The pathological cascade leading from primary storage to neural cell dysfunction and death in metachromatic leukodystrophy (MLD) has been poorly elucidated in human-derived neural cell systems. In the present study, we have modeled the progression of pathological events during the differentiation of patient-specific iPSCs to neuroepithelial progenitor cells (iPSC-NPCs) and mature neurons, astrocytes, and oligodendrocytes at the morphological, molecular, and biochemical level. We showed significant sulfatide accumulation and altered sulfatide composition during the differentiation of MLD iPSC-NPCs into neuronal and glial cells. Changes in sulfatide levels and composition were accompanied by the expansion of the lysosomal compartment, oxidative stress, and apoptosis. The neuronal and glial differentiation capacity of MLD iPSC-NPCs was significantly impaired. We showed delayed appearance and/or reduced levels of oligodendroglial and astroglial markers as well as reduced number of neurons and disorganized neuronal network. Restoration of a functional Arylsulfatase A (ARSA) enzyme in MLD cells using lentiviral-mediated gene transfer normalized sulfatide levels and composition, globally rescuing the pathological phenotype. Our study points to MLD iPSC-derived neural progeny as a useful in vitro model to assess the impact of ARSA deficiency along NPC differentiation into neurons and glial cells. In addition, iPSC-derived neural cultures allowed testing the impact of ARSA reconstitution/overexpression on disease correction and, importantly, on the biology and functional features of human NPCs, with important therapeutic implications. Sulfatide storage mainly affects oligodendrocytes and Schwann cells leading to progressive demyelination and dysfunction of the central and peripheral nervous system . Rapid motor and cognitive decline, and premature death are typical of the late-infantile forms of MLD. Juvenile forms display a slower progression of motor symptoms often preceded by cognitive and behavioral problems, which characterize the adult form8.Metachromatic Leukodystrophy (MLD) is a rare genetic lysosomal storage disorder (LSD) caused by the functional deficiency of Arylsulfatase A . ARSA catalyzes the desulfation of 3-O-sulfogalactosylceramide (sulfatide)2.Sulfatide occurs as a collection of cell- and tissue-specific molecular species that differ in the length of the acyl chain, the presence of saturated or unsaturated bonds and hydroxylation9 and in immature oligodendrocytes10, astrocytes, and neurons13. Preclinical studies in galactolipid-deficient and ARSA-deficient mice and neural cells15, as well as clinical observations16, suggest that sulfatide storage affects early oligodendroglial development and that sulfatide load beyond myelin might contribute to MLD pathology. Sulfatide is involved in oligodendrocyte survival and proliferation20, negatively regulates oligodendrocyte terminal differentiation17, delays myelin formation21 and has been proposed as a novel myelin-associated inhibitor of axon regeneration in CNS neurons22. Accumulation of sulfatide alters neuronal cell morphology and organization leading to axonal degeneration24 and possibly contributing to motor dysfunctions in MLD patients16. Finally, an excess of sulfatide has been associated with gray matter astrogliosis26. Overall, the molecular events that link the primary storage to the appearance and progression of cell-type-specific pathological phenotypes are poorly elucidated. Also, it remains unknown whether the observations made in murine systems recapitulate critical aspects of the human disease.In the nervous tissue, sulfatide species containing long-chain fatty acids (≥C24) are abundant in myelin. Short-chain fatty acid sulfatide species (C16−C18) are present in the cortical gray matter30. The establishment of MLD patient-specific iPSCs, the engineering of these cells to overexpress a functional human ARSA gene and their efficient differentiation in neural progenitors with therapeutic potential have been recently reported32. However, these studies were not focused on disease modeling and did not investigate the mechanisms of disease in MLD iPSCs and neural progeny.The derivation of patient-specific induced pluripotent stem cells (iPSCs) and their differentiation in cell types of interest have emerged as a powerful tool for human disease modeling and therapeutic screeningIn the present study, we took advantage of MLD iPSCs to model the impact of ARSA deficiency on sulfatide storage and secondary pathogenic events through the differentiation of iPSCs to neuroepithelial progenitor cells (iPSC-NPCs), neurons, astrocytes, and oligodendrocytes. We also highlighted the impact of ARSA reconstitution/overexpression  on disease correction as well as on the biology of human NPCs and neuronal/glial progeny.32. MLD iPSC clones expressing supraphysiological ARSA activity (MLD-ARSA) were obtained by lentiviral (LV)-mediated gene transfer of a functional human ARSA gene32. The iPSC clones used are listed in The normal donor  and patient-specific  iPSC lines/clones used here have been previously describedcis-Golgi matrix protein GM130 and of the early endosome antigen 1 (EEA1) protein 34 34 Fig. . Endo-ly)34 Fig. . The low)34 Fig.  suggest Overall these results suggest that ARSA deficiency is responsible for the moderate expansion of the endolysosome system and intracellular ROS production in MLD iPSCs without major impact on cell survival and proliferation.36  at different stages of differentiation/maturation32. NPCs were exposed to increasing concentrations of growth factors (GFs) promoting the differentiation of glial progenitors , oligodendroglial progenitors/immature oligodendrocytes  and mature oligodendrocytes . Furthermore, ≈5% and ≈10% of cells in these cultures expressed astroglial (GFAP) and neuronal (β-Tubulin III) markers, respectively  using a published protocolely Fig.  promotedIII Fig. . OverallIII Fig.  but supp+ proliferating cells in all the cell populations . However, only MLD cultures showed an exacerbated ROS production  in iPSC-NPC–derived astroglial, oligodendroglial, and neuronal progeny. To this end, we integrated IF and WB analysis evaluating the expression of markers of NPCs (NESTIN), glial progenitors , oligodendroglial precursors (PDGFRα), and mature (non-myelinating) oligodendrocytes , astrocytes (GFAP) and neurons .+ cells at d14; a decrease of GFAP+ cells and CNPase+ cells at d24; Fig. + cells in MLD as compared to ND cultures (significant difference at d24) suggested the occurrence of cell loss and/or delayed/reduced neuronal differentiation. This phenotype was rescued in MLD-ARSA cultures, which also showed higher percentages of β-tubulin III+ as compared to ND counterparts (d24), suggesting a boost of neuronal survival and/or differentiation due to ARSA overexpression . The anticipated/increased expression of β-tubulin III protein in MLD-ARSA cultures as compared to ND counterparts is in line with IF data and suggests an impact of ARSA overexpression on the iPSC-NPC differentiation program.The neuronal and glial defects highlighted by IF analysis were confirmed and extended by WB analysis Fig. . MLD culFig. . The ant+ astrocytes displayed ramified morphology without the long and thin processes that typically run in parallel to neuronal bundles in ND cultures . Interestingly, the supraphysiological ARSA mRNA expression in MLD-ARSA NPCs and differentiated progeny  was accompanied by increased expression of CGT, GALC, and GAL3ST1 enzymes, particularly at the late time points of differentiation  and composition  in ND, MLD, and MLD-ARSA iPSCs, NPCs (d0) and differentiated progeny (d24 and d34) by ultra-performance liquid chromatography-tandem mass spectrometryent Fig. . MLD iPSent Fig.  and sulfent Fig. . Co-locaent Fig. .Fig. 4PrThe C16−C18 sulfatide species were enriched in iPSC-derived cultures Fig.  and disp20 that was different from that of the correspondent iPSCs .All NPC populations displayed neural-like sulfatide compositionSCs Fig. . ND cultig. Fig. .In summary, the little sulfatide load in MLD iPSCs suggests that primary storage is not the direct cause of the lysosomal expansion and intracellular ROS production observed in this cell type. In contrast, the pluripotent to neural transition is accompanied by a significant increase of sulfatide content, which may account for and contribute to lysosomal dysfunction and oxidative stress observed in MLD NPCs. Finally, the progressive sulfatide storage and abnormal sulfatide composition may contribute to the defective glial and neuronal differentiation from NPCs observed in MLD cultures.C ≥ 24 or with hydroxyl groups were predominant in human brain tissues, despite a significant reduction observed in MLD as compared to ND samples .We sought to evaluate the appropriateness of the human iPSC-culture system to model the sulfatide storage and the defective neuronal and glial differentiation in MLD by a direct comparison with autoptic brain tissues of ARSA-deficient MLD patients and age-matched healthy donors  and stage-specific (NPCs vs. differentiated progeny) tolerability to enzymatic deficiency and toxic effects of sulfatide that likely account for the time-dependent progression of disease pathology. The mild elevation of LAMP1 expression and ROS levels in MLD iPSCs was not associated with detectable changes in sulfatide content and composition. Sulfatide metabolism has not been investigated in pluripotent cells but may play a role in protein trafficking, cell adhesion, and cell-to-cell communication59. The neural conversion of iPSCs triggered the acquisition of a neural-specific sulfatide composition20 and promoted sulfatide storage in ARSA-deficient NPCs. Lack of important cell death and functional impairment in MLD iPSC-NPCs despite the presence of lysosomal expansion might relate to the GF-dependent proliferative state of these cells and, possibly, to the proposed intrinsic tolerability of iPSC-NPCs to toxic storage61. The differentiation of MLD iPSC-NPCs into oligodendrocytes, neurons, and astrocytes exacerbated sulfatide storage and cell dysfunction, highlighting significant abnormalities at the morphological, molecular, and biochemical level in the three cell lineages. The increase of ARSA activity and total sulfatide measured in ND neuronal and glial progeny as compared to their NPC counterpart resembles the physiological ARSA increase during CNS development and oligodendroglial differentiation17. Interestingly, we showed similar sulfatide storage in MLD iPSC-derived neural cells and autoptic white matter CNS tissues from MLD patients62. The prevalence of short-chain fatty acid sulfatide  in iPSC-derived neural cultures is likely related to the presence of relevant percentages of non-myelinating oligodendrocytes, neurons, and astrocytes26, in which these species are abundant10. Interestingly, we show an enrichment of sulfatide with short-chain fatty acid (<C24) in the white matter of MLD patients as compared to unaffected individuals, in line with the loss of mature myelin-forming cells. Despite the low proportion of >C24 sulfatide species in the iPSC-derived cultures, their increase at the late stages of differentiation is in accordance with their known expression later in oligodendrocyte development. Moreover, the disease-specific storage recapitulates what observed in white matter tissues of MLD patients.The lysosomal expansion, Golgi abnormalities, accumulation of endo-lysosomes, and increased ROS production described in our cellular model are in line with previous studies describing human iPSC-based models of LSDs21. Here we contribute to clarify this pathogenic mechanism in MLD patient-specific oligodendroglial cells. We show that ARSA deficiency impacts on sulfatide composition beyond sulfatide load. We envisage that the unbalanced