Androgen receptor (AR) signals have been suggested to contribute to bladder tumorigenesis and cancer progression. Activation of epidermal growth factor receptor (EGFR) also leads to stimulation of bladder tumor growth. However, crosstalk between AR and EGFR pathways in bladder cancer remains uncharacterized. We have recently shown that androgens activate the EGFR pathway in bladder cancer cells. The purpose of this study was to investigate the effects of EGF on AR activity in bladder cancer. EGF increased AR transcriptional activity by 1.2-, 1.9- and 2.0-fold in UMUC3, 5637-AR and J82-AR cell lines, respectively, over mock treatment and a specific EGFR inhibitor, PD168393, antagonized the EGF effect. Combined treatment of EGF and dihydrotestosterone (DHT) further induced AR transactivation while an AR antagonist, hydroxyflutamide (HF), abolished the effect of not only DHT but also EGF. In growth assays, EGF alone/DHT alone/EGF+DHT increased cell numbers by 16/12/19%, 6/14/18% and 30/12/38% in UMUC3-control-shRNA, 5637-AR and J82-AR, respectively, whereas the effects of EGF were marginal or less significant in UMUC3-AR-shRNA (8%) or AR-negative 5637-V (<1%) and J82-V (17%) cells. HF treatment at least partially counteracted the EGF effect on the growth of AR-positive cells. Western blotting demonstrated that EGF, especially in the presence of DHT, upregulated the expression of the p160 coactivator TIF2 and HF again blocked this stimulation. Co-immunoprecipitation revealed the association between AR and estrogen receptor (ER)-β or Src in UMUC3 cells and stronger associations with EGF treatment, implying the involvement of the AR/ER/Src complex in EGF-increased AR transactivation and cell growth. Current results, thus, suggest that EGF promotes bladder cancer cell proliferation via modulation of AR signals. Taken together with our previous findings, crosstalk between EGFR and AR pathways can play an important role in the progression of bladder cancer. Epidemiological and clinical evidence has indicated a substantially higher risk of urinary bladder cancer in males yet there is a tendency showing more aggressive behavior in tumors from female patients ,2. RecenActivation of the epidermal growth factor (EGF) receptor (EGFR) family is known to involve the growth and progression of a variety of malignancies. In bladder cancer, EGFR/ERBB2 is frequently overexpressed, which correlates with higher tumor grade/stage and poorer prognosis –16. ExpeEGFR and ERBB2 gene expression, whereas activation of EGFR and ERBB2 modulates AR functions  and EGF from Sigma ; hydroxyflutamide (HF) from Schering ; and PD168393 from Calbiochem .Human bladder cancer cell lines, UMUC3, 5637 and J82, obtained from the American Type Culture Collection  were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% COCell lines stably expressing a full-length wild-type human AR (5637-AR and J82-AR) or vector only (5637-V and J82-V) were established, using a lentivirus vector (pWPI-AR or pWPI-control) with psPAX2 envelope and pMD2.G packaging plasmids, as we described previously ,26. SimiBladder cancer cells at a density of 50–60% confluence in 24-well plates were co-transfected with 250 ng of MMTV-luc reporter plasmid DNA and 2.5 ng of pRL-TK-luc plasmid DNA, using GeneJuice transfection reagent . Six hours after transfection, the medium was replaced with one supplemented with 5% charcoal-stripped FBS containing ethanol or ligands  for 24 h. Cells were harvested, lysed and assayed for luciferase activity determined using a dual-luciferase reporter assay kit  and luminometer .3) seeded in 96-well tissue culture plates were incubated with medium supplemented with charcoal-stripped FBS in the presence or absence of ligands . The media were refreshed every 24 h. After 96 h of treatment, 10 μl MTT (Sigma) stock solution (5 mg/ml) was added to each well with 0.1 ml of medium for 4 h at 37°C. The medium was replaced with 100 μl DMSO followed by incubation for 5 min at room temperature. The absorbance was then measured at a wavelength of 570 nm with background subtraction at 655 nm.We used the MTT (methyl thiazolyl diphenyl tetrazolium bromide) assay to assess cell viability, as described previously ,27. Brieμg) obtained from cell extracts were separated in a 10% sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membrane  by electroblotting using a standard protocol. Specific antibody binding was detected, using an anti-AR antibody , an anti-transcriptional intermediary factor 2 (TIF2) antibody , or an anti-GAPDH antibody , with horseradish peroxidase detection system .Protein extraction and western blotting were performed, as described previously  with minμg) from the cell lysates was incubated with 2 μg of anti-AR antibody (N20) or normal rabbit IgG (Santa Cruz Biotechnology) for 16 h at 4°C with agitation. To each sample we added 20 μl of protein A/G-agarose beads (Santa Cruz Biotechnology), incubated for 1 h and washed thrice with radio-immunoprecipitation assay buffer. Then, the complex was resolved on a 10% SDS-PAGE, transferred to the membrane and blotted with an anti-ERβ antibody  or an anti-v-Src antibody .UMUC3 cells were treated with mock (ethanol) or EGF for 24 h and protein . As shown in To further investigate how EGF influences AR signals, we examined AR expression by western blotting. In UMUC3, AR expression was increased by DHT (4.4-fold) and further enhanced by addition of EGF (6.4-fold), whereas no significant effect of EGF or HF was seen in the absence of DHT . HF cleaBecause EGF was shown to induce AR transcription by upregulating TIF2 expression in prostate cancer cells , we thenPrevious studies in prostate and breast cancers demonstrated that EGF induced AR/ER/Src association, resulting in activation of Src signaling ,28 and tDysregulation of the EGFR family is well known to associate with bladder cancer –16. AR sIn prostate cancer, accumulating evidence has indicated that EGFR/ERBB2 signals induce AR transactivation in an androgen-dependent and -independent manner –22. In bConsistent with previous findings shown by others and us ,7,9,26 aet al, such as prostate and breast cancers, AR/ER/Src association plays a crucial role in activation of Src signals triggered by EGF and/or sex hormones ,28. It wIn conclusion, EGF could increase AR transcriptional activity and cell proliferation in bladder cancer. These EGF effects were likely mediated through the AR pathway involving upregulation of TIF2 expression as well as activation of Src signals due to forming an AR/ER/Src complex. These results, together with our previous findings, not only shed light on crosstalk between the AR and EGFR pathways in bladder cancer but also enhance the feasibility of androgen deprivation interfering with this crosstalk as a potential therapeutic approach.


