Estrogen receptor β (ERβ) was first identified in the rodent prostate and is abundantly expressed in human and rodent prostate epithelium, stroma, immune cells and endothelium of the blood vessels. In the prostates of mice with inactivated ERβ, mutant phenotypes include epithelial hyperplasia and increased expression of androgen receptor (AR)-regulated genes, most of which are also upregulated in prostate cancer (PCa). ERβ is expressed in both basal and luminal cells in the prostate while AR is expressed in luminal but not in the basal cell layer which harbors the prostate stem cells. To investigate the mechanisms of action of ERβ and its potential cross-talk with AR, we used RNA-seq to study the effects of estradiol or the synthetic ligand, LY3201, in AR-positive LNCaP PCa cells which had been engineered to express ERβ. Transcriptomic analysis indicated relatively few changes in gene expression with ERβ overexpression, but robust responses following ligand treatments. There is significant overlap of responsive genes between the two ligands, estradiol and LY3201 as well as ligand-specific alterations. Gene set analysis of down-regulated genes identified an enrichment of androgen-responsive genes, such as FKBP5, CAMKK2, and TBC1D4. Consistently, AR transcript, protein levels, and transcriptional activity were down-regulated following ERβ activation. In agreement with this, we find that the phosphorylation of the CAMKK2 target, AMPK, was repressed by ligand-activated ERβ. These findings suggest that ERβ-mediated signaling pathways are involved in the negative regulation of AR expression and activity, thus supporting a tumor suppressive role for ERβ in PCa. The prostate is an androgen-responsive organ which is controlled by the androgen receptor (AR) both under normal physiological conditions and in malignancy. AR plays an important role in PCa as a strong driver of proliferation, and as such is the primary target for treatment of PCa . RecentlIn addition to AR, the prostate also expresses estrogen receptor β (ERβ), which is expressed in both basal and luminal cells . ERβ, a The LNCaP and 22Rv1 cell lines were obtained from the American Type Culture Collection (ATCC). LNCaP and 22RV1 cells were maintained in RPMI-1640  medium supplemented with 10% fetal bovine serum (FBS) , and Antibiotic-Antimycotic . All experiments used cells below passage 30. Compounds and concentrations used was R1881 (1 nM), 17β-estradiol (10 nM), , and LY3201 (10 nM) which was a gift from Elli Lilly Indianapolis, IN. All treatments were for 24 hours.LNCaP cells were infected with the lentivirus Lenti6-TOPO-V5-D, empty or containing cDNA for human ERβ at 2 m.o.i (multiples of infection). The lentivirus vector was obtained from Invitrogen Carlsbad, CA and the cloning of ERβ cDNA is described in Williams et al. . The conWhole-cell extracts, were prepared by washing cells twice with PBS, lysed in 2 times packed cell volume of lysis buffer  for 15 minutes on ice and then centrifuged at 14,000 x g for 10 minutes.Thirty micrograms of protein were loaded on an SDS-PAGE 10% Bis-Tris gel with Tris running buffer and transferred to a PVD membrane after electrophoretic separation. Membranes were blocked with 5% non-fat powdered milk in 0.1% TBST buffer and probed with anti-AR (sc-816), GAPDH-HRP (sc-47724), AMPK (2532S), pAMPK 2535S, FKBP5 122105 , CAMKK2 (H00010645-M01) , β-Actin (A1978) . Primary antibodies were used at 1:200–1000 dilutions, and secondary antibody was used at 1:10,000. The western blot experiments were minimally repeated two times.2 and 95% humidity. On the day of assay 10 μl MTS was reagent added to each well and incubated at 37C ° for 60 min and the color was measured at 490nM with SpectraMax spectrophotometer.Control and ERβ LNCaP cells were grown with blasticidin for 48 hrs in complete medium till subconfluency. Cells were trypsinized, washed with PBS, counted and suspended in 10% DCC-FBS. In a 96-well plate, 5000 cells/ well were added along with the indicated ligand and incubated for 5 days at 37C in 5% CO5’-TGACAACTTTGGTATCGTGGAAGG-3’ and R, 5-AGGCAGGGATGATGTTCTGGAGAG-3 (reference gene); AR; F, 5’-TCACCAAGCTCCTGGACTCC-3’ R, 5’-CGCTCACCATGTGTGACTTGA 3’; FKBP5; F, 5’- ATTGGAGCAGGCTGCCATTGTC -3’, R, 5’-CCGCATGTATTTGCCTCCCTTG-3’, CAMKK2; F, 5’-TCCAGACCAGCCCCGACATAG-3’, R, 5’-CAGGGGTGCAGCTTGATTTC-3’ 7500 Fast Real-Time PCR System (Applied Biosystems) using optimized conditions for SYBRGreen I dye system: 50 C for 2 minutes, 95 C for 10 minutes, followed by 40–50 cycles at 95 C for 15 seconds and 60 C for 50 seconds. Optimum primer concentration was determined in preliminary experiments, and amplification specificity confirmed by dissociation curve. The variance between the groups that are being statistically compared is similar.RNA extraction was performed with Qiagen mRNA extraction kit according to standard protocol. cDNA was synthesized from 1μg of total RNA with First Strand System according to standard protocol (Invitrogen Inc. NY). Real-time PCR was performed with SYBR Green iTaq master mix . Primers  were: GAPDH; F, Poly(A) mRNA was isolated using NEBNext poly(A) mRNA Magnetic Isolation Module. Libraries were prepared using NEBNext Ultra II RNA Library Prep Kit for Illumina. Sequencing was performed on NovaSeq 6000 with 150 bp paired-end reads. Treatments include three independent replicates.Reads were aligned to reference genome (GRCh38) indexes using STAR (v2.5). Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the cluster Profiler R package, in which gene length bias was corrected. GO terms with corrected P-values less than 0.05 were considered significantly enriched by differentially expressed genes. The RNA-seq data is available in NCBI’s Gene Expression Omnibus through accession number GSE144800.To determine the functions of ERβ in AR-positive PCa, we used RNA-seq to compare the transcriptomes of ERβ over-expressing and non-expressing LNCAP cells treated with vehicle (DMSO), and ERβ ligands estradiol (E2) and LY3201. Differential expression analysis consisted of comparing over- vs non-expressing ERβ cells within each treatment regime. DMSO-treatment had very little effect on gene expression  20–22]..22]. E2 Pre-ranked gene set enrichment analysis (GSEA) was used to identify pathways and mechanisms relevant to ERβ expression and activation in PCa. We examined those genes which are commonly regulated by LY3201 and E2 for enrichment analysis. The most positively enriched hallmark gene set in ligand-treated cells were genes responsive to estrogen; other highly enriched sets contained genes involved in MTORC1 signaling and the unfolded protein response. GSEA also revealed downregulated genes involved in several pathways associated with cancer hallmarks such as hypoxia and glycolysis . The mosSince AR-responsive genes were affected by ERβ activation see , we inveWe used AR-reporter constructs to investigate whether AR activity is directly affected by ERβ in ERβ-overexpressing LNCaP cells as well as 22Rv1 PCa cells. Ligand-treatment of transfected cells showed ERβ-dependent inhibition of AR-reporter activity compared to control transfected cells  and poorly differentiated intermediary cells  as well In the present study, we specifically examined the functions and potential mechanisms of action of ERβ in AR-positive LNCaP cells by RNA-seq. Consistent with its function as a ligand-dependent transcription factor, overexpression of ERβ elicited relatively small changes in the LNCaP transcriptome in a ligand-independent fashion, but treatments with ER ligands E2 or ERβ-specific ligand LY3201 resulted in altered expression of thousands of genes. As expected, GSEA results revealed significant enrichment of ER-regulated genes among those up-regulated by ligand treatment. Of particular interest is the down-regulation of genes involved in androgen response. GSEA results and follow-up studies on the expression and activity of AR and target genes shown herein provide evidence that the tumor suppressor actions of ERβ in PCa are due to its negative regulation of AR signaling. AR expression and/or activity is often increased in primary PCa, as well as in metastatic PCa. In the present study we found that in LNCAP cells overexpressing ERβ, AR activity is repressed by ERβ-ligands, as were the classical AR targets c-Myc, FKBP5 and CAMKK2. TBC1D4 is the most down-regulated AR-responsive gene and a major regulator of glucose uptake in the prostate by inducing membrane localization of Glut12. The two most up-regulated AR responsive genes FADS1 and KRT19 are likely to be targets of both AR and ERβ, where FADS1 has been shown to be regulated by estrogen , and KRTWe performed ChIP-seq in prostate cells but did not find ERβ binding around or within the AR gene, indicating that the regulation could be either on mRNA stability or protein stability of AR. A study by Nanni et al. showed that endothelial NOS, estrogen receptor β, and HIFs cooperate in the activation of a negative prognostic transcriptional pattern in aggressive human prostate cancer . We haveThe repression of AR by ERβ is not limited to LNCaP cells, but also occurs in another androgen-responsive cell line, 22Rv1, which expresses both normal AR as well as the AR variant AR7. In addition to regulating AR, GSEA and additional gene ontology analysis of genes differentially expressed following ERβ activation revealed an enrichment of genes involved in cancer-related processes, including apoptosis, response to hypoxia, KRAS signaling, and key metabolic pathways. As in many other studies we observe that expression of ERβ causes inhibition of proliferation , 25. TheS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 TableDMSO control Vs. LY ERβ Overexpression.(XLSX)Click here for additional data file.S2 TableLY values, Hallmarks gene sets.(XLSX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


