Similar results are seen when antagonizing Nrp1 using a dominant negative peptide. In genetic experiments, deletion of Nrp1 results in delay of mammary development. In addition, knockdown of Nrp1 inhibits MMTV-Wnt1 tumor growth in xenograft. Our data demonstrate that Nrp1 is critical for mammary development and tumorigenesis, revealing new insights into MaSC regulation and targeting stem cells in treatment of breast cancer.Wnt/β-catenin signaling is instrumental for the development of mammary gland and the properties of mammary stem cells (MaSCs). The Wnt signaling downstream effectors that engage in regulating MaSCs have not been extensively studied. Here, we report that Neuropilin-1 (Nrp1) expression is induced by Wnt/β-catenin signaling in MaSCs, and its function is critical for the activity of MaSCs. Nrp1 is particularly expressed in MaSCs that are marked by the expression of Protein C Receptor (Procr). Knockdown of Nrp1 by shRNA diminishes MaSCs’  Highly elongated basal cells and cuboidal luminal cells compose the two main cellular lineages of the nulliparous and non-pregnant mammary gland. The basal cell population  is able to generate new mammary glands in transplantation assays, thus representing a mammary stem cells (MaSCs)-enriched population3. More recently, study from our lab reveals a more refined MaSC population that is marked by the expression of Protein C Receptor (Procr). Procr+ MaSCs are composed of about 3–8% of total basal cells depending on the genetic background. Procr+ MaSCs have the highest reconstitution efficiency in transplantation assays compared to total basal cells and other known basal subpopulation4.The mammary gland is an epithelial organ, with a tree-like pattern of ductal networks. The majority of mammary development occurs postnatally. At the onset of puberty at around 3 weeks of age in mice, in response to ovarian hormones, the preexisting rudimentary ductal tree rapidly expands and extends across the fat pad, occupying the whole mammary fat pad by approximately 7 weeks of age9. In 3D Matrigel cultures, addition of Wnt3A or Wnt4 proteins to MaSC-enriched basal cell culture can maintain stem cell properties and promote MaSC expansion. The retention of stem cell properties is demonstrated by the ability of the cultured cells to efficiently reconstitute mammary glands in transplantation9. In an attempt to identify Wnt targets specifically expressed in MaSCs, microarray analysis of cultured MaSC-enriched basal cells was performed, leading to the discovery of the MaSC specific surface marker Procr4.The microarray analysis also suggests other new Wnt downstream target genes in mammary epithelial cells, which are potentially critical for the activities of MaSCs.Wnt/β-catenin signaling has been implicated in almost all stages of mammary development and is instrumental for MaSC self-renewal and expansion activities  and vascular endothelial growth factor (VEGF) family are well established ligands for Nrp113. Evidence has revealed that the Nrp1 also interacts with other growth factors11. Nrp1 and it close family member Nrp2 are mostly known for the regulation of cell motility, particularly with respect to neural and vascular development17. Nrp1 may play a role in epithelial cells as well. Robust Nrp1 expression has been found in human epithelial tumor cells derived from lung, breast, prostate, pancreatic, and colon carcinomas11. Nrp1 has also been implicated in the migration and survival of breast cancer cells20, however its potential role in MaSCs and in normal mammary development remains elusive. In this study, we identified Nrp1 as a novel target of Wnt/β-catenin signaling. We showed that the expression of Nrp1 is enriched in Procr+ MaSCs, and that Nrp1 plays an essential role in MaSC property maintenance and MMTV-Wnt1 mammary tumor growth.Neuropilin-1 (Nrp1) is a single-pass transmembrane glycoproteins, with a small cytoplasmic domain and multiple extracellular domainsin vitro culture system in which MaSC properties can be maintained using purified Wnt proteins8. In this culture system, mammary basal cells  were isolated using fluorescence-activated cell sorting (FACS) and cultured in 3D Matrigel in the presence or absence of Wnt3A proteins4. Microarray was performed using the cultured cells to identify downstream effectors of Wnt signaling in regulating MaSCs  assays. We used β-catenin antibody to immunoprecipitate chromatin DNA in mouse mammary cells and examined specific binding of the following: 9 separate fragments containing the TBE of Nrp1; a fragment without TBE at Nrp1 promoter region which acted as a negative control; and a fragment with one TBE at the Axin2 promoter as a positive control. As shown in Fig. Sequence analysis revealed six putative TCF-binding elements (TBEs) at the promoter and distal region and five putative TBEs at the first intron of rp1 Fig. . To test−, CD24+, CD29hi), luminal  and stromal cells  from 8-week old nulliparous mice, and analyzed Nrp1 levels by qPCR. We found that in the epithelial compartment, Nrp1 is markedly higher in basal cells than that in luminal cells, while it displays the highest level in stromal cells  and the other basal cells  were isolated from mature nulliparous female mice. We found that Nrp1 is expressed at a higher level (4.5 fold increase) in Procr+ MaSCs by qPCR analysis , whereas no Nrp1+ cells are found in luminal cells   mouse model, in which tdTomato serves as a reporter for Procr expression4. Immunostaining indicated that there is a substantial overlap of Nrp1 and tdTomato expression in basal compartment, in which about 34% of Procr+ basal cells were Nrp1+  . Together, these data suggest that in the epithelial compartment, Nrp1 is highly expressed in Procr+ MaSCs.To investigate the expression pattern of Nrp1 in the mammary gland, we isolated basal  Fig. . Of noteent Fig. , which ils) Fig. . This cain vitro. Isolated basal cells from nulliparous mice were infected with lentivirus containing GFP tagged Nrp1 shRNA. Infected cells were placed in 3D Matrigel culture and the formed colonies were visualized by GFP. To minimize potential off-target effect, two independent sh-RNAs were constructed for Nrp1 knockdown. The knockdown efficiency of sh-Nrp1 was confirmed by qPCR analysis . The mammary glands were harvested at 6-week old. In whole mount imaging, consistently we observed a delayed ductal extension phenotype in K14-Cre;Nrp1fl/fl;R26-mTmG when compared to the control  was administered in pubertal mice at 3-week old every other day for 3 times, the phenotype resulted from Nrp1 deletion was evaluated at 4 weeks later  had almost completed the epithelium extension and occupied the whole fat pad   Fig. . qPCR anion Fig. . These dMMTV-Wnt1 mammary tumor is a malignancy that has close association with MaSCs22. Considering the important role of Nrp1 in MaSCs, we examined its impact in MMTV-Wnt1 tumor growth. Immunofluoescent staining in tumor section suggested a sporadic pattern of Nrp1+ cells in MMTV-Wnt1 tumor epithelial compartment  are most likely contributed by the Nrp1 in mesenchymal compartment.The specific Nrp1 expression in Procr24. Nrp2 is expressed preferentially in the TEBs of developing glands. Deletion of Nrp2 using MMTV-Cre results in defects in branching morphogenesis24. VEGF165, an isoform of VEGF, induces Nrp2-dependent branching morphogenesis as demonstrated in vitro 3D culture model with a mouse mammary cell line24. In this study, we found that Nrp1 is particularly expressed in Procr+ MaSCs, distinct from the TEB enriched expression of Nrp2; Nrp1 is a Wnt-target gene in the mammary epithelium while Nrp2 is not (data not shown). Our data demonstrated the critical role of Nrp1 for the activities of MaSCs and the development of mammary gland, yet the mechanism by which Nrp1 functions in MaSC remains to be elucidated. Mammary epithelial cells express VEGF24, implying a possibility that VEGF is a potential ligand for Nrp1 in MaSCs. Considering that Nrps function as co-receptor for other growth factor, including fibroblast growth factor (FGF) family25, galectin-126, hepatocyte growth factor/scatter factor (HGF/SF)29, transforming growth factor-β1 (TGF-β1)31, platelet-derived growth factor (PDGF)32, and integrin receptors33, thus it is possible that Nrp1 also mediates the impact of other ligands in MaSCs.Nrp1 has a structural homolog, Nrp2, with 44% amino acid identity. They differ in their regulation and function . All experimental mice were treated according to the protocols approved by the Animal Care and Use Committee of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.The The fat pads were isolated from 8-week-old nulliparous or other specified-stage female mice. Fat pads were minced and placed in appropriate volume (1 g/10 ml) digestion mix  and digested for 2 h in a 100 rpm/min shaker at 37 °C, give a good shake every 15 min. Cells were pelleted and treated with red blood lysis buffer (Sigma) for 5 min, single cell suspension was obtained by subsequential incubation with 0.25% trypsin-EDTA