Rationale: Androgen receptor splice variant 7 (AR-V7) is a leading cause of the development of castration-resistant prostate cancer (CRPC). However, the regulation and function of AR-V7 at levels of post-translational modifications in prostate cancer therapy remain poorly understood. Here, we conducted a library screen of natural products to identify potential small molecules responsible for AR-V7 protein degradation in human prostate cancer cell lines.Methods: A natural product library was used to screen the inhibitor of AR-V7. Co-IP and biomass spectrum assays were used to identify the AR-V7-interacting proteins, whereas western blot, confocal microscopy, RNA interfering, and gene transfection were used to validate these interactions. Cell viability, EDU staining, and colony formation assays were employed to detect cell growth and proliferation. Flowcytometry assays were used to detect the distribution of cell cycle. Mouse xenograft models were used to study the anti-CRPC effects in vivo.Results: This screen identified rutaecarpine, one of the major components of the Chinese medicine Evodia rutaecarpa, as a novel chemical that selectively induces AR-V7 protein degradation via K48-linked ubiquitination. Mechanically, this effect relies on rutaecarpine inducing the formation of a GRP78-AR-V7 protein complex, which further recruits the E3 ligase SIAH2 to directly promote the ubiquitination of AR-V7. Consequently, the genetic and pharmacological activation of the GRP78-dependent AR-V7 protein degradation restores the sensitivity of castration-resistant prostate cancer to anti-androgen therapy in cell culture and animal models.Conclusions: These findings not only provide a new approach for overcoming castration-resistance in prostate cancer therapy, but also increase our understanding about the interplay between molecular chaperones and ubiquitin ligase in shaping protein stability. Prostate cancer (PC) is a common malignancy with high incidence and mortality among males worldwide Among multiple AR-Vs, AR-V7 is the most frequently expressed clinically, and is functionally relevant to CRPC development Evodia rutaecarpa, exhibited previously unknown activity in promoting AR-V7 protein degradation via the activation of the molecular chaperone glucose-regulated protein 78kD  and siah E3 ubiquitin protein ligase 2 (SIAH2). Importantly, targeting GRP78-dependent AR-V7 protein degradation via SIAH2 overcame CRPC in vitro and in vivo.Although the significance of AR-V7 has been well-demonstrated, the regulatory network controlling AR-V7 protein stability remains to be fully understood. There is also no specific inhibitor of AR-V7 available in the clinic, which hinders investigations as well as treatments for CRPC. The current study aims to identify potential small molecules responsible for AR-V7 protein degradation and further uncover the underlying mechanisms of action of their biomedical effects on overcoming CRPC. Based on a library screen of natural products, we found that rutaecarpine (Rut), an alkaloid extracted from the Chinese medicine Chemicals: natural product library (#L1400), rutaecarpine (#S2349), parecoxib (#S4656), bortezomib (#S1013), MG132 (#S2619), enzalutamide (#S1250), and bicalutamide (#S1190) were obtained from Selleckchem . Antibodies: anti-AR-V7 (#19672), anti-AR (#5153), anti-K48-ubiquitin (#12805), anti-IgG (#3900), anti-MDM2 (#86934), anti-CDK2 (#2546), anti-CDK4 (#12790), anti-CDK6 (#13331), anti-cyclinD1 (#2978), anti-p21 (#2947), anti-p27 (#3686), anti-p15 (#4822), anti-COX2 (#12282), anti-HA-tag (#2367), anti-FLAG-tag (#8146), anti-GAPDH (#5174), anti-GRP94 (#20292), anti-HSP90 (#4877), and anti-lamin B1(#13435) were obtained from Cell Signaling Technology ; anti-GRP78 (#ab21685), anti-HSP7C (#ab51052), anti-SIAH2 (#ab230532), anti-STUB1 (#ab134064), anti-AR-V7 , and anti-Ki67 (#ab15580) were obtained from Abcam .2, according to our previous reports The human prostate epithelial cell line WPMY-1; AR-positive PC cell lines LNCaP, C4-2, and 22Rv1; and AR-negative PC cell lines PC3 and DU145 were purchased from the American Type Culture Collection . The above cell lines were cultured in an incubator at 37 °C with 5% COThe RNA interfering assays using siRNA or shRNA were performed according to our previous reports For lentivirus shRNA transfection, lentivirus (pLKD-CMV-Puro-U6-shRNA) containing 2 pairs of shRNAs targeting AR-V7 or non-specific sequences (control shRNAs) were obtained from GeneChem . Additionally, 22Rv1 cells were seeded in 6-well plates for 24 h, and replaced with fresh medium containing 5 μg/ml polybrene . Lentiviruses were then added to the cells at a multiplicity of infection of 10. After incubation for 48 h, puromycin selection was performed to eliminate the unsuccessfully transfected cells at a concentration of 2 μg/ml. The sequences of AR-V7 shRNAs are listed below : AR-V7 shRNA-1: CcggCCGACTTTCCCTCTT ACATTTCAAGAGAATGTAAGAGGGAAAGTCGGTTTTTTg; AR-V7 shRNA-2: CcggGCCAGACTCAAATATTGTATTCAAGAGATACAATATTTGAGTCTGGCTTTTTTg.The following plasmids were constructed and purchased from GeneChem: plasmids (CMV-MCS-3FLAG-SV40-neomycin) containing the full length of human GRP78 CDS (gene ID: 3309) and its truncated mutants or control plasmids; the plasmids (CMV-MCS-HA-SV40-neomycin) containing the full length of human AR-V7 CDS (gene ID: 367) and its mutant form that lacks ΔCE3 on its C-terminal (AR-V7ΔCE3) or control plasmids; and the plasmids (CMV-myc-MCS) containing human SIAH2 CDS (gene ID: 6478). Lipofectamine 3000 (Invitrogen) reagent, RPMI opti-MEM (Gibco), and the plasmids were prepared in the following ratio: P3000 (2 μl): plasmids (1 μg): lipofectamine 3000 (2 μl). After incubation for 15 min, the mixtures were added to the cells seeded on plates or dishes and remained there for 48 h for further analysis, and fresh medium was replaced appropriately. The final concentration of the plasmids was 0.75 μg/ml.Immunofluorescence analysis was performed as previously described A PCR assay was used to determine the mRNA levels of AR-V7 in 22Rv1 cells exposed to Rut for 12 h, and this assay was performed as previously described This assay was performed according to the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit . Briefly, 22Rv1 cells were digested and centrifuged for 5 min at 800 ×g at 4°C, and then washed with cold PBS twice. The premixed mixtures of protease inhibitor cocktails, buffer A and buffer B (working solution 1) were then added to the collected cells and placed on ice for 30 min. After centrifugation for 10 min at 3000 ×g at 4°C, the supernatant was collected and considered as cytoplasmic proteins. The sediment was washed with working solution 1 twice at 800 ×g at 4°C, After discarding the supernatant, the premixed mixtures of protease inhibitor cocktails and buffer C (working solution 2) were added to the pellet, and followed by ultrasonic treatment with a Vibra-Cell Ultrasonic Liquid Processor  for 10 s and placed on ice for 30 min. After centrifugation at 14,000 ×g for 30 min at 4 °C, the supernatant was collected and considered as nuclear proteins. The extracted nuclear and cytoplasmic proteins were used in the following western blot and co-IP assays.This assay was performed as described before This assay was performed as described before Cell proliferation assays, including those for cell viability, colony formation, EDU staining and cell cycle, were performed as previously reported PC cells were seeded in 60 mm dishes and treated with indicated doses of Rut for 48 h. Cells were then digested and washed with PBS. For clonogenic assays, PC cells were resuspended and re-seeded on a 6-well plate and cultured for 2 weeks. Cells were then fixed with 4% paraformaldehyde for 10 min, and stained with 1% crystal violet solution for 5 min. Colonies > 60 μm were counted from three independent experiments. For cell cycle assay, PC cells were washed with PBS twice and resuspended with 500 μl PBS and 2 ml 70% ethanol at 4 °C overnight. Cells were then washed with PBS and incubated with the reaction mixtures containing PI , RNase A (100 μg/ml), and 0.2% Triton X-100 for 30 min at 4 °C. Cell cycle distributions of each group were ultimately analyzed with flow cytometry from three independent experiments.6 cells/100μl PBS/mice. After inoculation for 1 week, the mice were randomly divided into 9 groups (10 mice/group); 3 groups of them were treated with Rut at 20mg/kg/2d (i.p.) or 40mg/kg/2d (i.p.) or with vehicle for 15 days. Another 6 groups were used to determine the synergistic effects of Rut and Enza/Bica, and these mice treated with vehicle, Rut 20mg/kg/2d (i.p.), Enza 25mg/kg/2d (p.o.), Bica 25mg/kg/2d (p.o.), Rut + Enza, or Rut + Bica for 15 days. The nude mice sacrifice after 22 days of inoculation, the size and weight of tumors and body weight of mice were measured and calculated as reported previously All animal experiments were approved by institutional animal care and use committees. The 22Rv1 xenograft models were established as previously described t-tests or one-way ANOVA is used where appropriate. Statistical analysis was performed with GraphPad Prism5.0 software and SPSS 16.0. A p value of < 0.05 was considered statistically significant.Data are presented as mean ± SD from three independent experiments with multiple determinations for each experiment where applicable. To determine statistical probabilities, unpaired Student's Evodia rutaecarpaWe first determined the expression of AR-FL and AR-V7 in various human PC cell lines and a prostatic stromal myofibroblast cell line WPMY-1. Our western blot results showed that AR-FL and AR-V7 were expressed in 22Rv1, LNCaP, and C4-2, but not in WPMY-1, PC3, and DU145 cells. Compared to LNCaP and C4-2 cell lines, the 22Rv1 cell line had the highest level of AR-V7  To explore the underlying molecular mechanism of Rut-induced AR-V7 degradation, Co-IP combined with biomass spectrum assay was performed to identify the AR-V7 interacting proteins. The purified proteins from Co-IP using anti-AR-V7 antibodies were separated by SDS-PAGE, followed by silver staining Figure A. BiomasWe wondered whether this selective interaction was mediated by the specific C-terminal (CE3) of AR-V7, a deletion mutant of CE3 (HA-AR-V7 ΔCE3) and a wide-type HA-AR-V7 (HA-AR-V7 WT) plasmid were next engineered and co-transfected with FLAG-GRP78 into HEK293T cells. However, AR-V7 Δ CE3 more strongly interacted with GRP78 than HA-AR-V7 WT , AR-V7-positive , and AR-V7-negative (PC3 and DU145) cell lines following Rut treatment. Rut dose-dependently decreased the viability of AR-V7-positive cells, especially in 22Rv1 cells, which have the highest expression level of AR-V7. Meanwhile, normal prostate stromal (WPMY-1) and AR-V7-negative (PC3 and DU145) cell lines were not sensitive to Rut treatment at the concentration of 0~20 μM . To address this question, a cell viability assay was first performed and the combination index (CI) values were calculated in 22Rv1 cells exposed to Rut and Enza or Bica. The results showed that Rut synergistically enhanced the sensitivity of CRPC cells to Enza and Bica  Figure E-F. The in vivo, the 22Rv1 xenograft models were established and randomly separated as six groups, and treated with Rut, Enza, Bica alone or their combination. The results showed that the reduction of tumor sizes and tumor weights were more remarkable in combination groups than in the