AR, Gsα, Giα2 and GAPDH were examined by western-blot, at d35 and d70. Hearts were rapidly isolated and placed in a cold Tyrode solution composed as followed (in mM): NaCl, 137; KCl, 5.4; MgCl2, 1.2; Na2HPO4, 1.2; Hepes, 20; CaCl2, 1.0; pH 7.4 . LV and septum free walls were carefully separated and freezed in liquid nitrogen and stored at −80°C until used. LV and septum free walls samples were homogenized in 3 mL of Ripa buffer for 1 g of tissue plus 1X PMSF  and 2X protease inhibitor cocktail . Protein samples (25 µg) were submitted to electrophoresis on a 10% polyacrylamide/sodium dodecyl sulfate gel and then were run 150 min at 20 mA per membrane in TG-SDS buffer (Interchim). Gels and nitrocellulose membranes  were equilibrated in TG-SDS buffer with 20% ethanol, and protein fractions were transferred using an electroblotting apparatus . Nonspecific binding was blocked by incubating membranes in 5% non-fat dry milk in Tris-buffered saline (TBS)  with 0.1% Tween 20 added (TBS-T) and then membranes were incubated with the primary antibody. Membranes were washed in 5% non-fat dry milk in TBS-T and hybridized with the secondary antibody in 1% non-fat dry milk in TBS-T. Finally, membranes were washed with 5% non-fat dry milk in TBS-T then TBS, and antibody complexes were revealed by the enhanced chemiluminescence detection process (Bio-Rad). Chemiluminescence was visualized using an Amersham ImageQuant RT-ECL camera  and band signals were assessed by densitometry with ImageQuant TL software . For each lane, a ratio to the corresponding GAPDH band intensity was calculated; the use of GAPDH as reference was validated by checking the abundance stability of GAPDH protein between Ctrl and Dox-CM groups.The expressions of βAntibody references and conditions used in this study are summarized in via the aorta on a Langendorff apparatus (EMKA Technologies) and perfused with a Krebs-modified solution  continuously oxygenated with a 95% O2, 5% CO2 gas mixture to maintain a pH 7.4. Hearts were perfused at a constant flow-rate of 14 mL.min−1 at 37.0±0.4°C. After heart beatings became regular, left auricle was gently cut off to insert a small latex balloon, connected to a pressure transducer to record LV pressure, into the LV via mitral valves. Before starting record, the minimum pressure into the balloon was adjusted to 8–15 mmHg. Cardiac parameters were recorded using IOX1.5.7 software (EMKA Technologies): inotropism, lusitropism and chronotropism were respectively evaluated by measuring the contraction velocity (dP/dtmax), the relaxation velocity (dP/dtmin) and the heart rate (HR). After the stabilization period, specific β1-, β2- or β3-AR functions and AC stimulation were assessed by constructing concentration-response curves to different pharmacological agents. Thus, β1- and β2-AR functions were assessed by perfusing isoproterenol  in the presence of 10−6 M L-748,337 (a selective β3-AR antagonist) associated to 10−6 M ICI-118,551 (a selective β2-AR antagonist) or to 10−6 M CGP-20712A (a selective β1-AR antagonist), respectively. β3-AR function was assessed by perfusing growing concentrations (10−9 to 10−5 M) of SR 58611A (a β3-AR agonist) and AC response was assessed by perfusing growing concentrations (3.10−8 to 10−5 M) of forskolin (an AC activator). In order to assess the role of Gi in the β2-AR pathway, we used pertussis toxin (PTX) (a selective Gi inhibitor). Classically, PTX is administered to rats two or three days before experiments −1) in a closed system during 30 min, as described by other groups At d35, after removal, hearts were quickly flushed into a cold Tyrode buffer, and then were mounted on a cannula In vivo and ex vivo cardiac parameters in baseline as well as protein expressions were compared using Student’s t test for unpaired data. Concentration-response curves were compared with a two-way ANOVA  for repeated measures followed, when appropriated, by a Bonferroni's multiple comparisons test. Differences were considered significant if P<0.05.Data are presented as means ± standard error to the mean (sem) of n experiments obtained from n different animals. erythro--1--3-[(1-methylethyl)amino]-2-butanolhydrochloride), CGP-20712A, 1-[2-((3-Carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol dihydro chloride, L-748,337, N-[[3-[(2S)-2-Hydroxy-3-[[2-[4-[(phenylsulfonyl)amino]phenyl]ethyl]amino] propoxy]phenyl]methyl]-acetamide, forskolin and PTX were obtained from Tocris Bioscience , isoproterenol was obtained from Sigma-Aldrich  and SR 58611A, -(2)-2-(3-chlorophenyl)-2 hydroethanamide hydrochloride] was a generous gift from Sanofi-Synthélabo . All drugs were dissolved in distilled water, excepted isoproterenol which was solubilised in 1% acid ascorbic and L-748,337 and forskolin which were solubilised in dimethylsulfoxide (Sigma-Aldrich). The final concentration of the solvent in the organ bath was less than 0.1% v.v−1 and was used as controls for the effect of the active drug.ICI-118,551, ((±)-Dox-CM rats did not gain weight during the Dox-treatment, whereas the body weight of Ctrl rats increased regularly. After the end of Dox-treatment, the rat body weight increased again, but remained significantly lower than those of Ctrl