at 37 °C for 5 min and 0.1 mg/ml DNase I (Sigma) for 5 min with gentle pipetting, and then filtrated through 70-μm cell strainer.For cell labeling, the following antibodies were used: FITC conjugated CD31, CD45, TER119 (BD Pharmingen); CD24-PE-Cy7, CD29-APC (Biolegend), Procr-PE (eBioscience). Antibody incubation was performed on ice for 15 min in PBS with 5% fetal bovine serum. Before the FACS sorting, cells were filtrated through 45-μm cell strainer, and cells were sorted by using a FACSJazz (BD).To construct shRNA vectors, the annealed oligonucleotides were ligated to pLKO.1-EGFP, which was modified by replacement of the puromycin-resistance gene with EGFP. The target sequences of shRNAs were listed: Scramble shRNA: 5′-TCCTAAGGTTAAGTCGCCCTCG-3′; Nrp1 sh1: 5′-TGGCTGCAAGATAACAGATTA-3′; Nrp1 sh2: 5′-CCAGAGAATCATGATCAACTT-3′. The extracellular domain of mouse Nrp1 (NM_008737.2) were amplified by PCR from E14.5 cDNA, and then cloned into pHIV-EGFP to overexpress sNrp1 proteins. Recombinant lentiviral particles were produced in HEK293 cells by a polyethylenimine, linear  transfection system. Lentiviral particles were concentrated 38-fold by ultracentrifugation (Beckman Coulter) for 2 hours at 27,000 rpm, 4 °C.5 cells/ml overnight, and resuspended at a density of 4 × 105 cells/ml in chilled 100% growth-factor-reduced Matrigel (BD Bioscience), 50 μl/well of 24 wells cluster. The mixture was plated in 37 °C incubator for polymerization before being cultured with growth medium  and ITS ) which was changed the other day. Primary colony numbers were counted and the colony sizes were measured after 6 days culture. For passaging colonies, the medium was aspirated, and Matrigel was incubated in 500 μl of dispase (BD Bioscience) for 1 h  at 37 °C. Colonies were released from Matrigel and single cells were obtained by incubation in 0.05% Trypsin-EDTA for 5–10 min at 37 °C. Single cells were then resuspended in Matrigel as described above at the same density.FACS sorted cells were infected with lentivirus in a ultralow attachment cluster at a density of 3 × 10in vitro culture colony growth medium was aspirated, replaced with growth medium containng 0.2 mg/ml EdU (Life Technologies), followed experiment steps were performed according to the manufacturer’s protocol of Click-it EdU imaging kit with Alexa Fluo 594 (Life Technologies) prior to immunohistochemistry detection of EGFP.Half of Total RNA was extracted with RNAiso plus (Takara) or mini RNA preparation kit (Qiagen), and the reverse transcription reaction was performed by using PrimeScript RT Master mix kit (Takara). Quantitative RT-PCR was performed on StepOne Plus (Applied Biosystems) with FastStart Universal SYBR Green Master Mix kit (Roche). The RNA levels were analyzed by ΔΔCt method and normalized to Gapdh. qPCR product specificity was determined by melt curve analysis. The primers using in this study were listed:Gapdh-F: TGTGATGGGTGTGAACCACGAGAAGapdh-R: CTGTGGTCATGAGCCCTTCCACAAAxin2-F: AGCCTAAAGGTCTTATGTGGCTAAxin2-R: ACCTACGTGATAAGGATTGACTNrp1-F: ACAAATGTGGCGGGACCATANrp1-R: CCGGAGCTTGGATTAGCCATProcr-F: CTCTCTGGGAAAACTCCTGACAProcr-R: CAGGGAGCAGCTAACAGTGA6) were plated for 48 h and treated with vehicle or Wnt3A protein for 24 h before PFA crosslinking. Briefly, cells were cross-linked for 10 min with final concentration 1% PFA at room temperature. 125 mM final glycin was added to inactivate PFA at 37 °C for 5 min. Cells were scrapped and pelleted, cell pellet was incubated in chilled Mg-NI NP40 buffer for 10 min, then resuspended in lysis buffer with proteinase inhibitors. Genomic DNA was sonicated. Precleared sample (0.1 ml) was saved to assess input DNA. Anti-β-catenin antibody  was added to the precleared sample and gently mixed for O/N at 4 °C, followed by rotation with 50 μl protein G-agarose/Salmon sperm DNA (Millipore) for 2 h at 4 °C. Subsequent wash, elution, proteinase K treatment and de-cross-linking procedures were performed. TBEs enrichment was determined by qPCR analysis.Comma D β cells  for 2 min, mounted with prolong gold mounting medium. The primary antibodies used in this work were goat anti-mouse Nrp1 , Rat anti-K8  and rabbit anti-mouse Keratin 14 . IHC detection was performed with appropriate secondary conjugates (Life Technologies). Confocal images were captured by using Leica DM6000 TCS/ SP8 laser confocal scanning microscope.th pair of mammary glands were fixed in pre-chilled 4% PFA and washed with PBS twice. Tissues were incubated with carmine alum overnight, followed by destaining with solutions  for 2 h, dehydrated in 70%, 85%, 95%, 100% ethanol for 1 h, and treated with histoclear for 1 h. Images were captured by a dissection microscope (Leica).The 4http://www.R-project.org). Repopulating frequencies were estimated with a complementary log-log generalized linear model. Two-sided 95% Wald confidence intervals were computed, except in the case of zero outgrowths, when one-sided 95% Clopper-Pearson intervals were used instead.FACS Sorted cells were resuspended in 50% matrigel, PBS with 20% FBS, and 0.04% Trypan Blue (Sigma) and injected 10 μl volumes into the cleared fat pads of 3-week-old female nude mice. Reconstituted mammary glands were harvested 8 weeks after transplantation. Outgrowths were detected under a dissection microscope (Leica) based on fluorescence or carmine staining. Outgrowths with >10% of the host fat pad filled were scored as positive, and fat pad filled was marked as 4/4  filled, 3/4, 1/2, 1/4 and no growth. The repopulating frequencies were calculated using the “statmod” software package for the R computing environment  was calculated to indicate the variation within each experiment. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) were considered as significance.Student’s t-test was performed and the


Returning to sport is a key determinant of longer-term quality of life after ACL reconstruction . AlthougIt is possible that many rehabilitation programs are falling short in the later stages when it comes to physically and psychologically preparing an ACL reconstructed individual to return to sport. A primary aim of ACL rehabilitation is to restore physical knee deficits, yet restoration of knee deficits does not correspond to a successful return to sporting performance or prevention of further knee injury. Evidence is limited surrounding predictors of successful rehabilitation, return to sport, and reinjury after ACL reconstruction .The study by Arundale and colleagues, explored the benefit of adding perturbation training to a high level rehabilitation program designed to facilitate return to preinjury sport and minimize reinjury rates. The addition of perturbation training did not improve outcomes in this specific sample of ACL reconstructed men.Individuals who achieve dynamic knee stability after ACL rupture through rehabilitation alone, can return to sport with similar longer-term outcomes as those who underwent ACL reconstruction , 9. HoweInstead of seeking an ideal rehabilitation approach to improve outcomes for all ACL ruptured individuals, there is a need to identify common characteristics of patients who respond favorably to specific elements of ACL rehabilitation. This will help guide tailored rehabilitation recommendations, based on the physical and psychological characteristics of an individual with acute ACL injury.Too often ACL rehabilitation overlooks psychological barriers to returning to sport, including psychological readiness, low self-efficacy, knee confidence, and reinjury fears , 13. A gAdditionally, the KOOS-quality-of-life subscale is not ideal for assessing quality of life after ACL injury and reconstruction. An individual who is aware of their knee, or who modifies their lifestyle because of their knee, will have an impaired KOOS-quality-of-life score even if these are not negatively impacting upon their life quality. The ACL-quality-of-life score may be a more appropriate measure of quality of life following ACL injury and reconstruction .The rehabilitation journey should not end on return to sport. After returning to sport, the focus should shift to returning to preinjury performance, followed by a maintenance phase to reduce risk of further knee injury.Future studies delivering standardized ACL rehabilitation to participants should assure that rehabilitation strategies are described in reproducible detail. This should be done for preoperative rehabilitation, postoperative rehabilitation, and management with rehabilitation alone. This would enable future data pooling and meta-analysis, and advance current knowledge in this field.There is also a need for randomized controlled trials comparing the efficacy of different rehabilitation strategies within groups at risk of poor longer-term outcomes .ACL rehabilitation approaches may evolve through trialling new and novel interventions that extend beyond current practices and draw upon the neuroscience and psychological literature to address the neurophysiological and psychological impacts of ACL injury and reconstruction.