C18/C16 ratio might favor the maintenance of immature oligodendroglial progenitors and impair their differentiation. Indeed, CNPase expression is reduced and delayed in MLD cultures during differentiation, while PDGFRα fails to be properly downregulated. The involvement of PDGFRα downstream signaling cascades (e.g. AKT pathway)20 in determining this phenotype remains to be investigated.The role of excessive sulfatide in delaying oligodendroglial differentiation/maturation has been investigated in murine systems16 and from studies describing axonal degeneration and neuronal dysfunction in MLD murine models24. Our data showing reduced and delayed neuronal and astroglial differentiation from MLD NPCs strongly suggest that sulfatide storage in the early stages of human CNS development might contribute to MLD neuropathology. We are currently exploring the mechanisms underlying the early and persistent alteration of neurite organization observed in MLD neurons 64. In our cultures, the flawed neuronal development is accompanied by aberrant morphology and delayed/reduced differentiation of astrocytes, features that have never been described in murine systems. Astrocytes cooperate with neurons on several levels, including formation, maintenance and elimination of synapses; neurotransmitter trafficking and recycling; ion homeostasis; energy metabolism; defense against oxidative stress65. These dynamic cell-to-cell interactions are shaped early in CNS development. Therefore, their disruption might contribute to MLD pathology before overt oligodendroglial cell death and neurodegeneration, with important implications in terms of timing and efficacy of treatments. Additional studies are needed to highlight the mechanism(s) underlying the disease-specific defects in astrocytes and in astrocyte-neuron-oligodendrocytes communication and to dissect the potential cause/effect relationships.The impact of sulfatide accumulation on astroglial differentiation/maturation has been poorly investigated. A direct involvement of neuronal sulfatide storage to MLD pathology derives from clinical observations in MLD infantile patients66. Further studies are needed to assess how and to which extent glycosphingolipid homeostasis impacts on human NPC differentiation in a physiological vs. pathological background, and if such a mechanism contributes to the enhanced neuronal differentiation that we observed in MLD-ARSA cultures. Besides being important from a biological standpoint, this knowledge has important therapeutic implications. The iPSC-based system described here could represent a unique model to fully assess the safety of supraphysiological ARSA expression in human CNS cells in the context of gene therapy settings, complementing the studies performed so far in mice and large animals72 and providing crucial information for designing safe and effective CNS-directed gene therapy approaches.We provide proof of concept that normalization of sulfatide levels and composition achieved by LV-mediated ARSA overexpression in MLD iPSCs and neural progeny largely stabilizes the pathological cascade secondary to primary storage, rescuing the cell morphology and developmental expression pattern of glial and neuronal proteins. We also show that stable ARSA overexpression in MLD NPCs and differentiated progeny results in upregulation of GALC, CGT, and GAL3ST1 expression, suggesting an attempt of ARSA-overexpressing MLD cells to cope with the increased production of intermediate products consequent to enhanced sulfatide catabolism In vitro experiments were planned and executed in the context of controlled laboratory experiments and with the help of expert staff if required . For each of the indicated experiments/assays we performed: (i) multiple (minimum of two) experimental and biological replicates and (ii) multiple (minimum of two) independent experiments, in order to generate an adequate number of samples in each group. The experimental sample size was determined on the basis of previously collected data in the same experimental settings and by the interim analysis of results. No data were excluded from the analysis, unless in the presence of a clear technical flaw . Investigators conducting the experiments were not blinded to sample code. Investigator processing samples at the end of the experiment were blinded to sample code in some experimental settings  up to the stage of data analysis. For experimental details, please refer to the dedicated sections below and in the 32. The source and characteristics of the original ND and MLD fibroblast cell lines, as well as the list of ND, MLD, and MLD-ARSA iPSCs clones used in this study, is reported in the Supplemental Experimental Procedures.The normal donor (ND) and patient-specific (MLD) iPSC lines used in this study as well as the generation of ARSA-overexpressing MLD iPSCs by LV-mediated gene transfer have been previously described2) in MEF pre-conditioned iPSC medium (IPSCM) in the presence of 10 µM of the Rho kinase inhibitor Y-27632 (Sigma). When the cell culture reached ≈90% confluence , culture medium was replaced with KSR medium (KSRM) supplemented with 200 ng/ml of rhNOGGIN (R&D) and 10 µM of SB431542 (Sigma). The medium was changed daily for the next 3 days. Thereafter, it was switched every other day in order to gradually expose the cells to increasing  ratio of NPC/KSR medium. 2 days after the last switch the cells were detached using Accutase and plated on Matrigel-coated dishes in NPCM supplemented with 20 ng/ml bFGF and 20 ng/ml EGF. In these conditions, cells proliferated for a limited number of passages. For all the differentiation experiments we used cells between p2 and p4. Cells were detached using Accutase and plated on Matrigel-coated dishes  in NPCM (d0). During the first 4 days after plating (d4) we gradually replaced NPCM containing bFGF and EGF with increasing amount of GDM, supplemented with 10 ng/ml PDGF-AA, 10 ng/ml NT3, 10 ng/ml IGF-1, 5 ng/ml HGF, and 60 ng/ml T3. From d4 to d13 medium was changed every other day. From d14 to d23, PDGF-AA, NT3, IGF-1, and HGF were withdrawn from the medium . At d24, the medium was switched to GMM supplemented with 20 µg/ml of ascorbic acid (Sigma) until d34. For details on culture media, see the Supplemental Experimental Procedures.iPSC culturing and expansion has been performed as described in the Supplemental Experimental Procedures. For neural induction, ND, MLD, and MLD-ARSA hiPSC colonies were detached with Accutase (Life Technologies), suspended as single cells and plated on Matrigel (BD Pharmingen)-coated dishes . For details, see the Supplemental Experimental Procedures.Reactive oxygen species were detected using CellROXn = 3 experiments in triplicate.Samples were visualized with Nikon Eclipse Ni microscope using double laser microscopy with Nikon Plan- Fluor (×20 -air and ×40 -air magnification) objective lens . Images were acquired using a camera (Nikon DS Fi2) and NIS-Element F4.00.00 acquisition software (Nikon). Immunopositive area of acquired images (expressed in pixels) was calculated using the ImageJ software. ND samples were used to set the threshold. The immunopositive area was normalized to the number of nuclei in the same field. At least 10 fields/sample with 20–30 cells/field were quantified in Co-labeling between sulfatide (Sulf) and differentiation markers  was analyzed in differentiated cultures. Z-stacks were recorded utilizing a PerkinElmer UltraVIEW ERS Spinning Disk Confocal  using a ×63/NA1.4 Plan-Apochromat oil-immersion lens  with a 405 nm diode laser, 488 nm argon laser and a 568 nm krypton laser excitation wavelengths. Sequential confocal images were collected at 0.25 μm intervals covering 14 μm depth . The 3D signals and the orthogonal projection representations were obtained by means of Volocity Software . Images were imported into Adobe Photoshop CS4 (USA) and adjusted for brightness and contrast.41. For details see Supplemental Experimental Procedures.Analysis of sulfatide content was performed as previously describedn ≥ 3). Unpaired t test  was used to compare two independent groups. One-way ANOVA (followed by Bonferroni post-test) or Kruskal−Wallis test (followed by Tukey’s or Dunn’s post-test) were used for statistical analyses of more than two independent groups in the presence of Gaussian or non-Gaussian distribution of data (assessed by Kolmogorov−Smirnov test), respectively. The number of samples and the statistical test used are indicated in the figure legends.In vitro cell counts, data from qRT-PCR, western blot, mass spectrometry, enzyme activity, and data obtained following the quantification of the immunopositive area by the ImageJ software were analyzed with Graph Pad Prism 6.0c for Macintosh and expressed as the mean ± standard error of the mean (SEM, with Supplemental material


How humans make decisions in non-cooperative strategic interactions is a big question. For the fundamental Rock-Paper-Scissors (RPS) model game system, classic Nash equilibrium (NE) theory predicts that players randomize completely their action choices to avoid being exploited, while evolutionary game theory of bounded rationality in general predicts persistent cyclic motions, especially in finite populations. However as empirical studies have been relatively sparse, it is still a controversial issue as to which theoretical framework is more appropriate to describe decision-making of human subjects. Here we observe population-level persistent cyclic motions in a laboratory experiment of the discrete-time iterated RPS game under the traditional random pairwise-matching protocol. This collective behavior contradicts with the NE theory but is quantitatively explained, without any adjustable parameter, by a microscopic model of win-lose-tie conditional response. Theoretical calculations suggest that if all players adopt the same optimized conditional response strategy, their accumulated payoff will be much higher than the reference value of the NE mixed strategy. Our work demonstrates the feasibility of understanding human competition behaviors from the angle of non-equilibrium statistical physics. R (rock), P (paper) and S (scissors). In the simplest settings the payoff matrix is characterized by a single parameter, the payoff a of the winning action  game is a fundamental non-cooperative game. It has been widely used to study competition phenomena in society and biology, such as species diversity of ecosystems123457s over P , so no aa < 2 this NE is evolutionarily unstable with respect to small perturbations but it becomes evolutionarily stable at a > 2  was later quantitatively measured inEmpirical verification of non-equilibrial persistent cycling in the human-subject RPS game (and other non-cooperative games) has been rather nontrivial, as the recorded evolutionary trajectories are usually highly stochastic and not long enough to draw convincing conclusions. Two of the present authors partially overcame these difficulties by using social state velocity vectorsa > 2) and unstable (a < 2) discrete-time RPS games. We show strong evidence that the RPS game is an intrinsic non-equilibrium system, which cannot be fully described by the NE concept even in the evolutionarily stable region but rather exhibits persistent population-level cyclic motions. We then bridge the collective cycling behavior and the highly stochastic decision-making of individuals through a simple conditional response (CR) mechanism. Our empirical data confirm the plausibility of this microscopic model of bounded rationality. Our theoretical calculations also demonstrate that, if all the players adopt the same CR strategy and if the transition parameters of this strategy are chosen in an optimized way, this CR strategy will outperform the NE mixed strategy in terms of the accumulated payoffs of individual players, yet the action