rats, and reached a plateau from 5 weeks after the end of Dox-treatment day 49; . In addiThe mortality rate was 25.0% at d35 and 58.3% at d70 in Dox-CM when no mortality was observed in Ctrl . At d35,P = 0.427; d70: Ctrl: 368±6 bpm, Dox-CM: 337±17 bpm, P = 0.079). Representative images obtained by echocardiography in short axis view of a two-dimensionally directed M-mode at d35 are shown in Echocardiography-Doppler analyses were performed at d35 and d70. Heart rate, measured before all echocardiographic acquisitions, was monitored in order to be similar between both groups  and was significantly thinner at d70  . On the contrary, LVEDV was reduced in Dox-CM compared to Ctrl at d35  as at d70   . LVESV w= 0.050) .P<0.001) and LVSF , that were more pronounced at d70  (P = 0.001) as at d70  (P = 0.031) (P<0.001) as at d70    . MoreoveP<0.001)  whereas = 0.031) . In Dox-= 0.003)  albeit tctively) .P = 0.007) (P = 0.009) albeit dP/dtmax and dP/dtmin were unchanged (P = 0.216).As our study consists to identify molecular target at early stage of Dox-CM, we only performed LV catheterization at d35. Basal heart rate, measured before all pressure acquisitions, was increased in Dox-CM rats compared to Ctrl (= 0.007) . Dox-CM nchanged . LVEDP w3-AR protein at a band of 68 kDa (P = 0.192), whereas, at d70, β3-ARs were down-expressed in Dox-CM hearts compared to Ctrl hearts   (min only at the higher concentration of SR 58611A (10−5M) (P = 0.004) (3-AR stimulation in Dox-CM hearts produced a significant decrease in dP/dtmax only at the higher concentration of SR 58611A (10−5M) (P<0.001)   . In Ctrlctively)  and decr= 0.004) . CompareP<0.001)  and had ctively) .1-AR detected a band at 72 kDa (P = 0.002 vs Ctrl) (P<0.001 vs Ctrl)     and at dvs Ctrl) . In Ctrl1, each) . The heactively) .2-AR detected a band at 55 kDa (P = 0.039 vs Ctrl) (P<0.001 vs Ctrl) (2-AR stimulation induced a similar concentration-dependant increase of heart rate between Ctrl and Dox-CM hearts (P = 0.695)   and +155.3±26.8% (P<0.001), respectively   and at dvs Ctrl) . At d35,= 0.695) . In Ctrlctively)  but at aectively , and reaP = 0.941) (max (P<0.001) and dP/dtmin (P = 0.010) (max and dP/dtmin were increased by +43.2±10.3% (P = 0.004) and +61.1±10.7% (P = 0.002), respectively  . In Ctrl= 0.010) . In Dox-ectively .2 protein expression was detected by one band of 35 kDa (P = 0.184 vs Ctrl) (P = 0.009 vs Ctrl)    (Giαf 35 kDa  whose invs Ctrl) , but wasvs Ctrl) . Ex vivo= 0.346) . PTX-preP<0.001)  but inductively) .2-AR expression was increased at the early stage of the pathology, with an increase of β2-AR-induced contractility. Furthermore, β1-AR function was preserved in spite of decreased β1-AR protein expression and could be explained by an increase of AC expression and/or activity as illustrated by an increased forskolin-induced contractility in isolated hearts from Dox-CM rats.This study demonstrated for the first time in Dox-cardiotoxicity, βIn our study, the body weight of Dox-CM rats was smaller than in Ctrl one as previously described by other studies During echocardiographic acquisitions, heart rate was monitored in order to have similar heart rate in both groups, allowing a comparison of echocardiographic parameters. At d35, Dox-CM rats presented a slight alteration of systolic function as illustrated by a mild decrease in LVEF and LVSF and in LV longitudinal deformation. Dox-CM rats presented also a mild diastolic dysfunction as suggested by the increased IVRT and Tau values. At d70, decreases in LVEF, LVSF and LV longitudinal deformation were exacerbated whereas diastolic dysfunction was unchanged. As diastolic dysfunction frequently occurs before systolic dysfunction In human, Dox administered to adults  is well known to induce chronic cardiotoxicity where cardiac morphologic and functional alterations are close to those of dilated cardiomyopathy 3-AR protein expression was unchanged and a slight negative inotropic effect was obtained in Dox-CM hearts at the higher concentration of SR 58611A (10−5 M). Our data suggest that in early chronic Dox-cardiotoxicity, β3-AR could not play a major role in the cardiac alterations. At a later stage (d70), we observed a decreased β3-AR protein expression in Dox-CM hearts albeit another team reported an overexpression of β3-AR protein at the same stage vs Sprague-Dawley) and several studies reported that protein expression could be different between Sprague-Dawley and Wistar rats In our Dox-CM model, we reported a remodeling in β-AR expression and function. Three weeks after last Dox-injection (d35), β1-AR protein expression was decreased at both stages. This result is in agreement with those reported for β1-AR protein expression both in rabbits 1-AR expression at d35, the cardiac β1-AR responses were preserved. Several hypotheses could explain this feature. Firstly, a β1-AR reserve could be present and recruited in such conditions and, secondly, an increase in β1-AR signaling pathway can be evoked. We showed that Dox-treatment did not change Gsα protein expression, as it was reported in other study −1, respectively. Thus, the increased AC response to forskolin observed in our study could be a compensatory mechanism at early stage of