The reaction time was modified to obtain two samples (ZCO-6h and ZCO-12h) and the rest of the synthesis conditions were the same. The synthesized structures were systematically studied through various techniques: their crystalline characteristics were studied through XRD analysis, their morphologies were inspected through SEM and TEM, and the elemental distribution and oxidation states were studied by X-ray photoelectron spectroscopy (XPS). ZCO-12h sample has a larger surface area (55.40 m2·g−1) and pore size (24.69 nm) than ZCO-6h, enabling high-speed transport of ions and electrons. The ZCO-12h electrode showed a high-specific capacitance of 421.05 F·g−1 (31.52 C·g−1) at 1 A·g−1 and excellent cycle performance as measured by electrochemical analysis. Moreover, the morphologic characteristics of the prepared hierarchical materials contributed significantly to the improvement of specific capacitance. The excellent capacitive outcomes recommend the 3D-ZnCo2O4 hierarchical peony-like structures composed of 2D-nanoflakes as promising materials for supercapacitors with high-performance.The demand for eco-friendly renewable energy resources as energy storage and management devices is increased due to their high-power density and fast charge/discharge capacity. Recently, supercapacitors have fascinated due to their fast charge–discharge capability and high-power density along with safety. Herein, the authors present the synthesis of 3D-hierarchical peony-like ZnCo I2O4 , CuCo2O42O4 [2O4 , ZnCo2O42O4 [2O4 , NiMoO4  [2O4 [4 , Zn2SO4 [4 [2SO4  and NiFeSO4 [2O4  show fasow price ,26,27. Reduction ,29. ZCO eduction ,31. Furteduction . In receeduction ,34.Herein, polyvinylpyrrolidone (PVP)-supported ZCO 3D-hierarchical peony-like structures were fabricated by a hydrothermal route and used for pseudocapacitor with high-performance. The electrode had a 3D-structure consisting of 2D-nanoflakes with high specific capacitance and showed good cycle stability. The prepared materials exhibited notable electrochemical performances because the ZCO has hierarchical structures consisting of 2D-nanoflakes.All chemical reagents were procured from Sigma-Aldrich, St. Louis, MO, USA. First, 0.1 g of PVP (K-30) was added with 240 mL of deionized (DI) water and apply constant stirring to get a homogeneous solution. Then, 0.7 g (10 mmol) of zinc nitrate hexahydrate, 1.4 g (20 mmol) of cobalt nitrate hexahydrate and 0.7 g (48 mmol) of urea were mixed to the PVP solution with stirring. After stirring for a further 30 min, the mixed solution was shifted to a hydrothermal reactor  and maintained at 180 °C for 6 h in the furnace. After cooling the reactor, the precipitate was collected, cleaned with ethanol and DI water and lastly dried at 80 °C for 10 h. Afterward, the products were annealed at 400 °C under an air atmosphere for 4 h with a ramping rate of 5 °C /min to acquire crystallized flower-like ZCO (denoted as ZCO-6h). Flake-like ZCO (denoted as ZCO-12h) was produced under similar synthesis conditions, except for the reaction time (180 °C/12 h). The material characterization and electrochemical measurements are described in the XRD profiles  of the as-prepared ZCO-6h and ZCO-12h samples match well with the standard XRD profile of ZCO   coupled with EDS and TEM studies. The SEM images a,b of thFurthermore, PVP exhibits a substantial part in the formation of the peony-like morphology of ZCO structures. Due to the coordination effect of PVP, the morphologies of metal oxides were controlled . Becaused-spacing values of ZCO-6h, and ZCO-12h was calculated to be 0.466 nm  analysis. 0.466 nm c, and 0.0.466 nm g, respec1s core-level spectra . The survey of all elements in the ZCO-6h and ZCO-12h samples was studied in the order of 1350 to 0 eV, as revealed in  spectra a; as depectively b. The fo reports ,40,41. S3/22p and 1/22p states appeared at 779.6/779.6 eV (Co1) and 794.6/794.6 eV (Co3) for the Co3+ state of ZCO-6h and ZCO-12h, respectively, while the other peaks appeared at 780.8/781.1 eV (Co2) and 795.8/795.8 eV (Co4) for Co3+. For all the samples and the two states (+2 and +3 of Co), the energy alteration between 3/22p and 1/22p was nearly 15 eV, which agrees well with former reports [The asymmetric spectra exhibit three different regions of peaks, which are related to Co in the +2 and +3 states, along with a satellite peak. For comparison, we decomposed the spectra into two peaks for the +2 and +3 states and ignored the satellite peaks. The two peaks originating from the spin–orbit of Co  reports ,42. Ther2 adsorption–desorption isotherms of ZCO-6h and ZCO-12h and results are presented in 2·g−1, which is much bigger than that of the ZCO-6h sample (32.49 m2·g−1). ZCO-12h possesses more places and networks, thereby generating a greater interaction area among the electrolyte and the active material owing to its large active surface area. In addition, the pore volumes of the ZCO-6h and ZCO-12h samples are 0.19 and 0.28 cm3·g−1, respectively. The larger pore volume of ZCO-12h causes less resistance to the process of diffusion of electrolyte into the active material and mitigates volume expansion during the galvanostatic charge–discharge (GCD) method [The surface properties of the ZCO structures were further evaluated by Brunauer Emmett Teller (BET) investigations. N) method ,43. The −1 in the potential window of −0.2 to 0.6 V (vs. Ag/AgCl) using 6 M of KOH. For the two samples, a couple of redox peaks can be detected in the CV profiles, which implies the pseudocapacitive behavior of the two electrodes rather than an EDLC (rectangular shape) [i) and scan rate (v) using a power law [i’ is the peak current and ‘v’ is the scan rate, respectively, ‘a’ and ‘b’ are the appropriate constants. The value of the constant determines the charge storage process involved in the reaction. When b = 0.5, designates diffusion-controlled electrochemical process and when b = 1, denotes a non-diffusion-controlled surface redox process [−1 in the potential window of 0–0.4 V. As observed from the CD measurements of the two electrodes, the curves are symmetric in shape (voltage plateaus), which indicates an excellent pseudocapacitive behavior ( r shape) . The intower law .i = avb process . Figure ehavior  . ClearlyCs in F·g−1) values for ZCO-6h and ZCO-12h were calculated based on the following equation for a nonlinear charge/discharge curve [I is the current of discharge curve in mA, ∫Vdt is the area under the discharge curve in V.s, Vf − Vi is the potential difference between final and initial voltage in volts (v) and m is the active electrode mass in grams (g). Based on the above equation, the specific capacitances of ZCO-6h and ZCO-12h were calculated as approximately 359.85 F·g−1 (28.78 C·g−1) and 421.05 F·g−1 (31.52 C·g−1) at 1 A·g−1, respectively. The determined specific capacitance values for corresponding current densities are presented in 2O4 (ZCO-6h) and flake-like ZnCo2O4 (ZCO-12h) electrodes in this study is comparable to those of the spinel ternary metal oxide-based electrodes with a range of morphologies stated elsewhere.Specific capacitance (ge curve :(2)CS=2I−1 for 2000 cycles. Surprisingly, 88% of the maximum capacity is retained, which demonstrates the excellent cycle performance of the ZCO-12h.Rct) produced by the Faradaic reactions and a straight line in the low-frequency section, which shows the Warburg resistance (W) related with the flow of electrolyte into the electrode surface [Rs, Rct and W values of ZCO-6h and ZCO-12h are presented in the inset table of Rct decreases as a function of reaction temperature and the Rct of ZCO-12h (1.008 Ω) is lower than that of ZCO-6h (2.932 Ω); this indicates that ZCO-12h has excellent ionic conductivity and a fast charge–transfer rate [Furthermore, the electrochemical performances of the ZCO electrodes were determined through EIS analysis. The Nyquist plots  of the Z surface ,34,47. Tfer rate ,49. Besi2O4 structures with 2D-nanoflakes were effectively synthesized via the hydrothermal method. SEM analysis revealed that the prepared structures consist of a rough, porous surface, assembled with numerous nanoflakes and composed of nanolamella, which affords the highest specific capacitance of 421.05 F·g−1 (31.52 C·g−1) 1 A g−1 (for ZCO-12h). Furthermore, the maximum capacitance (88% retention for ZCO-12h) was attained after 2000 continuous charge–discharge cycles, signifies the better-cycled life of the prepared electrode martial.The 3D-hierarchical peony-like ZnCo


Finally, metabolism in vitro by rat intestinal flora and cell membrane immobilized chromatography technology were used to screen the effective components of AR in the treatment of lung cancer, and remaining components from the cell immobilized chromatography were collected and analyzed by ultra-performance liquid chromatography–electrospray quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The screening results of the integrated strategy showed that calycosin-7-O-β-D-glucoside, ononin, calycosin, astragaloside IV, astragaloside II, cycloastragenol, and formononetin may be effective components of AR in the treatment of lung cancer, and they may play a role in the treatment of lung cancer through autophagy and p53/AMPK/mTOR signaling pathway. The integrated strategy for effective-component discovery provided a valuable reference mode for finding the pharmacodynamic material basis of complex TCM systems. In addition, the prediction for targets and signal pathways laid a foundation for further study on the mechanism of AR in the treatment of lung cancer.Lung cancer is one of the most devastating diseases worldwide, with high incidence and mortality worldwide, and the anticancer potential of traditional Chinese medicine (TCM) has been gradually recognized by the scientific community. Astragali Radix (AR) is commonly used in traditional Chinese medicine in the treatment of lung cancer and has a certain clinical effect, but effective components and targets are still unclear. In the study, we established an integrated strategy for effective-component discovery of AR in the treatment of lung cancer based on a variety of techniques. First, the effective components and potential targets of AR were deciphered by the “component-target-disease” network using network pharmacology, and potential signal pathways on lung cancer were predicted by Gene Ontology (GO) biological function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Then, the therapeutic effects of AR in the treatment of lung cancer were evaluated  As a global health burden, lung cancer is the highest incidence cancer at present . Accordietc.  Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. has been used as a common clinical medicine in China for thousands of years. The accumulated data showed that AR was beneficial for the treatment of lung cancer in clinical practice  has been gradually recognized by the scientific community . In Chinetc. . Astragapractice . Howeverin vitro and in vivo.Network pharmacology is a discipline for investigating pathogenesis of disease through constructing and analyzing biological networks , and proAstragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.). Benzopyrene was purchased from Aladdin Reagent Co., Ltd. . Cisplatin  was gained from Nanjing pharmaceutical factory Co., Ltd. . 4% paraformaldehyde fixative was purchased from Nanjing Nanao Technology Co., Ltd. . Hematoxylin dyeing solution (D005) was purchased from Nanjing Jiancheng Bioengineering Institute . Eosin dye solution (KGA231), 0.25% trypsin, 1% Triton X-100, goat serum, 4’,6-diamidino-2-phenylindole, and DMEM high glucose medium were purchased from Nanjing KeyGen Biotech logical Co., Ltd. . Glyceraldehyde-3-phosphate dehydrogenase  antibody was purchased from Shanghai Kangcheng Biological Engineering Co., Ltd. . The antibodies against protein 53 , phosphorylated B-cell lymphoma-2 , mammalian target of rapamycin , AMP-activated protein kinase  and Beclin1 (ab133357) were obtained from Abcam . NP-40 lysis buffer (1210600) was purchased from Nanjing Shengxing Biotechnology Co., Ltd. . Polyvinylidene fluoride (k8JN62911) was purchased from Millipore Corporation . Pre-stained protein marker was purchased from Fermentas .AR was obtained from Anhui Jingquan Group Herbal Pieces Co., Ltd  and was identified by De-kang Wu  as the dried root of Astragaloside I (MUST-16012906) and astragaloside II (MUST-16031010) were purchased from manster biotechnology Co., Ltd. . Cycloastragenol (HHQC20170921) was purchased from Nanjing Spring and Autumn Biological Engineering Co., Ltd. . Astragaloside IV (1107781-201616), calycosin (111920-201304), calycosin-7-O-β-D-glucoside (111920-201505), formononetin (111703-200603), and ononin (111703-200501) were purchased from the National institute for Food and Drug Control of China . LC-MS grade methanol and acetonitrile were purchased from Merk Company . HPLC grade formic acid with a purity of 99% was purchased from Anaqua chemical supply . Purified water was prepared from a Milli-Q water purification system . Other chemicals (reagent grade) used were purchased from Nanjing Chemical Reagent Co, Ltd. .http://lsp.nwu.edu.cn/tcmsp.php) database (http://www.nrc.ac.cn:9090/ETCM/) (http://www.megabionet.org/tcmid/) (https://www.cnki.net/), PubMed (https://www.ncbi.nlm.nih.gov/pubmed), and SciFinder (https://scifinder.cas.org), and saved in SDF and Canonical SMILES structure format. All targets related to components of AR were collected from several databases, including PharmMapper (http://www.lilab-ecust.cn/pharmmapper/) (http://sea.bkslab.org/) (http://www.swisstargetprediction.ch/) (http://www.genecards.org/) (http://bidd.nus.edu.sg/group/cjttd)  , TCMID (/tcmid/) , CNKI (hmapper/) , similarab.org/) , and Swiion.ch/) , and werds.org/)  and Therp/cjttd)  to explohttps://string-db.org/), and was visualized with Cytoscape software (version 3.6.1). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of potential targets were performed using the plugin ClueGo (version 2.5.4) from Cytoscape software (version 3.6.1). The signal pathways closely related to lung cancer were statistically analyzed, and those with p < 0.01 after Benjamin's correction were considered to be significantly changed and selected for further research, named as potential signal pathways. Then, the potential signal pathways were evaluated by occurrence frequency of potential targets.The potential target-effective component network was constructed using Cytoscape software (version 3.6.1). In order to further explain the mechanism of AR in the treatment of lung cancer, the protein–protein interaction (PPI) network of lung cancer–related targets was explored by STRING  70% ethanol (v/v) in thermostatic water bath for 1.5 h. The two parts of extracts were combined, and the solvent was removed by rotary evaporation to obtain dried powder of AR. The dried powder of AR was stored in 4 °C refrigerator before use. The AR sample was analyzed by ultra-performance liquid chromatography–electrospray quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) to characterize chemical component, and main signals in chromatograph were identified, and compared with the reference substances. Eight components, including calycosin-7-O-β-D-glucoside, ononin, calycosin, formononetin, astragaloside IV, astragaloside II, astragaloside I, and cycloastragenol, were accurately identified. The component information and typical total ion chromatogram (AR and reference substances) were shown in A vacuum pump was used to simulate the human lung; a cigarette with the filter (containing 11 mg tar and 1.1 mg nicotine) was ignited and the end of the cigarette attached to a rubber hose. Smoke emitted from the burnt cigarette was dissolved in the serum-free culture medium through a glass pipette tip by a vacuum pump . The smoad libitum. All animal experiments were conducted in accordance with the guidelines of the laboratory animals and approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine.Forty male A/J mice, weighing 18–20 g, were purchased from Shanghai Experimental Animal Research Center (License number SCKK (HU) 2013-0056, Shanghai, China) and housed under pathogen-free environment with a 12h/12 h light–dark cycle and fed with food and water All the mice were randomly assigned to five groups: control group, model group, DDP group, 5.2 g/kg AR group, and 2.6 g/kg AR group. Except for the control group, the lung cancer model of several groups were created by intraperitoneally injecting with benzopyrene (100 mg/kg body weight dissolved in corn oil) twice a week for 4 weeks; the DDP group were intraperitoneally injected with DDP; mice in the 5.2 g/kg AR group and 2.6 g/kg AR group were orally administrated with AR at the dose of 5.2 g/kg/d and 2.6 g/kg/d (dose conversion according to the Chinese pharmacopoeia 2020 edition part one), respectively, while the control group was given normal saline solution by gavage. After that, all the groups continued to receive different treatments for 28 weeks and once a day. After 28 weeks, all mice were sacrificed, and the lungs were collected. Immediately, a portion of lung tissue was snap-froze in liquid nitrogen and stored at −70 °C for further analysis, and another portion was fixed in 4% paraformaldehyde for histopathological study.2O2 was added to block endogenous peroxidase activity. Finally, sections were blocked with normal goat serum for 30 min at room temperature and then incubated with anti-p-Bcl-2 and anti-p53 antibodies at 4 °C overnight. Sections were counterstained with hematoxylin and observed under light microscopy  using ×400 magnification.The lung tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated and paraffin-embedded. The paraffin-embedded lung tissues were sectioned and stained with hematoxylin-eosin (HE) and immunohistochemistry (IHC). For IHC analysis, the paraffin-embedded lung tissue sections were deparaffinized in xylene and rehydrated through graded alcohol. Then, 3% H2 at 37 °C. Culture medium was changed every two days, until NHBE cells reached 90% confluence. NHBE cells were digested by 0.25% trypsin and 0.02% EDTA solution, and prepared for the experiment with a density of 1×106 cells/mL. An MTT assay was used to assess the viability of NHBE cells exposed to different concentrations of CSE and AR  cells were purchased from Beina Chuanglian Biotechnology Co., Ltd. . NHBE cells were seeded into a plastic culture flask containing DMEM, and then placed in a humidified incubator containing 5% COE and AR . AccordiNHBE cells were digested with 0.25% trypsin to prepare single-cell suspension, centrifuged at 1000 rpm for 10 min, washed twice with prechilled PBS, and then fixed with prechilled 2.5% glutaric acid for 90 min at 4 °C. After washing in PBS, the cells were fixed with 1% osmium tetroxide at 4 °C for 30 min. After fixing, the cells were dehydrated with gradient ethanol and acetone, and then embedded in Epon812 resin (Sigma). The embedded blocks were cut into ultrathin sections using an ultramicrotome, and stained with uranyl acetate and lead citrate for ultrastructural examination under transmission electron microscopy .The NHBE cells were collected and used for Western blot analysis. The protein was extracted on ice using NP-40 lysis buffer . Lysates were centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatants were collected. Protein concentration was measured by using the DC protein assay (Bio-Rad). The separated protein samples were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membrane . Membranes was blocked with 5% nonfat dry milk in Tris-buffered saline with 0.05% Tween-20 (TBST) buffer, and then incubated with primary antibodies against p53 (1:500), p-Bcl-2 (diluted: 1:1000), Beclin 1 (diluted: 1:1000), AMPK (diluted: 1:1000), mTOR (diluted: 1:1000), and GAPDH  overnight at 4 °C. Subsequently, the membranes were incubated for 2 h at room temperature with secondary antibodies . Finally, the antigen–antibody complexes were visualized using enhanced chemiluminescence (Amersham) according to the manufacturer’s instructions, and visualized using Azure c600 imaging system . The protein levels were expressed as relative integrated intensity and were normalized to that of GAPDH.The NHBE cells were washed three times with cold PBS and fixed by 4% paraformaldehyde for 15 min at room temperature, followed by permeabilization process with 1% Triton X-100 (Fisher). NHBE cells were subsequently immuno-stained with primary antibody for 2 h at room temperature and followed by fluorescein isothiocyanate-labeled secondary antibodies for 30 min at room temperature. The samples were washed twice, adhered onto coverslips, and mounted with 4’,6-diamidino-2-phenylindole–containing mounting medium. Acquisition of images was performed using a fluorescence microscope .