marginal distribution of individual players is indistinguishable from that of the NE mixed strategy. Our work as a successful attempt of understanding competition dynamics from the perspective of non-equilibrium statistical physics may stimulate future more refined experimental and theoretical studies on the microscopic mechanisms of decision-making and learning in basic game systems1930313233In this work we investigate whether cycling is a general aspect even for the simplest RPS game. We adopt an improved cycle counting method on the basis of our earlier experiencesN = 6. Each population then carries out one experimental session by playing the RPS game 300 rounds (taking 90–150 minutes) with a fixed value of a. In real-world situations individuals often have to make decisions based only on partial input information. We mimic such situations by adopting the traditional random pairwise-matching experimental protocolt the players are randomly paired within the population and compete with their pair opponent once; after that each player gets feedback information about her own payoff as well as her and her opponent's action. As the experimental session finishes, the players are paid in real cash proportional to their accumulated payoffs (see Methods). Our experimental setting differs from those of two other recent experiments, in which every player competes against the whole population9a = 1.1, 2, 4, 9 and 100, respectively, in one-fifth of the populations so as to compare the dynamical behaviors in the evolutionarily unstable, neutral, stable and deeply stable regions.We recruited a total number of 360 students from different disciplines of Zhejiang University to form 60 disjoint populations of size a = 1.1 . We obtain very similar results for each set of populations of the same a value . This inertial effect is especially strong at a = 1.1 and it diminishes as a increases.We observe that the individual players shift their actions frequently in all the populations except one with alue see  online. a ≥ 2, an individual player's probability of making a clockwise action shift is equal to or just slightly different from that of making a counter-clockwise action shift (a = 2) and stable (a > 2) RPS games, in accordance with the NE theory. As shown in We notice that at on shift . There it is denoted as s(t) ≡ (Rn(t), Pn(t), Sn(t)) with qn being the number of players adopting action q ∈ {R, P, S}. Since Rn + Pn + Sn ≡ N there are (N + 1)(N + 2)/2 such social states, all lying on a three-dimensional plane bounded by an equilateral triangle (s(t) → s(t + 1) a rotation angle θ(t), which measures the angle this transition rotates with respect to the centroid c0 ≡  of the social state plane (see Methods)θ values signify counter-clockwise and clockwise rotations, respectively, while θ = 0 means the transition is not a rotation around c0. For example, we have θ(1) = π/3, θ(2) = 0, and θ(3) = −2π/3 for the three transitions shown in The social state of the population at any time triangle . Each poc0 during the time interval  is computed by Ct1, has an increasing trend in most of the 59 populations, indicating persistent counter-clockwise cycling. The cycling frequency of each trajectory in  is evaluated by f1,300 for all the 59 populations are listed in a = 1.1, mean ± SEM), 0.027 ± 0.008 (a = 2), 0.031 ± 0.008 (a = 4), 0.022 ± 0.008 (a = 9) and 0.018 ± 0.007 (a = 100). These mean frequencies are all positive irrespective to the particular value of a, indicating that behind the seemingly highly irregular social state evolution process, there is a deterministic pattern of social state cycling from slightly rich in action R, to slightly rich in P, then to slightly rich in S, and then back to slightly rich in R again. Statistical analysis confirms that f1,300 > 0 is significant for all the five sets of populations . The correlation between the mean cycling frequency f1,300 and the payoff parameter a is not statistically significant . We also notice that the mean cycling frequency in the second half of the game  is slightly higher than that in the first half  for all the five sets of populations , −0.0005 (a = 2), −0.0024 (a = 4), −0.0075 (a = 9) and −0.0081 (a = 100), which are all very close to zero and significantly different from the empirical values.Under the assumption of mixed-strategy NE , the social state transitions should obey the detailed balance condition. Therefore the observed persistent cycling behavior cannot be understood within the NE framework. Persistent cycling can also not be explained by the independent decision model which assumes the action choice of a player at one time is influenced only by her action at the previous time see  online. O the performance (output) of a player at a given game round, with O ∈ {W (win), T (tie), L (lose)}. Conditional on the output O, the probability that this player will decide to shift action clockwise or counter-clockwise or keep the same action in the next play is denoted as O−, O+ and O0 (≡ 1 − O− − O+), respectively. Most interestingly, we see from W0) of repeating the same action in the next play is considerably higher than her probabilities (W− and W+) of shifting actions. Furthermore, for payoff parameter a ≥ 2, if a player loses to her opponent in one play, she is more likely to shift action clockwise (probability L−) than either to keep the old action (L0) or to shift action counter-clockwise (L+).The action choices of different players must be mutually influenced. Our empirical data shown in O ∈ {W, T, L} and makes an action choice according to the corresponding three conditional probabilities . This model is characterized by a set Γ ≡ {W−, W+; T−, T+; L−, L+} of six CR parameters. Notice this CR model differs qualitatively from the discrete-time logit dynamics model24Inspired by these empirical observations, we develop a simplest nontrival model by assuming the following conditional response strategy: at each game round, every player review her previous performance rrn, ppn, ssn, rpn, psn and srn, respectively, as the number of pairs in which the competition being R–R, P–P, S–S, R–P, P–S, and S–R, in one game round t. Given the social state s =  at time t, the conditional joint probability distribution of these six integers is expressed as N − 1)!! ≡ 1 × 3 × … × (N − 3) × (N − 1) and m = n and = 0 if otherwise). With the help of this expression, we can then obtain an explicit formula for the social state transition probability crM[s′|s] from s to any another social state s′ (see Methods). We then compute numerically the steady-state social state distribution crf of this model is computed by θss′→ is the rotation angle associated with the social state transition s → s′, see Eq. (7).We can solve this win-lose-tie CR model analytically and numerically see  online. crf = 0.035 (a = 1.1), 0.026 (a = 2), 0.030 (a = 4), 0.018 (a = 9) and 0.017 (a = 100), agreeing well with the empirical measurements. Such good agreements between model and experiment are achieved also for the 59 individual populations /3 is the expected payoff of the NE mixed strategy, and crτ is the average fraction of ties among the N/2 pairs at each game round, with the expression crg depends on the CR parameters. By uniformly sampling 2.4 × 109 instances of Γ from the three-dimensional probability simplex, we find that for a > 2, crg has high chance of being lower than g0 (crg−g0) being −0.0085(a−2).  This is consistent with the mixed-strategy NE being evolutionarily stablecrg values (for the four cases of a ≠ 2) determined by the empirical CR parameters and the corresponding four mean payoffs of the empirical data sets all weakly exceed g0, indicating that individual players are adjusting their responses to achieve higher accumulated payoffs .Because of the rotational symmetry of the conditional response parameters, the CR model predicts that each player's action marginal distribution is uniform, identical to the NE mixed strategy  online.  than g0 , with th payoffs  online. crg. For population size N = 6 we give three examples of the sampled best CR strategies for a > 2: Γ1 = {0.002, 0.000; 0.067, 0.110; 0.003, 0.003}, with cycling frequency crf = 0.003 and crg = g0 + 0.035(a − 2); Γ2 = {0.995, 0.001; 0.800, 0.058; 0.988, 0.012}, with crf = −0.190 and crg = g0 + 0.034(a − 2); Γ3 = {0.001, 0.004; 0.063, 0.791; 0.989, 0.001}, with crf = 0.189 and crg = g0 + 0.033(a − 2). For large a these CR strategies outperform the NE mixed strategy in payoff by about 10%. Set Γ1 indicates that population-level cycling is not a necessary condition for achieving high payoff values. On the other hand, set Γ3 implies W0 ≈ 1, L0 ≈ 0, therefore this CR strategy can be regarded as an extension of the win-stay lose-shift  strategy, which has been shown by computer simulations to facilitate cooperation in the prisoner's dilemma game3738393.As shown in a. In a following paper we will study the effect of the payoff parameter a to the individual- and population-level behaviors in more detailIn game-theory literature it is common to equate individual players' action marginal distributions with their actual strategies11414243444692935484950N at given CR parameters (see W+/W− = T+/T− = L+/L− = 1), as long as W0 ≠ L0 . Informed consent was obtained from all the participanting human subjects.N = 6. The six players of each population carried one experimental session by playing the RPS game for 300 rounds with fixed payoff parameter a, whose value is chosen from {1.1, 2, 4, 9, 100}. During the game process the players sited separately in a classroom, each of which facing a computer screen. They were not allowed to communicate with each other during the whole experimental session. Written instructions were handed out to each player and the rules of the experiment were also orally explained by an experimental instructor. The rules of the experimental session are as follows:Each player plays the RPS game repeatedly with the same other five players. Each player earns virtual points during the experimental session according to the payoff matrix shown in the written instruction. These virtual points are then exchanged into RMB as a reward to the player, plus an additional 5 RMB as show-up fee. In each game round, the six players of each group are randomly matched by a computer program to form three pairs, and each player competes only with the pair opponent. Each player has at most 40 seconds in one game round to make a choice among the three candidate actions “Rock”, “Paper” and “Scissors”. If this time runs out, the player has to make a choice immediately . After a choice has been made it can not be changed. The 360 human subjects (referred to as players in this work) were distributed into 60 populations of equal size If you choose “Rock” and your opponent chooses “Scissors”, how many virtual points will you earn? (2) If you choose “Rock” and your opponent chooses also “Rock”, how many virtual points will you earn? (3) If you choose “Scissors” and your opponent chooses “Rock”, how many virtual points will you earn? (4) Do you know that at each game round you will play with a randomly chosen opponent from your group (yes/no)?Before the start of the actual experimental session, the player were asked to answer four questions to ensure that they understand completely the rules of the experimental session. These four questions are: (1) R, Paper as P, and Scissors as S).During the experimental session, the computer screen of each player will show an information window and a decision window. The window on the left of the computer screen is the information window. The upper panel of this information window shows the current game round, the time limit (40 seconds) of making a choice, and the time left to make a choice. The color of this upper panel turns to green at the start of each game round. The color will change to yellow if the player does not make a choice within 20 seconds. The color will change to red if the decision time runs out . The color will change to blue if a choice has been made by the player. After all the players of the group have made their decisions, the lower panel of the information window will show the player's own choice, the opponent's choice, and the player's own payoff in this game round. The player's own accumulated payoff is also shown. The players are asked to record their choices of each round on the record sheet /3. Therefore we set the exchange rate to be r = 0.45/(1 + a) to ensure that, under the mixed-strategy NE assumption, the expected total earning in RMB for a player will be 50 RMB irrespective of the particular experimental session. The value of the payoff parameter a, the numerical value of r, and the above-mentioned reward formula were listed in the written instruction and also orally mentioned by the experimental instructor at the instruction phase of the experiment.The reward in RMB for each player is determined by the following formula. Suppose a player s =  at game round t to another social state t + 1), if at least one of the two social states coincides with the centroid c0 of the social state plane, or the three points s, c0 lie on a straight line, then the transition c0, and the rotation angle θ = 0. In all the other cases, the transition c0, and the rotation angle is computed through x) ∈  from the social state s ≡  at time t to the social state t + 1) is expressed as: For the conditional response model, the transition probability SIv16Zhou