Podocyte injury is associated with progression of many renal diseases, including diabetic nephropathy. In this study we examined whether aldose reductase (AR), the enzyme implicated in diabetic complications in different tissues, is modulated by high glucose and osmolarity in podocyte cells. AR mRNA, protein expression, and activity were measured in mouse podocytes cultured in both normal and high glucose and osmolarity for 6 hours to 5 days. Hyperosmolarity acutely stimulated AR expression and activity, with subsequent increase of AR expression but decrease of activity. High glucose also elevated AR protein level; however, this was not accompanied by respective enzyme activation. Furthermore, high glucose appeared to counteract the osmolarity-dependent activation of AR. In conclusion, in podocytes AR is modulated by high glucose and increased osmolarity in a different manner. Posttranslational events may affect AR activity independent of enzyme protein amount. Activation of AR in podocytes may be implicated in diabetic podocytopathy. Aldose reductase , a member of the aldoketo reductase superfamily, catalyzes the reduction of broad spectrum of aldehydes, using NADPH as a cofactor. Specific AR substrates comprise saturated and unsaturated aldehydes such as progesterone, isocorticosteroids, aldehydes derived from biogenc amines, methylglyoxal, and other harmful metabolites . The enzκB-dependent expression of cytokines and chemokines [Glucose, with its apparent Km of 50–200 mM , is a poemokines , 12.Within the kidney, the highest expression and activity of AR is found in the medullary region. In renal cortex abundance of the enzyme is very low as compared to medulla, as well as to various other tissues , 13. NevWhereas it is accepted that aldose reductase is implicated in pathogenesis of diabetic glomerulopathy –22, its Conditionally immortalized mouse podocytes  were cultured as described previously . DiffereIt has been shown previously that cultured human embryonic kidney (HEK) cells rapidly utilize glucose, which within 24 hours may result in glucose starvation, endoplasmatic reticulum stress, and underglycosylation of numerous proteins . TherefoμL ice-cold lysis buffer containing (in mM) 50 HEPES (pH 7.2), 2 DTT, 5 EDTA, and 1 tablet/10 mL protease inhibitor cocktail  was added to each flask. The cells were scrapped, transfered into eppendorf tubes, and centrifuged at +4°C, 14,000 rpm for 30 minutes (Eppendorf Centrifuge 5810R). Resulting supernatant was stored at −80°C, except for an aliquot of 25 μL that was used for immediate protein assay according to the Bradford method [At indicated time, culture flasks were placed on ice, experimental media were discarded, and 400 d method . Bovine μg (nmol NADPH oxidized per minute per μg protein), based on a molar absorption coefficient of 6220 M−1·cm−1.Activity of AR was determined spectrophotometrically (Ultrospec 3000) at 37°C as described previously . In all α-smooth muscle actin  followed by alkaline phosphatase-labeled secondary antibodies . The complexes were visualised with 5-bromo-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium  and photographed in UVP BioImaging GDS-8000 system , using LabWorks 4.0 Image Acquisition and Analysis Software.Immunobloting was performed using standard techniques. In brief, trypsynized cells were centrifuged at 2,000 rpm for 10 minutes in +4°C; resulting pellet was lysed on ice in a buffer (pH 8.0) containing 1% Nonidet P-40, 20 mM Tris, 140 mM NaCl, 2 mM EDTA, 10% glycerol, and protease inhibitor cocktail  and centrifuged for 20 minutes at 14,000 rpm in +4°C. Fifteen micrograms of total protein were subjected to SDS-PAGE and transfer electrophoresis. The proteins on PVDF Immobilion membranes  were probed with primary antibodies to AR  and to μM antisense primer, 5 U/μL MuLV reverse transcriptase , 0.5 mM deoxynucleotide triphosphate mixture (dNTP), and 1 U/μL RNase inhibitor (Sigma-Aldrich). The PCR step was performed using 2.5 U Maxima Hot Start Taq DNA Polymerase (Fermentas Int Inc.), with 20 μL RT product and 80 μL PCR mix containing 0.1 μM sense primer. Primer sequences and PCR amplification conditions are shown in μL/lane) were resolved on ethidium bromide-stained 1.4% agarose gels, viewed and photographed on a UV transilluminator .Total RNA was extracted from each culture sample using TRI Reagent (Sigma-Aldrich) according to the manufacturer's instructions. RNA purity and concentration were determined by measuring the optical densities at 260 nm and 280 nm. Two micrograms of RNA were reverse transcribed at 37°C for 60 min using 0.1 β-actin (RT-PCR) or to α-smoth muscle actin (immunobloting).Semiquantitative analysis of the band optical densities was performed using Quantity One software  and normalized to 5 cells/tube were stained for 60 minutes at room temperature with 1 : 100-diluted rabbit antibodies to AR . Antigen-bound antibodies were visualized by 40 minutes incubation with 1 : 100 diluted Alexa488-conjugated donkey anti-rabbit IgG . Stained cells were washed with 1 mL WB, resuspended in 500 μL PBS and analyzed in flow cytometer . Basing on measurements of the single-cell fluorescence intensities, arbitrary selection of AR-positive cells was performed. For each selected population, the mean fluorescence index (MFI) was determined using FACS Diva software . Negative controls displayed negligible fluorescence of permeabilized podocytes.Following incubations, the cells were washed with PBS, trypsinized, suspended in PBS, and centrifuged two times for 7 minutes at 400 × g at room temperature. The pellets were then resuspended in 1 mL of a cold permeabilizing buffer , incubated for 30 minutes in room temperature, centrifuged and washed with 1 mL washing buffer (WB) . Finally, 1.5 × 10μg/mL 4′,6-diamidino-2-phenylindole dihydrochloride . All antibodies and stains were diluted in blocking solution. The cover slips were mounted on microscope slides using 15% Mowiol solution , and stained cells were analyzed under fluorescence microscope (Olympus IX51), using cellSens v.1.3 imaging software (Olympus).For immunocytochemical analysis, the podocytes were grown on collagen-coated round glass cover slips in 24-well culture plates containing experimental media, as indicated before. Immunocytochemistry was performed as described previously . Brieflyt-test for comparisons. Statistical significance is indicated by P < 0.05.All of the data are presented as means ± SEM from 3 to 5 independent experiments. The statistical analyses were performed using SigmaStat . Data were analysed using Student's μg in NG-Nosm, P < 0.05) already after 6 hours of incubation. Thereafter, a gradual decrease was observed, reaching the Nosm level after 48 hours. On the other hand, in HG medium after 6 hours only slight increase of AR activity was observed. After 5 days it reached the level that was only 1.2-fold higher than in the NG-Nosm group. Time course of changes in enzyme activities was similar in HG podocytes from both, normo- and hyperosmolar media which suggests that high glucose counteracted the stimulatory effect elicited by high osmolarity in NG cells.In NG-Nosm cells, AR activity remained constant during the whole incubation time . In contP < 0.05 versus NG-Nosm) (P < 0.01). Respectively, activity of the enzyme increased by 82 ± 2%, P < 0.05 as compared to the NG-Nosm cells  and prolonged (5 days) exposition to experimental conditions were further checked by flow cytometry. Densitometric quantification of obtained 37 kDa bands revealed that in hyperosmolar conditions, after 6-hour incubation AR expression in NG podocytes was markedly elevated  . QuantitNG-Nosm)  yielded sm cells . HoweverImmunofluorescent staining after 5 days of incubation confirmed that expression of AR in cytosol of NG-Hosm podocytes was upregulated, as compared to NG-Nosm and HG-Hosm cells . NG-NosmP < 0.025 versus NG-Nosm) as early as 6 hours after shifting to high glucose , while longer incubation seemed to reverse this effect.In podocytes cultured in HG-Nosm media, AR protein was markedly increased  of respective immunoblots to the RT-PCR products showed that expressions of AR mRNA and protein were correspondingly modulated. Similar to the enzyme activity in NG-Nosm cells, AR mRNA and protein expression remained at a constant level up to 5 days of incubation. Enhanced (NG-Hosm and HG-Nosm cells) or suppressed (HG-Hosm cells) expression of AR protein  conditions, the expression of AR mRNA in podocytes was relatively high, with abundance comparable to that of glyceraldehyde-3-phosphate dehydrogenase  and in vivo and in vitro [in vivo and in vitro [κB, which, in turn, can regulate the transcription of AR gene [in vivo and in vitro [In the present study we show that in cultured mouse podocytes, expression and activity of aldose reductase (AR) are modulated by high glucose and hyperosmolarity in a different manner. High osmolarity alone induced a rapid but transient increase of AR activity Figures , while oin vitro , 30, 31.in vitro , the benin vitro  and oxidin vitro , 34 strein vitro  and in ain vitro . In turnin vitro . In our in vitro , others in vitro . Anotherin vitro  that wasin vitro  accountein vitro . Multiplin vitro . In high AR gene . Since P AR gene , 45, thi AR gene . Both ox AR gene , 48. It  AR gene , 50 coul AR gene . In addiin vitro , 53. Thein vitro  as well in vitro  forms ofper se [+-dependent transport) or by glutamate/cystine antiporters [In contrast to high glucose, hyperosmolarity appeared to markedly stimulate AR activity in NG podocytes Figures . This waper se , 62. Howper se , the disper se , 65 whicper se –69. Sincper se , 71. Howper se . Transmeiporters  includiniporters , 75, andα (TNF α) or vascular endothelial growth factor (VEGF) in the kidney and vascular smooth muscle cells [α [Relatively high basal AR mRNA expression (le cells , 48, 78.le cells , TNF α [cells [α , as wellcells [α , 48.In summary, these results show that in cultured podocytes aldose reductase is regulated by both high glucose and high osmolarity. Furthermore, changes in AR protein expressed by the cells do not necessarily correspond to changes in activity of the enzyme. High glucose alone elevated AR protein level, which was not accompanied by respective enzyme activation. High osmolarity in turn stimulated both activity and expression of AR in podocytes already within the first hours of exposition. However, while AR protein remained increased over the whole time of incubation, the activity of the enzyme concomitantly decreased. This suggests that in podocytes posttranslational events may affect AR activity independent of the enzyme protein content. Modulation of AR-dependent metabolic pathway in podocytes may be implicated in the pathomechanisms of diabetic podocytopathy.