single-treatment groups  treatment . Rut, an alkaloid extracted from Evodia rutaecarpa, was proposed to exhibit bioactivity in cardiac hypertrophy prevention, adipogenesis/lipogenesis inhibition and atherosclerosis suppression Natural products are attractive sources for drug discovery Using Co-IP assays and biological mass spectrometry, various critical protein interacting partners of AR-V7 were identified in the current study. Because of the importance of molecular chaperones in the degradation of AR-FL, this study focused on the chaperones that may interact with AR-V7 and similarly influence its degradation. We confirmed that GRP78 strongly interacts with AR-V7, but weakly interacts with AR-FL. This observation was consistent with Rut down-regulating AR-V7, but not AR-FL in a certain range of doses. By using a specific AR-V7 C-terminal deletion mutant (AR△CE3), this study revealed that the CE3 was not required for binding AR-V7 to GRP78, suggesting that the difference of steric configuration between AR-V7 and AR-FL affected their binding capacities to GRP78. Interestingly, Rut increased the interaction between AR-V7 and GRP78 in CRPC cells. GRP78 is an endoplasmic reticulum (ER) chaperone that masters the unfolded protein response (UPR) which is just recently identified as an effective biomarker for treating hepatocellular cancer Chaperones are able to recruit E3 ligases to mediate the ubiquitination of their substrates in vitro and in vivo. Interestingly, our cell viability assay, clonogenic assay, and EDU staining assay showed that Rut suppressed the proliferation of PC cells in a different manner. These effects were more visible in AR-V7-overexpressed 22Rv1 cells, compared to AR-FL-expressed or AR-V7-deficient cells. These findings were consistent with the previous finding that Rut selectively down-regulates AR-V7. Further explorations showed that Rut arrested the G0/G1 phase of the cell cycle via the upregulation of the expression of inhibitors of the G0/G1 phase to S phase, including p15, p21, and p27, promoted by the increased formation of GRP78/SIAH2/AR-V7 complexes in the nucleus. Additionally, the knockdown of AR-V7 or overexpression of GRP78 increased the expression of p15, p21, and p27, while the knockdown of GRP78 decreased their expression. The restoration of AR-V7 rescued the Rut-induced cell growth suppression in vitro. Rut, exhibiting low cytotoxicity, also dramatically suppressed the tumor growth of CRPC models in vivo. Moreover, Rut could enhance the sensitivity of CRPC models to antiandrogen agents, including Enza and Bica, indicating that the use of Rut in CRPC may possess great translational value.By confirming that Rut enhanced formation of the GRP78-AR-V7-SIAH2 degraded complex and thereby promoted the degradation of AR-V7, we further explored its anti-CRPC effects in vitro and in vivo. Mechanically, this effect relies on the Rut-induced formation of the GRP78-AR-V7 protein complex, which further recruits the E3 ligase SIAH2 to directly promote the ubiquitination of AR-V7 (Figure In summary, this study has provided a novel anti-CRPC strategy by targeting AR-V7 with Rut Supplementary figures.Click here for additional data file.


These results imply that AR differs significantly among backflush needles and among companies depending on the shaft cross-sectional area.The present study aimed to identify the factors regulating aspiration rate (AR) of backflush needles and to compare those factors across various backflush needles from different manufacturers. The 27-gauge (27G), 25-gauge, and 23-gauge backflush needles from four different manufacturers, Alcon, MedOne, VitreQ, and DORC, were used for this experiment. AR was measured at four different aspiration vacuum levels: 100, 200, 400, and 650 mmHg. AR was significantly increased as the aspiration vacuum level increased when both aspirating balanced salt solution (BSS) and ethylene glycol; however, 27G products from VitreQ and MedOne were unable to aspirate ethylene glycol at the low aspiration vacuum level of 100 mmHg. When aspirating BSS at the high aspiration vacuum level of 650 mmHg, a smaller gauge number generally resulted in a significantly higher AR. AR, inner diameter, and cross-sectional area in Alcon products were significantly larger in any gauge, and the shaft length of Alcon products was significantly shorter than those of other manufacturers’ products in any gauge. Cross-sectional area negatively correlated with the degree of shaft deflection ( As a result, small self-sealing sutureless incisions, reduced rates of perioperative complications, and early social rehabilitation after surgery have been achieved4 and by developing a bi-blade vitreous cutter6. Although the debate on shaft stiffness is still ongoing, an increasing number of reports have found that these instruments pose no problem in surgical procedures11. There were concerns about inadequate light intensity with the light pipe, although the light source was changed from halogen to xenon and/or mercury vapor and chandelier lighting has also become commonplace13, thereby securing sufficient light intensity. There are virtually no problems now regarding brightness of the surgical field during surgery. Thus, many recent reports have found that 27G vitrectomy is sufficiently practical and useful in the treatment of various vitreoretinal diseases, which was initially regarded as difficult11.MIVS requires small gauge surgical instruments; however, the quality of the instruments should not be compromised in smaller gauges. For example, the vitreous cutter used in 27-gauge (27G) vitrectomy was expected to present problems like reduced surgical efficiency and shaft stiffness. However, the vitrectomy efficiency improved dramatically by changing the drive system of the vitrectomy machineHowever, regarding other surgery-related instruments, including the backflush needle, operation with a larger gauge is still considered advantageous. The backflush needle is a tool that performs aspiration of subretinal fluid and drainage out of the vitreous cavity during vitrectomy to restore retinal detachment and macular hole. Therefore, reducing the gauge size of these needles would reduce the fluid volume aspirated per time unit, which would decrease the surgical efficiency. In fact, when performing 27G vitrectomy, the aspiration rate (AR) of both passive and active suction by a 27G backflush needle was significantly inferior to the AR of a larger gauge needle. However, understanding the advantages and limitations of the different backflush needles and selecting the optimal one is complicated for surgeons because there have been no reports comparing AR across various types of backflush needles. To select the best instrument for a vitrectomy procedure, it is essential for the surgeon to have information about the unique properties of each product.This study aimed to compare the performance of backflush needles across various gauges of backflush needles from different manufacturers and to identify the factors that regulate AR of the active suction.First, we compared the AR for each aspiration vacuum level of each gauge from each company. As a result, when aspirating balanced salt solution (BSS), AR increased significantly as the aspiration vacuum level increased in each gauge from each company Fig. . SimilarWe compared AR between product gauges of each company. Except for the results of the comparison between the 27G and 25-gauge (25G) products from Company B, smaller gauge number resulted in significantly higher AR Fig. . Next, wTable r2 = 0.75, p = 0.0002; Fig. r2 = 0.21, p = 0.042; Fig. AR positively correlated with cross-sectional area , Company B, Backflush FlexTip , Company C, Brush Backflush Instrument , and Company D, Backflush instrument .The following parameters were measured for analysis: external diameter, internal diameter, full length, cross-sectional area, and shaft deflection. To measure the external diameter, the internal diameter, and the full length of the shaft, we first disassembled the shafts of each product. First, the length of disassembled shafts was measured using a digital caliper  with an accuracy of 0.1 mm, and this was defined as “full length of the shaft.” Next, the disassembled shafts were positioned vertically under a smartphone camera equipped with a microscope , which took images at 400 × magnification. Both the external and internal diameters were calculated by using an image analysis software . Afterwards, the external diameter measurement of each shaft was confirmed using the digital caliper . The squared internal diameter was calculated and used as the value to represent the cross-sectional area. We prepared three backflush needles for each gauge from all four companies, measured all parameters, and used it for analysis.14. The degree of deflection is proportional to the added force and the cube of the distance from the base to the point of the force. On the other hand, the degree of deflection is inversely proportional to Young's modulus and the moment of inertia of area. In this study, we kept the force and the distance from the base to the point of the force constant as described above. Thus, our results indicate that the greater the deflection, the more likely the material is to bend because both Young's modulus and the moment of inertia of area are numerical values related to the material and shape of the shaft material. The shaft displacement was measured using a digital caliper . We prepared five backflush needles of all gauges from all four companies, measured the shaft deflection, and used it for analysis.The shaft deflection, which can be a measure of the overall bendability of the metals that make up the shaft and is one of the contributing factors to the shaft stiffness, of each product was evaluated by measuring the tip displacement under a known force (0.5 N) at 20 mm from the shaft base as described previously19. Therefore, we used ethylene glycol, the viscosity of which was reported as 31.5 cps at 10° C, as an alternative material of subretinal viscous liquid vitreous for this study. Briefly, the 27G, 25G, and 23G products from each manufacturer were prepared. The beaker was put on an electronic scale, and each product was hung and set so as not to give any weight report on the electronic scale . BSS or ethylene glycol in a beaker was aspirated for 30 s by each product using the Constellation Vision System . BSS and ethylene glycol were aspirated at normal room temperature and 10° C, respectively. The amount of reduced liquid was measured using the electronic scale , and twice that amount was used as AR (g/min). We prepared three backflush needles of all gauges from all four companies, measured ARs, and used it for analysis.AR was measured at aspiration vacuum levels of 100 mmHg, 200 mmHg, 400 mmHg, and 650 mmHg on all products. Usually, during surgery, the backflush needle is used for the aspiration of BSS in vitreous cavity and/or subretinal viscous liquid vitreous. The viscosity of BSS is about 1 centipoise (cps). On the other hand, the mean maximum viscosity of liquid vitreous in the subretinal space of retinal detachment was reported as 29.3 cps at 37° CFor the 27G product, by enlarging and photographing the joint with a smartphone camera equipped with a microscope , the difference in the method of jointing the silicon chip and the shaft at each company was visually confirmed.Finally, we investigated the composition of the metal used for the shaft, the presence or absence of processing and/or coating on the outer and inner surfaces of the shaft.p < 0.05.Kruskal–Wallis H-test, followed by a post hoc analysis using Mann–Whitney U test with Bonferroni correction was used to compare AR and all parameters of the shaft. Spearman correlation analysis was then used to identify a significant correlation between shaft deflection and shaft cross-sectional area, and AR and cross-sectional area. Statistical analyses were performed using SPSS software . Statistical significance was inferred for