Dox-cardiotoxicity to maintain cardiac contractility and could explain, at least in part, the preserved β1-AR response. However, as β1-ARs are involved in pro-apoptotic effects, β1-AR could be also responsible for some Dox cardiotoxic effects as previously suggested In our Dox-CM model, β2-AR protein overexpression. At d35, in Ctrl isolated heart, specific β2-AR stimulation induced positive inotropic and lusitropic responses but to a lesser extent than β1-AR one. This lower response could be explained by a more compartmentalized cAMP signaling of β2-AR 2-AR inotropic effect was increased and reached a similar level than β1-AR stimulation. This increase could be due to several mechanisms. Firstly, we show that β2-AR protein expression was increased in Dox-CM LV and could lead to an increased β2-AR response. Secondly, cardiac β2-AR could be linked both to Gs and Gi proteins 2 protein expression was not modified at d35 but it was overexpressed at d70 of Dox-CM, as reported previously at this latter stage by Sun et al. 2-AR response in order to reach the same level that obtained in Dox-CM in the absence of PTX treatment, whereas Gi inhibition in Dox-CM hearts slightly increased β2-AR response. These data suggest that, in Dox-CM, β2-AR was mainly linked to Gs.Finally, we reported, for the first time, in chronic Dox-cardiotoxicity, a β2-AR/Gs signaling observed in our study is a beneficial compensatory mechanism or a detrimental effect is still to be determined. To support the hypothesis of a beneficial effect of β2-AR agonist, it has been shown that β2-AR/Gi signaling can mediate cardiac protective effects by activating cell surviving pathway in vivo studies reported beneficial effects of β2-agonist therapy in HF 2-AR, it could be proposed to use β2-AR blocker. However, it is important to note that β2-AR antagonists by blocking other physiological functions regulated by β2-AR, could also have detrimental effects: (i) a vasoconstriction because β2-AR is one of the most important vascular β-AR subtype whose activation induces a vasodilation in several vascular beds (including coronary vessels), (ii) a bronchoconstriction due to the reduction of bronchodilation induced by pulmonary β2-AR,…To know whether enhanced β2-AR pathway. Then, according to the results of this next important study, we hope that we could propose a new therapeutic target for Dox-CM.Thus, we rather suggest that further experiments must be performed to identify the most suitable effector of the cardiac β2-AR protein expression at early stage of the pathology, associated to an increased contractility. Furthermore, we observed a preserved β1-AR function albeit β1-AR was reduced. Clinical studies have suggested no beneficial effect of metoprolol, a β1-AR blocker, in Dox-treated lymphoma patients 1-AR blocker associated to a β3-AR agonistic properties against anthracycline-induced cardiomyopathy in patients with breast cancer 2-AR remain to elucidate in order to determine whether β2-AR activation or blockade could prevent cardiac alterations due to anthracycline.In conclusion, we have shown for the first time in rat Dox-CM, an increase of β


Visual loss following stroke impacts significantly on activities of daily living and is an independent risk factor for becoming dependent. Routinely, allied health clinicians provide training for visual field loss, mainly with eye movement based therapy. The effectiveness of the compensatory approach to rehabilitation remains inconclusive largely due to difficulty in validating functional outcome with the varied type and dosage of therapy received by an individual patient. This study aims to determine which treatment is more effective, a standardized approach or individualized therapy in patients with homonymous hemianopia post stroke.This study is a double-blind randomized controlled, multicenter trial. A standardised scanning rehabilitation program (Neuro Vision Technology (NVT) program) of 7 weeks at 3 times per week, is compared to individualized therapy recommended by clinicians.The results of the trial will provide information that could potentially inform the allocation of resources in visual rehabilitation post stroke.ACTRN12610000494033Australia and New Zealand Clinical Trials Register (ANZCTR):  Stroke is a leading cause of disability , and appVisual training in homonymous hemianopia post stroke is recognized as an important part of rehabilitation . The proThe second strategy is compensatory and aimed at enhancing eye movements and/or head movements. This takes a number of different forms including the training of oculomotor or saccadic movements, on a standardised computer screen, or devices such as the Dynavision , which iThe third strategy uses visual restitution therapy and aims to enhance neuroplasticity . This meIn Australia, most visual training employs compensatory eye and head movement based therapy but current delivery of vision rehabilitation is recommended by treating therapists and is thus subjective . TherapiIn response to the need for a structured therapy program of visual training in homonymous hemianopia post stroke, the Neuro Vision Technology (NVT) program has been developed which is a standardized therapy and