ΔΔCT method, and were automatically determined by the ABI 7500 Real-Time PCR System . The primers used for real-time PCR analysis were as follow: p-53, forward primer 5′-CAGACAGGCTTTGCAGAATG-3′, reverse primer 5′-GACCCTGGCACCTACAATGA-3’; p-Bcl-2, forward primer 5′-AAGCTGTCACAGAGGGGCTA-3′, reverse primer: 5′-CAGGCTGGA AGGAGAAGATG-3’; Beclin1, forward primer 5′-GTCCACGCTCGACCTTCTTAC-3′, reverse primer 5′-CACTTGCCAGTCTTAACCTCTG-3’; AMPK, forward primer 5′-TGCGTGTACGAAGGAAGAATCC-3′, reverse primer 5′-TGTGACTTCCAGGTCTTGGAGTT-3′; mTOR, forward primer 5′-CAGTTCGCCAGTGGACTGAAG-3′, reverse primer 5′-GCTGGTCATAGAAGCGAGAC-3′; and GAPDH, forward primer 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse primer 5′-TGTAGACCATGTAGTTGAGGTCA-3′.Total RNA was extracted using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. The RNA concentrations were determined using a spectrophotometer . cDNA was synthesized from RNA (2 μg) using First-strand cDNA Synthesis Kit . Real-time PCR analyses were conducted to quantitate p-53, p-Bcl-2, Beclin1, AMPK, and mTOR relative expression using SYBR Green real-time PCR kit  with GAPDH as an internal control. The cycle threshold values from all quantitative real-time PCR experiments were analyzed using 2-2 (0.2 g) and MgSO4.7H2O (0.2 g) were dissolved on 800 ml of distilled water. After that, K2HPO4·3H2O (1.0 g), KH2PO4 (1.0 g), NaHCO3 (10.0 g), NaCl (2.0 g), and resazurin solution  were added and stirred until dissolving, and then boiled distilled water was added to 1000 ml, mixed well, and cooled to room temperature. Then, tryptone (10.0 g), yeast extract (10.0 g), L-cysteine (1.0 g), and heme chloride solution  were supplemented. The pH was adjusted to 7.3 with NaOH test solution. All solution was autoclaved at 121 °C for 20 min and stored at 4 °C after cooling.CaClSix male Sprague–Dawley (SD) rats were provided by Shanghai Laboratory Animal Center (License No. SYXK (HU) 2013-0056, Shanghai China). Fresh intestinal contents (5.0 g) taken from SD rats were placed in a sterilized penicillin vial and mixed with normal saline at a ratio of 1 g:4 ml to make a suspension, and then the filtrate and anaerobic culture solution were mixed in a ratio of 1:9 to obtain enteric bacteria culture solution.AR  was added to intestinal flora culture medium (1 ml), which was then filled with nitrogen without oxygen. The reactions were terminated by adding 1 ml n-butanol and 1 ml ethyl acetate after incubation for 0, 4, 8, 12, 24, 48, and 72 h, respectively. Next, the mixtures were vortexed for 5 min after adding 10 μL internal standard solution  and centrifuged at 14,000 rpm for 5 min. Subsequently, the organic phases were collected and evaporated under nitrogen gas, and 200 μL of methanol was added, vortexed, and centrifuged again at 14,000 rpm for 5 min, respectively. The supernatant was passed through the 0.22 μm millipore filter before injecting into the UPLC-Q-TOF-MS.2 at 37 °C. The medium was replaced every day until A549 cells grew to 80–90% confluence. A549 cells were starved in serum-free medium for 3 h after washing with PBS. AR sample was incubated on intestinal bacteria (2 × 10−4 g/ml) for 0, 4, 8, 12, 24, 48, and 72 h, and then the incubated solution was separately added into the A549 cells for 90 min, and was washed repeatedly with PBS until without detected component. The dissociation solution (10.95 g/L Na2HPO4 and 12.91 g/L citric acid aqueous solution) was immediately added into the treated A549 cells, which were incubated at 37 °C and 5% CO2 for 30 min to inactivate the cell effect target. Finally, the dissociation solution was collected.A549 cells were purchased from Nanjing Kaiji biology Co., Ltd . A549 cells were seeded in a plastic culture flask containing DMEM, and then placed in a humidified incubator containing 5% CO7 cells/mL) was adjusted by PBS and then dissociated at room temperature for 1 h with dissociation solution. The cells were quickly placed at −80 °C for 20 min, and thawed in a thermostatic water bath at 37 °C for 10 min. The freezing–thawing process was repeated for 4 times and centrifuged at 2000 rpm for 20 min. Then, the dissociation solution and intracellular dissociation solution were collected and evaporated under nitrogen gas, and 200 μL of methanol was added, vortexed for 2 min, and centrifuged at 11,000 rpm for 10 min. Finally, the supernatant was passed through the 0.22 μm millipore filter before injecting into the UPLC-Q-TOF-MS.A549 cells were digested with pancreatin and suspended into DMEM/high glucose medium. The cell suspension was centrifuged at 3,000 rpm for 2 min at 4 °C; the cell density  equipped with hybrid quadrupole time-of-flight tandem mass spectrometry  coupled with electrospray ionization (ESI) source. Chromatographic separation was performed on a ACQUITY UPLC HSS T3 column . The flow rate was 0.3 ml/min, and the mobile phase consisted of solvent A  and solvent B (acetonitrile) with the optimized gradient elution: 0∼2 min, 2%∼8% B; 2∼8 min, 8%∼20% B; 8∼12 min, 20%∼35% B; 12∼18 min, 35%∼60% B; 18∼24 min, 60%∼70% B; 24∼28 min, 70%∼80% B; 28∼28 min, 80%∼2% B; and 28∼30 min, 2%∼2% B. The column temperature was set at 30 °C. In order to get better analysis results, the mass spectrometer was conducted in electrospray and multiple reaction monitoring scanning mode, in negative ion modes. The optimized parameters for mass spectrometer were as follows: capillary voltage, 0.5 kV; ion source temperature, 100 °C; cone gas flow rate, 50 L/h; desolvation temperature, 400 °C; and desolvation gas flow, 800 L/h. The information-dependent acquisition techniques and dynamic background subtraction were used to reduce the impact of matrix interference.t-test for pair-wise comparison. p < 0.05 was considered to be a significant difference, p < 0.01 was considered to be extremely significant difference, and p > 0.05 was considered to be no significant difference. All statistical analyses were performed using SPSS software .All data are presented as the mean ± standard deviation. Data from mice and NHBE cells were statistically evaluated using In this study, the computer virtual screening technology was used to provide a fast and efficient approach to obtain potential targets. At length, 160 potential targets of AR in the treatment of lung cancer were obtained, and the information of potential targets (degree ≥5) are listed in Based on the acquisition of potential targets, 36 potential effective components were obtained by matching potential targets, and their information is listed in Go enrichment analysis was performed on 160 potential targets, and limiting annotation was selected to homo sapiens. The top 10 terms of biological process , moleculp-value less than or equal to 0.01 were obtained . Besides, compared with the model group, the body weight of 5.2 g/kg AR group and 2.6 g/kg AR group were significantly increased , and the most significant effect was observed in 5.2 g/kg AR group. The appearance of all mice were observations throughout the experimental period, and the results showed that the mice in model group suffered from nose bleeding and sparse neck hair, and no obvious symptoms in other four groups were observed.The body weight changes of the animals in different groups were shown in p < 0.01) the number of lung tumor nodules (p < 0.01), and the p-Bcl-2 expression (p < 0.01) in the lung tissue of model group. The administration of AR could upregulate the p53 expression and downregulate the p-Bcl-2 expression in the lung tissue, which indicated that AR can reverse the expression of p53 and p-Bcl-2 in lung cancer mice.After 28 weeks, lungs were removed from the mice for analysis. Except for the control group, what were observed in the other groups showed obvious lung lesions, tumor-like proliferation, and tumor nodules. Compared with the model group, the mice by DDP, 5.2 g/kg AR, and 2.6 g/kg AR treatment were able to significantly reduce . As shown in p < 0.01). RT-PCR results (p < 0.01) and the relative mRNA level of nuclear p53 has been decreased (p < 0.01) by the treatment of 1000 µg/ml AR, respectively. The above results indicated that AR could promote p53 expression, thereby inhibiting autophagy and protecting cells from autophagy.The potential targets, p53, p-Bcl-2, and mTOR, predicted by network pharmacology technology, were verified at the protein and gene levels. As shown in  results  showed tp < 0.01). In addition, compared with model group, the relative mRNA level of p-Bcl-2 was significantly reduced (p < 0.01) in 1000 µg/ml AR group and 500 µg/ml AR group . RT-PCR results (p < 0.01). Immunofluorescence staining showed increased expression of mTOR expression in AR-treated cells at the concentration of 1000 µg/ml AR and 500 µg/ml AR (p < 0.01). mTOR is a major negative regulator of autophagy and a key protein for controlling autophagy. The above results showed that AR could increase mTOR expression and regulate cell autophagy to protect cells.As shown in  results  showed tp < 0.01) on AMPK and Beclin1 expression in NHBE cells after being treated with 1000 µg/ml AR. Besides, the relative mRNA level of AMPK and Beclin1 was significantly reduced (p < 0.01) in 1000 µg/ml AR group and 500 µg/ml AR group. It has been documented that p53 mediates autophagy through an AMPK/mTOR-dependent pathway  as shown in Ultimately, these effective components, that is, calycosin-7-O-β-D-glucoside, ononin, calycosin, astragaloside IV, astragaloside II, cycloastragenol, and formononetin, together form the material basis of AR for prevention and treatment of lung cancer, and they