Review of Radiation Oncology Physics: A Handbook for Teachers and Students, edited by Ervin B. Podgorsak, PhD Review of Radiation Oncology Physics: A Handbook for Teachers and Students, edited by Ervin B. Podgorsak, PhD, could not have occurred at a more opportune time in my career. I am currently teaching my first graduate‐level radiation therapy physics course as part of our newly initiated medical physics graduate program within the Department of Physics at the Virginia Commonwealth University in Richmond, Virginia. Although I have all the notes from my graduate courses in medical physics from UCLA and access to a host of other lectures and syllabi from other institutions, in my opinion this Handbook is ideal for anyone currently teaching a graduate‐level course in medical physics. All the chapters and sections have been very well organized and structured specifically from the viewpoint of presenting lectures on the fundamental concepts of modern radiation therapy physics.The opportunity for me to review the textbook The goal, as stated in the Foreword, is to provide the basis for the education of medical physicists initiating their university studies in the field. It is not designed to replace the large number of textbooks available, which will still be necessary to deepen the level of knowledge in specific topics presented in the Handbook. Rather, the intent is to serve as a factual supplement to the various textbooks on medical physics in the form of a syllabus covering all modern aspects of radiation oncology physics. In addition, the Handbook was developed as a teacher's guide in which the various topics in the syllabus could be expanded to form a detailed “bullet list” that contains the basic guidelines of the material to be included, so that the lectures can be prepared accordingly.The Handbook has a comprehensive bibliography within each section. I also found that the book successfully fills the gap in the teaching material for the specialty of medical physics, and does so in a single manageable volume with a logical, well‐thought‐out structure for presenting and learning modern radiation therapy physics.Basic Radiation PhysicsDosimetric Principles, Quantities and UnitsRadiation DosimetersRadiation Monitoring InstrumentsMachines for External Beam RadiotherapyExternal Photon Beam: Physical AspectsClinical Treatment Planning in External Photon Beam RadiotherapyElectron Beams: Physical and Clinical AspectsCalibration of Photon and Electron BeamsAcceptance Tests and CommissioningComputerized Treatment Planning Systems for External Beam RadiotherapyQuality Assurance of External Beam RadiotherapyBrachytherapy: Physical and Clinical AspectsBasic RadiobiologySpecial Procedures and Techniques in RadiotherapyRadiation Protection and Safety in RadiotherapyThe book has over 530 pages and a host of very well documented figures and tables to supplement the comprehensive listing of topics covered. As an indication of the breadth of coverage, see the titles for the 16 chapters:Handbook for Teachers and Students with coverage deeper than a simple guide.The genesis of this Handbook is described in the Preamble: in the late 1990s, the International Atomic Energy Agency (IAEA) initiated a systematic and comprehensive plan to support development of teaching programs in medical radiation physics to many of its member states. This effort was aimed at supporting countries to develop their own university‐based medical science programs in medical physics. In that initial effort, they developed a syllabus in radiation therapy physics with a goal to “harmonize” the various levels of training. With the development of the syllabus, a Teacher's Guide was developed in which the various topics in the syllabus were expanded to form a detailed bullet list containing the basics guidelines of the material to be included in each topic so that the lectures could be prepared accordingly. P. Andreo of IAEA led the initial Teacher's Guide, and later, in 2001, E.B. Podgorsak took the lead of the project. Dr. Podgorsak redesigned the contents so that the book is now a comprehensive The book was provided to me as a pdf file, which will of course greatly increase its circulation to IAEA member states, in accordance with its original goal. However, navigating a 500+ page document in this electronic format can be difficult. That said, since the Handbook will be provided in this electronic format, it will ideally lend itself to putting some of the text, figures, and tables into slide presentations and thus greatly improve the dynamic visuals of a typical graduate‐level medical physics course. Whereas the Handbook does provide a comprehensive listing of the topics that should be covered in their respective suggested order, it is important to again note that these are not comprehensively introduced, thus necessitating a more traditional textbook to accompany the students’ reading. There are also no example problems and solutions. My main request and suggestion to the authors for future editions is to include, as a supplement, several illustrative example problems and solutions. In my opinion, this well‐organized and well‐structured book, combined with a comprehensive series of example problems, will be a superb and welcome addition to any graduate course in radiation therapy physics.Review of Radiation Oncology Physics: A Handbook for Teachers and Students is an excellent review book dedicated to students and teachers involved in programs that train professionals for work in radiation oncology. It provides a compilation of facts on the physics as applied to radiation oncology and as such is very useful to graduate students in medical physics programs, residents, dosimetrists, and therapy technology programs. This book is expected to have wide dissemination by the IAEA; however, at this stage, it is published as working material seeking comments, corrections, and feedback.In summary, The working version of the book is available free of charge. It is a 14‐MB file that can be downloaded from either of two sites. The full book is available from the following site maintained by the editor:http://www.medphys.mcgill.ca/IAEABOOK/The full book or individual chapters can be downloaded from the following IAEA site:http://www‐naweb.iaea.org/nahu/dmrp/syllabus.shtm


Combination of these chemicals with antiandrogen had an additive suppressive effect on AR transcriptional activity. Our collective findings may pave the way in creating new strategies for the development and design of anti-AR drugs.Using bicalutamide-androgen receptor (AR) DNA binding domain-ligand binding domain as bait, we observed enrichment of FxxFY motif-containing peptides. Protein database searches revealed the presence of receptor-interacting protein kinase 1 (RIPK1) harboring one FxxFY motif. RIPK1 interacted directly with AR and suppressed AR transactivation in a dose-dependent manner. Domain mapping experiments showed that the FxxFY motif in RIPK1 is critical for interactions with AR and the death domain of RIPK1 plays a crucial role in its inhibitory effect on transactivation. In terms of tissue expression, RIPK1 levels were markedly higher in benign prostate hyperplasia and non-cancerous tissue regions relative to the tumor area. With the aid of computer modeling for screening of chemicals targeting activation function 2 (AF-2) of AR, we identified oxadiazole derivatives as good candidates and subsequently generated a small library of these compounds. A number of candidates could effectively suppress AR transactivation and AR-related functions  The androgen-androgen receptor (AR) pathway is important for physiological development and pathogenesis of diseases, such as prostate cancer . AR is aAnti-androgen withdrawal syndrome (AWS) describes the response to withdrawal of antiandrogen therapy , 13. A nIn the current study, we demonstrated enrichment of FxxFY motif-containing peptides that interacted with CDX-W741L mutant AR LBD and identified RIPK1 harboring a FxxFY motif as a novel AR coregulator. We further assessed the utility of AF-2-targeting peptidomimetics in blocking AR N-C and AR-cofactor interactions as a potential novel anti-AR strategy for treatment of prostate cancer.285FRPFY289). RIPK1 is a kinase that plays a crucial role in inflammation and cell death 2 × 0.5. Three to five mice were used per group . Mice were sacrificed 4 weeks after chemical injection [All experiments involving laboratory animals were conducted in accordance with the Guidelines for Animal Experiments of Chang Gung Memorial Hospital and approved by the Animal Research Committee at the hospital. LNCaP cells were harvested, washed twice with phosphate-buffed saline, and resuspended at a final concentration of 5 × 10njection  and tumot test.Data are presented as means ± SEM. Differences between two groups were assessed using the unpaired two-tailed Student’s