Several types of suppression phenomena have been observed in the visual system. For example, the ability to detect a target stimulus is often impaired when the target is embedded in a high-contrast surround. This contextual modulation, known as surround suppression, was formerly thought to occur only in the periphery. Another type of suppression phenomena is interocular suppression, in which the sensitivity to a monocular target is reduced by a superimposed mask in the opposite eye. Here, we explored how the two types of suppression operating across different spatial regions interact with one another when they simultaneously exert suppressive influences on a common target presented at the fovea. In our experiments, a circular target grating presented to the fovea of one eye was suppressed interocularly by a noise pattern of the same size in the other eye. The foveal stimuli were either shown alone or surrounded by a monocular annular grating. The orientation and eye-of-origin of the surround grating were varied. We found that the detection of the foveal target subjected to interocular suppression was severely impaired by the addition of the surround grating, indicating strong surround suppression in the fovea. In contrast, when the interocular suppression was released by superimposing a binocular fusion ring onto both the target and the dichoptic mask, the surround suppression effect was found to be dramatically decreased. In addition, the surround suppression was found to depend on the contrast of the dichoptic noise with the greatest surround suppression effect being obtained only when the noise contrast was at an intermediate level. These findings indicate that surround suppression and interocular suppression are not independent of each other, but there are strong interactions between them. Moreover, our results suggest that strong surround suppression may also occur at the fovea and not just the periphery. The detectability and appearance of a target are often suppressed in the presence of a superimposed or surrounding mask. Three types of suppression have been widely reported. The first, known as surround suppression, is observed when the target is surrounded by an annular mask Mechanisms underlying these phenomena of suppression are not completely understood. Early studies assumed that all the three suppression phenomena derived from inhibition exerted by a pool of cortical neurons The majority of previous studies have concentrated on exploring the characteristics of these suppressions or evaluating the differences between them How does surround suppression interact with interocular suppression? Although few studies have directly addressed this issue, related insights can be gained from studies on binocular rivalry, a phenomenon of the dynamic alternations in perception that occurs when the two eyes continuously view dissimilar images. It has been shown that the dynamics of binocular rivalry can be affected by contextual stimuli presented in proximity of rival targets Six participants (P1–P6) gave written informed consent to participate in the experiments. Five of them were university students and were naïve to the purpose of the study. The other (P4) was the first author. All observers had normal or corrected-to-normal eyesight. All procedures were approved by the Research Ethics Board of University of Electronic Sciences and Technology of China.2.Stimuli were generated by using Matlab and the Psychophysics Toolbox The stimuli and experimental design are illustrated in A previous study showed that interocular suppression could be released by interocular feature matching The Michelson contrast of the surround grating was fixed at 80% in all experiments. The Michelson contrast of the noise pattern was 15% in Experiment 1 and 2. In order to explore how the strength of interocular suppression influences interactions between surround suppression and interocular suppression, the noise contrast was systematically varied from 0 to 80% in Experiment 3.A black square frame  and 5 black points (0.09°×0.09° each) were presented continuously in each display to promote stable binocular alignment. One of the points was presented in the center of each display and served as fixation point; the other four were presented on the annular grating and arranged around the center stimuli in a diamond shape, each placed 1.02° away from the center fixation point .The target grating and the dichoptic mask were presented to the fovea of the non-dominant and dominant eyes, respectively. The annular grating was presented either to the same eye as the target (monoptic conditions) or to the opposite eye (dichoptic conditions), surrounding the center stimuli. We used a two-interval forced-choice (2IFC) procedure, in which the two test intervals each lasted for 800 ms and were separated by a 506 ms blank interval . Both thTarget detection thresholds corresponding to the correct rate of 75% were determined using a QUEST staircase procedure In this experiment, we explored how the detection of the target grating was affected by surround gratings when the target was undergoing interocular suppression. The center target grating, which was concurrently masked by a dichoptic stimulus =21.1, p<0.001). Post hoc comparisons with the Fisher's least significant difference (LSD) test showed that detection thresholds under all with-surround conditions, except the DO condition, were significantly higher than that under the no-surround condition , meaning that surround suppression was produced under these conditions.F=20.2, p<0.02) but no effect of eye-of-origin =1.1, p>0.35) and a significant interaction between the two variables =13.9, p<0.02). A post hoc Fisher's LSD test showed that the parallel surround always produced strong suppression, irrespective of the eye to which it was presented . However, the orthogonal surround could produce significant suppression only when it was presented to the same eye as the target grating . This indicates that the suppression from a parallel surround can transfer across the eyes, but the suppression from an orthogonal surround can not. This finding suggests that the parallel suppression originates from the site(s) beyond the binocular convergence, while the orthogonal suppression occurs at a monocular stage, which is consistent with a previous suggestion that the two kinds of surround suppression have distinct sources To evaluate the strength of surround suppression, a suppression factor, defined as the ratio of with-surround to no-surround thresholds, was calculated for each with-surround condition , especially for parallel surrounds  by which detection thresholds were elevated by nearly a factor of 3. This is in contrast to earlier studies demonstrating that surround stimuli only slightly impair the perceived contrast of a foveal target There is another possibility, however, that the large surround suppression effects observed here are not due to interactions between surround suppression and interocular suppression, but simply due to the specific stimulus configurations we used. For example, the detection of a target masked with a noise might be more prone to be influenced by contextual inputs, thereby allowing us to obtain stronger surround suppression. The effective contrast of the target might be reduced by the dichoptic mask to low levels at which surround suppression has been reported to be stronger In this experiment, we aimed to examine the effects of surround gratings when interocular suppression was abolished or weakened. Meese and Hess (2005) have reported that the suppression induced by a binocular mask is much weaker than that induced by a dichoptic mask In this experiment, a black ring was superimposed on the target grating to match the same ring on the dichoptic noise pattern . With thF=4.9, p<0.01). A post hoc analysis (Fisher's LSD test) showed that, compared with the no surround condition, the target detection threshold in the MP condition was significantly elevated (p<0.02), whereas other with-surround conditions had no significant effect . A two-way ANOVA analysis revealed that the suppression factors in this experiment =36.9, p<0.005). In particular, for parallel surround conditions, the introduction of the binocular matching ring led to a remarkable decrease in the surround suppressive effect from a factor of about 3 to below 1.5 . These results demonstrate that the manipulation of interocular suppression can greatly influence the effect of surround suppression. Even though this experiment had very similar stimulus configurations to those in Experiment 1, surround suppression was substantially decreased by releasing the interocular suppression. This suggests that the robust surround suppression effects found in Experiment 1 can not be attributed to the specific surround and center stimuli, but are mainly due to the involvement of interocular suppression.Taken together, Experiments 1 and 2 indicate that surround suppression and interocular suppression are not two independent suppression phenomena, but they interact with one another. In this experiment, we further investigated whether these interactions depend on the strength of interocular suppression. To address this question, we examined how surround suppression varied with increasing strength of interocular suppression, which was achieved by systematically increasing the contrast of dichoptic noise. Six noise contrast levels  were tested. Other aspects were the same as in Experiment 1, except that no black ring was presented.F=43.4, p<0.001; MO: F=4.5, p<0.03; DP: F=11.8, p<0.001; DO: F=1.3, p > 0.3; One-way ANOVA with repeated measures). When the noise contrast was zero , no surround suppression was observed for each surround configuration, consistent with a previous suggestion that surround suppression is absent in the fovea The individual and averaged results for the three participants are shown in Previous studies reported that surround suppression is strong only in the periphery, but weak or even absent in the fovea. In the study presented here, we tested the effect of a surround mask on the detection of a foveal target that was simultaneously interfered with by an interocular mask. We observed strong surround suppression, especially in parallel surround conditions under which the target detection threshold could be elevated by about a factor of 3. However, when interocular suppression was released by presenting a fusion ring to the two eyes, the strong surround suppression effect was no longer observed, suggesting that the interocular suppression plays a critical role in producing the robust surround suppression. In another experiment, we further found that the strength of surround suppression depends on the contrast of the interocular mask. The strongest surround suppression was found only when the dichoptic mask was at a medium contrast level. These findings suggest that surround suppression and interocular suppression are not two independent suppression phenomena, but that there exist interactions between them.How can we explain the present finding that the otherwise weak foveal surround suppression is enhanced to a high level when it is accompanied by interocular suppression? Because no neurophysiological study, to our knowledge, has ever investigated the two forms of suppression in a single experiment, the neural mechanisms underlying their interactions remain unknown. At this point, we can only offer some speculations.Both interocular suppression and orientation-specific surround suppression have been proposed to originate from the visual cortex and to be generated via inhibitory GABAergic interneurons Alternatively, our result may be attributed to high-level cognitive processes. A psychophysical study has shown that surround suppression is dramatically affected by attention: compared with the full attention condition, the suppression effect in the poor attention condition is increased by about a factor of 4 At which stage in the visual pathway do the surround stimuli exert their influence on interocular suppression? A previous study suggested that interocular suppression originates from a stage prior to complete binocular combination Furthermore, in a preliminary experiment, we found that when the noise mask was superimposed onto the target grating in the same eye (overlay suppression), the effects of surround suppression on target detection were very limited see . This isWe have argued above that surround suppression exerts its effects on interocular suppression prior to binocular combination. The question then can be raised of the stage at which the surround suppressive signal originates. In the present study, the most robust suppressive effects were caused by the parallel surround grating, regardless of whether it is presented to the same eye as the target or to the other eye, i.e., the iso-orientation surround suppression can transfer across the eyes. This strong interocular transfer of surround suppression has also been reported in previous psychophysical In the present study, the center target and the dichoptic mask were briefly presented, and the detectability of the target was impaired by the mask because of interocular suppression. However, if the dissimilar stimuli were continuously presented to different eyes, we would then observe another suppression phenomenon – binocular rivalry, in which perception alternates between the images of the two eyes every few seconds.Because binocular rivalry and interocular suppression are produced under very similar stimulus condition, they are usually thought to involve common neural mechanisms. One piece of evidence has been provided by Baker and Graf. They reported that the mean dominance durations in binocular rivalry correlate with the magnitude of interocular suppression within, as well as between, observers. Here, we found that interocular suppression was influenced by surround inputs, especially when the surround and the target had the same orientation. If binocular rivalry shares common mechanisms with interocular suppression, it should also be greatly affected by contextual stimuli. In line with this prediction, Paffen et al. (2005) have reported that the discrimination of a speed probe, which was presented on a drifting grating undergoing binocular rivalry suppression, is further impaired when a surround motion is added, suggesting that binocular rivalry suppression can be deepened by contextual stimuli Furthermore, the dynamics of binocular rivalry has also been found to be influenced by contextual stimuli. For example, the predominance of a rival stimulus is greatly decreased by the presence of a context sharing the same properties  as the rival target We have shown that the detection of a foveal target that is simultaneously undergoing interocular suppression is further strongly impaired by the presence of surround stimuli. This surround suppression effect is influenced by the manipulation of the interocular suppression and varies with the strength of interocular suppression. These results suggest that surround suppression and interocular suppression are not independent of one another, but that there are interactions between their underlying neural mechanisms. In addition, our results also suggest that surround suppression does not only prevail in the periphery, but can, under specific conditions, also exhibit a strong effect in the fovea.