The dynamics of a spatial quantum formulation of the iterated Samaritan’s dilemma game with variable entangling is studied in this work. The game is played in the cellular automata manner, i.e. with local and synchronous interaction. The game is assessed in fair and unfair contests, in noiseless scenarios and with disrupting quantum noise. Beyond γ=1.460 it is The pay-off dependence of ΠQQ, generates ΠQD, ΠDQ and ΠDD, respectively. The pay-off dependences of γ in the SD of the pure strategies with full noise may be checked in μ=1. In particular, in the  context, With maximum noise it isK1 Kraus operator, deferring in ΠQQ becomes diagonal as with amplitude damping, but with μ=0.5 phase damping, ΠQQ also turns out diagonal  in a two-dimensional N×N lattice. In order to compare different types of players, two types of players, termed A and B, are to be considered. A and B players alternate in the site occupation in a chessboard form, so that every player is surrounded by four orthogonal adjacent partners , and four diagonal adjacent mates, i.e. players with the same role, either Samaritan or beneficiary . The game is played in the cellular automata (CA) manner, i.e. with uniform, local and synchronous interactions [i,j) will adopt the parameters of his mate player  with the highest pay-off among their mate neighbours.In the spatial version of the quantum SD (QSD-CA), we deal with each player occupying a site  parameter values in each scenario. In the subsequent figures, the results regarding the charity player A have been marked red, and those regarding the beneficiary player B have been marked blue. With the exception of T=200 with variable entanglement factor γ at three noise levels. These figures show in their left frame the mean pay-offs across the lattice  –21 and tγ up to just passed π/8, γ=0 (or if αA=αB=0), Π becomes factorizable as in the classic game with independent strategies  pQQB=2 and γ>γ*=π/4 and (ii) pQQA=3 and rategies , i.e. Π=Q,Q) with high entanglement. The latter reverses the SD structural trend in favour of the beneficiary player, inducing the highest possible sum of pay-offs. One may say that the Q strategy resolves the dilemma, though the sceptical reader might argue that as Q has no real component (θ=0), the dilemma is resolved in the imaginary … world.Thus, the main finding derived from γ=0 (a), γ=π/4 (b) and γ=π/2 (c). As a result of the initial random assignment of the parameter values, it is initially in both frames: γ=0, after a short transition time, both the parameters and mean pay-offs stabilize their values fairly soon, the latter close to the pay-offs of NE . In the γ=π/2 dynamics in figure 4c, both π, and both Q strategy and in consequence the mean pay-offs of A and B players stabilize at  in a straightforward manner. Remarkably, the parameter tendencies heavily emerge from the very beginning, despite the full range of parameters initially accessible in the CA local interactions. In figure 4b, with γ=π/4, the threshold for the emergence of Q, the parameter stabilization is achieved also in fast manner, with the exception of T=100 in a rather unexpected way which determines the sudden leap of γ=0 a, γ=π/4 ), γ=π/4 b and γ=πnd γ=π/2 c. As a rαA=0 and αB=π/2. γ and select the NE pair with θA=3π/4 and θB=π/4 for high entanglement. The most distinctive difference of γ.The seminal reference  analysesorted in  in the fΠ may be generated from independent strategies  in the classic context, without resorting to any quantum approach. Thus, for example, an ad hoc method based in the external parameter 0≤k≤1 is given in [Let us point out here that non-factorizable given in , and shok* emerges from the x≤1 restraint. The pay-offs of both players in the SDG with strategy pairs in NE under the model 2), pB=((3−2k)(1+2k))/(4−2(2k−1)2) for k≤k*=0.89.With the joint probabilities generated as in , the paihe model  are plotb,c shows the mean pay-offs (b) and mean values of x and y (c) in five simulations at T=200 of a SDG-CA. The form of the pay-offs curves in the CA simulations mimic remarkably well that achieved with strategies in NE in conventional (non-CA) simulations (a) for threshold k<k*. For k≥k* the CA simulations produce the exact values π11=(2k−1)2, π12=π21=0, π22=4k(1−k) and consequently the pay-offs of the  pair, i.e. pA=3(2k−1)2, pB=2(2k−1)2, which show a fairly linear aspect for k>k*, reaching pA=3,pB=2 for k=1 (as achieved with k=0).Figure 6pay-offs b and mea x and y c in fiveulations a for thr2.1.μ=0.5 noise. The general form of the graphs of γ=π/4 with μ=0.5 noise. From approximately γ=π/4, μ=0.5 generates the joint probabilities: γ. In consequence, the pair μ=0.5 is in NE for γ>γ*=π/3. As a reflection of this fact, in γ>π/3=1.047 both players resort again to the Q strategy (as shown in its figure 7b), and the form of both pay-offs emerging in the CA simulations is that shown in figure 2a under the μ=0.5 label, where the fairly locally linear functions intersect at γ=1.460 giving pA,B=1.777, as was demonstrated in the previous section.of noise  remains K2 varies), the pair  is not in NE with phase-damping noise for high values of γ. Consequently, in QSG-CA simulations with μ=0.5 phase-damping (not the shown here) the discontinuity observed with μ=0.5 amplitude-damping noise in γ approaches π/3 does not emerge in the pay-offs graph. The parameter graph shows in this scenario how the Samaritan player drifts to the Q strategy γ increases beyond π/3, but the beneficiary player does not accompany the Samaritan in this trend.Despite the structural proximity of both amplitude and phase-damping quantum noises mentioned at the end of §μ=1.0 noise. Full noise seems to impede the emergence of the  pair, so that the oscillations of both pay-offs not far from  shown in γ, remain here for higher entanglement.θ,α) parameters of both players tend to approach their middle levels . In this scenario, it is γ increases: pA from 0.25 down to −0.5, pB from 1.5 down to 1.0. The actual pay-offs γ=π/2 in figure 8a turn out to be over the expected . This is due to spatial effects that make it difficult to estimate the actual mean pay-offs from the mean parameters. An example of spatial structure is given in maze-like aspect. Figure 8a shows also the mean-field pay-offs (p*) achieved in a single hypothetical two-person game with players adopting the mean parameters appearing in the spatial dynamic simulation, those given in figure 8b. Namely,a, the mean-field pay-offs  are marked , somehow following the  colours of the actual mean pay-offs  pair, they are not particularly relevant, because the mean-field estimations do not appear in those figures. In the subsequent figures, the same criterion will be applied.Although spatial effects arise in 3.Let us assume the unfair situation: a type of players is restricted to classical strategies ,α) ones ,28. Sincθ,α)-player A (red) versus a classic θ-player B (blue) in a QSD-CA with variable entanglement factor γ. The structure of the asymptotic mean pay-offs across the lattice  game [b.CA) game , or θ¯A≃θ-player A versus -player B, the structure of the mean pay-offs versus γ (not shown here) resembles that shown in θ,α)-player A versus θ-player B scenario, although the quantum player B gets pay-offs slightly over 1.5, whereas the pay-offs of the classic player A stand slightly negative. The advantage of the quantum player (B) facing a classic player (A) is foreseeable; what may surprise here is that the quantum player B does not take a relevant advantage of his privileged role, getting pay-offs not far from those achieved in the opposite unfair scenario where he is the classic one.In simulations in the scenario of 3.1.αB=0 unfair scenario with μ=0.5 noise (not shown here) produce roughly the same results as in the noiseless unfair scenario of αB=0, μ=0.5) simulations, so that the mean-field pay-offs estimations fit well to the actual ones in these unfair scenarios.The simulations in the αB=0 unfair scenario of μ=1.0 noise. The results regarding both the pay-offs and mean parameters shown in μ=1.0 scenario of maze-like aspect compared to those in the fair scenario of α instead to θ as in the unfair simulations just before considered. In such a scenario, the  pair emerges in NE regardless of γ in the noiseless and μ=0.5 scenarios, as the beneficiary player can not resort to strategies such as D (or Loaf) which demands θ>0. As a result, in the noiseless figure 12a it is μ=0.5 noise (figure 12b), the c, where α parameters. This is so because θ=0 makes 4.β parameter is available.This section deals with the full space of strategies SU(2), operating with three parameters (3P) as given in , so thatγ variation, albeit in a lower degree as γ grows. For low γ, the mean-field pay-off estimations fit fairly well the actual mean pay-offs, particularly in respect of player A, but as γ grows heavy spatial effects emerge, so that the actual mean pay-offs p*, particularly in respect of player A. No particular structure becomes apparent in the parameter patterns in figure 13b, which corresponds to a rather erratic behaviour of the mean-field estimations.4.1.μ=0.5 noise (not shown here) roughly similar results as in the noiseless 3P scenario of In three-parameter strategies QSD-CA simulations with γ variation, much as in π/2.π/2, and π/4. Besides, high spatial effects emerge in θ-player A versus -player B, the classic charity player A gets negative pay-offs regardless of γ, but the pay-offs of the beneficiary player B keep not far from 1.5 for all γ, thus, surprisingly, getting smaller pay-offs than those achieved in the opposite unfair scenario of The form of the graphs in the noiseless unfair three-parameter simulations shown in T=100, together with the parameter and pay-off patterns at T=100 in a simulation with γ=π/2 in the QSD-CA unfair scenario of Figure 16a indicates that both the quantum parameters and the pay-offs quickly reach their fairly permanent values, without a relevant initial transition time. As a result, the actual (and mean-field) values shown at T=200 in T=100 (and even before) in maze-like aspect that is in the origin of the notable discrepancy between the actual and mean-field pay-offs, particularly that of the charity player A.μ=0.5 noise (not shown here), although the general form of the graphs of the pay-offs and parameters versus γ is similar to that in the noiseless scenario of μ=0.5 noise the actual mean pay-offs of both players increase their values as γ increases at a lower degree compared to those achieved in the noiseless simulations.In three-parameter strategies QSD-CA unfair simulations with μ=1.0 unfair 3P scenario of αB=βB=0, if θA=θB=π/2 and αA=βA=π/4 it is: pB=1.5, pA(γ=0)=0.25, down to pA(γ=π/2)=−0.50. Figure 17b indicates that the just described quantum parameters scenario applies with high entanglement in the simulations of this figure, and in consequence the mean–mean field pay-off estimations in figure 17a correspond with the theoretical values approximately from γ=π/4. Spatial effects minimally influence the actual mean pay-offs of the beneficiary player A in the CA simulations, whereas they support those of the charity player A so that he achieves no negative pay-offs.In the ρi=(|00〉〈00|+|11〉〈11|)/2 and ρi=(|01〉〈01|+|10〉〈10|)/2. The strategies in NE in these scenarios have in common the parameters θA=θB=π/2 and αB+βB=π/2; with the first density matrix it is αA+βA=π and with the second one αB+βB=0. The results of their corresponding CA simulations are not shown here, but they are able to detect such NE in a straightforward manner, i.e. free of spatial effects, reporting in both cases non-negative pay-offs to the Samaritan player. In the particular case of full entangling, it is In , two NE 5.solve dilemmas, by allowing for NE strategies out of the scope of the classic approach as summarized below.A spatial formulation of the iterated QSD game with arbitrary entangling is studied in this work. The game is played in the cellular automata (CA) manner, i.e. with local and synchronous players’ interaction. The evolution is achieved via imitation of the best-paid neighbour of the two players confronted in the SD: the charity and the beneficiary player. The paper considers both the general case of players accessing the full space of quantum strategies, which allows for three parameters (3P), and the particular case of strategies restricted to a subset of them allowing for only two parameters (2P). Although the restriction to two parameters may be criticized, the 2P model is a good and widely used test-bed to show how the quantum approach in game theory may Q strategy is adopted by both players in a dramatically defined manner, somehow resolving the dilemma as mutual Q provides the pay-offs of the  choice.In fair contests (facing two quantum players), the CA simulations allow for the emergence of the NE strategies. Thus, with low entanglement the strategy in NE in the classic game dominates the scene preserving the structural SD trend favouring the beneficiary player. But with high entanglement, the so-called Q,Q) pair. In our study  emerges from γ>π/4 in noiseless simulations, and from γ>π/3 with amplitude damping quantum noise at middle level. In the extreme case, full noise impedes the emergence of the  pair. Besides, noise notably alters the pay-offs of mutual Q.With quantum noise, the dynamics demands higher entanglement for the emergence of the (In the unfair quantum versus classic scenario, the beneficiary player over-scores the charity player regardless of the degree of entanglement and the degree of noise. This is so even if the beneficiary player is the classic one, albeit in this case the charity player may achieve relatively high positive pay-offs in noiseless simulations with high entangling.Mean-field approaches of the actual mean pay-offs fail as a tool for estimation due to spatial effects in unfair contests and in simulations with high noise.General pay-off matrices  and otheDeviations from the canonical cellular automata paradigm adopted here may lead to more realistic models. Particularly, structurally dynamic CA, asynchronous updating, spatial dismantling and, last but not least, dynamics with embedded tuneable memory of past pay-offs and parameter values .