Androgen receptor (AR) is a validated drug target for prostate cancer based on its role in proliferation, survival, and metastases of prostate cancer cells. Unfortunately, despite recent improvements to androgen deprivation therapy and the advent of better antiandrogens with a superior affinity for the AR ligand-binding domain (LBD), most patients with recurrent disease will eventually develop lethal metastatic castration-resistant prostate cancer (CRPC). Expression of constitutively active AR splice variants that lack the LBD contribute toward therapeutic resistance by bypassing androgen blockade and antiandrogens. In the canonical pathway, binding of androgen to AR LBD triggers the release of AR from molecular chaperones which enable conformational changes and protein–protein interactions to facilitate its nuclear translocation where it regulates the expression of target genes. However, preceding AR function in the nucleus, initial binding of androgen to AR LBD in the cytoplasm may already initiate signal transduction pathways to modulate cellular proliferation and migration. In this article, we review the significance of signal transduction pathways activated by rapid, non-genomic signaling of the AR during the progression to metastatic CRPC and put into perspective the implications for current and novel therapies that target different domains of AR. Approximately 30% of patients will relapse after primary therapy . AndrogeAR gene or overexpression of AR protein, synthesis of intratumoral androgens, stimulation of ligand-independent AR activity by epidermal growth factor (EGF) or interleukin-6 (IL-6) or by the mitogen-activated protein kinase (MAPK) cascade, phosphoinositide 3-kinase (PI3K)/Akt, and protein kinase A pathways, and the expression of constitutively active AR splice variants such as ARv567es and V7 , a DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD). These domains operate as individual folding units that contribute to the transformation of a transcriptionally active receptor. Unlike the LBD and DBD which are structured domains, the AR NTD is intrinsically disordered and mediates protein–protein interactions that are required for transactivation. During AR transactivation, the activation function 1 region (AF1) of the AR NTD acquires transient folding structures to bind transcriptional coactivators to bridge the AR to basal transcriptional machinery. Classic deletion analyses of the AR NTD identified two transcriptional activation units within AF1 (Tau1 and Tau5), which represent surfaces that mediate ligand-dependent and ligand-independent transactivation, respectively , 13.In the canonical model, inactive AR is maintained in the cytosol by molecular chaperones that include heat shock proteins, co-chaperones, and cytoskeletal proteins . BindingIn response to androgen, a rapid association of AR with the non-receptor tyrosine kinase Src is responsible for enhancing cell proliferation through activation of the MAPK/ERK cascade. AR interacts with Src by binding of a polyproline sequence between residues 371 and 381 of the AR NTD to Src homology domain 3 (SH3). This association facilitates unfolding of Src and autophosphorylation to activate the Src kinase domain , 18. TheActivation of the Ras-Raf-MAPK/ERK cascade is the primary mitogenic stimulus initiated by non-genomic AR effects observed in androgen-sensitive prostate cells. Levels of phospho-(p-) ERK1/2 peak within 5–30 min of exposure to DHT in LNCaP cells, PC-3 cells stably expressing wild-type AR, and primary prostatic stromal cells, but this is not detected in primary human genital skin fibroblasts until 16 h, implying cell-type specificity , 29. Actin vitro and demonstrate favorable antitumor activity in vivo using prostate cancer xenografts  signaling lipids to induce activation of Akt kinase, which leads to regulation of transcription factors to inhibit apoptosis pathways and promote cell survival. Accumulation of Akt p-S473 is detected over 10–30 min upon androgen stimulation of non-tumoral VDEC cells or PC3 cells when stably expressing AR, supporting its role as a non-genomic AR stimulus (AR promoter (3 to PIP2 by phosphatase and tensin homolog (PTEN), which is a commonly lost tumor suppressor in prostate cancer. Indeed, genomic alterations affecting the PI3K/Akt pathway are detected in 42% of primary prostate tumors and 100% of metastatic tumors  still have low levels of testosterone in their serum (less than 0.1 nM), which is within the range of AR to mediate non-genomic responses , 58. LevAntiandrogens bind the AR LBD to inhibit full-length AR and thus have no effects on constitutively active AR splice variants, such as ARv567es and V7, which do not express a functional LBD. Expression of ARv567es and V7 is repressed by androgen and increased in the absence of androgen, thereby mediating transcription of target genes when the full-length AR is not transactivated by androgen under castrate conditions. ARv567es is predominantly nuclear , whereasInhibitors targeting the AR NTD might be promising candidates for blocking non-genomic AR signals that enhance tumor growth or survival. To date, two families of compounds have been discovered that bind to the AF1 region of the AR NTD and inhibit transactivation. These are sintokamide A and EPI compounds , 70. BotAlong with androgen deprivation therapy, non-genomic AR signaling substrates may need pharmacological inhibition to provide maximum benefit to CRPC patients. Several inhibitors of Src family kinases have been tested in a clinical setting for prostate cancer, notably inhibitors that target kinase activity, dasatinib BMSS-354825) and saracatinib (AZD0530); and KX2-391, a peptidomimetic that blocks the substrate binding site of Src. Clinical studies indicate that targeting Src or inhibiting activated downstream kinase pathways in isolation is ineffective for CRPC , 76–79. 5 and sarJL and MS both provided substantial contributions to the conception of the work and interpretation of literature. JL provided the first draft and MS revised the work for important intellectual content. JL and MS both approved the version submitted. JL and MS agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.MS is a director and officer of ESSA Pharma Inc. that has licensed EPI and sintokamide technologies. MS has equity and receives consulting fees from ESSA Pharma Inc. JL declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Triple-negative breast cancers comprise 15% of breast cancers and are more common in women with BRCA1 mutations. Although most have basal gene expression signatures, others resemble luminal tumors with expression of androgen receptor-related genes and some express the immunoinhibitory protein programmed death-ligand 1 (PD-L1). Given the availability of androgen receptor-targeted and immune therapies for triple-negative breast cancers, determining predictors of these biomarkers is important.To determine the prevalence and predictors of androgen receptor and PD-L1 expression in BRCA1-associated and sporadic triple-negative breast cancer.We studied 197 triple-negative breast cancers: 78 (39.6%) from BRCA1 mutation carriers and 119 (60.4%) from noncarriers. Tumor pathology was reviewed and tissue microarray sections were immunostained for androgen receptor and PD-L1.P=0.01). Twenty-six percent of cancers expressed PD-L1 with no significant difference in frequency between carriers and noncarriers. Factors predicting androgen receptor expression were lower histologic grade (odds ratio (OR) 4.6; 95% confidence interval (CI) 1.1–19.7), older age at diagnosis  and PD-L1 expression . PD-L1 expression was significantly more common in cancers with lymphocytic infiltrates  and androgen receptor expression , and less common in tumors with lymphovascular invasion .Androgen receptor expression was seen in 18% of tumors and was significantly less common in tumors from BRCA1 mutation carriers than noncarriers (9.2 vs. 23.7%; These results identify predictors for androgen receptor and PD-L1 expression among triple-negative breast cancers that may lead to better treatment selection and participation in clinical trials. TNBC is an aggressive subtype of breast cancer and its treatment is challenging in part owing to the heterogeneity of the disease. Recently, at least six molecular subtypes of TNBC have been identified by gene expression profiling with different clinical outcomes and responses to therapy.4 LAR TNBCs express high levels of nuclear AR and luminal cytokeratins. There is some evidence that TNBCs that express the AR  are less sensitive to chemotherapy but can respond to AR antagonists.2 Six-month clinical benefit rates of 19% and 29% in metastatic AR+ TNBC have been reported with bicalutamide (TBCRC 011) and enzalutamide, respectively.6 A higher frequency of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations has also been reported in the AR+ compared with other subtypes of TNBC suggesting another potential therapeutic strategy for these cancers.7One subtype of TNBC is the luminal androgen receptor (LAR) group. While lacking the estrogen receptor, these TNBCs are heavily enriched in hormonally regulated pathways including androgen metabolism and androgen receptor (AR) signaling.8 Recent successes with immune checkpoint inhibitors in TNBC have been reported. Overall response rates of 19% with pembrolizumab monotherapy and MPDL3280A (atezolizumab) monotherapy have been reported in advanced TNBCs that express PD-L1 (PD-L1+), with durable responses observed.10 Although responses to PD-1 and PD-L1 blockers are not restricted to tumors that express PD-L1, higher response rates have been observed in cancers with PD-L1 expression.11–13In addition, some TNBCs express programmed death-ligand 1 (PD-L1), a transmembrane protein expressed on both cancer cells and tumor-infiltrating inflammatory and immune cells. PD-L1 binding to programmed death 1 (PD-1) on T cells is one potential mechanism of tumor immune evasion.BRCA1 mutations. Seventy percent of the breast cancers that develop in women with inherited BRCA1 mutations are TNBC and 10–20% of women with TNBC have a BRCA1 mutation.14–16 Although outcomes after standard anthracycline-based adjuvant chemotherapy are similar for BRCA1 mutation carriers and noncarriers with TNBC, the BRCA1-associated TNBCs appear to be more responsive to platinum chemotherapy, likely due to their defect in homologous recombination.17–19 It is not known whether the frequency of AR and PD-L1 expression differs among TNBC in women according to BRCA1 status.Another group of TNBC can develop in women with germline BRCA1 mutation may facilitate the use of appropriate targeted therapies. To address this, we assessed the frequency of AR and PD-L1 expression in a cohort of primary TNBCs and determined whether the prevalence differed between TNBC from BRCA1 mutation carriers (herein referred to as BRCA1 carriers) and noncarriers. In addition, we evaluated whether any clinical or tumor pathologic features predicted for AR+, PD-L1+, or BRCA1-associated TNBC.Owing to the heterogeneity of TNBC, identifying factors that predict for AR and PD-L1 expression and the association of a germline BRCA1 mutation carriers were significantly younger at diagnosis than noncarriers . The most common histologic type was invasive ductal carcinoma among both carriers (92.2%) and noncarriers (90.2%). Compared with TNBC in noncarriers, BRCA1-associated TNBC significantly more often had high histologic grade as well as some degree of stromal lymphocytic infiltration (P=0.03). There was no difference in axillary nodal involvement between TNBC in BRCA1 mutation carriers and noncarriers . There was no significant difference in CK14 or EGFR expression among TNBC in women with or without a BRCA1 mutation.The results of IHC staining of the tissue microarrays (TMAs) are presented in BRCA1-associated TNBC significantly less often had any degree of AR expression  or stronger AR expression  expressed the AR, with at least 1% of cancer cells staining, whereas 22 (11.3%) demonstrated >10% of cancer cells staining. Compared with sporadic TNBC, BRCA1 carriers and noncarriers . When PD-L1 positivity on cancer or inflammatory cells is considered, of the 177 TNBC evaluable, 163 (92.1%) were PD-L1+ with no significant difference in frequency between BRCA1-associated and sporadic TNBC  showed ⩾1% staining by IHC. There was no significant difference in PD-L1 cancer cell expression between TNBC from  P=0.11: .BRCA1 carriers, 3 (4.1%) expressed both, with one cancer having weak AR staining (1–10% cells) and 2 having >10% AR staining. Twelve (10.3%) of the 117 TNBC from noncarriers had co-expression of AR and PD-L1 on cancer cells, 5 with weak AR staining and 7 with >10% AR staining (data not shown).Of the 190 TNBCs for which both AR and PD-L1 staining results were available, 15 (7.9%) expressed both AR and PD-L1 in cancer cells by IHC. Of the 73 TNBC from BRCA1 mutation status was no longer significantly associated with AR expression ⩾1 or >10%  and lower tumor grade . In addition, PD-L1 positivity in cancer cells significantly predicted AR expression on ≥1% of cancer cells . In the multivariable model, after adjusting for age, tumor