TGFB1) +869 T/C and +915 G/C polymorphisms on AR risk. However, the results obtained are inconclusive.Transforming growth factor-beta 1(TGF-β1) is involved in the development of acute rejection (AR) episodes in solid organ transplant recipients; and a number of studies have been conducted to investigate the combined effects of human TGF-β1 gene . In addition, subgroup analysis by transplant types demonstrated a similar association in patients receiving heart transplant .Fourteen eligible studies with 565 AR cases and 1219 non-AR cases were included. Overall, a significantly decreased risk was detected in patients carried with intermediate producer (IP) haplotypes  and/or low producer (LP) haplotypes  compared with high producer (HP) haplotypes (T/T G/G and T/C G/G; IP vs. HP: OR = 0.75, 95% CI, 0.58–0.96, P TGFB1 HP haplotypes were significantly associated with an increased risk for AR in solid organ transplant recipients, particularly patients receiving cardiac allograft.The current meta-analysis and systematic review indicated that recipient  Transforming growth factor-beta 1 (TGF-β1) is a multifunctional cytokine ubiquitously produced by a wide variety of cells, including T lymphocytes, monocytes, vascular endothelium, and renal tubular cells TGFB1) is mapped on the chromosome 19q13.1–13.3 with seven exons and six introns, whose regulation and expression is influenced by the presence of single nucleotide polymorphisms (SNPs) TGFB1 have been the focus of extensive researches and donor TGFB1 +869 T/C polymorphism has been proven to be significantly associated with AR risk in vitro production levels: high producer (HP) (T/T G/G and T/C G/G), intermediate producer (IP)  and low producer (LP)  TGFB1 +869 T/C and +915 G/C polymorphisms with AR risk in kidney transplant recipients The human TGF-β1 gene (TGFB1 +869 T/C and +915 G/C polymorphisms on AR risk after solid organ transplantation and 2) the potential influence of covariants such as ethnicity, transplantation types, and immunosuppressive protocols.In this meta-analysis, we integrated the data from all eligible studies to explore 1) the combined effects of recipient TGFB1 +869 T/C and +915 G/C polymorphisms with AR risk in solid organ transplantation, a comprehensive electronic search of PUBMED, EMBASE, China National Knowledge Infrastructure (CNKI), and Wanfang databases was performed until November 29, 2013. To search and include as many related studies as possible, we applied various combinations of the following medical subject headings and key words: transforming growth factor beta-1, TGFβ-1, or TGFB1; acute rejection, early allograft outcome, or graft rejection; transplantation or organ transplantation; polymorphisms or variants. Furthermore, the reference lists of reviews and retrieved articles were manually screened for additional studies No restrictions were placed on language, and only published studies with full-text articles were included.This meta-analysis was conducted and reported in accordance with the PRISMA  guidelines  [30]. ToTGFB1 haplotypes (at position +869 and +915) with AR risk; and 3) providing sufficient data to calculate the odds ratio (OR) and its corresponding 95% confidence interval (CI). When several studies with overlapping data were eligible, those with smaller sample size or less reliability were excluded. In addition, studies without detailed information were excluded, after the efforts to extract data from the original paper or contact the corresponding authors failed.The studies identified from the above mentioned databases were screened by two independent authors (Yu-Zheng Ge and Ran Wu) according to the following predesigned inclusion criteria: 1) case-control design; 2) evaluating the correlation of Data of eligible studies were extracted by two reviewers (Yu-Zheng Ge and Tian-Ze Lu) independently and in duplicate according to the predesigned data-collection form. The following information was extracted: last name of first author, publication year, country of origin, ethnicity, transplantation type, immunosuppressive protocol, number of both ARs and non-ARs and phenotypic distribution in both groups. Different ethnic descents were categorized as Asian, Caucasian, African and Mixed (which included more than one ethnic descent). Transplantation types were characterized as renal, liver, and heart transplantation. Based on different calcineurin inhibitors (CNIs) within the immunosuppressive protocol, the studies were divided into two subgroups: Cyclosporine A (CsA) group and CsA/tacrolimus (FK506) group. Discrepancies occurring during the process of study inclusion and data extraction were resolved by discussion with a third reviewer (Wen-Cheng Li), and consensus on each item was achieved eventually.TGFB1 gene and AR risk. The HP haplotypes (T/T G/G and T/C G/G) were used as the baseline for calculation of ORs in three different comparisons . Stratified analyses were also conducted based on ethnicity, transplant types, and immunosuppressive protocols. The statistical significance of the pooled OR was assessed with the Z test, and P<0.05 was considered significant.Crude OR and its corresponding 95% CI were used to assess the strength of association between the haplotypes of +869 T/C and +915 G/C polymorphisms in Chi-square based Q-test was performed to measure between-study heterogeneity, and the presence of heterogeneity was considered significant if P<0.10 All statistical analyses were performed with STATA software  and Review Manager .Fourteen eligible articles were identified according to the predesigned selection criteria, and the detailed screening process was shown in TGFB1 +869 T/C and +915 G/C polymorphisms with AR risk of solid organ transplantation. Overall, a significantly decreased risk was detected in two comparisons . TGFB1 haplotypes (at position +869 and +915) and AR risk in transplant recipients.A total of 14 studies including 565 AR and 1219 non-AR cases were identified to assess the haplotypic association of No significant between-study heterogeneity was observed in this meta-analysis . SensitiTo examine the publication bias of the currently available literature, both Begg's funnel plot and Egger's test were conducted. The shape of the funnel plots did not reveal any evidence of obvious asymmetry in all comparison models , S2, S3.TGFB1 +869 T/C and +915 G/C polymorphisms on AR risk. In the current study, we provide evidence that patients carried with TGFB1 HP haplotypes (T/T G/G and T/C G/G) are more likely to suffer from AR after solid organ transplantation , which could be utilized to identify patients predisposed to AR and potentially benefiting from tailored immunosuppressive protocol.Organ transplantation has been recommended as the most optimal treatment choice for patients suffering end-stage disease. However, AR still remains a crucial determining factor which influences the short-term function and long-term outcome of both recipients and allografts TGFB1 IP and/or LP haplotypes were less likely to suffer from AR than those with HP haplotypes . Considering that ethnic background could influence the frequency of genotypes and haplotypes in some context, we conducted subgroup analysis based on ethnicities but failed to detect any significant association. Further stratified analysis by transplant types demonstrated a remarkably decreased AR risk for heart transplant recipients carried with TGFB1 IP and/or LP haplotypes, which was in agreement with the study conducted in a cohort of 111 pediatric cardiac transplant recipients TGFB1 haplotypes and AR risk. However, FK506 has never been administrated as a unique CNI in any included studies. Therefore, we divided the studies into two subgroups: CsA arm and CsA/FK506 arm, and then conducted a subgroup analysis. As a result, patients with TGFB1 IP haplotypes were less likely to be affected by AR in CsA arm , which could be interpreted as a clue that the recipients with TGFB1 IP haplotypes can benefit from CsA after organ transplantation.After pooling the data from 14 eligible studies, we shown that patients with TGFB1 +869 T/C HP genotype (TT) was significantly associated with decreased AR risk TGFB1 +869 T/C and +915 G/C polymorphisms on actual production levels of TGF-β1. The effects of the nine potential haplotypes on TGF-β1 expression levels have been previously studied in vitro and among patients without organ transplant TGFB1 SNPs and TGF-β1 plasma levels or intragraft mRNA levels was detected in a cohort of renal transplant recipients TGFB1 haplotypes on AR risk. The current study is confined to recipient SNPs; however, in some context, donor polymorphisms may contribute much more to AR risk than recipient SNPs TGF-β1 is a pleiotropic and multifunctional cytokine with immunosuppressive and fibrogenic properties, which may play a central role in both the initiation and propagation of AR and chronic rejection (CR) TGFB1 +869 T/C and +915 G/C polymorphisms rather than one single SNP on AR risk TGFB1 SNPs in the development of AR episodes. However, several limitations should be considered when interpreting the results. First, considering the unavailability of other detailed information, we did not conduct stratified analysis based on some cofactors such as follow-up time, gender, age, panel reactive antibodies level, human leukocyte antigens mismatch and donor source, which may influence the results. Second, the limited number of AR cases and non-AR cases may lead to a relatively small power. Third, only published studies with sufficient data were included, thus, publication bias may have occurred even though results of both Begg's test and Egger's test did not detect it. Last but not least, the meta-analysis is retrospective due to the methodological limitations.The current meta-analysis focused on the combined effects of TGFB1 HP haplotypes of +869 T/C and +915 G/C polymorphisms (T/T G/G and T/C G/G) might be a possible genetic susceptibility locus for AR after solid organ transplantation, which could be utilized to identify patients predisposed to AR and potentially benefiting from personalized immunosuppressive protocol. In addition, monitoring TGFB1 could help manage CR to some extent, as TGFB1 triggers fibrogenesis linked to chronic rejection (CR). Further well-designed and unbiased studies with larger sample size, diverse ethnicities, donor-recipient pairing and various applications of CNIs should be conducted to verify our findings. Furthermore, functional studies of TGFB1 gene polymorphism are warranted to understand the underlying mechanisms.In summary, this meta-analysis suggested that recipient Figure S1Begg's funnel plot for publication bias test (LP vs. HP for TGFB1 haplotypes).(TIF)Click here for additional data file.Figure S2Begg's funnel plot for publication bias test (IP vs. HP for TGFB1 haplotypes).(TIF)Click here for additional data file.Figure S3Begg's funnel plot for publication bias test (LP/IP vs. HP for TGFB1 haplotypes).(TIF)Click here for additional data file.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.Table S1Statistical analyses of publication bias for TGFB1 haplotypes at +869 T/C and +915 G/C polymorphisms.(DOC)Click here for additional data file.