The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer, as is evident from the efficacy of androgen-deprivation therapy, AR is also the most frequently mutated gene, in castration resistant prostate cancer (CRPC). AR has therefore become an even more attractive therapeutic target in aggressive and disseminated prostate cancer. To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, a toolkit of AR expressor and androgen response element (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence assays. Interestingly, the background reporter activity in androgen-depleted medium was significantly higher in LNCaP cells compared to the prostate transit amplifying epithelial cell lines, EP156T-AR and 957E/hTERT-AR with exogenous AR. The androgen-induced signal to background was much higher in the latter benign prostate cells than in LNCaP cells. Androgen-independent nuclear localization of AR was seen in LNCaP cells and reduced ARE-signaling was seen following treatment with abiraterone, an androgen synthesis inhibitor. The ARE reporter activity was significantly stronger when stimulated by androgens than by β-estradiol, progesterone and dexamethasone in all tested cell types. Finally, no androgen-induced ARE reporter activity was observed in tumorigenic mesenchymal progeny cells of EP156T cells following epithelial to mesenchymal transition. This underscores the observation that expression of the classical luminal differentiation transcriptome is restricted in mesenchymal type cells with or without AR expression, and presence of androgen. Androgen receptor (AR) plays a critical role in the normal development and function of the prostate gland . AR, alsProstate cancer is the most frequently diagnosed cancer and the second most common cause of cancer death in men . AR playin vivo and in in vitro three-dimensional (3D) tumor models.In our study, we developed ARE based assays that are useful to understand the AR activity in different conditions. The reporter assays are helpful in the evaluation of potential drug candidates in AR-directed therapies. The assays provide much faster ways to test the AR activity on a large scale. Additionally, they can be useful to study therapeutic efficacy and development of drug resistance The prostate transit amplifying basal epithelial cell line EP156T was grown in MCDB153 medium  with 1% fetal calf serum (FCS) and supplemented with growth factors and antibiotic . The proAR gene into destination vectors according to Invitrogen user manual (MAN0000682). The AR expression clone AR GC-E2325  was transferred to the destination vectors pLenti6.3/V5-DEST and pLenti7.3/V5-DEST  vectors, and the AR expression clone with V5 tag AR GC-E0060-CF  was transferred to pLenti6.3/V5-DEST destination vector. The schematic maps of AR expression vectors are shown in AR expression clones were generated by performing LR recombination reactions to insert the rd generation lentiviral destination vector pEZ-Lv152 . Three ARE reporter vectors CS-GS241B-gLUC-Lv152, CS-GS248B-gLUC-Lv152 and CS-GS249B-gLUC-Lv152 with Gaussia luciferase reporter signals and three ARE reporter vectors CS-GS241B-mCHER-LV152, CS-GS241B-mCHER-LV207-01 and CS-GS241B-mCHER-LV207-02 with mCherry fluorescent reporter signals were prepared and supplied by Labomics S.A. . The schematics of ARE reporter vectors are shown in ARE reporter vectors were developed by inserting ARE consensus binding artificial promoter combinations in the 3™ Packaging Mix according to the manufacturer’s guideline . Lentivirus was harvested from the supernatant 48 hours and 72 hours post transfection. EP156T cells were infected with harvested lentiviral particles combined with polybrene at final concentration 8 μg/ml and incubated for 24 hours. Used medium was replaced with androgen-free complete MCDB153 medium containing growth factors, 1% charcoal stripped FCS for 48 hours. The infected target cells were cultivated with 2 μg/ml blasticidin, and stably transduced EP156T-AR cells were selected. EP156T-AR cells were grown and maintained in androgen-free MCDB153 medium containing growth factors and charcoal stripped 1% FCS.The virus particles were generated by transfecting 293FT producer cells with pLenti6.3/AR-GC-E2325 AR expression vector and ViraPower™, Cat. No. HPK-LvTR-20). The lentiviral particles in the supernatant were harvested 48 and 72 hours post transfection and treated with Lenti-Pac™ Lentivirus Concentration Solution according to the manufacturer’s guideline . EP156T, EP156T-AR, 957E/hTERT-AR, EPT3-PT1-AR and LNCaP cells were infected with harvested lentiviral particles combined with polybrene at a final concentration of 8 μg/ml. Used medium was replaced with fresh medium and cells were incubated at 37°C for 48 hours. The infected target cells were grown with 12.5 μg/ml hygromycin and stably transduced EP156T-241B, EP156T-AR-241B, 957E/hTERT-AR-241B, EPT3-PT1-AR-241B and LNCaP-241B cells were selected. In addition, LNCaP cells were transduced with CS-GS241B-mCHER-LV207-01 and CS-GS241B-mCHER-LV207-02 vectors following the same protocol to obtain LNCaP-207-01 and LNCaP-207-02 cells, respectively.The virus particles were generated by transfecting CS-GS241B-mCHER-LV152 and HIV packaging mix in HEK 293Ta lentiviral packaging cell line following the manufacturer’s instructions  transfection reagent was used. The cells were maintained in androgen-free DMEM medium  with 10% charcoal-stripped FCS , treated and were assayed for Firefly and Renilla luciferase activities in Synergy H1 Hybrid Reader  using appropriate luciferase substrate from Dual-Glo® Luciferase Assay System . All luciferase assays were performed at least in triplicate.The HEK 293FT producer cells were grown in white walled, flat surface 96-well plates and co-transfected with Cignal Androgen Receptor Reporter (CCS-1019L) and AR expression vectors according to the manufacturer’s guideline . Lipofectamine™Gaussia Luciferase Assay Kit (GeneCopoeia. Cat. No. SPGA-G100). Afterwards, cell proliferation assays were performed using CellTiter 96® AQueous One Solution Cell Proliferation Assay . All the experiments were performed at least in triplicate.The 293FT producer cells were grown in white walled, flat surface 96-well plates and were co-transfected with AR expression vector and ARE reporter vectors according to the manufacturer’s instruction . The cells were maintained in androgen-free DMEM medium with 10% charcoal-stripped FCS, treated and the supernatant was monitored for Gaussia luciferase activity in a Synergy H1 Hybrid Reader  using appropriate luciferase substrate from Secrete-PairOne day before the treatment, ARE responsive reporter cells were grown in androgen-free medium in 6-well plates. The cells were treated and harvested after 24 hours, washed twice with cold (4°C) PBS and immediately analysed on the LSRFortessa cytometer . All subsequent analyses were performed with FlowJo software . One percent events were accepted as false-positive in the negative controls throughout the experiments. All the experiments were performed at least in triplicate.One day before the treatment, ARE responsive reporter cells were grown in androgen-free medium in black walled, flat surface 96-well plates. The cells were treated and analyzed for fluorescence signals by the Leica DM IRBM microscope  using Qcapture software (QImaging) and Synergy H1 Hybrid Reader  using Gen5 software (BioTek). The excitation was set at 584 nm and emission at 610 nm for mCherry signals.The cells were grown on 12 mm glass coverslips  in 24 well plates. The cells were washed with PBS and fixed with 4% formaldehyde solution in PBS for 20 min at room temperature (RT). After fixation, cells were washed twice with PBS and subjected to membrane permeabilization with 0.5% Triton X-100 for 5 min. After permeabilization, cells were washed twice with PBS and treated with 100 mM glycine in PBS for 10 min to quench free aldehyde groups and reduce background fluorescence. The cells were then washed with PBS and blocked with 0.5% BSA in PBS for 15 min at RT. Primary Anti-AR antibody  was added with 1:100 dilution in 0.5% BSA in PBS and incubated for an hour at RT. Following 3 times washing with PBS, the cells were incubated with Texas red-labelled secondary anti-rabbit IgG  for 30 min at RT in dark. The cells were than washed twice in PBS and the coverslips were mounted in Prolong Gold with DAPI  on glass slides and were analyzed.t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001. Z´-factor values were calculated as described by Zhang et. al. [p+σn)/|μp-μn| where σp and σn are the standard deviations of positive and negative controls, respectively, and μp and μn are the means of positive and negative controls, respectively. Fluorescence intensity data from the three independent experiments were used to calculate Z´-factor where 1 nM R1881 induced samples were used as positive controls and untreated ethanol (ETOH) samples as negative controls.Mean values of three independent experiments with standard error of the mean (SEM) or standard deviation (SD) are displayed. Significance was confirmed by using unpaired two-tailed Student’s  et. al.  using thAR remains critically important in prostate cancer as shown by the fact that although CRPC may be independent of androgen levels, it continues to rely on AR signaling. The factors that promote AR signaling in CRPC involve AR upregulation, mutations, co-activator protein alterations and tumor microenvironment effects –30. Thusi.e. pLenti6.3/AR-GC-E2325, pLenti6.3/ARV5-GC-E0060 and pLenti7.3/AR-E2325. These vectors were co-transfected with Cignal AR reporter in AR negative 293FT cells. The Cignal reporter consists of AR responsive Firefly luciferase and a constitutively expressing