come from the two-type components, flavone and saponin.In this study, an integrated strategy for effective-component discovery of AR in the treatment of lung cancer was established. The results indicated that the integrated strategy can be applied to the efficiently screen effective components in complex systems. In our research on the bioactivity of AR, we found that the administered doses were high compared with single component drug. Therefore, the screening and confirmation of effective components in this article will help to reduce the dose by removing the ineffective components.Modern pharmacological studies have shown that the activity of drugs is closely related to their cell membrane affinity and permeability. An important step in the role of TCM is the binding of effective components to cell membranes, specific enzymes, or receptors in cells. In this study, A549 cells were used as the separation carrier, AR was taken as the research object, and the specific affinity between each component in AR and cells was determined by cell membrane–immobilized chromatography. It is worth noting that the screening results show that calycosin-7-O-β-D-glucoside, ononin, calycosin, astragaloside IV, astragaloside II, cycloastragenol, and formononetin may be effective components of AR in the treatment of lung cancer, and which is consistent with the previous research results of AR in the prevention and treatment of cancer . Throughvia sequestration of Beclin1 away from class III PI3K; and the interaction between Bcl-2 and Beclin1 is related to mTOR kinase-dependent phosphorylation of Bcl-2 (Autophagy has been implicated in a wide range of human diseases, including lung disorders such as lung cancer, chronic obstructive pulmonary disease, and lung infection diseases . The artof Bcl-2 . The prein vitro/vivo studies, it still could not avoid some false positives. Moreover, we found that the dosage of AR was very high compared with single-component drug. Therefore, it is necessary to further knock out invalid components of AR to reduce dosage. Finally, the preliminary research results indicated that AR in the treatment of lung cancer may be through p53/AMPK/mTOR signaling pathway; certainly, further experimental validation should be needed to confirm this hypothesis.In this study, an integrated strategy for effective-component discovery of AR in the treatment of lung cancer was established, which provides a valuable reference mode for finding the effective components of TCM. In addition, preliminary research results indicated that AR in the treatment of lung cancer may through p53/AMPK/mTOR signaling pathway, which laid a foundation for further in-depth study of the mechanism of AR in lung cancer. Despite some promising results were obtained in the study, there are still several potential limitations to improve. First, we have to admit that network pharmacology virtual screening has some limitations, and the predicted effective components and potential targets may need to be further comprehensively verified through a variety of different technologies. Even though the study adopts an integrated research strategy combining network analysis and


In this note, we introduce excess strategic entropy—an entropy-based measure of complexity of the strategy. It measures complexity and predictability of the (mixed) strategy of a player. We show and discuss properties of this measure and its possible applications. In every stage game, each player uses a mixed strategy . Generally, in the literature, it is assumed that players can carry out any strategy in a specified strategy set, should they choose to play it. While this latter assumption may seem innocuous in a model where few strategies are available to each player, it may be criticized as being unrealistically rational in more complex models where a theoretical definition of strategy leads to a strategy set that contains a large number of choices, many of which are impractically complex. This is usually the case in repeated games—at every stage, players are playing a history (in-)dependent game and therefore the set of all strategies can be huge. Thus, it is reasonable to consider the complexity of the strategy  interactions have been modeled as formal games. In particular, repeated games play the central role in models of the long-term competition in economic theory and modeling interactions which are repeated frequently ,2. In sue, e.g., ). In adde, e.g., . This muIn general, restricting the set of feasible strategies having regard to complexity of the strategy seems to be particularly relevant. A popular approach to the problem of complexity of strategies is to assume bounded rationality of an agent. There have been many attempts to model feasible (implementable) sets of strategies that reflect some aspects of the bounded rationality of players. Finite automata, bounded recall, and Turing machines are a few of the approaches taken. These models are useful because they provide us with quantitative measures of complexity of strategies, e.g., number of states of automata and the length of recall. This approach, used e.g., in ,6, descrIn the seminal article, Neyman and Okada introduced entropy-based measures of uncertainty for mixed strategies of repeated games—strategic entropy and strategic entropy rate . They stTo provide a proper and more precise insight into the structure and complexity of strategy, we propose taking the next step—looking at convergence rates of the entropy. It is commonly agreed that a slow entropy convergence rate is a sign of complex structure of a process . ObviousWe propose to use the concept of excess entropy, which measures an entropy convergence rate. It can result in better understanding of strategies played—measuring not only randomness of the strategy but its structure, regularity, and predictability as well. To this end, we will construct a measure based on the quantity widely used in information theory and physics.complexity measures. To reduce confusion, it has become convenient to refer to them instead as statistical complexity measures. In doing so, they are immediately distinguished from deterministic complexities. In contrast, statistical complexity measures discount for randomness, and so provide a measure of the regularities present in an object above and beyond pure randomness.Structure and correlation are not completely independent of randomness. It is generally agreed that both maximally random and perfectly ordered systems possess no structure . Neverthexcess entropy  to study complexity of chaotic signal. It made a huge impact in particular on statistical mechanics and information theory (Google Scholar records over 1600 citations of this article), and in economics as well [n-block entropy asn and stored information, effective measure complexity, Grassberger’s complexity, and predictive information). Among others, it measures the excess randomness of the system, and therefore tells us how much additional information must be gained about the sequences in order to reveal the actual per-symbol uncertainty h. Thus, it can be interpreted as a measure of statistical complexity.In 1983, Grassberger and Procaccia in their seminal paper  introduc as well . The mosn stage games of the repeated game converges to the limit that is to the (strategic) entropy rate of the strategy. To this aim, we propose an analogue of excess entropy, which we will call excess strategic entropy (shortly: excess s-entropy). We will use it to measure statistical complexity and unpredictability of strategies. Natural interpretations of this measure impose its applicability e.g., in the case of bounded recall and reputation models. Undertaken research and obtained results fit into the growing field of applications of entropy-based concepts in economics, and game theory in particular (see [The aim of this note is to incorporate the concept of excess entropy into the game theory setting. It can be used to answer the question how fast the strategic entropy of the (first) lar  is a sequence S and T to be endowed with the product topologies with the discrete topology on each factor. S and T, respectively. A mixed strategy of Player 1 (resp. 2) is then a probability on Every pair of pure strategies random playNote that Remark 1.Ω can be represented as the full shift Ω is the set of all one-sided sequences on Ω. Moreover, the partition imposed by n-equivalence classes is the partition into cylinders; see To simplify discussion, one can identify A (and B) with the set of non-negative integers X be a random variable which takes values in In this subsection, we recall well-known quantities and facts from information theory. Let Definition 1.The entropy The notion of entropy can be naturally extended to an arbitrary finite dimensional vector of random variables or probability distributions.Definition 2.The conditional entropy Lemma 1(see ). H(Y|X)Definition 3.