The increasing challenges and complexity of business environments are making business decisions and operations more difficult for entrepreneurs to predict the outcomes of these processes. Therefore, we developed a decision support scheme that could be used and adapted to various business decision processes. These involve decisions that are made under uncertain situations such as business competition in the market or wage negotiation within a firm. The scheme uses game strategies and fuzzy inference concepts to effectively grasp the variables in these uncertain situations. The games are played between human and fuzzy players. The accuracy of the fuzzy rule base and the game strategies help to mitigate the adverse effects that a business may suffer from these uncertain factors. We also introduced learning which enables the fuzzy player to adapt over time. We tested this scheme in different scenarios and discover that it could be an invaluable tool in the hand of entrepreneurs that are operating under uncertain and competitive business environments. A decision is a (conscious) choice of a move (or action) from among a well-defined set of alternatives and an individual decision maker is motivated to act in such a manner that the expected value to him of the outcome is as high as possible Shubik . The indThe decision maker is an individual at the simplest level and a decision process must have a purpose in so far as it only exists to further a particular objective or goal of the decision maker Oderanti . When falinguistic approach  in capturing uncertainties that may surround his business environments. This will help the entrepreneur to have competitive advantages over his competitors. The models also include learning procedures that enable the agents to optimize the fuzzy rules and their decision processes. This is another contribution of the paper: a set of fuzzy models that include learning, and can be used to improve decision making in business.As the complexity of a system increases, the utility of fuzzy logic as a modeling tool increases Oderanti . For verFuzzy logic provides a framework that attempts to define a natural way of dealing with problems in which the source of imprecision is the absence of sharply defined criteria of class membership rather than the presence of random variables Zadeh . It is aIn general, a fuzzy decision making system (FDMS) uses a collection of fuzzy membership functions and decision rules that are solicited from experts in the field to reason about data , reserved or generated wealth (W) and aggressive marketing efforts (M). These resource allocations will be done simultaneously by both players. Only the opponent’s move history will be known, but without knowledge of the opponent’s current choice of strategy. The allocations are denoted as a vector  for each player and constitute the strategy of that player.In our experiment, each player is given five units of initial resources which may represent capital, time, personnel or other business resources. In this case study, we assume capital (say £5M). In each round, the players may choose to allocate their resources to one of three roles: C refer to the proportion of resources that are spent to retain existing customers (if any) such as various customer service improvements, customer care, satisfaction, delight and customer retention initiatives. Marketing aggressiveness M denotes the part of these resources that are allocated to various advertising, marketing and promotional campaigns. These are principally targeted towards getting new customers. Reserved wealth W refers to part of the resources that are kept unused in the firm’s coffer.Consolidation efforts Y that is a new entrant into a market. Y does not have existing customers to consolidate at the start of the game and therefore has C = 0. It may then decide to allocate all or most of its resources on advertising (marketing) campaigns M. If it chooses to allocate all to marketing M = 5, then its strategy  becomes . This is considered to be the strongest strategy. We refer to it in Section "Fuzzy inference system for business decisions (FISBD)" Step 11, as globally optimizing player (Geq).As examples of players’ strategies, consider a firm Y enters the market with a much reduced price Esp (probably as a result of new technology which leads to reduced production cost Cp). If it is economically impossible for the incumbent (existing) firm G to cut its price to the same level due to its high production cost Cp, G may decide to devote most of its resources (say £4M) to consolidate its existing customers in order to retain its market share. It may then decide to allocate the remaining resources Mg to market new customers. Therefore, G’s strategy  becomes .Assume C,W,M] out of his available total resources (say £5M). This is how firms allocate resources to their core strategies for competition (such as advertisement) and how these allocations could affect their payoffs in an uncertain or fuzzy market environment.The difference between strategies of different players is the proportion, number or amount that each player decides to allocate to each component of his own strategy  for each player.Our FISBD involves two players (firms) in a typical duopoly market which we shall represent as green (D) and the production costs(CP).List all uncertain (fuzzy) factors that will be considered in taking the business decision: the uncertain or fuzzy information (factors) we are taking into consideration in this illustration are anticipated market demand information  represented as green (g) with strategy represented as  and yellow (y) with strategy represented as .Determine the strategies of the players: Here, we are adopting three strategies for each player and these strategies are consolidation effort, wealth created or reserved and aggressive marketing efforts denoted as a vector with three elements  and yellow player strategy is denoted as  where: Play the game: The procedure for playing the game is as follows: The game state is represented as vector ) Initial stage of the game is  . We define Ew = Esp - CP, where Where Esp represents the expected selling price of the product.and r = 0 and if pence is greater than zero (pence > 0), the green player wins, if less than zero (pence < 0), then the fuzzy agent player (yellow) wins else, the game is draw (i.e. if pence = 0). The game ends when  This 2-player game is a zero sum game and therefore, yellow loses whenever green wins and vice versa and since our aim is to develop an agent that would win as much as possible, maximize his payoff and minimize that of the opponents, Nash equilibrium : Using Matlab fuzzy toolbox, all the fuzzy inputs are passed into the Mamdani type FIS as shown in Figure Get the defuzzified output from the FIS: The crisp output for the FISBD is computed using centre of gravity method (COG) and sampled results are as shown in Figure r reaches 1 counting down from 5 (i.e. when r = 1).Determine whether the conditions for the end of the game have been met: In this case study, the condition for the end of the game is when the number of rounds fminsearch function in Matlab having considered other optimization algorithms such as gradient descent and genetic algorithm. Fminsearch uses the Nelder-Mead Simplex Search Method for finding the local minimum x of an unconstrained multivariable function f(x) using a derivative-free method and starting at an initial estimate.Training and performance evaluation: training and learning , using the notation of (Braathen and Sendstad S was used as in (Braathen and Sendstad strongest opponent choice of strategy (say an opponent with strategy [0 0 5] as in iteration 12 in Table Geq).Consider two players G and Y playing the game, the expected outcome or payoff of a game can be denoted as C W M], this results in FISBD fuzzy agent winning the game with highest payoff and we regard this as equity against a locally optimizing opponent (Leq). Another extreme opponent which may be regarded as weakest opponent will be that which reserves all his resources i.e. an opponent with strategy [0 5 0]  are as listed in the steps below: All these steps that are necessary for playing this game are as summarized in the flowchart shown in Figure n-player games that represent perfect market competitions with many players. Our fuzzy player is still represented as yellow, the n-th player, who faces n - 1 opponent players (competitors). The n-player games also follow the procedural steps of 2-player FISBD general illustrations in Section "Fuzzy inference system for business decisions (FISBD)" with exceptions to steps 2 and 7 which are modified as follows:In this section, we examined n players. For j = 1 to n - 1, the opponents P(j) strategies are denoted as  and the fuzzy agent (yellow) strategy as . Step (2) Determining the strategy: as an example of a perfect market competition, we have S = . Where P1 to Pn-1 represent opponent players’ (competitors) amount of resources, Pn represents fuzzy agent player (yellow) amount of resources, Aw represents opponents’ accumulated wealth (profit) and r is the number of rounds the game is played. Both the competitors and fuzzy player strategy are as stated in step 2 above.  