Renilla luciferase construct. The AR expression levels were detected in 293FT cells when induced with synthetic androgen 1 nM R1881 for 24 hours by using a dual-luciferase assay. This androgen-induced AR response effect was significantly higher with the pLenti6.3/AR-GC-E2325 vector construct 2-TATA Luc Reporter [KLK3 promoter region. AREI (AGAACAgcaAGTGCT) was inserted in four repeats and AREII (GGATCAgggAGTCTC) was inserted in two repeats, thereby having 109 base pairs with six ARE tandem repeats in total [The DBD of AR consists of two zinc fingers-like motifs that bind to the 15 base pairs palindromic repeat ARE consensus sequence , 31. AREReporter , therebyReporter . The AREin total . The seqThe ARE reporter vectors were co-transfected with the previously selected AR expression vector pLenti6.3/AR-GC-E2325 in 293FT cells. These cells were treated with 1 nM R1881 for 24 hours and the Gaussia luciferase response efficiencies of ARE reporter vectors were compared. MTS proliferation assays were performed to normalize the values. The luciferase reporter response in the co-transfected cells treated with R1881 showed a high AR specificity when compared to the cells that were only transduced with ARE reporters and treated with R1881. Additionally, the luciferase reporter response of the 241B consensus promoter showed a significantly higher sensitivity to androgen-induced AR activity in co-transfected cells treated with R1881 when compared among ARE reporter vectors . The 241Afterwards, the 241B consensus promoter was cloned into pEZ-Lv152 with a mCherry fluorescent reporter, CS-GS241B-mCHER-Lv152 . This veTo generate ARE reporter responsive prostate cell lines, the AR positive and androgen responsive prostate cancer cell line, LNCaP , and theThe generated LNCaP-241B and EP156T-AR-241B cell lines showed androgen-dependent reporter responsiveness when treated with 1 nM R1881 according to fluorescence microscopy, flow cytometry and multi-well fluorescent recording . FluoresNext, we evaluated the robustness of our ARE-reporter based AR activity assay using a statistical parameter Z´-factor assay . Z´-factIn order to develop a fast track ARE reporter system that will facilitate the investigation of AR functional activity, an internal normalization system of ARE driven mCherry fluorescent signals was developed. For this purpose, the constitutively expressing eGFP reporter vector, CS-GS241B-mCHER-Lv207-01 was transduced into LNCaP cells to generate LNCaP-207-01 reporter cells. In this vector the 241B ARE promoter sequence was cloned into a 3rd generation lentiviral mCherry reporter vector pEZ-Lv152 with the eGFP and hygromycin resistance genes driven by the SV40 promoter and separated by an internal ribosome entry site (IRES) as shown in Drosophila, and prevent external enhancers from improper activation of reporter gene promoters [In an effort to further increase the mCherry fluorescence signal to noise ratio. LNCaP cells were transduced with the CS-GS241B-mCHER-Lv207-02 ARE reporter vector (LNCaP-207-02) that contained DNA core insulator enhancers (CIEs) in addition to constitutively expressing eGFP and hygromycin resistance genes as shown in romoters , 45. Theromoters . The addromoters .i.e. progesterone, β-estradiol and dexamethasone. This demonstrates a high androgen-specificity of the ARE reporter. Studies suggest that the most frequent mutation of the AR gene in androgen-independent human prostate cancer LNCaP cells found is the substitution of amino acid T877A in the AR LBD. This mutant AR increases the binding affinity of other steroids such as progesterone, β-estradiol and glucocorticoid. It results in inappropriate activation of AR transcriptional activity [PSA gene transactivation [Ligand binding to AR is the primary and critical step of ligand-dependent transcription of AR target genes. The three-dimensional structure of AR LBD shares some commonalities with other ligand-bound steroid receptors . Steroidactivity , 51. Inttivation , 53, ourWith the introduction of the ARE reporter system in different prostate cell lines, we have observed some basal level of mCherry fluorescence activity in these cell lines. This basal fluorescence level varies among the generated prostate reporter cell lines when they were grown in androgen-free conditions. The degree of basal fluorescence level in the cell lines can range from a background and/or bleed-through fluorescent signals to a certain level of AR activity in androgen depleted conditions.Some degree of background fluorescence and bleed-through may exist in the generated prostate reporter cell lines. These signals can also be observed when AR negative DU145 prostate cancer cells and EP156T cells were transduced with 241B ARE-reporter system (data not shown). Thus, our findings suggest that in spite some background noise signals low AR activity signals might persist in androgen-depleted conditions.We also investigated the ARE reporter response in the mesenchymal type EPT3-PT1 cells. We have previously published the stepwise generation of malignant prostate, mesenchymal EPT3-PT1 cells from benign epithelial EP156T cells by physiological adaptation and selection , 57–59. The pLenti6.3/AR-GC-E2325 androgen receptor expression vector was selected in initial screenings on the basis of high-level exogenous AR expression. This AR expression vector was further used in co-transfection and co-transduction experiments in AR negative cells. The ARE promoter sequence 241B was found to have high sensitivity reporter response to an androgen-dependent AR activity among several generated ARE promoter sequences. When the 241B ARE promoter was linked to the mCherry fluorescence reporter, the signals were measured and quantified using flow cytometry and multi-well fluorescence reader on the basis of AR transcriptional activity. The prostate cell lines with the 241B ARE promoter mCherry reporter easily detected AR transcriptional activity in a multi-well fluorescent recorder. The AR antagonists or drugs affecting AR signaling activity can be analyzed by the relative quantification of the mCherry reporter response. The addition of GFP as an internal control in the reporter abolished the need to use other means for normalization such as MTS which requires more experiments and can be biased by drugs or stimulations that affect cell metabolism. The developed system demonstrated a strong specificity to androgen-mediated AR transcriptional activity compared to other steroid hormones such as β-estradiol, progesterone and dexamethasone. Expression of exogenous AR in epithelial type cells induced an androgen-dependent ARE reporter response, however, exogenous AR expression were unable to induce ARE reporter response in mesenchymal type cells. These findings also conclude that exogenous AR expression was able to induce a classical androgen-dependent AR transcriptome in, such as KLK3, KLK2 FKBP5, in epithelial type cells but not in mesenchymal type cells. This developed reporter response system can be useful in drug screening and will also help to investigate the role of the AR in terminal differentiation of prostate cells both in two and three-dimensional cell cultures.S1 Fighygromycin resistance gene. (B) The ARE sequences 241B, 248B or 249B fused to the mCMV promoter drive mCherry expression, and the SV40 promoter facilitates constitutive expression of the hygromycin resistance gene. (C) The ARE sequence 241B fused to mCMV promoter drives mCherry expression, and SV40 promoter facilitates constitutive expression of eGFP and hygromycin resistance genes that are separated by an internal ribosome entry site (IRES). (D) The ARE sequence 241B fused to mCMV promoter drives mCherry expression. The core insulator enhancers (CIE) are added on each side. SV40 promoter facilitates constitutive expression of eGFP and hygromycin resistance gene that are separated by an IRES.(A) The ARE promoters 241B, 248B or 249B fused to the mini-cytomegalovirus (mCMV) promoter drive Gaussia luciferase expression and Simian virus 40 promoter (SV40) facilitates constitutive expression of the (TIF)Click here for additional data file.S2 Figblasticidin resistance gene driven by the SV40 promoter. (B) The pLenti7.3/ARV5-GC-E2325 vector contains the CMV promoter that ensures constitutive high level expression of the downstream AR gene GC-E2325. The vector contains the eGFP marker driven by the SV40 promoter.(A) The pLenti6.3/ARV5-GC-E0060 vector contains the human cytomegalovirus (CMV) promoter that ensures constitutive high level expression of the downstream AR-V5 tag gene GC-E0060. The vector contains the (TIF)Click here for additional data file.S3 FigImmunofluorescence (A) EP156T cells were transduced with the pLenti7.3/AR-E2325 vector that allows constitutive exogenous expression of AR with eGFP as a marker protein to generate EP156T-AR cells. After transduction and eGFP selection, the cells were maintained in regular MCDB153 medium for several passages. The cells were plated and treated with ± 1 nM R1881 for 48 hours. Texas red (Tx-Red) fluorescent signals indicate AR. (B) EP156T cells were transduced with pLenti6.3/AR-E2325 vector that allows constitutive exogenous expression of AR to generate EP156T-AR cells. After transduction and blasticidin selection, the cells were maintained in regular MCDB153 medium for several passages. The cells were treated with ± 1 nM R1881 for 48 hours. FITC fluorescent signals indicate AR.(TIF)Click here for additional data file.S4 FigFluorescence microscopy of mCherry fluorescent signals in LNCaP-241B cells grown in androgen free medium or supplemented with R1881. Treatment with 10 μM enzalutamide or 10 μM abiraterone was for 24 hours.(TIF)Click here for additional data file.