grade and PD-L1,  or >10% .or inflammatory cells.Factors that significantly predicted PD-L1 expression in cancer cells in the multivariable model included the presence of a lymphocytic infiltration , and ≥ 1% AR expression  whereas the presence of lymphovascular invasion significantly decreased the odds that the cancer cells would express PD-L1 , presence of lymphocytic infiltration , and CK5/6 expression . While high histologic grade and lack of AR expression (<1% of cells staining) significantly predicted a BRCA1 mutation on univariable analysis, they were not significant in the multivariable model . Gucalp et al.5 found 10% of 424 ER-negative breast cancers were AR+ (>10% cells staining) in the metastatic setting. Three other studies have evaluated AR positivity in TNBC specifically; McGhan et al.22 found 23% of 94 primary and recurrent TNBC expressed the AR (>10% staining). Luo et al.23 reported that 28% of 137 primary TNBC were AR+ (≥1% cells staining) and Collins et al.24 reported 31% AR positivity (>10% cells staining) in 216 primary basal breast cancers, defined as ER, PR and HER2-negative with either CK5/6 or EGFR expression by IHC staining. Our finding that 18% of primary TNBC express AR is slightly lower than other studies and may reflect that 40% of our cohort consisted of TNBC from BRCA1 carriers for which the frequency of AR expression was lower. Our results are consistent with a smaller study that found that 21% of 30 BRCA1-associated ER-negative breast cancers expressed the AR using the same criteria of at least 1% of tumor nuclei staining.25Few studies have assessed the frequency of AR expression in early TNBC. In primary ER-negative breast cancer, the prevalence of AR expression has ranged from 10% to 32%,26 and histologic grade24 among breast cancers this is the first study to our knowledge to report this finding in ER-negative breast cancers or TNBC specifically. McGhan et al.22 also found that older age predicted for AR expression in TNBC. Identifying older patients with TNBC who may respond to AR antagonists could be particularly important as they may not be optimal candidates for chemotherapy. Indeed, AR inhibitors are showing promise in ER-negative breast cancer. In a phase II trial using bicalutamide, a nonsteroidal antiandrogen, in women with AR+, ER− and PR-negative metastatic breast cancer, 19% of 21 evaluable patients achieved response or stable disease for at least 6 months.5 Similarly, a phase 2 study in advanced AR+ TNBC using enzalutamide, another AR antagonist, demonstrated a 29% 6-month clinical benefit rate in 75 evaluable patients.6 New agents such as selective androgen receptor modulators are also now in clinical trials. Although androgen receptor expression was significantly less common in TNBC from BRCA1 carriers than noncarriers, this may reflect the younger age at diagnosis and higher grade of BRCA1-associated TNBC, as BRCA1 mutation status was no longer significant on multivariable analysis.It is important to identify TNBC that express the AR as drugs targeting the AR are demonstrating activity. We found that older age and lower grade increased the likelihood that a TNBC expresses the AR. Although AR expression has been associated with lower proliferative rateet al.27 who reported that 19% of 105 primary TNBC showed cancer cell PD-L1 positivity. We used the 9A11 mouse monoclonal antibody that recognizes an epitope in the cytoplasmic domain of PD-L1 and gives good membrane staining; Mittendorf et al. used the 5H1 antibody that recognizes an extracellular epitope. One conundrum is that different studies have used different criteria for considering a tumor PD-L1+. There is no consensus regarding whether cancer cells, inflammatory infiltrates or both should be considered; the threshold for PD-L1 positivity; whether to use qualitative or quantitative methods; or the optimal antibody. When PD-L1 positivity was defined as ⩾1% of cancer or inflammatory cells staining, 92% of all TNBC in our cohort were considered PD-L1+, making this cutoff value less useful. Other studies have reported PD-L1 positivity in 30–45% of breast cancers by varying criteria of epithelium and stromal cell staining using quantitative analysis of various monoclonal antibodies, and have reported that expression is higher in TNBC.29Our finding that 26% of primary TNBC express PD-L1 on the cancer cell surface is consistent with the findings of Mittendorf 11–13 We found that the presence of lymphocytic infiltration significantly predicted PD-L1 expression on cancer cells. Denkert et al.30 also found that mRNA expression of 12 immune-related genes, including PD-L1 was higher among 314 TNBC with higher levels of stromal tumor-infiltrating lymphocytes (TILs) in the GeparSixto trial. Wimberly et al.28 found a positive correlation between TILs and PD-L1 expression in 105 primary breast cancers and Mittendorf et al.27 found that CD8+ T cells were more frequent in PD-L1+ versus PD-L1-negative TNBC. PD-L1 expression may be expressed constitutively on cancer cells or may be induced by cytokines such as gamma interferon secreted by activated T cells.31 Indeed, classification of tumors into four groups based on the presence or absence of TILs and PD-L1 expression on cancer cells has been proposed to guide immune therapy approaches.32 It is likely that further characterization of the subset of TILs will also be important in planning tumor specific immune approaches.Since PD-L1 expression enriches for response to anti-PD1 and anti-PD-L1 agents, it is useful to identify predictors of PD-L1 expression among TNBC.BRCA1 carriers than noncarriers, one might have expected that BRCA1-associated TNBC also would have a higher frequency of PD-L1 expression. We did not find this to be true. It has been proposed that tumors with higher somatic mutational burdens may be those that are more likely to induce an immune response and respond to immune therapy as a result of neoantigen creation. Recently, Le et al.33 reported that colorectal and other cancers with germline or somatic loss of DNA mismatch repair (MMR) genes have a significantly higher response rate and 12-week clinical benefit rate to pembrolizumab than MMR-proficient colorectal tumors. It is possible that as the somatic mutational signature induced by BRCA1 mutations is different than that associated with MMR deficiency, the nature of the immune response induced is also different.34As lymphocytic infiltration was significantly more common in TNBC from We found that TNBCs that express the AR are also threefold more likely to express PD-L1 on cancer cells. Overall, 7.9% of the TNBC in our cohort coexpressed AR and PD-L1 with 10.3% of TNBC from noncarriers demonstrating expression of both receptors. If confirmed, this could provide the basis for combination therapy with AR inhibitors and immune therapy for this subset of TNBC.BRCA1 mutation as therapies such as PARP inhibitors and platinum agents are more active in this subset of TNBC.35–37 The National Comprehensive Cancer Network now recommends genetic testing for any woman diagnosed with TNBC by age 60 years.38 However, identifying BRCA1-associated TNBC by pathologic features and/or biomarker expression may increase recognition of these tumors. We found that younger age at diagnosis as well as lymphocytic infiltration and expression of the basal cytokeratin 5/6 in the tumor significantly increased the likelihood that a TNBC was associated with a BRCA1 mutation. Our findings are consistent with other studies that demonstrated that younger age16 and basal cytokeratin expression39 are significantly more common in BRCA1-associated than sporadic TNBC. To our knowledge, this is the first report that among TNBC, lymphocytic infiltration is significantly more common in BRCA1-associated cancers.It is useful to identify patients with TNBC who have a A potential limitation of this study is that the tumor present on cores in the TMA may not be representative of the entire tumor if particular cancers are heterogeneous. In addition, assessment of immunostains in this study was qualitative, which introduces the possibility of inter-pathologist variability. However, the extent of any variability should differ with respect to any of the outcomes assessed in this study. In addition, if qualitative interpretation of AR and PD-L1 stains can be standardized, as they have been for steroid receptors and HER2, then more widespread use of these biomarkers may be feasible.BRCA1 mutation status, AR and PD-L1 expression all provide important information that predicts response to an increasing number of targeted therapies. Confirmatory studies will be important and may lead to better selection of patients with TNBC for treatment and participation in clinical trials.In conclusion, our study confirms the heterogeneity of TNBC and contributes to understanding the factors that increase identification of various subtypes of TNBC. Germline BRCA1 testing were identified through the clinical databases and annotated Specialized Program of Research Excellence (SPORE) specimen bank at Beth Israel Deaconess Medical Center and Dana–Farber Cancer Institute. ER, PR, and HER2 status, assessed as part of the routine clinical evaluation, was abstracted from institutional pathology reports. We excluded 9 TNBCs from BRCA2 mutation carriers, 3 TNBCs for which a previous cancer from that individual was included and 21 cancers with insufficient remaining tumor for research use. The final study cohort, therefore, consisted of 78 TNBC from BRCA1 carriers and 119 TNBC from noncarriers. BRCA1 mutation status was established for 120 of these women by high-throughput heteroduplex detection from a banked research blood sample as described previously.40 The other 77 women had genetic testing through commercial laboratories through high-risk clinics.Two hundred thirty Stage I-III TNBC diagnosed from 1989 through 2008 from women who had BRCA1-associated and sporadic TNBC were reviewed by the two study pathologists (SJS and LC). Each cancer was scored for the following pathologic features: histologic type; Nottingham combined histologic grade, with each of the three components of grade  recorded separately; extent of stromal lymphocytic infiltrate ; and presence of lymphovascular invasion.Clinical characteristics were abstracted from medical records, and included age at diagnosis and clinical stage . Histologic sections of 41TMAs were constructed in the Dana–Farber Harvard Cancer Center Tissue Microarray Core Facility by obtaining three 0.6-mm cores from each tumor after study pathologists identified representative areas of invasive cancer. Sections cut from the tissue microarrays were immunostained using mouse monoclonal antibodies for basal cytokeratins , epidermal growth factor receptor (EGFR), AR and the 9A11 mouse monoclonal antibody to PD-L1  and 95% confidence interval (CI) and used Firth estimation to deal with sparse data, as necessary.42 The outcomes assessed were BRCA1 carrier status, PD-L1 cancer positivity and AR staining with two cutoffs for positivity: ⩾1 and >10%. For BRCA1 carrier status, PD-L1 cancer positivity and AR staining ⩾1%, we built a multivariable logistic regression model including all predictor variables that were significantly associated with the outcome in the crude models. For AR staining >10% we used the same variables as in the multivariable model for AR staining ⩾1%. All tests were two-sided and a P<0.05 was considered statistically significant. All statistical analyses were conducted with SAS 9.4 .Descriptive data are presented as mean±s.d., median (interquartile range), or frequency and proportion. Continuous data were compared using a This study was approved by the institutional review board of Dana Farber-Harvard Cancer Center (DF-HCC 07-334). Given the retrospective nature of the study, subject consent was not required.