Therapies for lethal castration-resistant prostate cancer (CRPC) are an unmet medical need. One mechanism underlying CRPC and resistance to hormonal therapies is the expression of constitutively active splice variant(s) of androgen receptor (AR-Vs) that lack its C-terminus ligand-binding domain. Transcriptional activities of AR-Vs and full-length AR reside in its N-terminal domain (NTD). Ralaniten is the only drug proven to bind AR NTD, and it showed promise of efficacy in Phase 1 trials. The peptidyl-prolyl isomerase Pin1 is frequently overexpressed in prostate cancer. Here we show that Pin1 interacted with AR NTD. The inhibition of Pin1 expression or its activity selectively reduced the transcriptional activities of full-length AR and AR-V7. Combination of Pin1 inhibitor with ralaniten promoted cell cycle arrest and had improved antitumor activity against CRPC xenografts in vivo compared to individual monotherapies. These findings support the rationale for therapy that combines a Pin1 inhibitor with ralaniten for treating CRPC. Leung et al. find that the peptidyl-prolyl isomerase Pin1 targets the intrinsically disordered N-terminal domain of the androgen receptor (AR). They show that combining Pin1 inhibition with ralaniten compounds that bind to the AR N-terminal domain has enhanced antitumor activity on castration-resistant prostate cancer in xenografts, suggesting therapeutic potential. Most CRPC continues to depend on AR transcriptional activity to maintain tumor growth and many involve the expression of constitutively active AR splice variants (AR-Vs) that lack the LBD. The most clinically relevant AR-V is AR-V7, which is detected in one-third of CRPC patients, associated with bone metastases, and resistance to abiraterone and enzalutamide3. Common to both full-length AR and AR-Vs is the N-terminal domain (NTD) that contains all of the AR’s transcriptional activity5. Thus, identifying drugs that disrupt the AR NTD should block the transcriptional activities of both full-length AR and AR-Vs. One hurdle in finding these drugs is the intrinsic disorder of this domain.Globally, there were 1.3 million new cases of prostate cancer in 2018. Approximately 30% of these patients will develop recurrence after primary treatment and require hormonal therapies. Currently, all FDA-approved hormonal therapies such as abiraterone acetate and enzalutamide target the C-terminal ligand-binding domain (LBD) of full-length androgen receptor (AR). About 20–40% of castration-resistant prostate cancer (CRPC) patients have de novo resistance to these agents, and virtually all patients will acquire resistance to AR-LBD-targeted therapy within 2–3 years6. Ralaniten binds to Tau-5 in AR NTD7 and inhibits the transcriptional activities of full-length AR and AR-Vs9. Proof-of-concept validation for the AR NTD as a drug target and the ralaniten scaffold for developing drugs to treat CRPC was provided by the first-in-human clinical trial (NCT02606123). A second-generation ralaniten analog (EPI-7386) is currently in Phase I clinical trial (NCT04421222).The first and currently only drug to be tested in clinical trials that directly binds to an intrinsically disordered protein is ralanitencis/trans isomerase Pin1. This enzyme is commonly overexpressed in prostate cancer, and its expression in patient biopsies correlates with recurrence12. Pin1 regulates the conformation of proteins by catalyzing the isomerization of proline bonds at specific phosphorylated motifs 14. Numerous putative binding sites for Pin1 are found within the intrinsically disordered AR NTD. Pin1 is composed of a WW domain that recognizes phosphorylated substrates joined by a flexible linker to a peptidyl-prolyl isomerase (PPIase) domain to carry out its catalytic function16. In the absence of Pin1, the rate of isomerization between the cis- and trans proline is inherently slow since bond rotation is impeded by the adjacent phosphoryl group17. Proline-directed Ser/Thr kinases (CDKs and MAPKs) and phosphatases (PP2A) that recognize and modify Pin1 sites are specific to the trans-proline conformation20. Thus, Pin1 is the only human isomerase that recognizes a proline-directed phosphorylation site, and Pin1-mediated cis/trans isomerization is proposed as a conformational switch for signal transduction21.To identify approaches that would disrupt the transcriptional activity of AR NTD, we considered the peptidyl-prolyl There is interest in Pin1 as a potential prognostic marker for prostate cancer, but there are no reports on whether Pin1 expression in prostate cancer influences AR signaling. Here we determine whether targeting Pin1 would be beneficial for treating CRPC. We investigated the mechanism and effect of genetic and pharmacological inhibition of Pin1 activity in CRPC and assessed the feasibility of combining a Pin1 inhibitor with ralaniten compounds using in vitro and in vivo models of CRPC.12.The AR NTD contains several putative binding sites for Pin1 Fig. . The lev22, there was no association between levels of PIN1 and AR  and polo-like kinase 1 (PLK1), which inactivates the isomerase domain and enhances protein stability, respectivelyThe impact of the loss of Pin1 expression on AR transcriptional activity was evaluated in Pin1 knockdown experiments using two Pin1-targeting siRNAs that target separate regions of the human PIN1 transcript Fig. . Transcrtrans retinoic acid (ATRA). Consistent with previous reports26, both juglone and ATRA inhibited the isomerase activity of Pin1 in a dose-dependent manner  did not inhibit the isomerase activity of Pin1  for PSA and 6.45 µM  for PB, whereas ATRA was more potent and had an IC50 of 4.60 µM  for PSA and 3.57 µM  for PB. The 13cisRA conformer was less potent than ATRA  within the AR hinge region, which harbors a bipartite nuclear localization signal required for AR nuclear translocationent Fig. . In the  29 Fig. . Pin1 in 29 Fig. . These r30. The loss of AR-LBD shifts the location of transcriptional activity from Tau-1 with androgen, to Tau-5 in the absence of LBD30. To determine whether Pin1 impacts transactivation of AR NTD, we employed a transactivation assay that consists of a construct encoding the human AR1–558 of the NTD fused to a Gal4DBD  that lacks AR-LBD. Forskolin- and interleukin-6 (IL-6)-induced transactivation of AR NTD32 were both blocked by treatment with an AR NTD inhibitor, ralaniten , as previously reported34 , but their fold-inductions were overall reduced by 33–48%  were involved in transactivation, we assessed the activity of Gal4-AR48% Fig. . Juglone31. This overlaps with the region required for Pin1 interaction . Juglone did not prevent phosphorylation of MAPK (isoforms p44 and p42) or STAT3 (Tyr705 and Ser727)  Fig. . These d35.AR drives the proliferation of most prostate cancer cells. To determine whether blocking AR transcriptional activity by Pin1 inhibitors leads to decreased proliferation, BrdU incorporation assays were utilized in a battery of cell lines. Androgen-induced proliferation of LNCaP cells depends on functional full-length AR. LN95 cells express full-length AR and AR-Vs, including AR-V7, but their proliferation is driven by AR-Vs and is not increased by androgen and thus are resistant to AR-LBD inhibitors. PC-3 and DU145 cells are devoid of functional AR and are androgen-independent. The effects of Pin1 inhibitors were compared to enzalutamide (AR-LBD inhibitor) and ralaniten (AR-NTD inhibitor) as controls. Pin1 inhibitors attenuated androgen-induced proliferation of LNCaP cells similar to validated AR antagonists Fig. . Pin1 in3-luciferase reporter that is driven by three tandem repeats of an AR V7-specific promoter element from the UBE2C gene36 was tested. As expected, V7BS3 luciferase was highly induced by ectopic expression of AR-V7, which could not be blocked by AR-LBD inhibitor enzalutamide  on dose–response curves for ralaniten and EPI-7170 for inhibiting androgen-induced PSA(6.1 kb)-luciferase activity was determined. The IC50 of ralaniten for PSA(6.1 kb) luciferase was 7.91 ± 1.15 μM (R2 = 0.94), and the curve shifted to the left with ATRA and an improved IC50 of 4.45 ± 1.28 µM (R2 = 0.83)  as monotherapy and an IC50 of 0.42 ± 1.16 μM (R2 = 0.93) with ATRA  Fig. . A similTRA Fig. . For bot 38 Fig. . Excludi39. AR-V7 is reported to regulate the expression of a distinct subset of cell cycle-related genes40, including UBE2C. To determine whether ATRA alters the expression of cell cycle proteins, we performed western blot analyses from LN95 cells treated with ATRA (0–10 µM) or the inactive 13cisRA conformer. ATRA binds directly to the catalytic pocket of Pin1, which inhibits the PPIase domain and induces degradation of Pin1 protein25. Consistent with this mechanism, a dose-dependent decrease in Pin1 levels was measured with ATRA , but not cyclin B1 (M cyclin), and selectively increased the levels of cyclin-dependent kinase (CDK) inhibitor p27Kip1 but not p21Cip1  activity42. ATRA significantly increased the percentage of SA β-gal-positive cells for LNCaP and LN95 cells, but not for PC-3 and DU145 cells  and phospho-Rb (serine residues 807 and 811) were also decreased by both monotherapies and combination therapy ). Moreover, we measured levels of serum PSA, a widely used and well-characterized clinical biomarker for CRPC progression and patient responses to therapy. PSA expression in sera was reduced on average by 27% in animals treated with ATRA compared to placebo, albeit the responses varied between animals and did not reach statistical significance  pellet and then treated daily with either EPI-7170 (30 mg/kg) or vehicle (CMC). After 21 days of treatment, tumor growth was significantly reduced in the animals treated with ATRA, EPI-7170, and combination, relative to the vehicle/placebo-control Fig. . The comXenograft tumors harvested at the end of the study were variable in appearance, but several of the tumors from the combination group were remarkably reduced in size and white in appearance Fig. . Western48. This has led to an interest in finding therapies that target the AR NTD. Unfortunately, this domain is intrinsically disordered. Only one drug, ralaniten, has been tested clinically that directly binds to any intrinsically disordered target, thereby emphasizing the hurdles in developing drugs to these difficult targets. Ralaniten showed clinical signs of efficacy but had poor pharmacokinetics that led to excessive pill burden49. A second-generation ralaniten analog with improved stability, EPI-7386, is currently in Phase I evaluation. Ralaniten binds residues 354– 4487 predominantly within Tau-5 (residues 360–485). In response to androgen, transcriptional activity of full-length AR resides in Tau-1 (residues 101–370). The loss of AR-LBD with AR-Vs shifts the site of transcriptional