Step (7) Play the game: procedures for playing the game are as follows: The game state is represented as vector r are arbitrary. In a real system, any number that suitably represents the process can be chosen.As explained in Section "Players’ strategies", our choices of the number five in Equation P1,P2,···,Pn-1,Pn,Aw,r]. Initial state of the game is  according to the vector , for j = 1 to n - 1, the opponents P(j) choose their moves (strategies)  where: C(j) + W(j) + M(j) = P(j) = 5 and yellow who is the fuzzy player chooses his strategy . At every state  and when they both chose . In both cases and some other cases, before training, it was green that won the game while after training, it was the fuzzy agent (yellow) that won. Moreover, in all cases, even when green wins, his payoff , before leaning of the fuzzy player, green’s payoffs were 351.6, 136.8 and 1054.5 for those three iterations respectively. However, after learning, these were reduced (minimized) by the From the results explained above, it can be observed that training (learning) of the fuzzy agent was really important and the training algorithm was very effective because it enables the agent to learn and reach the performance criteria.Esp = Ew + CP).At the end of the game, the estimated price for the commodity can be forecast with Equation n-player simulations shown in Table From the n = 3), very interesting cases are seen in those iterations where one expected the fuzzy player to lose because he started the game with weaker strategies than those of his competitors (as it happens in 2-player games), but because the player reasons in accordance with the fuzzy engine (rule base) and changes his strategies accordingly, the fuzzy player wins in those cases, and better than he wins in the 2-player game results shown on Table As shown in Table n ranging from 1 to 100, the results of the n-player FISBD game show that the larger the number of opponent players (competitors), the better the fuzzy player performs, as illustrated in graphs of Figure This shows that the fuzzy player performs better in the 3-player game than in the 2-player games where he won only eight iterations out of thirteen iterations as shown in Table n-player games that represent perfect market structure. A fuzzy inference system for business decisions was designed and implemented using Matlab software. Fuzzy rules were constructed in developing the FISBD model using the Matlab toolbox and the implementation of this model heavily depends on expert knowledge and experience to facilitate the development of a reasonable fuzzy rule base for the determination of the if-then rules that denote the relationship between inputs and the output variables.We have modelled decision making processes under uncertainty in business games, using fuzzy logic concepts and game theory. Our model was termed fuzzy inference system for business decisions (FISBD). We illustrated this for 2-player games that represent duopoly market structure and Furthermore, we have applied a learning algorithm to the decision processes which enables the decision agent to optimize his performance in the decision processes as the games were played so as to meet the set criteria. To do the learning, the Nelder-Mead simplex method for finding the minimum of an unconstrained multivariable function was used.Results of the learning showed that the learning algorithm works very effectively and efficiently as the fuzzy player (yellow) was able to perform much better after learning with higher payoffs and this enables him to reach the set criteriaWe verified these results by designing a control experiment (simulation) in which the fuzzy player does not change his moves in accordance with the fuzzy rule base. The payoff of the fuzzy player in the control experiment (where he did not use fuzzy rule base) are far less than what he got when he used fuzzy rule base to make his business decisions.Our FISBD procedure has practical uses in business contexts as it can serve as very useful tools in the hands of an entrepreneur to: Advise him on certain marketing strategic decision policies that can keep his business in strategic advantage over his competitors in the market. Give him insight on how his firm can successfully compete with its peers in the market by determining how much of its available resources or efforts could be dissipated on our three adopted strategies of marketing in such a way that his profit  will be maximized.D) information, cost of producing a commodity (CP) and other fuzzy information at his disposal to achieve the set goal of his business. Effectively utilize the uncertain (fuzzy) and prevailing or anticipated market demand  nature of most information available to business decision makers. While the traditional laws of demand and supply address the nature of decision processes by consumers and suppliers respectively, our own approach extends them further. This is to address the nature of decision processes by an intending entrepreneur or manufacturer to forecast the prospect of the proposed business through profit prediction from estimated selling price given the fuzzy market or industry information available to him. This allows him to determine price and marketing strategies in function of a very low, medium, high, very high, etc. demand.n-player games that represent perfect market structure. The results of our n-player simulations showed that an entrepreneur needs not to worry about the proliferation of competitors in the industry because by adopting this FISBD model, the results of the n-player games show that the larger the number of opponent players (competitors), the better the fuzzy player performs in the games as shown in Table We have demonstrated that our model works well with large number of players. We illustrated this by using In arriving at our results, the simulations are based on assumptions and conditions that the players involved in the decision processes are rational players (Section "Introduction") and that only the fuzzy player (yellow), at the moment, uses fuzzy moves  and less to the operation department (C) of the organisation. The organisation will then change these roles until desirable results are attained in the business.For example, rather than competing with capital resources (say £5M), the organisation’s competing resources may be in terms of roles assigned to personnels in the organisation. For instance, due to persistent reduction in sales over the last few weeks, an organisation may decide to assign more personnels to the marketing department  of the fuzzy system will also help robot to learn in making better decisions using fuzzy inference systems and also to deal with systems requiring advanced decision making in unpredictable environments Saridis .Also, future work may be carried out to test the system behaviour toward other fuzzy inference techniques. In our model, we have used Mamdani-type fuzzy inference system. However, there are other inference techniques that can be tested on the system and evaluate its performance. other popular common methods of deductive inference for fuzzy systems Ross  that can Sugeno systems Tsukamoto modelsOther areas of future work may also be channelled toward trying other optimization algorithms on the system and evaluate the performance of the models. Other optimization techniques that may also be tried on the models.


At nanomolar concentrations, idebenone protected the rodent RGC cell line RGC-5 against complex I dysfunction in vitro. Consistent with the reported dosing and observed effects in LHON patients, we describe that in mice, idebenone penetrated into the eye at concentrations equivalent to those which protected RGC-5 cells from complex I dysfunction in vitro. Consequently, we next investigated the protective effect of idebenone in a mouse model of LHON, whereby mitochondrial complex I dysfunction was caused by exposure to rotenone. In this model, idebenone protected against the loss of retinal ganglion cells, reduction in retinal thickness and gliosis. Furthermore, consistent with this protection of retinal integrity, idebenone restored the functional loss of vision in this disease model. These results support the pharmacological activity of idebenone and indicate that idebenone holds potential as an effective treatment for vision loss in LHON patients.Leber’s hereditary optic neuropathy (LHON) is an inherited disease caused by mutations in complex I of the mitochondrial respiratory chain. The disease is characterized by loss of central vision due to retinal ganglion cell (RGC) dysfunction and optic nerve atrophy. Despite progress towards a better understanding of the disease, no therapeutic treatment is currently approved for this devastating disease. Idebenone, a short-chain benzoquinone, has shown promising evidence of efficacy in protecting vision loss and in accelerating recovery of visual acuity in patients with LHON. It was therefore of interest to study suitable LHON models  Leber’s hereditary optic neuropathy (LHON) is a rare inherited mitochondrial disorder with an estimated prevalence in Europe of 1∶45000 LHON is caused by well characterized mitochondrial DNA (mtDNA) mutations, among which three so called primary mtDNA mutations account for more than 95% of all LHON cases: 11778G>A (ND4 subunit) Idebenone, a short-chain benzoquinone, is a potent antioxidant and inhibitor of lipid peroxidation Here we describe the pharmacological effects of idebenone in an RGC cell line and a well-established mouse model of LHON and demonstrate the protective effect of idebenone on retinal toxicity and visual impairment induced by complex I dysfunction.For mouse dietary treatment, idebenone (2-(10-Hydroxy-decyl)-5,6-dimethoxy-3-methyl- 2, 90% humidity) in DMEM , Penicillin-Streptomycin-Glutamine (PSG)). For experiments, 3×103 RGC-5 cells per well were seeded in DMEM  into BD Falcon™ 96-well plates. RGC-5 cells were constantly exposed to either idebenone or vehicle from one day after seeding (pre-incubation), together with rotenone (co-incubation) or after the rotenone stress (post-incubation). For the 1 day post-incubation period, cells were cultured in Hanks’ BSS .RGC-5 cells were cultured under standard conditions  was combined with 10 µl cell lysate to start the reaction. Idebenone-mediated rescue in cell viability was defined as the percentage of idebenone-mediated increase in cell viability (based on ATP content) relative to the rotenone-induced reduction in cell viability (based on ATP content).As a measure of cell viability, the cellular ATP content was quantified by luminescence from the ATP-dependent enzymatic oxidation of luciferin by luciferase