Γ. The entropy rate of the process Let Stationary processes (and induced by them stationary strategies) will be an important tool in our considerations. In this section, we recall the concept of a stationary process and show the connection between stationary process, and existence of a shift-invariant measure (an in-depth discussion of these connections can be found in ).B.We say that process In this note, we will focus our attention on stationary strategies.Definition 4.A strategy profile For stationary processes, the upper limit in Formula  can be rt if Player 1 plays a mixed strategy We define the strategic entropy rate of the strategy posed in . It is an, that is,n stages. Therefore, n that Player 2 faces when Player 1 uses t. The dual interpretation is that it is the amount of information on the play of the game, which Player 2 can obtain using t when Player 1 uses For each found in :Lemma 2.1.2.For each 3.For each 4.Moreover, if Now, we can define the strategic entropy rate.Definition 5.If and the strategic entropy rate of the strategy σ is the supremum of In general, the limit of We start this section with a discussion on the existence of the limit in Formula  for statLemma 3.Sequences If additionally σ is stationary, then they are decreasing and have a common limit.Proof. For any process, we have that they are non-negative sincee μ (see ). To sim entropy , we obtaTherefore, the sequence Corollary 1.If Naturally, we can ask whether we can consider limits instead of the upper limits in Formula . UnfortuExample 1.Let at every even stage, Player 1 chooses 1;at every stage at every stage Then,For every Thus, s-entropy). One may ask if the assumption of stationarity isn’t too restrictive. Obviously using only stationary strategies may seem to be quite restrictive, but we know that, for many types of games, there exists an optimal stationary strategy [We can see that the assumption that the strategy is stationary gives a nice theory of the strategic entropy . Example 3Let andMoreover,In addition, if Thus,(Independent actions). s-entropy doesn’t respond to the unpredictability of a one-stage game. In both examples, a player plays in an unpredictable way (playing mixed strategy) but not in a complex way. Nevertheless, it detects unpredictability of a long-run play that is how predictable is the change in a player’s mixed strategy (probability distribution).From Examples 2 and 3, we can see that excess Example 4Suppose that σ is a periodic strategy of period p. Then, (Periodic strategy). Example 5 (Infinite excesss-entropy).Let s-entropy when changes of strategies are frequent, while, if changes are rare, the excess s-entropy should be finite. Now, we show another example which catches this intuition.We can see that we should expect infinite excess Example 6.Let us consider a two-player matrix game, whereThe first player plays “Top” as long as Player 2 chooses “Left”. If Player 2 plays “Right” at the stage m for the first time, then Player 1 chooses a mixed action with distribution Therefore, for every or there exists If the first condition is fulfilled, then Therefore,ands-entropy can be used in more complex games, we observe that we are building on the theory of entropy convergence rates. Therefore, we are able to use tools and ideas designed to study these rates. First, looking through the lens of information theory, Feldman et al. showed that excess entropy is the proper tool to detect and analyze patterns produced by a process [s-entropy of a strategy t can be used to detect and quantify patterns produced by a play One may ask how this concept works and how this quantity can be calculated in more sophisticated and complex problems. Although this is not the aim of this article, we shortly discuss this here. The purpose of this section was to give illustrative examples, showing the intuition behind the introduced quantity. Nevertheless, to describe how excess  process ,24. It is-entropy agrees with the intuition of the complexity of the strategy. The natural question to ask is how we can bound excess s-entropy. Here, we will concentrate on stationary strategies. First, from Corollary 1, if a play induces stationary process, thenAs we can see from the previous section, excess s-entropy.In Theorems 2 and 3, we give few properties of the excess Theorem 2.Let σ be a stationary strategy. Then, the following conditions are equivalent:1.2.3.Proof. We will prove equivalence of conditions showing implications t. Condition (3) is an immediate consequence of (2). Noticing that by (3) we have To show implication from s-entropy:Moreover, for stationary strategies, we have a simple lower bound for excess Theorem 3.If σ is stationary, then Proof. First, let n to infinity results inConverging with In this article, we proposed a new measure of statistical complexity and unpredictability of a strategy—excess strategic entropy. Because of its entropy-based origin, we were able to catch intuition behind this quantity by understanding how entropy and entropy convergence rates work in the context of game theory. We showed and discussed its properties. To simplify discussion, we introduced this quantity for a two-player game, but it can be easily extended to games with more players. Lastly, in this section, we look at two possible applications of this quantity.s-entropy (and complexity of a strategy) will be equal to zero, as the player won’t be eager to change his strategy. Nevertheless, if a player is not in the Nash equilibrium from the beginning, he may prefer to choose one of the mixed strategies which is not too complicated—for instance that doesn’t force him to change a one-shot strategy too much. Thus, restricting the set of strategies to those with bounded excess s-entropy seems to be a natural choice. Even if the player wants to “converge to the Nash equilibrium”, he may prefer those Nash equilibria to which his sequences of one-shot strategies converge using strategies with bounded excess s-entropy. Moreover, results obtained by Neyman et al. [s-entropy will impose high predictability of the mixed strategy (probability distribution which he uses is predictable), and will impact the possibility of choosing the best response strategy by the player with an unrestricted set of strategies. Therefore, bounded excess s-entropy strategies should be thoroughly explored.It is natural to restrict strategies of a player to those of finite statistical complexity. Obviously starting from the Nash equilibrium excess n et al. ,25,26,27s-entropy may give a better insight are reputation models. The notion that commitment is valuable has long been a critical insight of non-cooperative game theory, and has deeply affected a number of social science fields, including macroeconomics, international finance, and industrial organization. It seems natural to expect that agents playing in the long-run competition will base their decisions on the reputation of the opponent. Moreover, if the opponent is more predictable (in the sense that the probability distribution which he uses is predictable), then the player can find a better response to the predicted strategy, or may prefer to play with the more predictable player. It should be added that using entropy-based measures in reputation models is not a new idea. For instance, in [Another research area where studying excess ance, in , Ekmekciance, in ,30.Methods used in article are standard methods and are thoroughly discussed in


Primary care practitioners (PCPs), being the front liners, play an important role in treating allergic rhinitis (AR). As there is no proper tool to assess their perception, attitude, and practice in utilizing the guidelines, we aimed to develop and validate a new questionnaire for such purpose.The development phase consists of both literature and expert panel review. The validation phase consists of content validity, face validity, and construct validity. Cronbach's alpha was used to verify internal consistency. The development phase produced a questionnaire with 3 domains: perception, attitude, and practice consisting of 60 items (PAP-PCP questionnaire). Item response theory analysis for perception demonstrated the difficulty and discrimination values were acceptable except for 3 items. Exploratory factor analysis for attitude and practice domains showed the psychometric properties were good except for 3 items in practice domain. Experts judgement was used to decide on the final selection of questionnaire which consists of 59 items.The final validated questionnaire has 3 domains with 59 items. All domains had Cronbach's alpha above 0.65 which was reliable. 302 physicians completed the questionnaire. 98% PCPs diagnosed AR based on clinical history. Although, majority agree AR guidelines is useful (67%), they had difficulty in using it to classify AR (54.9%) and determine AR severity (73.9%). Oral anti-histamines (first and second generation) were the most prescribed (>75%) followed by intranasal corticosteroids (59%) and combined intranasal corticosteroid and oral anti-histamine (51%). Majority agreed that treatment efficacy (81.8%), adverse effects (83.8%), fear of adverse effects (73.5%), route of administration (69.4%), dosing frequency (72.5%), taste (64.6%) and cost (73.5%) affect treatment compliance.The newly developed and validated questionnaire is a promising instrument in understanding the treatment gap in AR. Although further testing and refinement are needed, it provides an initial means for evaluating knowledge and understanding of PCPs in treating AR. An allergen exposure of allergic individuals results in an immunoglobulin E (IgE)-mediated inflammatory response, which can be manifested clinically as nasal congestion, postnasal drainage, rhinorrhea, nasal itching, sneezing, and itchy or watery eyes.As many patients rely on their PCPs for diagnosis and treatment, primary care practice represents a crucial and important profession to be evaluated as part of the management strategy of AR.The study was a non-interventional prospective observational study. Ethical approval was obtained from the local human ethics committees. Written consent in English was obtained from each participant prior to conducting the survey.This study was conducted amongst PCPs in the out-patient setting of hospitals, health clinics, and private clinics from April 19, 2018 until December 18, 2018. Sample size was determined using the factor analysis methodIn the first phase, the PAP-PCP questionnaire, a self-administered questionnaire was developed from consultation among experts and adaptation from literature reviews.Perception is defined as the way in which something is regarded, understood, or interpreted. In short, it is the result of a cognitive process. The perception questions explored concepts of existence of AR practice guidelines, diagnosing AR, common symptoms of AR, and classification and severity of AR . An attiIn the second phase, the validation of PAP-PCP questionnaire was done based on 3 levels of evidence which are content validity, face validity, and construct validity. The Content Validity Index (CVI) assessed relevance and representability of each item to a specific domain by the panel of experts.,The