activity from Tau-1 with androgen to Tau-5 in the absence of androgen30. Multiple hits against the AR NTD that encompass both Tau-1 and Tau-5 are proposed to yield better therapeutic responses for CRPC that express both full-length AR and AR-Vs6.Increasing evidence suggests that AR-Vs underlie resistance of CRPC to second-generation AR-LBD inhibitorscis/trans-proline isomerase is frequently elevated in prostate cancer and associated with disease recurrence12. We found that Pin1 interacted with AR NTD predominantly within Tau-1 (residues 234–391) and that genetic and pharmacological inhibition of Pin1 selectively disrupted several key aspects of AR signaling. These included inhibition of: (1) transcriptional activities of full-length AR and AR-V7; (2) transactivation of AR NTD; (3) protein–protein interactions; and (4) AR- and AR-V-dependent proliferation of prostate cancer cells. A combination of pharmacological Pin1 inhibitor with ralaniten compounds was synergistic for blocking cell cycle progression and promoted senescence of enzalutamide-resistant LN95 cells whose proliferation is driven by AR-Vs. Consistent with in vitro data, this combination therapy had superior antitumor effects in vivo compared to monotherapies.Pin1 motifs are predicted across the entire AR NTD and could potentially impact transactivation of AR NTD. This 1 to S phase of the cell cycle and increased cellular senescence50.Here we provide evidence that genetic and pharmacological inhibition of Pin1 selectively impeded AR transcriptional activity and AR-driven proliferation. Support for selectivity was drawn from clinical data supporting the association between PIN1 levels and the levels of AR and its target genes KLK3/PSA, TMPRSS2, and NKX3.1. Non-AR-driven reporters were not blocked by Pin1 inhibitors or knockdown contrary to reporters driven by full-length AR and AR-V7. Gene expression analyses showed a sensitivity of AR-regulated genes to inhibiting Pin1 activity. On-target activity against Pin1 was measured in harvested CRPC xenografts. Similarly, inhibiting Pin1 induced known biological responses that occur with the loss of AR function that included attenuating progression from G1 to S phase of the cell cycle52. Consistent with these reports, both ralaniten compounds and ATRA decreased the S phase population and increased G1, whereas enzalutamide had no effect on AR-V-driven proliferation. G1 arrest was accompanied by increased levels of p27Kip1 and the loss of cyclins D1 and A2 by ATRA which were expected in response to blocking AR function52. Similarly, ATRA and ralaniten both increased cellular senescence with concomitant losses of Skp2 and pRb which are known responses to a loss of AR function51. A combination of ATRA with the more potent ralaniten analog EPI-7170 was more effective than the individual monotherapies in inhibiting the in vivo growth of LN95-D3 xenograft tumors, reducing serum PSA, and was not toxic to the animals. This xenograft model expresses PSA in castrated animals and demonstrated robust growth in vivo with a tumor volume doubling time of approximately 3 days; therefore, it is a representative model for aggressive, AR-V7-positive CRPC. ATRA (tretinoin) is the only clinically used drug known to inhibit Pin1 and is FDA-approved to treat acute promyelocytic leukemia. These studies overall support that repurposing ATRA as a combination therapy with a ralaniten analog could provide a significant near-term clinical impact for the treatment of CRPC.Functional AR is required for the progression of cells from Gl-glutamine and 1 mM of sodium pyruvate; VCaP cells in DMEM  with 10% FBS, 2 mM l-glutamine, and 1 mM sodium pyruvate. All cell lines were maintained at 37 °C in a humidified incubator containing 5% CO2 for not more than ten passages. Cell lines were routinely tested with the Venor GeM Mycoplasma Detection Kit (Sigma-Aldrich) to ensure that they were mycoplasma-free.Cell lines were obtained from the following sources: LNCaP cells from Dr. Leland Chung ; LNCaP95 (LN95) cells from Dr. Stephen Plymate ; VCaP cells and PC-3 cells from the American Type Culture Collection ; and DU145 cells from Dr. Victor Ling . LNCaP, VCaP, PC-3, and DU145 cells were authenticated by short tandem repeat analysis and tested to ensure they were mycoplasma-free by DDC Medical . Cell lines were maintained in the following culture media: LNCaP cells in RPMI-1640 media with 5% fetal bovine serum (FBS); LN95 in RPMI-1640 media with 10% charcoal-stripped serum (CSS); PC-3 and DU145 cells, in DMEM  with 5% and 10% FBS, respectively, and supplemented with 2 mM cis-retinoic acid (13cisRA), and juglone were purchased from Sigma-Aldrich. Ralaniten (EPI-002) was provided by NAEJA  and second-generation analog EPI-7170 was synthesized by Dr. Raymond Andersen (University of British Columbia). Enzalutamide was purchased from OmegaChem  and bicalutamide was a gift from Dr. Marc Zarenda . Synthetic androgen methyltrienolone (R1881) was purchased from Perkin-Elmer , IL-6 from R&D Systems , and forskolin from EMD Millipore . Antibodies against various proteins were from the following sources, mouse monoclonal antibodies: Pin1 (8C10), Cyclin D1 (DCS-6) from Santa Cruz Biotechnology; STAT3 (124H6), Cyclin A2 (BF683), Rb (4H1) from Cell Signaling Technology; GAPDH (6C5) from Invitrogen; β-actin (A5441), polyhistidine (HIS-1) from Sigma-Aldrich; rabbit monoclonal: DAPK1 (#3008), PLK1 (208G4), Cyclin B1 (D5C10), p16 INK4a (D3W8G), p21 Waf1/Cip1 (12D1), p27 Kip1 (D69C12), PSA/KLK3 (D11E1), phospho-Rb  from Cell Signaling Technology; rabbit polyclonal: AR (N-20) from Santa Cruz Biotechnology; phospho-STAT3 , p44/42 MAPK (9102), phospho-p44/42 MAPK (Thr202/Tyr204), Skp2 (D3G5) from Cell Signaling Technology.ATRA, 13-32. All point mutations were made by site-directed mutagenesis using QuikChange II . Mutations were verified by Sanger sequencing from the UBC Nucleic Acid Protein Service Unit. Transfections with siRNA were performed using 10 nM of Pin1-targeting siRNA  or non-targeting control siRNA (4390843) with lipofectamine RNAiMAX reagent diluted in Opti-MEM media (Invitrogen). For reporter assays, transfections were performed in serum-free media with Lipofectin (Invitrogen) or FugeneHD . Luciferase activity was measured for 10 s with the Luciferase Assay System (Promega) and normalized to total protein concentration determined by Bradford assay.The PSA(6.1 kb)-luciferase and probasin-luciferase plasmids and transactivation assays for AR-(1–558)-Gal4DBD, AR-(1–233)-Gal4DBD, AR-(234–391)-Gal4DBD, AR-(392–558)-Gal4DBD have been previously describedThe effect of inhibitors on Pin1 isomerase activity was evaluated using a fluorometric Pin1 Assay Kit , according to the manufacturer’s protocol. In brief, compounds (0–50 µM) were incubated with 500 ng of recombinant human Pin1 protein  in a black 96-well, clear-bottom plate , and the reaction was started by adding the fluorogenic substrate. Fluorescence intensity was measured with excitation wavelength at 490 nm and emission wavelength at 520 nm on an Infinite M1000 plate reader  at 37 °C. Measurements were recorded every 5 min for up to 1 h.33. In brief, cell lysates were precleared with a non-specific rabbit IgG antibody and immunoprecipated with an antibody against a STAT3 (124H6). For the interaction of full-length AR and Pin1, co-immunoprecipitation assays were performed from LNCaP cells treated with androgen  or vehicle for 3 h. Approximately 500 µg of whole-cell lysate was precleared with a non-specific rabbit IgG antibody, incubated with 1 µg of Pin1 (H-123) antibody for 1 h at 4 °C on a rotating rack, and then incubated for an additional hour with 50 µL of µMACS protein A/G magnetic beads . Pin1 was bound to µ columns (Miltenyi Biotec), washed four times with lysis buffer, and then eluted with SDS sample buffer. For western blot analyses, membranes were blocked with 5% non-fat milk in phosphate-buffered saline containing 0.1% Tween-20 (PBS-T) and then incubated overnight at 4 °C with primary antibody (1:200–5000) in PBS-T containing 5% non-fat milk or 2% BSA. After incubation with the primary antibody, the membrane was washed three times with TBS-T, incubated with a horseradish peroxidase-conjugated antibody secondary antibody  for 1–2 h at room temperature, and washed three times with PBS-T and two more times with PBS before chemiluminescence was detected with ECL Prime Reagent . Western blot images were captured using the ChemiDoc MP Imaging System  and quantified using ImageJ software.Immunoprecipitation of STAT3 complexes has been previously describedLNCaP cells were transfected with 1 μg of Pin1 plasmid and 8–11 μg of an expression plasmid encoding his-tagged AR-(1–558), AR-(1–233), AR-(234–391), or AR-(392–558). The following day, cells were treated with compounds and stimulated with IL-6 (50 ng/mL) for 6 h. When harvesting, cell pellets were washed in PBS, flash-frozen in liquid nitrogen, and stored at −80 °C. For protein extraction, thawed cell pellets were resuspended in 1 mL of lysis buffer . The lysates were passed several times through a 28 gauge needle and then cleared by centrifugation. Lysates were incubated with 200 μL of Ni-NTA agarose beads for 1 h at 4 °C, washed twice with HEPES/NaCl buffer (pH 8.0) containing 60 mM imidazole, and eluted with HEPES/NaCl buffer (pH 8.0) containing 300 mM imidazole. The eluted samples were resolved on a 12.5% SDS-PAGE along with input samples  and analyzed by probing for Pin1 (8C10) and polyhistidine (HIS-1).9. Approximately 2.8 × 105 LNCaP cells were grown on coverslips in six-well plates and transiently transfected with 2 μg of an expression vector encoding a YFP-AR fusion protein. On the following day, cells were pre-treated with the indicated compounds for 1 h and stimulated with R1881 (1 nM) or a vehicle containing ethanol for 3 h. After incubating with compounds, cells were fixed with 4% paraformaldehyde, washed twice with PBS, and then counterstained with DAPI. Slides were examined on an Axiovert 200 Fluorescence Microscope . The ratio of the YFP signal in the nucleus relative to the cytoplasm was quantified with ImageJ software. For each image, background fluorescence was measured and subtracted from experimental values. A total of at least 50 cells from nine images containing six or more cells per field were quantified to determine the nuclear to cytoplasmic YFP ratio for each treatment group.AR translocation assays were performed by a modified protocol from Myung et al.BrdU incorporation was determined with the colorimetric BrdU ELISA kit (Roche Diagnostics), according to the manufacturer’s protocol. In brief, cells were seeded in 96-well plates  in media with reduced serum (0.5–1%). On the following day, cells were pre-treated with test compounds for 1 h and then incubated with 0.1 nM of R1881. After the indicated incubation times, cells were labeled with 10 μM of BrdU for 2 h and then fixed for the assay. BrdU incorporation was quantified with a VersaMax ELISA microplate reader  by measuring absorbance at 370 nm with a reference wavelength of 492 nm.3-luciferase plasmid, which contains three tandem repeats of an AR-V7-specific promoter element of the UBE2C gene, was a gift from Dr. Stephen Plymate (University of Washington) and has been described36. LNCaP cells seeded in 24-well plates were co-transfected with the V7BS3-luciferase reporter (0.25 μg/well), an expression vector