Frailty is an important clinical syndrome that is consistently associated with adverse outcomes in older people. The relevance of frailty to chronic respiratory disease and its management is unknown.To determine the prevalence of frailty among patients with stable COPD and examine whether frailty affects completion and outcomes of pulmonary rehabilitation.1% predicted 48.9 (21.0)) were recruited between November 2011 and January 2015. Frailty was assessed using the Fried criteria  before and after pulmonary rehabilitation. Predictors of programme non-completion were identified using multivariate logistic regression, and outcomes were compared using analysis of covariance, adjusting for age and sex.816 outpatients with COPD (mean (SD) age 70 (10) years, FEV209/816 patients  were frail. Prevalence of frailty increased with age, Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage, Medical Research Council (MRC) score and age-adjusted comorbidity burden . Patients who were frail had double the odds of programme non-completion , often due to exacerbation and/or hospital admission. However, rehabilitation outcomes favoured frail completers, with consistently better responses in MRC score, exercise performance, physical activity level and health status . After rehabilitation, 71/115 (61.3%) previously frail patients no longer met case criteria for frailty.Frailty affects one in four patients with COPD referred for pulmonary rehabilitation and is an independent predictor of programme non-completion. However, patients who are frail respond favourably to rehabilitation and their frailty can be reversed in the short term. What is the prevalence of frailty in stable COPD, and does frailty affect the completion and outcomes of pulmonary rehabilitation?Frailty affects one in every four patients with COPD entering pulmonary rehabilitation, is associated with favourable outcomes, but is also strong risk factor for non-completion.This is the first characterisation of the frailty phenotype in stable COPD and demonstrates that physical frailty is amenable to treatment with pulmonary rehabilitation.Frailty describes a clinical syndrome characterised by multisystem decline that leads to reduced functional reserve and increased vulnerability to dependency or mortality following minor stressor events.The relevance of frailty to chronic respiratory disease has not been fully dissected. In retrospective cohort studies, self-reported frailty is more common in older people with COPD than without it, and markers of frailty have identified those at increased risk of subsequent hospital admission or death.10Another important but unstudied topic is the interplay between frailty and pulmonary rehabilitation. Pulmonary rehabilitation is highly effective at improving symptom burden, physical function and health status, although patient response is heterogeneous.Study objectives were to determine the prevalence of frailty among patients with stable COPD, describe the clinical characteristics of the COPD frailty phenotype and examine whether frailty affects completion and clinical outcomes of pulmonary rehabilitation. We hypothesised that frailty would independently predict non-completion of pulmonary rehabilitation.Participants were recruited to this prospective cohort study from respiratory outpatient and pulmonary rehabilitation clinics at Harefield Hospital  between November 2011 and January 2015. Eligible patients were aged 35 years or above, with a physician diagnosis of COPD consistent with the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteriaFrailty was defined using the Fried phenotype model,10.1136/thoraxjnl-2016-208460.supp1Supplementary dataBioelectrical impedance analysis  was used to estimate skeletal muscle mass index (SMI).34Pulmonary rehabilitation was an 8-week outpatient exercise and multidisciplinary education programme, comprising two supervised and at least one additional home-based session each week and organised according to the British Thoracic Society Quality Standards for Pulmonary Rehabilitation.Our sample size was based on the precision to which the overall prevalence of frailty could be estimated. Assuming it was within the range 10%–60%, prevalence could be estimated to within ±3.5% using 800 participants with a large sample normal approximation (nQuery Advisor V.6.0). Analyses were performed using SPSS  and graphs produced using Prism 5 . Data were presented as proportions (95% CIs) or mean (SD).2 for trend. Baseline characteristics as well as pulmonary rehabilitation uptake, attendance and completion rates were compared across groups  using one-way analysis of variance or χ2 for trend with a Bonferroni correction applied to post hoc pairwise comparisons. Uptake was defined as the proportion of assessed patients who attended the first supervised session, adherence was assessed using the number of supervised sessions attended and completion was defined as the proportion of patients who had documented attendance at a minimum of eight supervised sessions, representing 50% attendance.36The prevalence of frailty was determined overall and then compared across groups according to age, GOLD spirometry stage, MRC dyspnoea score and age-adjusted Charlson comorbidity score using χUnivariate logistic regression was used to assess the relationships between non-completion, frailty status (not or prefrail/frail) and candidate explanatory variables informed from existing literature and clinical judgement.1 48.9 (21.0) % predicted, MRC score 3.3 (1.0))  age 70 (10) years, FEV3 (1.0)) . The ove3 (1.0)) . Of the 3 (1.0)) . Frailty3 (1.0)) .Patients who were frail with COPD had significantly reduced SMI but not body mass index as compared with prefrail or robust patients . Reduced1% predicted, Charlson Index, ISWT, QMVC, ADO, CAT score, HADS anxiety and depression, and frailty status were associated with non-completion. Each of the frailty characteristics was individually associated with non-completion though the relationship was strongest when frailty status was considered overall  and 70.3% (95% CI 67.1% to 73.4%), respectively, and mean (SD) attendance was 11.4 (4.2) of 16 supervised sessions . Rates w overall . In the  overall . When ex overall . Among p overall  S2.Following completion of rehabilitation, significant improvements were observed across all groups for SMI, QMVC, CRQ dyspnoea, fatigue, emotional and mastery domains, and physical activity  and in pIn this prospective cohort study, we identified that one-quarter (25.6%) of patients with stable COPD referred for pulmonary rehabilitation were frail according to the Fried phenotype criteria. Patients who were frail demonstrated high levels of impairment as compared with prefrail or robust patients, one consequence of which was more difficulty engaging in pulmonary rehabilitation as a mainstay of their disease management. Adjusting for all known confounders, being frail was associated with over double the odds of programme non-completion. Nonetheless, in those who completed rehabilitation there was a gradient of treatment response, in favour of patients who were frail, for outcomes relating to symptom burden, exercise performance and health status. Furthermore, in a large proportion of those who completed pulmonary rehabilitation, the frailty phenotype was reversed, at least in the short term.1 79.6 (25.2) % predicted) and minimal evidence of functional impairment (only 2.5% of patients with COPD had a slow gait speed).Our point prevalence estimate for frailty is within the range previously reported in COPD (10.2% to 28.0%),1 79.6 2.2 % predThe detailed phenotypic data highlight the extent to which frailty adversely impacts patients with chronic respiratory disease. Others have associated frailty with poor exercise capacity and physical disability.40Irrespective of cause, frailty denotes an increased state of risk related to falls, disability, hospitalisation and mortality.The impressive outcomes seen following programme completion provide strong grounds to explore how better to support patients who are frail through rehabilitation, potentially through organisation changes or by supplementing supervised exercise with novel strategies, for example, neuromuscular electrical stimulation.There are limitations to consider. We purposefully selected the Fried model which mainly reflects physical frailty and incorporates measures that are effort dependant or rely on patient recall. However, the model has proven construct and predictive validity and is the most established criterion measure of frailty.In conclusion, frailty affects one-quarter of patients with stable COPD assessed for pulmonary rehabilitation. Frailty is an independent risk factor for programme non-completion but appears to result in favourable rehabilitation outcomes. Future research should identify how best to support patients who are frail through pulmonary rehabilitation.