A market research company (Nielsen) reported that consumers in the Asia-Pacific region have become the most active group in online shopping. Focusing on augmented reality (AR), which is one of three major techniques used to change the method of shopping in the future, this study used a mixed design to discuss the influences of the method of online shopping, user gender, cognitive style, product value, and sensory channel on mental workload in virtual reality (VR) and AR situations. The results showed that males’ mental workloads were significantly higher than females’. For males, high-value products’ mental workload was significantly higher than that of low-value products. In the VR situation, the visual mental workload of field-independent and field-dependent consumers showed a significant difference, but the difference was reduced under audio–visual conditions. In the AR situation, the visual mental workload of field-independent and field-dependent consumers showed a significant difference, but the difference increased under audio–visual conditions. This study provided a psychological study of online shopping with AR and VR technology with applications in the future. Based on the perspective of embodied cognition, AR online shopping may be potential focus of research and market application. For the future design of online shopping platforms and the updating of user experience, this study provides a reference. The use of virtual reality (VR) technology and augmented reality (AR) technology in network marketing practices is on the rise. Especially, a market research company (Nielsen) reported that consumers in the Asia-Pacific region have become the most active group in online shopping. Online shopping also represented the fourth retail revolution. AR technology is one of three major technologies identified to be likely to change the future of shopping (the others are QR codes and mobile payment). Based on the perspective of embodied cognition, AR online shopping may be potential focus of research and market application. However, studies based on the technology were less relevant with psychological phenomena. Under the condition of VR and AR, this study aims to investigate the difference of mental workload. For the future design of online shopping platforms and the updating of user experience, this study provides a reference.Virtual reality technology represents a model of the real world produced by a computer . AR techThrough both social mechanisms (via community identification) and psychological mechanisms (via trust and satisfaction), virtual community participation has greatly promoted loyalty intentions . Users’ The Vuzix company developed Vuzix Star 1200 to enhance the glasses of the average consumer. This technology can fuse real world images and intelligent mobile phone information. Recently, Microsoft developed an AR projector. This technology uses 4 Kinect to realize the aim of interactions between participants and the image. Google is improving AR glasses, in which the scenery will stack corresponding intelligent information. IBMLabs stated that they developed a mobile shopping application prototype based on AR technology. The Mattel and Fox Films sales department launched a joint series of Avatar toys with AR. HoloLens, made by Microsoft, represents the industry’s confidence in the development of AR. The preliminary AR application of Taobao and Amazon can portray AR as a new technology into the field of consumption. To accelerate the development of technology, relevant research on internal psychological mechanisms is needed.Early research on the mental workload can be traced back to the study of Knowles in 1960s . The stuThe “Flow” system from Amazon combines online shopping and AR. To reduce the mental workload, the role of AR technology based on experience marketing characteristics should not be ignored. Mental workload was one of the important themes for human factor engineering and the man-machine design field. To achieve job satisfaction, the control of job enrichment and mental workload are necessary . In modeUnder the condition of VR and AR, this study aims to investigate the difference of mental workload. For the future design of online shopping platforms and the updating of user experience, this study provides a reference. The use of VR and AR technology in network marketing practices is on the rise. However, studies based on the technology were less relevant with psychological phenomena. Based on the AR technology, from the perspective of the user experience of online shopping, the study tested the mental workload about VR and AR. The study investigated the differences in the users’ gender and cognitive style, product value, and sensory channel in the VR situation and the AR situation. From the perspective of AR, psychological research on online shopping helps shoppers to perceive commodity information more accurately, reduce the time investment, and attract the eyes of consumers. Males would not be too keen on price factors, paying more attention to the product itself and the efficiency of the shopping experience . A high H1The property of high value is the main influencing factor of high mental workload for males.H2In the VR situation, mental workload of users decrease for field dependent when perceptual information is added through sound.H3In the AR situation, mental workload of users increase for field dependent when perceptual information is added through sound.Focusing on the AR, which is one of three major techniques used to change the method of shopping in the future, this study used a mixed design to discuss the influences of the method of online shopping, user gender, cognitive style, product value, and the sensory channel on mental workload in VR and AR situations.Mage = 29.81, SDage = 2.61, 16 field-independent) participated in the experiment. All the participants were right-handed, had normal or corrected-to-normal vision and no color blindness or color weakness, and had no similar experimental experiences before. First, the experimenter randomly selected shoppers near the exit of the furniture store as the possible participants. The experimenter asked the participants the following two questions: (1) Have you ever shopped online? (2) Are you willing to participate in an online shopping experiment for a sofa and mirror? The researcher recruited those who agreed as formal participants. Second, this study used snowball sampling technology by asking the initial participants to recruit related individuals to participate in the experiment. After being recruited, the participants were asked finished their task in their own homes (area is 25 to 35 m2).Thirty-six participants who intended to purchase furniture or visited a furniture exhibition hall , the acceleration induction module, and the triaxial gyro, which could simultaneously determinate six directions, positions, movements, and accelerations. In addition, the iPad was equipped with GPS, cellular data, gravity sensors, and a distance sensor.Figure 1 shows a VR online shopping rendering. Using the iPad 2, the user viewed a stack effect between virtual products and indoor facilities using a real-time viewfinder of SnapShop software, ultimately achieving the goal of AR .SnapShop Showroom (software developed by SnapShop Inc.) for the iPad 2 determined whether the furniture was decorated in the rendering. After choosing a product, the product image floated on the camera and could be added to the interior space. The simulated rendering could also be saved and shared with friends. This experiment used a virtual background room that belonged to the software and an authentic depiction of the house for participants. Downsizing recording software compatible with the iPad 2 could easily record, export, and broadcast data. The experiment was conducted in the audio–visual integration channel condition. The participants were asked to view IKEA products in the network advertisement and listen to an IKEA advertisement, which was produced by downsizing the recording software  at the same time.The embedded figure test (EFT) was the measurement used to test cognitive style (field dependence or field independence), which was revised by the Department of Psychology, Beijing Normal University . In thisThis self-reported scale  requiredThis experiment used a 2 (online shopping method: VR vs. AR) × 2  × 2 (user cognitive style: field dependence vs. field independence) × 2  × 2  mixed design. The online shopping method, product value, and sensory channel were independent variables, and the mental workload was the dependent variable.2.The experimenter entered the subject’s apartment, conducting a mental workload experiment for online shopping after a brief conversation. The experiment was conducted in the sitting room. Because of the high homogeneity of customers in the market, the areas of their sitting room ranged between 25 and 35 mFirst, participants completed the informed consent form and basic information form. Second, participants conducted a cognitive style test by EFT. Third, the items were prepared. The iPad 2 was connected to the Internet (WLAN). The experimenter demoed the online shopping process using APP (SnapShop Showroom software) to participants.Fourth, the experiment conducted a psychological experiment of online shopping. There were eight experimental treatments for each participant, including the following: VR – low value – visual conditions, VR – low value – audio and visual integration conditions, VR – high value – visual conditions, VR – high value – audio and visual integration conditions, AR – low value – visual conditions, AR – low value – audio and visual integration conditions, AR – high value – visual conditions, AR – high value – audio and visual integration conditions. In the experiment, four experimental treatments for VR (randomized experiment order) were conducted by participants; then, four experimental treatments for AR (randomized experiment order) were conducted by participants. The participants themselves entered the main page of APP and were classified. In the visual experiment, the model of product chosen by participants and the software’s own indoor scene model could be added together. By operating the touch screen, participants could move the furniture to any position. After choosing it, if the participant decided to purchase it, they placed it in their shopping cart; otherwise, they gave up. In the AR experiment, the model of product choosing by participants and the authentic scene of the house could be added by the cameras of the iPad 2 to combine the virtual and real scenes. By operating the touch screen, participants could move the furniture to any position. After choosing it, if the participant decided to purchase the furniture (with a price tag), they placed it in their shopping cart; otherwise, they gave up. Study participants were recruited from visitors to a furniture store. The experimenter compared the price of a sofa and mirror in the store. In this experiment, the sofa was defined as a high-value product; the mirror was defined as a low-value product. The visual shopping interface was produced from SnapShop Showroom software. Audio advertisement was broadcasted by downsizing recording software . In audio–visual integration, the experiment synchronized the visual interface and recording auditory advertisement (loop). Participants could store or share photos added together with the model of the product chosen by participants and the authentic scene of the house. Under the eight experimental treatments, all the participants were tested by NASA-TLX.F = 4.20, p < 0.05, F = 0.44, p > 0.05]. The main effects of product value , sensory channel , and cognitive style were not significant . The interaction between the user gender and product value was significant [F = 6.08, p < 0.05, F = 4.22, p < 0.05, Figure 3) showed that for the males, high-value products’ mental workload was significantly higher than that of low-value products . H1 is confirmed. In the VR situation , the visual mental workload of field-independent and field-dependent consumer showed a significant difference (38.56 vs. 49.00), but the difference was in reducing in audio–visual condition (39.83 vs. 47.87). Mental workload of users for field dependent was difference in visual and audio–visual conditions (49.00 vs. 47.87). H2 is confirmed. In the AR situation, the visual mental workload of field-independent and field-dependent consumers showed a significant difference (40.40 vs. 48.65), but the difference was an increase in audio–visual conditions (38.88 vs. 49.46). Mental workload of users for field dependent was difference in visual and audio–visual conditions (48.65 vs. 49.46). H3 is confirmed.Researchers performed data management using SPSS 17.0 software. The study used ANOVA, multiple comparisons and analysis of simple effects. The repeated-measures ANOVA revealed that the main effects of gender were significant [Virtual reality technology represents a model of the real world produced by a computer . In the In the VR situation, the visual mental workload of field-independent and field-dependent consumers showed a significant difference, but the difference was in the reduction of audio–visual conditions. Field-dependent consumers relied more on the surrounding