encoding AR-V7 (0.2 μg/well), and a filler plasmid . On the following day, the cells were treated with the indicated compounds for 24 h. Luciferase activity was measured for 10 s using the Luciferase Assay System (Promega) and the data were normalized to total protein concentration determined by a Bradford assay. Western blot analyses were performed to verify the expression of AR-V7.The V7BS5 of fixed cells were labeled with fluorescein (FITC)-conjugated anti-BrdU antibody  and the DNA content was stained with 7-AAD (Sigma). The data were collected with a BD FACSCalibur flow cytometer (BD Biosciences). FITC and 7-AAD fluorescence were detected in the FL1 and FL3 channels, respectively, using CellQuest Pro and analyzed by FlowJo V10 software . Pre-gating was performed on all samples by plotting FL3-W × FL3-H to exclude doublets from the analysis.Cells were grown in 10 cm dishes in 10% CSS for 3 days before the media was changed to 1.5% CSS and ATRA and/or EPI-002 were added at the indicated concentrations. After treatments, the cells were labeled with BrdU (10 μM) for 2 h and then trypsinized, washed, and fixed in 70% EtOH/PBS at −20 °C. Approximately 2.5–5.0 × 1042. Cells were seeded in four-well chamber slides at a density of 8000-10,000 cells. On the following day, the media was changed to low serum media (1.5%) and cells were treated with EPI-002 or ATRA. Three days later, the media was removed and cells were fixed with 2% formaldehyde/0.2% glutaraldehyde for 10 min at room temperature, washed with PBS, and then incubated with freshly prepared β-Gal staining solution  titrated to pH 6.0. Staining was optimal after incubating with the β-Gal solution at 37 °C for 16–18 h. After staining, the slides were washed twice with PBS, once with absolute methanol, and then mounted with 50% glycerol/PBS. The percentage of β-galactosidase-positive cells were counted with ImageJ using the Cell Counter plugin. A total of nine images containing 100–300 cells were scored to determine the percentages of positive cells.β-galactosidase activity at suboptimal pH (6.0) was used as a marker to detect cellular senescence2. Six–eight-week-old male (NOD-scid IL2Rgammanull) mice were maintained in the Animal Care Facility at the British Columbia Cancer Research Centre. Mice were castrated 2 weeks before they were subcutaneously inoculated in the right flank with two million LN95-D3 cells in a 1:1 volume of matrigel . Tumor volume was measured twice a week by digital calipers and calculated by the formula: length × width × height × 0.5236. ATRA  or a matching placebo pellet purchased from Innovative Research of America  was subcutaneously implanted in the left side of the neck of mice 3 weeks after inoculating the cells or when tumor volume was about 33 mm3. Starting 3 days after the implants, animals were treated daily by oral gavage with 30 mg/kg body weight of EPI-7170 or vehicle (3% DMSO/1.5% Tween-80/1% CMC). Tumors were excised 1 day after the last dose and prepared for western blot analysis and immunohistochemistry.All experiments involving animals conform to the relevant regulatory and ethical standards and were approved by the University of British Columbia Animal Care Committee (A18-0077). Metacam  was administered subcutaneously prior to any surgery. Animals were anesthetized with isoflurane and euthanized by COSections were cut from formalin-fixed, paraffin-embedded tumor tissue at a thickness of 5 µm, deparaffinized in xylene, and then rehydrated in alcohols and distilled water. Endogenous peroxidase was blocked by incubating with a 3% hydrogen peroxide solution for 5 min. Sections were incubated overnight at 4 °C with antibodies recognizing the Ki-67 antigen  or antibodies specific for the AR N-terminus  at a dilution of 1:50. PSA was detected using a monoclonal mouse anti-human PSA antibody concentrate . A negative control without the primary antibody was included for each sample on the same slide. Antigen was detected with 3,3-diaminobenzidine (DAB) and hematoxylin was used for counterstaining. Images were captured on an Axio Imager M2 microscope (Zeiss) at ×200 magnification. The percentage of Ki-67-positive cells was determined with ImageJ using the Cell Counter plugin. At least 1000 cells were scored from three images of representative tumors for each treatment group. The intensity of DAB staining for PSA was quantified by ImageJ using color deconvolution and measuring the mean gray value for the DAB image, where optical density = log(max intensity/mean intensity), and a max intensity of 255 for an 8-bit image.Serum levels for total PSA were measured by a PSA ELISA kit  with a lower limit sensitivity of ~1 ng/mL. Whole blood was sampled from the tail vein on days indicated or by cardiac puncture immediately after euthanasia. The serum fraction was separated by centrifugation at 12,000 rpm for 3 min at room temperature and then stored at −20 °C until further analysis. Serum samples were diluted tenfold for the assay and were compared to calibration standards, according to the manufacturer’s protocol. The concentration of total PSA was determined by a VersaMax ELISA microplate reader by measuring absorbance at 450 nm with a reference wavelength of 650 nm.Fa and Fb are the fractional responses for treatment A and B at a given dose38. The CI was determined by the equation Fab represents the fractional response for the combination of A and B. Drug combinations were considered to be synergistic if CI < 1, additive if CI = 0, and antagonistic if CI > 1. Statistic differences were determined by GraphPad Prism 8  by analysis of variance (ANOVA) unless stated otherwise. We used an alpha level of 0.05 for all statistical tests, where *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant. Experiments were repeated with independent biological replicates at least three times.Synergistic interactions between compounds were evaluated according to the Bliss independent model for a predicted additive effect,Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Chronic arsenic exposure is known to be related to the progression of atherosclerosis. However, the pathogenic mechanisms of arsenic-induced atherosclerosis have not been fully elucidated. Because disruption of the blood coagulation/fibrinolytic system is involved in the development of arteriosclerosis, we investigated the effect of arsenite on fibrinolytic activity in human vascular endothelial EA.hy926 cells in the present study. Fibrinolysis depends on the balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) secreted from vascular endothelial cells. We found that arsenite reduced fibrinolytic t-PA activity by inhibiting its synthesis without affecting PAI-1 production. The inhibitory effect of arsenite on t-PA expression was partially recovered by the reactive oxygen species (ROS) scavenger Trolox. The nuclear factor erythroid 2 related factor 2 (NRF2) pathway is known to be activated by arsenite via ROS production. We confirmed that arsenite activated the NRF2 pathway, and arsenite-induced inhibition of fibrinolytic t-PA activity was abrogated in NRF2-knockdown EA.hy926 cells. These results suggest that arsenite inhibits the fibrinolytic activity of t-PA by selectively suppressing its synthesis via activation of the NRF2 pathway in vascular endothelial cells. Arsenic is a ubiquitous metalloid found in the Earth’s crust. Arsenic pollution of groundwater has been reported in several countries across the globe ,2,3. RecIn animal experiments using apolipoprotein E-deficient mice as a model of human atherosclerosis, it has also been shown that exposure to arsenite accelerates the progression of atherosclerosis ,13,14,15The human-derived endothelial cell line EA.hy926 was established by hybridization . Cui et 2) against endothelial EA.hy926 cells by morphological observation and 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assays. After a 24-h incubation, arsenite did not alter the morphology of cell layers , a pentavalent arsenic compound, only slightly decreased the mRNA expression of t-PA (III), but not arsenate (AsV), significantly inhibits the fibrinolytic activity of vascular endothelial cells via the selective suppression of t-PA synthesis and secretion.Fibrinolytic activity depends on the balance between t-PA and PAI-1 . Thus, t of t-PA a; the mR of t-PA b. Theref2), which stimulates the cyclic AMP pathway via activation of adenylate cyclase from endothelial EA.hy926 cells into the conditioned medium, did not change after arsenite treatment  inhibits t-PA synthesis through NRF2 activation in cultured human vascular endothelial EA.hy926 cells without altering PAI-1 synthesis, resulting in decreased fibrinolytic activity , and phase-III transporters (such as multidrug resistance-associated proteins)  and nuclear lysis buffer (20 mM HEPES-KOH (pH 7.9), 400 mM NaCl, 1.5 mM MgCl2, 1 mM DTT), 0.2 mM PMSF, 5% glycerol) containing protease inhibitors . Protein concentration was determined using a detergent compatible (DC) protein assay kit . Protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to an Immobilon-P membrane (Merck KGaA). The membrane was incubated with primary antibodies against NRF2  and Lamin A/C  and horseradish peroxidase (HRP)-coupled anti-rabbit IgG (Thermo Fisher Scientific) and anti-mouse IgG  secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence using Immobilon Western Chemiluminescent HRP substrate (Merck KGaA) and detected with a LuminoGraph I Imaging System . The band intensities were analyzed using ImageJ 1.53 g .Western blotting was performed as described previously . Endothet-test as appropriate. Differences between groups were considered significant at p < 0.05.All statistical analyses were performed in Excel  with the Statcel3 add-in . The data were expressed as the mean ± standard deviation (S.D.). The statistical significance of data was determined using one-way analysis of variance (ANOVA) with the post hoc Bonferroni/Dunn multiple test or Student’s