Candida albicans, the PKA regulatory subunit Bcy1 plays a critical role in the regulation of cell differentiation and death. It has long been considered that Bcy1 is essential for cell viability in C. albicans. In the current study, surprisingly, we found that Bcy1 is not required for cell growth, and we successfully generated a bcy1/bcy1 null mutant in C. albicans. Deletion of BCY1 leads to multiple cellular morphologies and promotes the development of filaments. Filamentous and smooth colonies are two typical morphological types of the bcy1/bcy1 mutant, which can undergo spontaneous switching between the two types. Cells of filamentous colonies grow better on a number of different culture media and have a higher survival rate than cells of smooth colonies. In addition, deletion of BCY1 significantly increased the frequency of white-to-opaque switching on N-acetylglucosamine (GlcNAc)-containing medium. The bcy1/bcy1 null mutant generated herein provides the field a new resource to study the biological functions of the cAMP signaling pathway in C. albicans.The conserved cAMP-dependent protein kinase (PKA) is composed of the regulatory and catalytic subunits and acts as the central component of the cAMP signaling pathway. In the human fungal pathogen  Candida albicans, this pathway plays a central role in the regulation of morphological transitions, carbon source utilization, quorum sensing, cell death, and virulence , serum, and elevated temperatures], cells of C. albicans activate the cAMP signaling pathway and undergo morphological changes , and white, gray, and opaque cell types  was added to the solid medium for the filamentation and white-opaque switching assays. Media used for spot serial dilution growth assays: YPD, Lee's (Lee's medium without sugar)  using the same strategy as described below. The two alleles of BCY1 were deleted using the fusion PCR strategy  was used for transformation of the bcy1/bcy1 mutant. The two BCY1-related fragments were amplified from genomic DNA of C. albicans (SC5314) with primer pairs .TPK1 and TPK2-overexpressing plasmids , cells from a filamentous or smooth colony grown on Lee's glucose medium for 3 days were resuspended and cultured in liquid Lee's glucose for 24–96 h at 25°C. A small aliquot of cells was collected at the time point indicated and replated on YPD plates. After 5 days of growth at 30°C, colonies of the smooth and filamentous types were counted. The switching frequency =  × 100%.Lee's glucose, Lee's GlcNAc, and YPD media were used for the filamentation assays. The cells were cultured at 25° and 37°C as indicated in the figure legends. For quantitative filamentous-smooth colony type switching assays (The cells were grown in liquid Lee's glucose medium for 48 h at 25°C and collected for propidium iodide (PI) and 4′-6-diamidino-2-phenylindole (DAPI) staining assays as described previously  at two temperatures (25 and 37°C). These culture conditions were used because the three media exhibit different levels of filamentation induction in C. albicans. A high temperature (37°C) promotes filamentation, whereas a low temperature (25°C) favors yeast cell growth. Therefore, a combination of these conditions would facilitate the discrimination of the filamentation ability of different strains. At 25°C  on all three media. Cells of the smooth colonies were swollen and looked unhealthy, whereas filamentous cells had a much healthier appearance. On Lee's GlcNAc medium, a portion of the bcy1/bcy1 mutant cells exhibited an opaque-like phenotype  induced the formation of an opaque-like cell type on Lee's GlcNAc medium. Therefore, we deleted BCY1 in a white-opaque switchable MTLa/Δ strain . The colony and cellular morphologies of the WT, BCY1/bcy1, and bcy1/bcy1 mutants (MTLa/Δ) are shown in Figure BCY1/bcy1 mutant exhibited similar colony and cellular phenotypes to those of the WT strain. Both the smooth and filamentous types of the bcy1/bcy1 mutant could undergo white-opaque switching on Lee's glucose medium. However, both types underwent hyperfilamentation on Lee's GlcNAc medium. Replating and cell type-specific gene expression assays indicated that on Lee's GlcNAc medium, the filamentous cells had an opaque identity  are not essential for cell viability (Cannon and Tatchell, bcy1/bcy1 null mutant generated in C. albicans, we re-evaluated the roles of Bcy1 in the regulation of filamentation, cell growth, and carbon source utilization. We also found that Bcy1 regulates white-opaque switching in C. albicans.The cAMP/PKA pathway plays a critical role in the regulation of a number of features of the human fungal pathogen CYR1, the sole gene encoding the adenylyl cyclase in C. albicans, completely blocked filamentation in response to several potent inducers including serum, CO2, and bacterial peptidoglycan (Rocha et al., PDE2 results in hyperfilamentation in C. albicans (Feng et al., BCY1 in C. albicans promotes filamentation under conditions favoring yeast cell growth (such as at low temperature and in rich media, Figures pde2/pde2 mutant (Jung and Stateva, bcy1/bcy1 mutant. Moreover, the phenotypes of the bcy1/bcy1 mutant are highly similar to the hyperactive CYR1 mutant (Bai et al., Deletion of S. cerevisiae causes a variety of phenotypes (Cannon et al., C. albicans, we found that deletion of BCY1 also resulted in multiple colony and cellular phenotypes including yeast, filamentous, and opaque-like forms (Figure bcy1/bcy1 mutant exhibited different cell growth and carbon nutrient utilization abilities (Figure bcy1/bcy1 mutant. These results suggest that deletion of BCY1 in C. albicans promotes cell death, potentially due to constitutive activation of the cAMP-PKA pathway. Consistent with this idea, Phillips et al. (bcy1/bcy1 mutant could be a strategy of cells to avoid cell death or to improve their anti-stress abilities. Consistent with our study, Laprade et al. (C. albicans provides protection against antifungal-induced programmed cell death (Laprade et al., Mutation of the PKA regulatory subunit in s Figure . Interess Figure . Filamens Figure . Filamens et al.  demonstrs et al. . Filamene et al.  recentlyC. albicans and is involved in the regulation of virulence, sexual mating, and stress responses (Slutsky et al., CYR1 suppresses GlcNAc and CO2-induced white-to-opaque switching, whereas activation of this pathway by ectopic expression of the activating form of RAS1, RAS1V13, or deletion of PDE2 promotes the opaque phenotype (Huang et al., BCY1 promotes white-to-opaque switching in C. albicans (Figure BCY1 leads to a moderate increase in this switch and deletion of both alleles causes a mass conversion on GlcNAc-containing media. GlcNAc is a potent inducer of the opaque phenotype. Consistent with the phenotype in the bcy1/bcy1 mutant, Huang et al. (PDE2 also results in a mass white-to-opaque conversion on GlcNAc-containing media (Huang et al., White-opaque switching is another important feature of s Figure . This prg et al.  reportedbcy1/bcy1 mutant in C. albicans, which clarifies the essential role of this PKA regulatory subunit and provides a new avenue to study the cAMP-PKA pathway in this medically important pathogen. Our study also provides new insights into the functional roles of Bcy1 in the regulation of filamentation, carbon source utilization, and white-opaque switching. The results reported herein further confirm the conserved features and central role of the cAMP-PKA pathway in the regulation of a variety of biological features of C. albicans.In summary, we successfully generated a XD and GH designed the study. XD, CC, and QZ performed experiments. GH, XD, CC, and QZ analyzed data. GH, XD, CC, and QZ wrote the manuscript.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.


Fast, nongenomic androgen actions have been described in various cell types, including neurons. However, the receptor mediating this cell membrane–initiated rapid signaling remains unknown. This study found a putative androgen receptor splice variant in a dopaminergic N27 cell line and in several brain regions  from gonadally intact and gonadectomized (young and middle-aged) male rats. This putative splice variant protein has a molecular weight of 45 kDa and lacks an N-terminal domain, indicating it is homologous to the human AR45 splice variant. Interestingly, AR45 was highly expressed in all brain regions examined. In dopaminergic neurons, AR45 is localized to plasma membrane lipid rafts, a microdomain involved in cellular signaling. Further, AR45 protein interacts with membrane-associated G proteins Gαq and Gαo. Neither age nor hormone levels altered AR45 expression in dopaminergic neurons. These results provide the first evidence of AR45 protein expression in the brain, specifically plasma membrane lipid rafts. AR45 presence in lipid rafts indicates that it may function as a membrane androgen receptor to mediate fast, nongenomic androgen actions. Evidence has been building for the existence of a membrane androgen receptor, but the nature of this receptor remains elusive. We predicted that the membrane androgen receptor is a splice variant that is present in lipid raft microdomains within the neuronal plasma membrane. Indeed, an androgen receptor that is lacking an N-terminal domain and complexed to Gαq and Gαo G proteins was highly expressed in lipid rafts in dopaminergic neurons. This putative splice variant provides a potential target for mediating androgen’s nongenomic actions.2 terminal domain  is expressed in many areas of the brain, including brain regions not involved in regulating classic endocrine functions  has been accumulating since the 1990s. Investigators have found that even cell-impermeable androgens can have fast cellular effects . Rapid m2 regulatory domain. For example, both AR splice variants (AR8 and AR45) have truncated NTD . This deletion decreases the protein molecular weight of AR from 110 to 45 kDa . FunctioTo determine whether the putative mAR could be AR45 in different brain regions , we measured protein expression of AR using antibodies targeting the CTD and NTD of the AR. Because neuronal mAR has been associated with intracellular calcium signaling , we exam1 (sc-391), Gαi2 (sc-13534), and Gαi3 (sc-262) antibodies were obtained from Santa Cruz Biotechnologies. Flotillin (3253) and caveolin-1 (3267) antibodies were obtained from Cell Signaling Technology. Alexa Fluor 594 antibody was purchased from Jackson ImmunoResearch Laboratories. GAPDH (GT239) antibody was obtained from GeneTex. Biotinylated anti–rabbit IgG (BA-1000) was purchased from Vector Laboratories. Androgen R/NR3C4 (AB58761) was obtained from R&D Systems. DMSO was purchased from VWR. RPMI 