involves both a static scanning training and a dynamic mobility component. The static scanning component uses the NVT device,   after thThe secondary outcome assessments will provide a broader picture of function and quality of life following a stroke and include Visual scanning analyzer (VSA) , Pepper Sample size has been calculated based on data reported in a pilot study validating the primary outcome measurement tool, the Mobility Assessment Course . The minData will be entered into an SPSS database with all identifying details removed. An ANCOVA will be performed to compare outcomes between the intervention groups, controlling for initial scores as covariates in the analyses.Statistical analyses will be undertaken using SPSS version 17.0 Statistical software. All analyses will on an intention to treat basis and will be blinded (i.e. groups identified by number only) and results published according to the Consort Statement formal.Visual rehabilitation training is routinely provided for people with hemianopia following stroke however the benefits of visual rehabilitation training on functional outcomes have not been well evaluated. Despite us knowing it is important to rehabilitation as it impacts negatively on independence following stroke .The goal of visual rehabilitation involves making the most of a person's residual vision, specifically to try to overcome the visual disabilities that are most troublesome to them. Part of the difficulty in evaluating the effectiveness of vision rehabilitation in homonymous hemianopia is the varied type of strategies used by therapists. At the moment the details of the rehabilitation program are negotiated between each patient and their rehabilitation therapist. No robust evidence currently exists to identify the most effective way to deliver vision rehabilitation in people with homonymous hemianopia after a stroke or the optimum time required .There is evidence that providing more exercise therapy early after stroke improves peoples' ability to recover  but the This project will compare the effectiveness of current visual rehabilitation protocols which vary in type and dosage according to individual clinicians' recommendations, to a new standardized technology (NVT). Functional outcome measures, such as performance on a mobility assessment course and scanning ability will be employed as well as patient reported outcomes such as, vision specific health related quality of life to allow a meaningful assessment of this rehabilitation intervention.A debate exists around the mechanisms which underlie the remedial approach in visual rehabilitation following stroke, that is compensatory or neuroplasticity . The outAs in many areas of rehabilitation more rigorous research is needed to identify which remedial approaches should be included in clinical practice for visual loss following stroke.Allison Hayes has participated in the development of the device and currently involved in the commercialisation.All authors contributed to the design and conduct of the clinical trial. SG carried out data analysis, and drafted the manuscript. AH conceived of the study and participated in its design and coordination. AD and MC participated and oversees the trial process. CC is involved in the neuro-ophthalmic assessment and data acquisition of all participants and helped draft the manuscript. All authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/11/87/prepub


The steroid hormone androgen mediates a wide range of developmental, physiological and malignant responses by acting as a ligand for the androgen receptor (AR). AR in turn functions as a nuclear receptor transcription factor and executes specific gene expression programs in an androgen-dependent manner. Since the growth of prostate cancer cells initially depends on androgen, cancer therapy uses hormone-deprivation approaches to reduce the levels of serum androgen. While prostate cancer (PC) initially responds to androgen-ablation, most tumors progress to a castration resistant (CR) state insensitive to treatment. Both the androgen-dependent and the CRPC state depend on AR, which is probably activated by alternative molecular pathways in CRPC in response to low androgen levels.Here, we assess the role of the transcriptional co-regulator TRIM24/TIF1α in mediating AR-dependent gene expression and growth. We find that TRIM24 expression is increased in different states of PC, particularly in CRPC. When we induce the drug-regulated knock-down of TRIM24, we find that cell growth in both our androgen-dependent (LNCaP) and in our CRPC model (LNCaP-abl) is affected. Therefore, we hypothesize that TRIM24 is involved in mediating both ligand-dependent and ligand-independent activation of AR. This is further consistent with our finding that TRIM24 and AR can physically interact in both conditions. We are currently identifying TRIM24-dependent gene expression programs by combining genome-wide binding analyses with microarray studies.This will help us shed light on how the transcriptional co-regulator TRIM24 may reprogram AR activity in its progression from an androgen-dependent to a castration-resistant state. Current results from this study will be presented at the meeting.