perception domain was analysed by two-parameter logistic item response theory (2-PL IRT) analysis, using R software , as it consists of unidimensional items with dichotomous responses. Difficulty in the range of −3 to +3 and discrimination in the range of 0.35–2.5 were considered acceptable.The exploratory factor analysis and Cronbach's alpha were used to measure construct validity and internal consistency of the remaining 2 domains in the questionnaire (attitude and practice).The data were tabulated using Microsoft Excel and analysed using the Statistical Package for Social Sciences, SPSS 24 for Windows. Descriptive statistics was used to summarise the socio-demographic characteristics of subjects. Numerical data was represented as mean (SD) and frequency (n-%). Questions were coded based on a Likert-scale, on which a more desirable answer was given a higher score than a less desirable answer. Factor loadings were used to extract factors and items in exploratory factor analysis (EFA). Cronbach's alpha coefficient was used as an estimate of the internal consistency of the questionnaire.A total of 302 PCPs participated in this study consisting of 118 men and 184 women. The age ranged from 28 to 73 years old with mean age of 36.4. The majority of them were Malays 271 (89.7%), followed by Chinese 23 (7.6%), Indian 7 (2.3%), and others 1 (0.3%) . The meaa.Perception domainDo you know the common symptoms of AR?), P-Q8  and P-Q9  which were out of the acceptable range (>2.5). However, following expert consensus and judgement, all questions were retained because the contents of the items were deemed important and they had acceptable difficulty values with a reliable Cronbach's alpha (0.7).b.Attitude domainBased on the two-parameter logistic item response theory (2PL-IRT) analysis, the psychometric properties of the perception domain were good. For the difficulty parameter, all the perception items were within acceptable range of −3 to +3. In terms of discrimination, most of the items were within acceptable range (0.35–2.5), except for question P-Q7 (c.Practice domainKaiser-Meyer-Olkin Measure of Sampling Adequacy for attitude domain was 0.77 (adequate) and Barlett's test of the sphericity was significant . Three factors were extracted in attitude domain, which were factor A1 with 13 items , factor A2 with 8 items  and factor A3 with 7 items . All items were retained as their factor loadings were acceptable (>0.3). Reliability for each factor in attitude domain were above 0.65. Reliability of factor A1 was 0.83, factor A2 was 0.78 and factor A3 was 0.87 .c.PractiI diagnose my patients with AR by clinical history), PR-Q19f  and PR-Q20a  had low factor loadings. Following experts consensus and judgement, 2 items PR-Q18a and PR-Q20a were retained while item PR-Q19f was removed. Reliability for each factor in practice domain were 0.90, 0.86, and 0.66 respectively  and Barlett's test of the sphericity was significant . Similarly, 3 domains were extracted; factor PR 1 with 8 items , factor PR 2 with 8 items  and factor PR 3 with 4 items . All items in the 3 domains had acceptable factor loadings (>0.3) except for 3 items. Three items, PR-Q18a  which had lower scores. More than 50% of PCPs answered incorrectly for those items and these findings suggest a knowledge gap amongst PCPs. PCPs recognised ARIA and Global Initiative for Asthma (GINA) guidelines, able to differentiate AR from non-AR, aware of the role of appraising and diagnosing asthma, recognised common symptoms of AR, not aware of other AR practice guidelines, and had difficulties in classifying and determining the severity of AR  (43%), combined oral antihistamine and oral decongestant (20%), intranasal corticosteroid as a monotherapy (17%), and combined intranasal corticosteroid and oral antihistamine (12%). For physicians who did not find immunotherapy safe, they prescribed oral antihistamine (first and second generation) (45%), combined oral antihistamine and decongestant (21%), intranasal corticosteroid as a monotherapy (18%), and combined oral antihistamine and intranasal corticosteroid (17%). For physicians who never used allergy tests, they prescribed oral antihistamine (first and second generation) (47%), combined oral histamine and decongestant (20%), and combined oral antihistamine and oral decongestant (18%).,,AR with its increasing prevalence, represents a major global health concern. As the front liners in managing AR patients, PCPs play an important role in providing the appropriate care. There are evidences that unmet needs of patients have not been addressed despite AR guidelines such as ARIA have been revised and disseminated to health care workers worldwide.Do you know the common symptoms of AR?), P-Q8 (Do you know how to classify AR) and P-Q9 . However, based on the content validity, the 3 items were found to be relevant and adequately represent the domain. Content validity is a prerequisite for any other forms of validity; thus, they should be given the highest priority in the development of any new inventory.I diagnose my patients with AR by clinical history) was retained as clinical history is the best way of diagnosing AR by the PCPs while item PR-Q20a  was retained, because it is the first line treatment for mild and moderate AR according to ARIA guidelines. Item PR-Q19f  was removed as the experts felt that the item did not fit that domain (under presented construct).The initial questionnaire following the content and face validation, had 3 domains  with 60 items. Overall, the perception domain showed good psychometric properties based on the difficulty and discriminatory parameters of the items. The discrimination parameters were mostly within the acceptable range except for items P-Q7 , the results suggested that PCPs knew GINA better than ARIA guidelines. This probably reflects their concerns about asthma as most perceived asthma as a life-threatening condition and should be properly treated. They knew that asthma must be evaluated in AR (P-Q5) but surprisingly despite knowing the AR symptoms (P-Q7), the PCPs have low awareness of AR classification (P-Q8) and the severity of the disease (P-Q9). On the other hand, the results for the attitude domain, suggested that more than half of PCPs agree that ARIA is useful in categorizing AR patients and acknowledge classifying patients based on severity . The discord between perception and attitude reflect the knowledge gap among PCPs in the significance and proper use of ARIA guidelines to classify AR and determine AR severity for proper treatment selection in their practice. The other interesting finding is despite only 201 respondents knew about ARIA guidelines, but 222 respondents agreed or strongly agreed that ARIA was useful for categorizing patients. We believe this is due to social desirability bias. The respondents might be pressured to answer that they agree about the usefulness of the guidelines to avoid being perceived as ignorant even though they have been reassured that their answers would be confidential. Majority of the PCPs (67.2%) agreed that the diagnosis of AR patients should be based on clinical history and allergic testing (A-Q13). When assessed for their attitude towards AR treatment , most PCPs agreed that oral anti-histamines, both first generation (72.1%) and second generation (83.8%), oral antihistamines and decongestants (85.4%), leukotriene antagonists (50%), and intranasal corticosteroids (87.1%) were effective and safe in treating AR patients. This may explain their preference (PR-Q20) of prescribing oral anti-histamines, first generation (78.5%) or second generation (85.8%), oral antihistamines and decongestants (67.9%), intranasal corticosteroids (59.3%), and oral antihistamines and intranasal corticosteroids (51.7%). The choice of medications in their practice reflects their consideration for patient's treatment compliance (A-Q17). Most PCPs agreed that treatment efficacy (81.8%), adverse effects (83.8%), fear of adverse effects (73.5%), route of administration (69.4%), dosing frequency (72.5%), taste (64.6%), and cost (73.5%) affect treatment compliance. Those factors might explain their choice of first line therapy using oral antihistamines for all AR patients to be followed by combination therapy only when patients do not response to first line treatment despite ARIA recommending treatment based on the severity of AR. The use of single modality in AR treatment also could be due to the cost factor as most patients attending PCPs clinic are cash paying and PCPs need to ensure their treatment is affordable. The overall results indicate that there was a knowledge gap among PCPs which require more educational program among PCPs to create better recognition and understanding in better utilization of ARIA guidelines.For the perception domain  and the results might be different with the involvement of older age group. Hence, we recommend more studies to validate the questionnaire in larger representative samples to ascertain its generalizability.The newly developed and validated questionnaire is a promising instrument for the evaluation of the perception, attitude, and practice of PCPs towards AR. This is an important step towards understanding the treatment gap in AR. Although further testing and refinement are needed, it provides an initial means for evaluating the knowledge and understanding of PCPs towards ARIA guidelines.None.BA, RK, NFHNH, AFI, ZWM, DYW participated in the design of the study, drafted the manuscript, collected the data, analyze the data and revised the manuscript for critical content. All authors read and approved the final manuscript.Ethical approval was obtained from Human Research Ethics Committee USM (USM/JEPeM/17110581) and Medical Research & Ethics Committee (NMRR-17-3342-38820). Written consent in English was obtained from each participant prior to conducting the survey.All authors have approved the manuscript for submission.The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.The authors declare that they have no competing interests.