environment as a frame of reference. Because of the effect of the cognitive information on visual advertising, the mental workload of field-dependent consumers was increasingly affected by the visual information. However, if the visual information added auditory content, consumers would have a more comprehensive knowledge of products, and this situation reduced mental workload. Field-independent consumers relied more on themselves as a frame of reference. The audio–visual integration information increased their mental workload.Augmented reality technology is one of three major technologies identified as likely to change the future of shopping (the others are QR codes and mobile payment). In the AR situation, males’ mental workload was greater than females’ mental workload. Considering that males shop online less frequently than females , they woIn this experiment, participants combined the product with their own home because the AR combined the real situation and virtual situation. The AR technology adds computer technology to promote human–computer interactions to enhance the user’s perception toward the realistic world . This kiField-dependent consumers relied more on the surrounding environment as a frame of reference. Information perception has become an important factor in AR shopping . BecauseThe user’s mental workload is different for AR and VR. The sensory channel and cognitive style have become the main factors for inconsistency. Firstly, field dependence is easily affected by the shopping environment. In the VR environment, multiple modalities (vision and auditory) decrease the mental workload for field-dependent users because most of the senses are constructed by themselves, not shopping environment. The construction of feeling is accelerating cognitive process. In the AR environment, multiple modalities increase mental workload for field-dependent users because most of the senses are generated based on the real world. The machining process consumes a lot of cognitive resources. However, field-independent users are not affected by the shopping environment. Secondly, the formation of embodied cognition is through the body’s experience and its activity. The cognition  of AR online shopping is through the body’s online experience and self. The cognition of AR online shopping essentially reflects the characteristics of embodied cognition. In fact, the relationship of embodied cognition and social consumption has become the important research area . AR is uUsers’ mental workload and SA are the keys to the supervisory control of safety–critical systems . SimilarThe AR shopping platform should pay attention to psychological differences, physical differences and gender differences. The shopping platform should include three aspects of improvement, namely gender difference, personality difference and product value features. Male and female shopping platforms should highlight these differences. The mental workload of users of different genders was not the same. The marketing of differently gendered products should employ a diversified strategy. Through shopping data, we can establish a shopper’s personality model. Based on the shopper’s personality model, the retailer could make personalized APP experiences. The study suggested that AR shopping should try to use visual advertising.Although most of our findings support our hypotheses, other features of the shopping experience may have influenced the results. One factor may be the participant selection. Because household surveys are difficult, the number of participants was small. The sample size can be further expanded. Second, the choice of just one item (the sofa) as high-priced item and of just one item (the mirror) as a low-priced item is a limitation. The choice of the item may be one of the influencing factors. The third factor is the contention in cognitive neuroscience. The study results should be verified through neuromarketing methods. Wireless wearable physiological equipment is available for future research. Fourth, AR online shopping will become the mainstream in future studies.In online shopping, the males’ mental workload was greater than the females’ mental workload. The property of high value was the main influencing factor for the high mental workload of the males. The psychological effect of AR technology and VR technology differs in online shopping. The method of online shopping, sensory channels and cognitive style interacted in online shopping.The study was approved by the academic and ethics committee of school of education in Hebei University. The participants who were intend to purchase furniture or visited in the furniture exhibition hall participated the experiment. The participants were asked finished their task in participants’ apartment. All participants provided their written informed consent to participate in this study.Conceived and designed the study: XZ, CS, and XY. Performed the study: XZ and CS. Analyzed the data: XZ, CS, and CZ. Wrote the paper: XZ, CS, XY, and CZ.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.


Background. Allergic rhinitis (AR) significantly impairs the quality of life of the patients; however, a questionnaire alone is an insufficient and subjective measure of this condition. Obtaining an objective clinical assessment of the level of impairment will be valuable for its treatment. β-Endorphin is one of the most important mediators of both mental state and specific immunity. Thus, we investigated the possibility of using β-endorphin as a biomarker for evaluating the impairment level in AR. Methods. This study included 48 patients with AR and 32 healthy volunteers. The serum β-endorphin level was determined by enzyme immunoassay, and the serum-specific IgE and total IgE levels were determined by immunoblot assay. The Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) was used to assess the impairment level in the symptom duration. Results. The β-endorphin concentration was significantly decreased in AR patients compared to the healthy controls . There was significant negative correlation between the impairment level and serum β-endorphin level , but there was no association between the serum β-endorphin and total IgE levels . Conclusion. β-Endorphin is a systemic biomarker that has the potential to assess the impairment level in AR and may therefore be a novel therapeutic target for the treatment of AR. Allergic rhinitis (AR) is a symptomatic disorder involving the nose and eyes and is caused by exposure to allergens. It is characterized by sneezing; itching; nasal congestion; rhinorrhea; and generalized symptoms including fatigue, mood changes, depression, anxiety, and impaired ability to work or study. These bothersome symptoms often impair the quality of life (QOL) of the patients . The QOLβ-endorphin is one of the most important mediators of both the immune response [β-endorphin is an important peptide for maintaining the homeostasis of behavioral, cognitive, and neuroendocrine functions and mediating the Th1-Th2-type response switch [β-endorphin has potential as a biomarker for evaluating the QOL in AR patients. In this study, we investigated the serum β-endorphin level in patients with AR and in healthy controls and evaluated the associations between the serum β-endorphin level and RQLQ scores as well as total IgE grade in the symptomatic stage in AR patients.The immunopathology and psychological state of AR are the main factors affecting the QOL in patients with AR. The opioid peptide response , 4 and mresponse , 6. Datae switch . Thus, wParticipants were recruited from the Clinic of Otorhinolaryngology, Head and Neck Surgery of our hospital. Patients with AR were selected according to the diagnostic criteria proposed by ARIA  and were in the age range of 18–65 years [β-endorphin levels. One patient did not complete the procedure. The study was approved by the Ethics Committee of our Hospital (IRB: 2014ks033), and all the participants gave their informed consent.Thirty-two healthy volunteers  and forty-eight patients with AR  were recruited for participation in this study. Serum samples were collected from all patients and healthy volunteers between 8 AM and 10 AM and stored at −80°C within 1 h until the assay for IgE and The QOL impairment level was assessed using the RQLQ. The RQLQ contains 28 items in 7 domains, including activity, sleep, nose symptoms, eye symptoms, non-nose/eye symptoms, practical problems, and emotional state. Each item was scored from 0 (not troubling) to 6 (extremely troubling) . The totβ-endorphin (pg/mL) in the serum was assayed using a commercially available ELISA Kit  according to the manufacturer's instructions. The total IgE was detected using an AllergyScreen Test Kit , which indicates if the total IgE content is less than 100 KU/L, between 100 and 200 KU/L, or higher than 200 KU/L.The concentration of t-test was used to compare the β-endorphin concentrations between the AR and healthy control subjects. One-way analysis of variance (ANOVA) for the three groups and Spearman's rank correlation coefficient (as continuous variable) were used to assess the relationship between the serum β-endorphin level, the RQLQ score, and the total IgE grade. A two-tailed value of p < 0.05 indicated statistical significance.Data were analyzed using SPSS 17.0 . An unpaired β-endorphin level was significantly  reduced in the AR patient group (25.93 ± 13.87 pg/mL) compared to healthy controls (50.424 ± 20.729 pg/mL) (The serum 9 pg/mL) .n = 19). In the moderate group, the mean score was 79.70 ± 16.19 (n = 23). In the severe group, the mean score was 126 ± 19.18 (n = 5). The mean β-endorphin concentrations in the mild, moderate, and severe groups were 33.65 ± 15.73, 22.27 ± 9.59, and 11.64 ± 2.64 pg/mL, respectively  and the scores . However, there were no correlations between the β-endorphin concentration and total IgE grade , moderate (scores 57–112), and severe (scores 113–168). In the mild group, the mean score was 38.47 ± 11.93  molecule, is associated with the activity of the stress-sensitive hypothalamic-pituitary-adrenal (HPA) axis [β-endorphin is widely known in the regulation of immune function [β-endorphin levels have been reported in human rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, gout, ankylosing spondylitis, pseudogout, and psoriatic arthritis [β-endorphin level was significantly decreased in patients with AR compared to healthy controls. Results from a previous colorectal hypersensitivity study showed that mice that developed hypersensitivity have reduced expression of the β-endorphin gene and suggest that β-endorphin acts as a peripheral mediator in hypersensitivity [β-endorphin is an independent biological marker for allergic symptoms and disease severity [β-endorphin level in patients with atopic dermatitis [β-endorphin response has been shown to decrease the activation of respiratory muscles and change the pattern of breathing to a more rapid and shallow one [β-endorphin may play a crucial role in allergic diseases such as AR, which is similar to the findings of our study. Another study found a statistically significant reduction in the β-endorphin concentrations after histamine provocation in patients with asthma [The opioid PA) axis , 13. Thefunction . Reducedrthritis  as well rthritis . In our sitivity . Reportsseverity –20. In trmatitis . Investillow one . These sh asthma .β-endorphin induces an increase in nasal congestion through the direct neuroendocrine receptor of mast cells that enhances the mediator response to nasal allergen challenge by intranasal β-endorphin challenge [β-endorphin level as a biomarker and found its statistical significance; however, this study only measured olive and grass pollen immunotherapy in children [One study found that hallenge . In an ichildren .β-endorphin by neuroendocrinology mechanisms, including interaction with sensory nerves [β-endorphin is not only a mediator of immune responses but also a predictor of mental health problems [β-endorphin has received considerable attention for both its immunological function in allergic disease and its potential to predict the mental state of patients, few studies have investigated the relationship between β-endorphin and QOL, which are associated with these two conditions. These previous studies provided evidence that β-endorphin is associated with impaired QOL in AR, and our study further explored this possibility. Our results revealed that serum β-endorphin and RQLQ scores were highly negatively correlated between both the classified groups and the scores.These studies focused on the suppression of immunological function of y nerves , mast cey nerves , blood Ty nerves , and evey nerves . It is kproblems –32. Mentproblems –35. AlthThis study has some limitations. First, the sample size of our study is small. Second, other covariates such as age, gender, and psychological factors of the participants who filled out the RQLQ were not accounted for. Addressing these limitations in future studies will help verify the findings of the present study.β-endorphin level was obviously reduced in the patients with AR compared to the healthy controls. Second, the β-endorphin level was significantly related to the RQLQ severity of the AR.In conclusion, our study revealed two important findings. First, the serum