and using a multimode plate reader . Briefly, RGC-5 cells were washed in PBS and lysed in 100 µl lysis solution  for five minutes at room temperature. In 96-well plates, 100 µl of ATP measurement buffer  obtained from Janvier (France) were single-housed in Makrolon type II cages under a regular 12-hours light-dark cycle (8 am to 8 pm) with free access to food and water. All procedures were performed in accordance with the Swiss regulation on the use and care of animals and under the required license approved from the Kantonales Veterinäramt Basel-Land (permission Nr. 415).Mice were treated daily with dietary doses of idebenone or vehicle for 21 days or were treated orally by gavage with a single 60 mg/kg dose of idebenone. Mice were sacrificed and biological fluids were sampled 17 to 20 hours following dietary treatment and at time intervals from 5 min to 6 hours after gavage dosing. Briefly, mice were anesthetized by 5% isofluran inhalation in an induction chamber and sacrificed by cervical dislocation followed by decapitation. Blood was collected from the head into EDTA-containing tubes for plasma preparation. Individual plasma samples  or pooleIntravitreal injection of rotenone (5 and 15 mM in DMSO) or DMSO (vehicle) was performed as previously described Mice were anesthetized by i.p. injection of ketamine/xylazine  7 days after rotenone injection and transcardially perfused with 4% PFA in PBS 0.1 M (pH 7.4). The left eyes (injected) and the right eyes (uninjected controls) were rapidly removed and the corneas were eliminated at limbus level; vitreous humor and crystalline lens were discarded. Tissue samples were labeled with random numbers by a person not involved in the study to ensure blinding during the analyses. This person kept the ‘treatment allocation’ list to himself and persons involved in tissue processing and data analysis were blind to the treatment allocation at all times. The samples thus obtained were post-fixed with 4% PFA in PBS 0.1 M (pH 7.4) overnight at 4°C. Samples were soaked in a PBS 0.1 M, 30% sucrose solution for 4 days at 4°C, embedded in freezing resin , cryostat-cut in longitudinal sections (14-µm thick), placed on glass slides and stored at -20°C until stained. Sections were blocked and permeabilized in 0.1 M PBS, 0.3% Triton X-100 (Sigma) and 5% heat-inactivated horse serum  for 1 h at room temperature. Slices were then incubated overnight at 4°C with primary antibodies in 0.1 M PBS, 0.3% Triton X-100 and 5% HS. The following primary antibodies were used: mouse monoclonal and rabbit anti-Brn3a  visual recovery by counting the number of clockwise head movements per 2 min session prior to rotenone injection and at different time intervals following rotenone injection, 2) number of mice showing clockwise head movements at different time intervals following injection and expressed as the percentage of responder mice, and 3) visual acuity of the non-injected eye used as a control by counting the number of counterclockwise head movements per 2 min session prior injection and at different time intervals following injection. The overall experimental process was blinded at each step to ensure that the persons involved in optomotor testing, counting optomotor responses and analyzing the data were not aware of the treatment given to the animals. At the end of the analysis, the study was unblinded and the treatment allocation was matched to the data.Optomotor testing) was used to control for successful intraocular rotenone injection. In response to the injection of rotenone, a total loss of visual acuity was generally observed within 24h in the optomotor test system. Consequently, animals not showing complete loss of optomotor response at 24h post rotenone injection were excluded from histological and behavioral analyses.To correct for possible deviations in the procedure that could lead to erroneous conclusions, immunolabeled sections were evaluated for staining quality. Specifically, samples were excluded from the quantitative analysis in case of weak or unspecific fluorescence signal defined as relative fluorescence units (RFU) ≤20% of standard values or ≤120% of the background fluorescence. In addition, functional quantification of visual acuity  was used to compare non-normalized data. Statistical significance was set at p≤0.05(*), p≤0.01(**) and p≤0.001(***).in vitro.To assess a cytoprotective effect of idebenone, we recapitulated the situation of mutated mitochondrial complex I in LHON RGC cells by exposing the retinal ganglion cell line RGC-5 We previously reported that next to its antioxidant activity, one important function of idebenone is to bypass a dysfunctional complex I. This mechanism involves an alternate electron flux directly to complex III and can restore cellular ATP levels in vitro, we next assessed whether such concentrations of idebenone could be achieved in the mouse eye following oral administration of idebenone. Following treatment with idebenone given in the diet over a period of three weeks or after a single oral administration (gavage) in the adult mouse, the concentrations of idebenone in the aqueous and vitreous humor and plasma were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). After administration of idebenone through the diet of male mice from 20 to 2000 mg/kg of body weight/day, concentrations of idebenone in aqueous and vitreous humor increased approximately dose proportionally, similar to the increase observed in the plasma  of 37.4 ng/ml (111nM) in the aqueous humor at the first sampling time 5 min post-dose (tmax)  , S3. Thee (tmax) , suggeste (tmax) , suggestn plasma . Similarin vivo. Mice were pre-treated for 3 weeks with idebenone or vehicle in the diet before rotenone was injected intravitrealy into the left eye. Retinal ganglion cell number, retinal thickness and reactive gliosis were quantitated based on immunohistochemical analysis of retinal sections 1 week after rotenone challenge  is caused by mutations in complex I of the mitochondrial respiratory chain, which leads to complex I dysfunction, decreased ATP production and oxidative stress. The pathology of LHON is characterized by swelling of the retinal nerve fiber layer (RNFL) which precedes loss of retinal ganglion cells (RGC). This dynamic process evolves in parallel with functional impairment and atrophy of the nerve in vitro and in vivo models of LHON described here. This can be achieved by comparing the dose effective in the prevention of rotenone-induced cell death in the in vivo mouse model and in the clinical trial on a mg/kg and/or mg/m2 basis. The clinical dose of 900 mg/day idebenone (Catena®) was administered as 300 mg three times a day (tid) which corresponds to 15 mg/kg/day or 5 mg/kg tid for an average patient weight of 60 kg in vitro  Photograph of the experimental set up used for assessing visual acuity. (b) Visual acuity was also evaluated by counting the ratio between clock-wise and counterclock-wise head movements (CW/CCW) within 2 min. 7 days prior injection (day -7) and 1 to 70 days after injection  of 5 mM rotenone. Quantification following vehicle treatment and rotenone injection , and idebenone 2000 mg/kg treatment and rotenone injection . Data are expressed as mean ± SEM.(TIF)Click here for additional data file.Table S1Concentrations of idebenone in plasma, aqueous and vitreous humor following repeated administration of idebenone in the diet to male mice. Data are expressed in ng/ml (± SEM). Idebenone was administered at 20, 40, 200, 400, and 2000 mg/kg in the diet. Idebenone concentration was measured at least 8 hours post last dose. blq: below limit of quantification (2 ng/ml); n = number of mice used for sampling; samples were pooled as outlined in Material and Methods section.(DOCX)Click here for additional data file.Table S2Pharmacokinetic profile of idebenone in plasma, aqueous and vitreous humor following single oral administration of idebenone at 60 mg/kg to male mice. Data are expressed in ng/ml (± SEM). Idebenone concentration was measured 5, 15, 30, 60, 120, 240, and 360 min post-dose. blq: below limit of quantification (2 ng/ml); n = number of mice used for sampling. Samples for aqueous and vitreous humor were pooled as outlined in the Material and Methods section.(DOCX)Click here for additional data file.Table S3Pharmacokinetic parameters of idebenone in plasma, aqueous and vitreous humor following single oral administration of idebenone at 60 mg/kg to male mice. Data are expressed as mean for plasma based on n = 20 mice per time point. Samples for aqueous and vitreous humor were pooled as outlined in the Material and Methods section.(DOCX)Click here for additional data file.Table S4Effect of idebenone (2000 mg/kg) treatment on head movements of mice with rotenone (5 mM)- and sham (DMSO)-injected eyes. Optomotor analysis of the rotenone (5 mM)-injected eye and the sham (DMSO)-injected eye in C57BL/6J mice was performed by counting clockwise (CW) and counterclockwise (CCW) head movements. Head movements were evaluated at a velocity of 3 rpm 7 days prior injection (day -7) and after injection . Optomotor data expressed as mean number of head movements/2 min. ± SEM. Statistical significance relative to vehicle + rotenone group: p≤0.05 (*) based on raw numbers; n = number of mice per group. CW: clockwise head movements; CCW: counterclockwise head movements; vehicle + rotenone: vehicle treated and injected with 5 mM rotenone; IDE2000+ rotenone: idebenone (2000 mg/kg) treated and injected with 5 mM rotenone.(DOCX)Click here for additional data file.Video S1Normal clockwise (CW) and counter-clockwise (CCW) head movements during the optomotor response of a representative mouse, which was sham (DMSO)-injected into the eye.(WMV)Click here for additional data file.Video S2Absence of clockwise (CW) head movements during the optomotor response of a representative mouse, which was rotenone-injected into the eye.(WMV)Click here for additional data file.