Non-genetic heterogeneity is emerging as a crucial factor underlying therapy resistance in multiple cancers. However, the design principles of regulatory networks underlying non-genetic heterogeneity in cancer remain poorly understood. Here, we investigate the coupled dynamics of feedback loops involving (a) oscillations in androgen receptor (AR) signaling mediated through an intrinsically disordered protein PAGE4, (b) multistability in epithelial–mesenchymal transition (EMT), and (c) Notch–Delta–Jagged signaling mediated cell-cell communication, each of which can generate non-genetic heterogeneity through multistability and/or oscillations. Our results show how different coupling strengths between AR and EMT signaling can lead to monostability, bistability, or oscillations in the levels of AR, as well as propagation of oscillations to EMT dynamics. These results reveal the emergent dynamics of coupled oscillatory and multi-stable systems and unravel mechanisms by which non-genetic heterogeneity in AR levels can be generated, which can act as a barrier to most existing therapies for prostate cancer patients. Phenotypic plasticity, i.e., the ability of cells to switch back and forth reversibly among different states (phenotypes), is a universal feature of adaptation to varying environments encountered by various biological systems . This thStochasticity in phenotypic plasticity and/or heterogeneity is a direct consequence of limited number effects of molecules involved in various biochemical reactions, including transcription, translation and cell-cell communication signaling ,33,34. SIn prostate cancer (PCa), androgen-deprivation therapy (ADT) has been a standard-of-care treatment for over 75 years. Resistance to ADT eventually occurs in most patients, leading to metastatic castration-resistant prostate cancer (CRPC) ). Progresee Methods). While a strong negative feedback leads to sustained oscillations of AR  . ZEB canNext, in the following sections, we investigate how different couplings between the AR and EMT circuits can modify their standalone circuits’ dynamics and give rise to different states. First, to gain an understanding of how the AR circuit responds to external signals, we study a generic coupling between AR and an external node (X). Next, we explicitly couple the PAGE4 and EMT circuit through AR-ZEB mutual inhibition.Before coupling the PAGE4-AR circuit with the EMT circuit, we first add a single node X to the PAGE4-AR circuit to understand how perturbations to AR signaling can modify its oscillatory dynamics A. We alsAt high ck loops ,53 ; specifically, a Hill coefficient (n) quantifies how nonlinearly or steeply the fold-change depends on inducer/inhibitor level. We observed that the higher the value of n for AR-X coupling , while SNAIL (S) is an external EMT-inducer A. To invAs a first step, we study the effects of weak bidirectional coupling, i.e., tability .Next, we consider the case where ZEB inhibits AR strongly while AR only weakly inhibits ZEB  E. This d= 180 K) F. Thus, = 180 K) C also diNext, we examine the case when we interchange the interaction strengths of both arms of the feedback loop between AR and ZEB1, i.e., when AR inhibits ZEB strongly, but ZEB inhibits AR weakly  by +10% or −10%. We quantify parameter sensitivity in terms of variation for amplitude and period of AR oscillations . GeneralTo summarize the results, we drew phase plots showcasing the accessible EMT states  can drive EMT through Notch signaling mediated activation of SNAIL, subsequently affecting the dynamics of EMT and PAGE4-AR circuits. We have previously shown that the dynamics of EMT circuit remain largely unaltered upon including miR-34 [Previously, we have studied the coupled dynamics of EMT and Notch that were capable of exhibiting a maximum of four distinct states . Drawingg miR-34 ; thus, wg miR-34    can assume up to four distinct phenotypes when induced with varying levels of external Delta ligand (Dext) . In partext (Dext = 100), the cell assumes an epithelial phenotype, and Notch receptors and ligands relax to a constant level (ext level (Dext = 400) induces a co-existence of two epithelial phenotypes—one of them  does not exhibit oscillations in terms of miR-200 and Jagged and the other one  does (ext = 900), the epithelial Sender phenotype is not observed, perhaps because of strong activation of Notch signaling and consequent inhibition of Delta by Notch Intra-Cellular Domain (NICD). In this scenario, the epithelial Receiver phenotype is maintained which continues to exhibit oscillations in both miR-200 and Jagged  does . On furtd Jagged . OverallNext, we consider the opposite case, i.e., weak AR-to-ZEB signaling and strong ZEB-to-AR signaling , oscille states  suggestsThe bidirectional coupling between AR signaling and EMT offer a possible mechanistic link associating the progression of cells towards a partial or full EMT and gain of therapy resistance in prostate cancer. In particular, our model suggests that the epithelial phenotype usually co-occurs with PAGE4 oscillations, whereas transitions to hybrid E/M or mesenchymal phenotypes quench these oscillations and promote low AR levels. From a clinical standpoint, low levels of AR suggest an androgen independent (AI) phenotype that is potentially less susceptible to androgen deprivation therapies. Therefore, EMT induction can potentially promote therapy resistance by stabilizing an androgen independent PCa phenotype through the ZEB1-AR signaling axis. A partial and/or full EMT has been associated with resistance to various therapies in multiple cancers ; howeverOur model predicts that besides the EMT circuit, Notch signaling pathway can also exhibit oscillations. This pathway has been shown to display oscillatory dynamics, but mostly in the context of somite segmentation clock . The oscConsidered together, our work highlights how coupling between Notch, EMT and PAGE4/AR signaling pathways can give rise to non-trivial dynamics and non-genetic heterogeneity in a cancer cell population. Thus, our results add to the growing literature on investigating coupled dynamics of EMT and other regulatory modules ,76,77,78X) obey the chemical rate equations that describe their interactions with other species in the circuit. The generic form of such equations is:In our model, the temporal dynamics of micro-RNAs or proteins . In case of regulation on X from multiple species, those terms are multiplied. The second term on the RHS  to take the following form:SH takes the following form:Here, the first term in RHS (A(t)) is number of molecules of the species A at any given time (t). Furthermore, in the PAGE4 circuit, we consider that case of delay in the inhibition of CLK2 by AR. In this particular case, the levels of CLK2 at time t depend on the levels of AR at The first argument in the Shifted Hill function (Detailed equations for all species in the PAGE4, EMT and Notch circuits are presented in the