Scaphoideus titanus were collected by yellow sticky traps inside and outside the vineyard over the period July 10th–September 9th, 2015. They were counted and singly analyzed for the presence of FD phytoplasmas by PCR. Multifactorial correlations among vector population level, prevalence of infected insects inside and outside the vineyards, disease prevalence in cultivated and wild Vitis plants, and location of wild Vitis plants with respect to the vineyard were analyzed. Abundance of S. titanus adults significantly decreased from the end of July onwards, particularly inside the vineyard (average range 22.7 ± 2.5 insects/trap). Percentage of FD-positive S. titanus was significantly higher outside the vineyard (up to 48% on average) compared to inside the vineyard (up to 34% on average), and increased during the season in both compartments.Quantitative estimates of vector populations and their infectivity in the wild and in cultivated compartments of agroecosystems have been carried out to elucidate the role of the wild compartment in the epidemiology of Flavescence dorée (FD). Seven sites were selected for the investigations in the Piedmont Region of Italy. They were characterized by a high variety of agricultural and ecological landscape features, and included a vineyard surrounded by wild vegetation. In order to describe abundance and prevalence of FD-infected vectors in the cultivated and wild compartments of the vineyard agroecosystem, adults of  Candidatus Phytoplasma” mainly based on 16S rRNA gene phylogeny. By definition, FD phytoplasmas (FDp) are those belonging to the 16SrV-C and -D ribosomal subgroups transmitted by the American grapevine leafhopper, Scaphoideus titanus Ball [S. titanus is the main vector of FDp, as it feeds and breeds on Vitis species and can transmit FDp following acquisition on either cultivated grapevine inside the vineyard or from infected, naturalized Vitis rootstock plants outside the vineyard, thus sustaining both secondary (within vineyard) and primary (from outside the vineyard) infections. Abandoned (or untreated) vineyards and wild Vitis rootstock plants in the areas surrounding vineyards are frequently infected and represent a reservoir of both FDp and S. titanus [Vitis spp. do not show symptoms but are susceptible to FDp [S. titanus, which coevolved with them in the Nearctic Region. Besides S. titanus, other leafhopper and planthopper species have been identified as FDp vectors, among these Orientus ishidae [Dictyophara europaea, Allygus spp. [Phlogotettix cyclops [S. titanus is regarded as the main vector associated with all the major epidemics of the disease [Vitis and associated S. titanus, was the major source of infection. To clarify the epidemiology of FD in the area, genetic tracking of phytoplasmas in the vineyard agroecosystems was carried out [Vitis, as well as in S. titanus collected inside and outside the vineyards, largely overlap, further proof of the wild compartment role in the FD epidemiology. The comparative analysis of population level and of proportion of infected S. titanus in the cultivated and wild compartments of the vineyard agroecosystem is almost unexplored and represents essential information for management of FD. The aim of the work is to fill this knowledge gap by conducting systematic investigations in representative sites of the Piedmont Region, where FD is a major problem for viticulture. Our results substantially improve the understanding of the epidemiology and contribute to the design of rational and effective control programs of FD and its vector.Flavescence dorée of grapevine (FD) is a phytoplasma-associated disease present in several European countries. The disease has a major impact on viticulture because inflorescences and berries dry up, shrivel, and cannot be further processed. Other symptoms include downward leaf rolling with yellowing (in white varieties) or reddening (in red varieties), leaf vein necrosis, lack of lignification, presence of black spots on the new canes, and premature leaf fall ; on the nus Ball . The vec titanus ,8. Amerie to FDp  and are  ishidae , Dictyopgus spp. , and Phl cyclops . However disease ; soon afVitis vinifera . The sites were named after the villages closest to them using the following abbreviations: AT, CI, CR, LM, MO, PA, and PC, as previously detailed , and were managed according to guidelines for organic viticulture (based on three applications of pyrethrins in June–July).The vineyard in Asti , AT was à d’Alba , MO, theS. titanus populations were monitored at each site both inside and outside the cultivated vineyards by means of yellow sticky traps (YSTs), 25 × 40 cm (0.1 m2)   during sTotal nucleic acids were extracted from single leafhoppers according to the method of Pelletier , then suSchematic maps were produced with the software QGIS v 3.2.3 ‘Bonn’  Figure .S. titanus through yellow sticky traps hung at fixed places inside or outside each vineyard  with a log link function was used. Fixed covariates were Trap position , and Time period . The interaction terms were Trap position × Time period. To incorporate the dependency among observations of the same vineyard, we used Vineyard as a random intercept.To model the number of Model assumptions were verified by plotting residuals versus fitted values, for each covariate in the model and for each covariate not in the model. We assessed the residuals for temporal dependency. Model validation did not raise any significant concern with normality of residuals and linear relationship among variables , Time period . The interaction terms were Trap position × Time period. To incorporate the dependency among observations of the same vineyard, we used Vineyard as a random intercept. Overdispersion was accounted for by using a quasi-GLM model and correcting the standard errors accordingly (To model the proportion of FDp-positive The package lme4  and glmmS. titanus and proportion of infected grapevines measured inside the vineyards was estimated using nonparametric Spearman’s rank correlation (cor.test in stats R package) [Correlation between proportion of infected package) .S. titanus numbers trapped inside vs. outside the vineyard at each time period . Z-test was used to compare the proportion of infected S. titanus collected in the same compartments of the vineyard agroecosystems . Plots were constructed using package ggplot2 [Wilcoxon rank-sum test was applied to the comparison of  ggplot2  and lemo ggplot2  in the sS. titanus adults were collected in all sites during July and August both inside and outside the vineyards , but differed for later sampling periods (August–early September), with traps located outside collecting more insects compared to the ones located inside the vineyards , and the sampling time. More than 40% of infected leafhoppers were recorded at Cisterna, La Morra (outside the vineyard only), Montà, and Portacomaro, while at Paderna, about 10% of leafhoppers (both from inside and outside vineyard traps) were FDp carriers. Similarly, inside the vineyard of La Morra, only 7% of tested leafhoppers were infected . OverallS. titanus was significantly higher for individuals trapped outside than inside the vineyards, irrespective of the period of trapping  or not properly treated with insecticides (CR) may also host high populations of S. titanus adults. The selected sites were characterized by the presence of FD-infected cultivated S. titanus adults were collected with YSTs in July (likely at the very end of July), and then captures declined more or less gradually, both within the vineyards and in the wild vegetation compartment. In most reports, S. titanus populations peak in the first half of August [S. titanus development .The highest numbers of f August ,22,23. Telopment  and the ucharest . Howeverosystems ,25,26, wS. titanus and this can account for the high population of the insect. As for the LM vineyards, no specific factors  could be evoked to account for this high S. titanus density which is, to some extent, unpredictable. Among the vineyards, the highest population levels were recorded at LM and CR. In the latter vineyard, only pyrethrins were applied against S. titanus [S. titanus between the cultivated and wild compartments of the vineyard agroecosystem. On the contrary, at LM, the two populations were apparently separated. In fact, very few insects were FDp carriers inside the vineyards, and many were infected in the wild compartment. Interestingly, at LM, the wild vegetation was present in a large area standing downhill and below the level of the vineyard, with a woodland shield protecting the vineyard from major air flows. It is then possible that leafhoppers, in the absence of ascendant air flows, are unable to fly upwards and reach the vineyard. Indeed, without prevalent wind conditions, S. titanus does not move far [S. titanus dispersal has been noticed and considered in pest risk assessment of FD [Our GLMM model showed that a higher proportion of infected insects was recorded for the leafhoppers collected in the wild compartment compared to those from within the vineyard. This evidence is consistent with data of Lessio et al. , confirm titanus . Howevermove far . Where tnt of FD . S. titanus, demonstrated by Bressan et al. [Vitis were greater. Actually, Vitis tested at random were FDp-infected, while percentages of infected plants within the vineyards were generally lower. Therefore, the increase in FDp-positive insects may be partly explained by the increasing load of FDp in the insects due to multiplication over time. This multiplication of FDp in most insects that fed on infected source plants would overcome the detection threshold of the PCR assay. The chosen real-time PCR assay detects phytoplasmas well before the completion of their latent period . However, FDp could also be acquired by adults [S. titanus correlates with phytoplasma load in the plant [2 of the model was equal to 0.57, presumably other factors, besides the proportion of infected leafhoppers inside the vineyard, may account for the spread of the disease within a vineyard . Actually, the vineyards were cultivated with different varieties, and these may show different levels of susceptibility to FD [The proportion of FDp-infected vectors slightly increased over the summer, in line with the data of Lessio et al. ; it is wn et al. . Increasy adults ,31, and y adults  and thathe plant . We can ty to FD ; differeS. titanus adults were collected from the wild vegetation compartment of several sites, and vector population levels of this compartment were higher than those measured inside the corresponding vineyard. The pattern of vector population decrease over August and the beginning of September was significantly different inside and outside the vineyard (population decreased faster inside the vineyard), thus confirming the effects of the insecticides applied against the adults in the vineyards. As expected, the proportion of FDp-infected vectors increased over the summer, even though the proportion of FDp-positive leafhoppers (possibly not infectious yet) was already high in July, indicating that grapevines are exposed to infectious leafhoppers for a long period of time. A higher proportion of FDp-infected leafhoppers was recorded for the insects collected in the wild compartment compared to those from the vineyard, thus indicating the important role of outside FDp sources in the epidemiology of the disease. This study provides valuable information on the role of the wild compartment in the epidemiology of Flavescence dorée disease, and represents one of the few studies conducted at the level of the vineyard agroecosystem as a whole. Further research should be devoted to the evaluation of FD spread reduction following removal of wild Vitis in the surroundings of vineyards.High numbers of