Past theory and research view reciprocal resource sharing as a fundamental building block of human societies. Most studies of reciprocity dynamics have focused on trading among individuals in laboratory settings. But if motivations to engage in these patterns of resource sharing are powerful, then we should observe forms of reciprocity even in highly structured group environments in which reciprocity does not clearly serve individual or group interests. To this end, we investigated whether patterns of reciprocity might emerge among teammates in professional basketball games. Using data from logs of National Basketball Association (NBA) games of the 2008–9 season, we estimated a series of conditional logistic regression models to test the impact of different factors on the probability that a given player would assist another player in scoring a basket. Our analysis found evidence for a direct reciprocity effect in which players who had “received” assists in the past tended to subsequently reciprocate their benefactors. Further, this tendency was time-dependent, with the probability of repayment highest soon after receiving an assist and declining as game time passed. We found no evidence for generalized reciprocity – a tendency to “pay forward” assists – and only very limited evidence for indirect reciprocity – a tendency to reward players who had sent others many assists. These findings highlight the power of reciprocity to shape human behavior, even in a setting characterized by extensive planning, division of labor, quick decision-making, and a focus on inter-group competition. Reciprocity refers to various patterns by which individuals exchange favors, support, goods, or other valued resources. Past research has identified various forms of reciprocity, including direct While past research suggests that fundamental motivations lead humans to engage in direct reciprocity, and perhaps also indirect and generalized reciprocity, few studies have explored whether patterns of reciprocity might help explain behavior in highly-structured group settings where resource sharing is largely a product of planning, strategy, and division of labor. However, such organizational contexts are both ubiquitous and socially significant. Further, documenting reciprocity in a setting such as this – where reciprocity is neither readily apparent nor explicit, and where benefits of reciprocity are not clearly evident for either the individual or group – would offer more convincing evidence that engagement in these forms of behavior is in fact based in strong motivations.To this end, we investigate whether patterns of passing in professional basketball games exhibit the same patterns of reciprocity found with other resource sharing. One might reasonably wonder whether reciprocity plays any role at all in this domain, as passing in professional basketball games is heavily structured as a result of carefully planned strategy and an explicit division of labor on the court prescribing who passes to whom. Further, passing to someone on the basis of past passing patterns, rather than an assessment of what is the most productive pass to make in a given situation, does not clearly benefit either the player or team. Nonetheless, given theory and research suggesting the fundamental nature of reciprocity, it is possible that these dynamics in fact structure passing behavior, producing hidden patterns that would not be immediately observable without systematic analysis. Below we present each form of reciprocity, identifying the type of resource exchange it describes, and highlighting the social psychological mechanisms thought to drive it.Direct reciprocity involves an actor, A, repaying B for benefits received from him/her in the past. This pattern of reciprocal resource sharing is depicted in Indirect reciprocity occurs when a benefactor is rewarded by third parties for behaving generously towards others, i.e., when A rewards B for having given to some third party, C, in the past. This pattern is depicted in Generalized reciprocity involves A repaying benefits to B that A received from some third party, C, in the past. This pattern is portrayed in Passing in basketball can be viewed as a form of resource sharing and thus might reasonably be subject to the same causal forces shaping exchange in other settings. Thus, in the present study we investigate whether these three fundamental forms of reciprocity identified by past research might help explain patterns of passing in professional basketball games.Unfortunately total passing data is not available for NBA basketball games. As a result, here we study patterns of assists, passes determined to lead directly to a made basket. Note that this provides an imperfect measure of passing behavior, however there is some reason to think that assists might offer better insight on reciprocity dynamics in this context. One justification of this measure lies in the fact that assists are especially valuable passes, as they lead directly to scoring which benefits the individual and team. Thus, there is reason to think that assists are more likely to be viewed by players as the sort of “valued resources” that forms of reciprocity apply to. As a result, “receiving” assists should feel like receiving valued rewards, more than receiving other passes. In addition, there is good reason to think that players often know that a pass is likely to lead to a scoring opportunity, such as passes to players who are undefended or nearer to the hoop. Thus, it is also likely that benefactors perceive that “giving” assists entails greater value than making other sorts of passes.For each assist, we know, 1) who has given and who has received the assist, and 2) the exact game time of the event. These data allow us to test the following hypotheses:Direct reciprocity hypothesis: A focal player, A, will be more likely to give an assist to another player, B, if A has received assists from player B in the past.Indirect reciprocity hypothesis: A focal player, A, will be more likely to give an assist to another player, B, if B has given assists to some other players, C, in the past.Generalized reciprocity hypothesis: A focal player, A, will be more likely to give an assist to another player, B, if A has received assists from some other players, C, in the past.http://www.basketballgeek.com and www.dougstats.com. The latter provided season-wide statistics for each player while the former provided play-by-play accounts for more than 1,000 games of the season, including time-stamped records of all assisted baskets. In our reduced dataset, each assist was represented by a set of four player dyads. The dyads included the player who gave the assist, paired with each of the four other players on the floor at the time. A dyad was coded as “1” if an assist occurred between the two players and “0” otherwise. In all, the dataset included 170,756 such dyads. In what follows, we refer to the player giving the assist as “player A” and the potential recipients as “player B.”In order to test our hypotheses, we analyzed assists occurring in the 2008–09 NBA season, using data published at We analyzed the data using conditional logistic regression models. Conditional logistic regression models are appropriate for predicting the choice among a set of alternatives as a function of different attributes of the choice set iy refers to individual i's choice, m refers to a particular outcome that could be selected, iz refers to a set of predictor variables, and γ refers to the estimated coefficients associated with each predictor variable.Formally, the model is specified as:Coefficients estimated from this model refer to the effect of a unit change in the independent variable on the log odds that player A will choose a particular player B, rather than other potential recipients of an assist.The key independent variable in this analysis was a count of the number of assists A had received from another player, B, but had not yet repaid; i.e., the number of assists A had received from B to that point in the game, minus the number of assists A had given to B. We experimented with different versions of this variable  but ultimately decided to use the continuous variable because models using this variable fit the data best according to BIC statistics. Because the motivation to reciprocate likely attenuates over time Indirect reciprocity corresponds to the desire to help someone who has exhibited helping behavior toward others in the past. In this context, if a focal player were motivated by indirect reciprocity, he would be more likely to assist a player who had frequently assisted others, even if that player had not assisted the focal player. Accordingly, we measured drivers of indirect reciprocity with a count of how many assists player B had given to others, not including A. We also interacted this with the (logged) number of minutes player A and player B had been on the floor together since player B last assisted a player other than A. If player B had never assisted anyone or had never assisted anyone other than player A, we included the total amount of time A and B had been on the floor together to that point in the game.Generalized reciprocity represents the idea that a person may be motivated to give to others if he or she has received favors in the past, even if he does not give back directly to those who have given to him or her. To test for generalized reciprocity, we counted how many assists A had received from everyone on the floor, excluding B. Similar to our time-based interaction terms for direct and indirect reciprocity, we included a variable to measure the time that A and B had been on the court together since A last received an assist from anyone besides B.We controlled for a variety of factors that might cause a player to be chosen as the recipient of an assist more frequently than others. In order to capture the fact that a player's position is a major driver of the role he plays on the team with respect to assisting behavior, we included indicators for B's position . We also controlled for player B's field goal percentage (shots made per attempt), assists per game, shots attempted per game, and points per game for the 2008–9 season. We included the average number of minutes played per game (logged) by player B. To capture the idea that players who are perceived as having a “hot hand” might tend to receive more assists, we controlled for the number of shots player B had made so far in the current game. To account for the possibility that players might “take turns,” tending to pass to those who had not recently had an opportunity to take a shot, we also controlled for the number of shots player B had attempted in the current game. We also controlled for the number of minutes player A and player B had been on the court together to that point in the current game.We also controlled for player A's position, average minutes played per game (logged), and average assists per game for the 2008–9 season. Because these characteristics of player A do not vary within a possession, we are unable to estimate their effects by including them in the model as is. As a result, in order to ensure variation on these variables within a given possession, we chose to interact player A's characteristics with the number of minutes A and B have overlapped on the court so far in the current game. Finally, we accounted for possible differences in teams' strategies with respect to assists by including team indicator variables, again interacted with the number of minutes A and B have overlapped on the floor until the current point in the game. b = 1.581, p<.001). Moreover, the greater a player's average shots attempted per game for the season, the more likely he is to receive an assist .0.101−1) higher that player A will assist player B. The negative interaction term indicates that this effect diminishes over time, consistent with our expectation.In model 2, we tested for direct reciprocity by including the count of how many assists player A “owes” player B. This variable is not significant. However, the results of model 3 provide evidence of a direct reciprocity effect once we account for the fact that the motivation to reciprocate is likely to decline over time. Model 3 includes the interaction of the count of assists owed and the (logged) time since B last assisted A. Conditional on giving an assist to anyone, for each additional assist received from B that has not yet been repaid, odds are 10.6% . Thus, being responsible for an assist may not be seen as a strong indicator that one is generous and deserves to be rewarded by third parties. Still, given the robustness of past research on indirect reciprocity, the prospect that more generous basketball players are subsequently rewarded by their teammates – even those they did not directly benefit – deserves further attention. The lack of evidence for generalized reciprocity may be a product of the subtlety of this effect. While past research has documented tendencies for people to “pay forward” favors received, these effects appear to be much smaller than corresponding direct reciprocity effects.In the setting we studied, individuals tended to repay assists received from teammates with direct reciprocity, though neither individual nor team performance was clearly served by such behavior. But while testimony to the power of reciprocity, our findings cannot speak to what psychological mechanism(s) – e.g., internalization of cultural norms, feelings of indebtedness, a hope that reciprocity might lead to future benefits for oneself – might drive these effects, providing a potentially fruitful avenue for future investigation. These findings underscore the strength of human motivations to engage in direct reciprocity, demonstrating that it obtains even in a setting where individual performance is highly salient and rewarded, player roles are clearly defined, and within-game strategy and coaching prescribes much passing behavior.