1640, penicillin-streptomycin (PS), and trichloroacetic acid (BDH9310) were purchased from VWR. Fetal bovine serum (FBS) and PBS were obtained from Corning. Charcoal-stripped FBS (CS-FBS) was purchased from Atlanta Biologicals. Mounting medium was obtained from Vector Laboratories (H-1200). SuperSignal West Pico/Femto chemiluminescent substrates, dithiothreitol (DTT), and Clean Blot IP Detection Reagent (PI21230) were obtained from Thermo Fisher Scientific. Deoxycholic acid (D-6750), inactin (T133), horse serum (H1138), and Triton X-100 were purchased from Sigma-Aldrich. Tris, Any KD polyacrylamide gel, Tris-glycine buffer, and PVDF membranes were purchased from Bio-Rad. Total testosterone ELISA (RTC001R) was purchased from BioVendor. Testosterone was made using an ethanol vehicle .Testosterone (A6950-000) was obtained from Steraloids. Goat anti-rabbit (sc-2004), androgen receptor C-19 (sc-815), androgen receptor N-20 (sc-816), AR441 (sc-7305), AR N20P (sc-515856), AR C19P (sc-515863), Gαq (sc-393), Gαs (sc-823), Gαo (sc-393874), Gαiad libitum access to food and water. Animals were weighed at the time of surgery and at death. All experimental procedures were approved by the University of North Texas Health Science Center IACUC in accordance with the guidelines of the Public Health Service, the American Physiologic Society, and the Society for Neuroscience for animal care and use.Experiments were conducted on young adult (3 mo) or middle-aged retired breeder (9–12 mo) male Sprague-Dawley rats (Charles River). Animals were either gonadally intact or were gonadectomized to remove circulating gonadal hormones. Rats were individually housed in a temperature-controlled environment on a 12:12-h light-dark cycle. All rats had Under 2.5% isoflurane, a midline scrotal incision was made to expose the spermatic cord. The spermatic cord was tied off with sterile sutures, and the cord was cut distal to the thread to remove the testes. The incision was closed with sterile absorbable sutures .One week after surgery, each rat was anesthetized with 2.5% isoflurane and decapitated. The brain was removed from the skull, rinsed in ice-cold PBS, and placed into a brain matrix (Braintree Scientific) on ice. Using razor blades, the brain was cut into 1-mm coronal sections. The razor blades were placed on dry ice to freeze the freshly cut brain sections. Punches were obtained from the SN pars compacta (–5.30 mm from bregma), second layer of the entorhinal cortex , and the CA1 layer of the dorsal hippocampus (–4.52 mm from bregma) using 1-ml syringes with a 20-gauge blunt needle . Samples27 (N27), derived from fetal rat mesencephalic tissue, is positive for tyrosine hydroxylase expression  or vehicle control for 18 h and collected for protein. The testosterone concentration used in this study was 100 nM, representing the high end of the normal testosterone range in men  on ice. For in vivo preparations, brain region micropunches were incubated with RIPA homogenization buffer with DTT (1 µM), EDTA (1 mM), and protease and phosphatase inhibitors (1:200) for 30 min on ice, sonicated (QSonica) at 20% amplitude, and pulsed 3 times for 3 s. Next, lysates were centrifuged at 4°C for 20 min at 12,000 × g. Protein concentrations were determined using the BCA assay (Thermo Fisher Scientific) according to the manufacturer’s instructions.For 4 cells per plate. After treatments, cells were washed with PBS and lysed with a cocktail of hypotonic homogenization buffer and phosphatase inhibitors (1:100) on ice. Each sample (n) consisted of two 100 × 20-mm plates. For micropunches, a total of 0.025 g of SN tissue was homogenized in hypotonic homogenization using a sonicator to homogenize the tissue. After homogenization, cellular fractionation followed by sucrose density analysis of membrane lipid rafts was performed  using a swing bucket rotor . After the high-speed centrifugation, equal-volume fractions were taken from the top layer of the gradient, resulting in nine fractions (low-density proteins at the top gradient layers to high-density proteins at the bottom gradient layers). Protein was precipitated using the trichloroacetic acid (TCA) method  method . The pelpression A.+/N20– AR protein expression at 45 kDa was observed only in the membrane fraction (not the cytosol or nuclear fractions), whole-cell lysates were used. Protein (25 μg) was incubated overnight at 4°C in a cocktail containing RIPA lysis buffer and 1 μg primary antibody (AR C-19 or Gαq). A Sepharose bead slurry was coupled to each sample by incubating at 4°C overnight. Samples were washed, eluted, and resuspended in 4× Laemmli buffer. To avoid IgG band interference, the blot was incubated with Clean Blot IP Detection Reagent HRP, per the manufacturer’s instructions. Coimmunoprecipitation experiments were replicated four times. Only frozen samples were used to decrease AR fragment protein expression  in TBS-Tween with 1% nonfat milk for 2 h or overnight at 4°C. Afterward, the membranes were washed every 10 min for 30 min and incubated with secondary antibodies at 1:1000 in TBS-Tween with 1% nonfat milk for 30 min at room temperature. Protein bands on the membrane were visualized using an enhanced chemiluminescence detection assay (Thermo Fisher Scientific). Protein band intensities were imaged using GeneSys software corresponding with the G:Box Chemi XRQ system (Syngene). Protein band densities were quantified by NIH ImageJ densitometer software and normalized to GAPDH for whole-cell lysates, using the equation / × 100. All antibodies used in this study are commercially available. The specificity of the antibodies was assayed by using primary antibodies that were preabsorbed with blocking peptides  at 4°C overnight on representative blots. Previous immunohistochemical studies have shown specificity for AR-C19 and AR N-20  of denatured whole cell lysates, micropunch tissues, cellular fractions, or lipid raft fractions were loaded into a Bio-Rad Any KD polyacrylamide gel, electrophoresed in Tris-glycine buffer, and transferred onto a PVDF membrane. Membranes were blocked for 30 min with 5% nonfat milk in TBS-Tween at room temperature. After blocking, membranes were incubated with specific primary antibodies , transcardially flushed with 0.1 M PBS (100–200 ml), and then perfused with 4% paraformaldehyde in 0.1 M PBS , 7.4% dihydroxyandrosterone, and <0.1% for androstenedione, androsterone, epiandrosterone, dihydroandrosterone, estrone, estradiol, estriol, cortisol, 11-deoxycortisol, progesterone, and 17OH-progesterone.During the first 2 h of the circadian light phase, each rat was anesthetized with 2.5% isoflurane and decapitated. Prostate and seminal vesicle wet tissue weights were measured. Trunk blood (5–7 ml) was obtained in EDTA-coated tubes  on ice. The blood was centrifuged  at 2000 × pAnalysis was performed using IBM SPSS Statistics, version 21. Data were expressed as mean F1,45 = 233.284, p < 0.05). One week after gonadectomy (GDX), plasma testosterone levels were significantly decreased compared with gonadally intact rats, regardless of age . No differences in testosterone levels between young and middle-aged gonadally intact rats were observed. Consistent with a decline in testosterone, GDX rats exhibited a significant decrease in weights for androgen-sensitive accessory organs: prostate  and seminal vesicles . Middle-aged retired breeders, regardless of hormone condition, had significantly heavier prostate  and seminal vesicle  weights than young sexually naive rats. These results are consistent with prior studies showing that sexual experience increases testosterone and androgen-sensitive accessory organ weights , second layer of the entorhinal cortex (ETC), and CA1 layer of the dorsal hippocampus from young and middle-aged male rats that were either gonadally intact or GDX . Frozen Widespread CTD AR immunoreactivity (and not NTD AR–positive cells) was observed throughout the SN in young and middle-aged rats, respective of hormone and age status A, B. AR N27 cells and SN brain tissue were split into membrane, cytosol, and nuclear fractions. The membrane portion of both N27 cells and SN brain tissue was further separated into nine fractions using a sucrose gradient –5. Full-–RT-PCR for AR was performed to determine whether full-length transcripts were present in SN and N27 cells . Several1-3 and Gαs were not expressed in any of the membrane fractions. Interestingly, the G protein Gαo was expressed only in SN tissue and not in the N27 cell line  or G protein immunoreactivity. In N27 cells, AR45 interacted with Gαq, regardless of testosterone exposure A. FurtheThis study found AR protein expression at 45-kDa molecular weight in all samples examined: N27 cells, SNpc, hippocampus, ETC, and testes. Furthermore, this AR variant is present in plasma membrane lipid rafts from N27 cells and SN brain tissue from young and middle-aged rats with and without sex hormones. The 45-kDa membrane protein was evident only using a CTD-targeted antibody for AR, consistent with the AR45 splice variant. Interestingly, protein expression of AR45 was not altered by steroid hormones, age, or temperature. Although full-length AR mRNA was found in all brain samples examined, little to no AR immunoreactivity using the NTD-targeted AR antibody was observed in SNpc tissue, unlike the ETC and hippocampus. Expression profiles for AR immunoreactivity for these brain regions are different, indicating different mechanisms of androgen action in SN versus hippocampus and ETC. This result, coupled with a lack of a detectable exon 1–containing transcript in N27 cells, suggests that the immunoreactive AR45 may represent a bona fide splice variant rather than posttranslational processing of the full-length AR.+  commonly occur in caveolin proteins and steroid receptors  are female-derived cell lines, indicating that these fast, nongenomic androgen actions in neurons are (1) applicable to both males and females and (2) dependent on the hormonal and AR expression milieu.Because AR45 appears to be the predominant AR in the SNpc, it may have clinical relevance in the progressive motor disorder Parkinson’s disease (PD), resulting from the loss of THthe SNpc . Men havthe SNpc . Similarthe SNpc , which mthe SNpc . It is uthe SNpc . Furtheric cells , which aic cells . InteresThis is the first study to show the presence of a putative AR splice variant protein in the SNpc, hippocampus, and ETC brain regions. Specifically, our results show that AR45 localizes in the membrane lipid rafts from N27 cells and SN dopaminergic neurons. Furthermore, AR45 interacts with Gαq and Gαo G proteins, which can impact intracellular calcium signaling . More re