In this work, new evidence for the photodegradation reactions of acetaminophen (AC) is reported by photoluminescence (PL), Raman scattering and FTIR spectroscopy. Under excitation wavelength of 320 nm, AC shows a PL band in the spectral range of 340–550 nm, whose intensity decreases by exposure to UV light. The chemical interaction of AC with the NaOH solutions, having the concentration ranging between 0.001 and 0.3 M, induces a gradual enhancement of the photoluminescence excitation (PLE) and PL spectra, when the exposure time of samples at the UV light increases until 140 min, as a result of the formation of p-aminophenol and sodium acetate. This behavior is not influenced by the excipients or other active compounds in pharmaceutical products as demonstrated by PLE and PL studies. Experimental arguments for the obtaining of p-aminophenol and sodium acetate, when AC has interacted with NaOH, are shown by Raman scattering and FTIR spectroscopy. Acetaminophen (AC) is an active compound of different drugs marketed under the name of Paracetamol, Theraflu, Coldrex, Parasinus, Algopirin, and so on. The therapeutic effects for which AC is administered are: (i) the reduction of headaches during migraines ; (ii) thFor the quantitative determination of paracetamol in biological fluids and tablets, a sustained effort was carried out in the development of analytical techniques such as high-performance liquid chromatography (HPLC) , UV-VIS The chemical compounds, namely AC and NaOH, were purchased from Sigma–Aldrich. The drugs containing AC as an active compound, marked under the name Paracetamol , Parasinus  and Pararemin , were bought from a local pharmacy. The composition of a tablet of: (i) Paracetamol from Magistra C&C contains 500 mg AC and excipients of the type povidone, corn starch, croscarmellose sodium and stearic acid; (ii) Parasinus contains as active compounds 500 mg AC, 3 mg chlorpheniramine maleate, 30 mg pseudoephedrine hydrochloride and excipients of the type pregelatinized starch, talc, povidone, stearic acid, magnesium stearate; and (iii) Pararemin contains as active substances 250 mg AC, 150 mg propyphenazone, 50 mg caffeine and excipients of the type pregelatinized starch (whole), pregelatinized starch , polyvinylpyrrolidone, croscarmellose sodium, stearic acid, isomalt, talc, anhydrous colloidal silicon dioxide.−3, 10−2, 10−1 or 0.3 M.To study the behavior of AC in the absence of excipients, an aqueous solution of AC 0.33 M was prepared. The influence of the alkaline solutions on AC was analyzed using aqueous solutions of NaOH with various concentrations. Thus, in all experiments, 2 mL of AC 0.33 M interacted with 1 mL NaOH 10A tablet of each drug was dispersed into 10 mL distilled water, under ultrasonication, for 15 min. In order to obtain a clear solution, a successively filtration was carried out. For each drug containing AC, i.e., Paracetamol, Parasinus and Pararemin, 2 mL of pharmaceutical compounds interacted with 1 mL NaOH 0.3 M.The solid-state interaction of AC with NaOH, at a non-hydrostatic pressure equal to 0.58 GPa for 5 min has involved: (a) the preparation of three mixtures of AC and NaOH as follows: (i) 0.168 g AC and 0.04 g NaOH; (ii) 0.1 g AC and 0.2 g NaOH; and (iii) 0.16 g AC and 0.4 g NaOH. The weight ratios of the three mixtures of AC and NaOH are equal to 4.2, 0.5 and 0.4. Homogenization of each mixture of AC and NaOH was performed by grinding for 1 min.The samples were exposed successively at UV light, for 140 min, using a mercury-vapors lamp with the power of 350 W, by the selecting with an UG5 filter of the spectral range 200–380 nm, that contains the Hg spectrum line of high intensity at 253 nm.PL and PLE spectra of the AC aqueous solution in the absence and in the presence of NaOH were recorded, in the right-angle geometry, with a Fluorolog-3 spectrophotometer, FL3-2.2.1 model, from Horiba Jobin Yvon, endowed with a 450 W Xe lamp.Raman spectra of AC before and after the interaction with NaOH in the solid state were recorded with a FT Raman spectrophotometer, RFS100S model, from Bruker, endowed with a YAG:Nd laser (excitation wavelength 1064 nm).IR spectra of AC in the initial state and after the interaction with NaOH in the solid state were recorded with an FTIR spectrophotometer, Vertex 80 model, from Bruker, in the attenuated total reflection geometry.Additional information concerning the products resulting from the interaction of AC with NaOH are shown by X-ray photoelectron spectroscopy (XPS), using a SPECS spectrometer endowed with a PHOBIOS 150 analyzer and an Al Kα source.6 to 6.1 × 105 counts/sec when AC is exposed to UV light for 140 min  in −3, 10−2, 10−1 and 0.3 M, the intensity of the PL spectra is equal to 6.09 × 105, 3.1 × 105, 7.5 × 104 and 2.3 × 104 counts/sec, respectively.unts/sec a. As obs−3 M  a gradual decrease in the intensity of the Raman lines situated in the spectral range 1100–1400 cm−1 as well as the Raman lines assigned to the vibrational modes of C-N-H bending in the amide group and C=O stretching in the amide group peaked at 1562 and 1649 cm−1, respectively; (iii) a down-shift of the Raman line from 3066 to 3043 cm−1; (iv) a gradual increase in the intensity of the Raman line, assigned to the vibrational mode of C-N stretching + C-O stretching + C-C stretching in aryl group, which is peaked at 1608 cm−1; and v) a change of the ratio between the intensities of the Raman lines peaked at 1649 and 2931 cm−1 from 4.35  to 2.69 and 1.12, when samples contain the AC: NaOH mass ratios equal to 0.5 and 0.4, respectively. A puzzling fact is that the aniline-derived compounds and carboxylic acids show in the Raman spectra, lines at 1605 [−1 [−1 originates in the formation of p-aminophenol and sodium acetate, respectively.In ctively.  The inte at 1605 ,20 and 21605 [−1 , respect−1, belonging to the vibrational modes of: the phenyl deformation + C-C stretching in amide group, phenyl ring deformation + C-C rocking in amide group, C-C-H bending in aryl group, N-C=O bending in amide group, C-C-H bending in amide group, C-C rocking + C-O-H bending in aryl group, C-O-H bending + C-C-H bending in aryl group, C-N stretching + C-C stretching in amide group, C-N-H bending in amide group + C-O stretching + C-O-H bending in aryl group, C-O-H bending + C-H rocking + C-C stretching in aryl group, C-H rocking in amide group, C-O-H bending in aryl group + C-N-H bending in amide group, C-H rocking in amide group, C-N-H bending in amide group, C-N stretching + C-O stretching + C-C stretching in aryl group, C=O stretching in amide group, N-H stretching in amide group and O-H stretching, respectively. [−1, simultaneous with the decrease of the absorbance of IR band attributed to the phenyl ring deformation + C-C rocking in amide group vibrational mode; (ii) the decrease in the absorbance of the IR bands localized in the spectral ranges 900–1200 and 1373–1379 cm−1 and the down-shift of the IR bands from 968, 1107 and 1171 cm−1 to 940, 1093, 1164 cm−1, respectively; (iii) the increase in the absorbance of the IR bands situated in the spectral range 1400–1500 cm−1 and the down-shift of the IR bands from 1504 and 1562 cm−1 to 1492 and 1525 cm−1; and (iv) the gradual decrease in the absorbance of the IR bands localized in the spectral ranges 1590–1660 and 3300–3700 cm−1. The absence of the IR bands of 1329, 1373 and 3327 cm−1 as well as the diminution of the absorbance of IR bands localized in the spectral ranges 1510–1700 and 3500–3600 cm−1 simultaneous with the increase of the absorbance of IR band at 1492 cm−1 attributed to the C=C stretching vibrational mode in compounds of the type aniline [The IR spectrum represented by black curve in ctively.  As incre aniline  indicate1, the XPS C1s spectrum of AC is characterized by the five peaks at 284, 285, 286, 287.4 and 290 eV, assigned to the following bonds: C=C, C-N, C-O in phenolic compounds, C=O in amide and C=O/C=C in aromatic ring, respectively. [1) at 400.4 and 399.7 eV which were attributed to the –NHCO and –NH– bonds. [1,b2), and iii) the presence of the Na2s peaks at 62.9 and 64 eV in 2, which were attributed to CH3COONa and un-reacted NaOH [1)—the peak at 1073 eV attributed to NaOH and another one at 1072 eV attributed to CH3COONa. Due to the large difference in kinetic energy of the photoelectrons from Na2s and Na1s orbitals, it is proper to assume that the Na1s photoelectrons describe the first layers of the sample’s surface while the Na2s photoelectrons describe the deeper layers. Comparing the peak intensities in the two spectra we can deduce that the unreacted NaOH is only on the surface of the sample . All these changes plead for the formation of p-aminophenol and sodium acetate. Based on these results, the most important advantages and drawbacks of the PL in comparison with other methods used in the assessing of the photodegradation processes of the pharmaceutical compounds are shown in According to ctively.  Two peak– bonds.  In contrted NaOH ,25, respe sample c1,c2. Al−1 and the change of the ratio between the relative intensities of the Raman lines at 1649 and 2931 cm−1 in the favor of the last one; the Raman lines at 1608 and 2931 cm−1 correspond to the C=C stretching vibrational mode in the aniline-derive compound and carboxylic group, respectively; and (v) diminution of the amide group weight in AC as a consequence of the interaction with NaOH was highlighted in IR spectra by the absence of the bands at 1329, 1373 and 3327 cm−1, the decrease in the absorbance of the IR bands situated in the spectral ranges 1510–1700 and 3500–3600 cm−1 and the presence of the IR band at 1492 cm−1 attributed to the C=C stretching vibrational mode in aniline-derive compounds. A diminution of the amide group simultaneous with the formation of the amine and carboxylic groups, as a consequence of the interaction of AC with NaOH, was also highlighted by XPS spectroscopy.Using photoluminescence (PL), Raman scattering and FTIR spectroscopy, new evidence concerning the interaction of AC with NaOH was reported in this work. Our results lead to the following conclusions: (i) photoluminescence represents an alternative method to UV-VIS spectroscopy to monitor the reaction of AC with NaOH under UV light; this reaction is highlighted by the up-shift of the PLE and PL bands from 317 and 394 nm to 338 and 432 nm, respectively, simultaneous with an intensity increase of these spectra; (ii) the PLE and PL studies have demonstrated that this photochemical reaction is not influenced by the excipients existing in pharmaceutical compounds or the other active compounds such as those used in antipyretic drugs; (iii) the changes invoked in the emission spectra are a consequence of the fact that the interaction of AC with NaOH results in the formation of p-aminophenol and sodium acetate; (iv) the main evidence shown in Raman spectra for the formation of p-aminophenol and sodium acetate consist of the presence of the line at 1608 cm


Weiner’s attribution theory posits that it is adaptive to ascribe challenges to controllable causes  and maladaptive to ascribe them to uncontrollable causes . This is supported by our prior research that showed a heightened risk of mortality when mobility challenges were attributed to old age. The present pilot study randomly assigned older adults (N=36) in a day hospital to either an attributional retraining (AR) intervention group that viewed a video intended to shift causal thinking regarding mobility challenges (uncontrollable→controllable causes), or to a comparison group (No-AR). Participants completed a Time1 survey, the AR intervention (one week later), and a Time2 follow-up survey two weeks later. A manipulation-check revealed that AR was effective in shifting causal thinking away from maladaptive causes; a decline in the endorsement of the old age attribution was observed in the AR group , but not in the No-AR group . The AR and No-AR groups were equivalent at Time 1 on two quality-of-life outcomes: helplessness and perceived control (PC) over health. However, helplessness declined (Time1-Time2) in the AR group , whereas it was relatively stable in the No-AR group . Moreover, PC increased marginally in the AR group , but declined in the No-AR group . Together, these findings suggest that attributions can be shifted away from uncontrollable causes and that this shift can have a protective effect that benefits quality-of-life.


Allergic rhinitis (AR) is believed to be a complex genetic disease. The last decade has been marked by the publication of more than 20 genome-wide association studies (GWASs) of AR and associated allergic phenotypes and allergic diseases, which have shown allergic diseases and traits to share a large number of genetic susceptibility loci. The aim of present study was therefore to investigate the highly replicated allergy related genes and variants as candidates for AR in Han Chinese subjects.A total of 762 AR patients and 760 control subjects were recruited, and a total of 58 susceptible variants previously reported to be associated with allergic traits were choose for replication.P = 0.029, OR = 1.469 for GG vs. AA); rs6554809 in DNAH5 ; rs1438673 in WDR36-CAMK4 loci , rs7775228 in HLA region , rs7203459 in CLEC16A .Logistic regression analyses revealed that in the co-dominant-effect model as assessed by the AIC, compared with wild-type carriers, significant AR risk were associated with rs9865818 in LPP (We replicated Han Chinese AR-specific susceptibility loci in LPP, DNAH5, HLA, CLEC16A and WDR36-CAMK4. Further understanding the molecular mechanisms underlying these associations may provide new insights into the etiology of allergic disease. Allergic rhinitis (AR) is a major chronic respiratory disease induced by an immunoglobulin E (IgE)‐mediated reaction in allergen‐sensitized subjects. It is believed that AR and the comorbid conditions including allergic asthma, eczema, or any other allergic disease, are complex genetic diseases resulting from the effect of both multiple genetic and interacting environmental factors on their pathophysiology. Moreover, Barnes  has prop. [The last decade has been marked by the publication of more than 20 genome-wide association studies (GWASs) of AR, atopy and the associated allergic phenotypes and allergic diseases and traits . Andiapp.  reported. , suggest. , 16. TheThe study protocol was approved by the Ethics Committee of Beijing TongRen Hospital and performed in accordance with the guidelines of the World Medical Association’s Declaration of Helsinki. All subjects involved were adults (≥ 18 years) of Han Chinese ethnic origin from the Beijing region, China, and provided written informed consent prior to entry in the study.Der f and Der p); seasonal grass pollens ; animal hair (dog and cat); molds  and cockroach. Subjects were also considered to be sensitized to allergens when the serum sIgE was greater than 0.35 kU/l. AR subjects with (i) co-morbid asthma, eczema, or any other allergic disease; (ii) hypertension, diabetes or other chronic diseases; or (iii) tumor in the nasal cavity or any other inflammatory nasal disease were excluded. The diagnosis of asthma was confirmed by a physician according to Global Initiative for Asthma (GINA) guidelines [Two rhinology specialists (Y. Z. and L. Z.) were responsible for the screening the study subjects in outpatient clinic of Allergy department at Beijing TongRen Hospital, during the study period from February 2010 to February 2011. Finally, a total of 762 consecutive adult subjects with physician-diagnosed clinical AR were recruited. The flow diagram of recruitment was shown in Additional file idelines .A total of 760 adult healthy control volunteers were also recruited during the study period. For matching with AR groups, health control group had similar age distribution, gender ratio, and also Han Chinese ethnic origin from the Beijing region for an ethnically similar local population to determine background population allele frequencies. None of the control subjects had a history of allergic or any nasal disease, nor demonstrated any abnormal clinical features in the nasal cavity or positive serum sIgE by Phadiatop  testing.A 2-ml volume of venous blood samples from each participant was taken in citrate-anticoagulated glass tubes, and were frozen at − 40 °C. Total genomic DNA of the leucocyte was extracted from 1 ml of peripheral blood using the Whole Blood DNA Extraction Kit , according to the manufacturer’s instructions. Genomic DNA extracted was dissolved in 0.1× TE buffer  and stored at − 20 °C.58 susceptible single nucleotide polymorphisms (SNPs) in 55 gene/regions previously reported as highly associated with allergic traits –13, 15 wSNPs were typed using iPLEX chemistry on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer  . In brie2-test. Mann–Whitney test was used to compare serum total IgE level between case and control groups, and between different genetic models in dominant or recessive models. Kruskal–Wallis test was used to compare serum total IgE level among different genotypes in co-dominant model.Values are expressed as mean ± standard deviation or as numbers and percentages. Differences in age between case group and control group were evaluated using the t-test. Differences in gender and frequencies of the alleles and genotypes between case group and control group were evaluated using the χhttp://ihg.gsf.de/cgi-bin/hw/hwa1.pl). It was tested in the control group by the Chi square test for goodness of fit, and a P-value of < 0.05 was considered to be statistically significant. Akaike’s information criteria were used to select the most parsimonious genetic model for each SNP [P-value of 0.05 after corrections with 100,000 permutations. All P values are two tailed. These analyses were conducted with Stata statistical package .Hardy–Weinberg Equilibrium (HWE) was tested by the Chi square test for goodness of fit with a Web program (each SNP . Odds raP = 0.167) or the ratios for males/females (P = 0.372) between the control and AR groups. The median total serum IgE measurements were significantly increased in AR group than control group . Furthermore, 273 (35.8%), 168(22.0%), and 321 (42.2%) of AR subjects, respectively, were found to be allergic to HDMs alone, seasonal pollens alone, and mixed allergens.The demographic characteristics of the study population are shown in Table P < 0.0001). The minor allele frequencies (MAF) of four selected SNPs, such as rs6673480, rs17513503, rs7032572 and rs1250761 could not satisfy association study (MAF < 0.01). The four SNPs, rs3860069, rs11680788, rs9303280 and rs12973620 failed in assay design. Thus, we finally enrolled 47 SNPs to process the next analysis , rs1438673: T>C (P = 0.029), rs7775228: T>C (P = 0.001), and they remain associated after 100,000 permutations (P < 0.05). The distribution of the genotypes of rs6554809, rs1438673 and rs7775228 showed significant difference between cases and controls.The genotype distributions of 47 selected SNPs in the case group and control group are summarized in Table P = 0.029, OR = 1.469, 95% CI 1.041–2.074 for GG vs. AA); rs6554809 in DNAH5 ; rs1438673 in WDR36-CAMK4 loci , rs7775228 in HLA region . However, it was a protective factor with the SNP in rs7203459 in CLEC16A . The detailed data was shown in Table Logistic regression analysis showed significantly related to AR risk compared with wild-type carriers in the codominant effect model assessed by AIC. The result showed that it was a risk factor with the SNP in rs9865818 in LPP  and in rs7775228 , while protective effect was found in rs7203459 in CLEC16A . In addition, under recessive model, rs7617456 in TMEM18 was also revealed with protective effect , and rs1438673 was evaluated with AR risks . The detailed data was shown in Table Under dominant model, the significant AR risks were observed in rs6554809 in DNAH5  and reviewed in Table The functions and effects of these positive SNPs were retrieved from HaploReg v4.1 tool (P < 0.0001). No significant association with serum total IgE was observed under recessive model for rs7775228. The co-dominant model analysis for rs7775228 found a significant decrease of serum total IgE among TT carries (median 49.8 KU/L) compared to CC  and TC carries , respectively , as well as under co-dominant model, significant elevation of serum total IgE was detected among CC carries (median 60.50 KU/L) comparing with TC carries (median 54.6 KU/L) (P = 0.0413)  and it cis-regulates HLA-DRB410, similar with our present findings.Actually, it is almost 4 decades since an association between genetic variants in the HLA region and specific allergen sensitization was reported , and varP = 3.3 × 10−6) was firstly detected as a candidate gene for AR and grass sensitization through a genome-wide meta-analysis by Ramasamy et al. [DNAH5 . Bønnelykke et al. [P = 3.4 × 10−6). Here we also replicated rs9865818 of LPP using diagnosed AR population. Several new allergy-associated loci are in or near genes involved in T-helper cell differentiation. The association between LPP and allergy may be mediated by an effect on the expression of BCL6 (B cell lymphoma 6), a transcription factor that represses the STAT6-mediated response to IL-4 and IL-13 and IgE class switching [Hinds et al.  conductee et al.  performewitching  and inhiwitching . Conerniwitching , while twitching .In this study, we found that the SNPs in rs1438673 and in rs7775228 had strongly association with serum total IgE in AR group. The variant near the HLA-DQB1 was regarded as a predictor of total serum IgE levels in multiple race-ethnic groups, which was identified by an independently meta-analysis . The serIt is striking that in the present study, variants in only 5 of 58 previously identified candidate genes demonstrated significant association with Han Chinese AR. Of these variants, rs6554809-T allele in DNAH5 was the risk factor for AR in our study, which was opposite to Ramasamy et al. . These oHowever, the findings of our study are slightly limited, particularly in view of associations in only 6 genome-wide studies due to a limited number of SNPs in two genotyping assays. Furthermore, this study is somewhat limited in the sample size and the results did not exclude the potential false positive and negative associations, although we enrolled well-diagnosed AR to avoid population stratification as well as adjustment for multiple testing involved in 47 SNPs. Further studies would also be performed in larger cohorts and assess associations in a larger number of loci for identification of susceptibility to AR.Intriguingly, whereas, it’s indubitable that the analysis driven by statistical and knowledge-based evidence represents a promising approach for identifying new genes or targets involved in complex traits and an important next step will be to more carefully characterize the extent to which indi-vidual associations lead to a global predisposition to allergy compared to effects on specific target tissues, such as skin, lung or mucosa.In summary, here we replicated shared and AR-specific susceptibility loci in LPP, DNAH5, HLA, CLEC16A and WDR36-CAMK4 which were also plausible candidates in terms of their biological function in the development of AR. Further understanding the molecular mechanisms underlying these associations may provide new insights into the etiology of allergic disease in Han Chinese.Additional file 1. Additional tables and figures.