Sulfatide-liposome binding to P-selectin and sulfatide/P-selectin-dependent platelet aggregation was blunted by peptide antagonists, carrying the EWVDV motif within N-terminal extensions, such as CDVEWVDVSC , but not by the EWVDV core motif itself (IC50 > 1000 μM), albeit both being equally potent inhibitors of PSGL-1/P-selectin interaction (IC50= 7–12 μM). Conclusions: Our data suggest that the sulfatide/P-selectin interaction implicates multiple binding pockets, which only partly overlap with that of PSGL-1. These observations open ways to selectively interfere with sulfatide/P-selectin-dependent platelet aggregation without affecting PSGL-1-dependent cell adhesion.Background: Membrane-exposed sulfatides are proposed to contribute to P-selectin-dependent platelet aggregation. Here, we demonstrated that P-selectin-mediated platelet aggregation on a collagen-coated surface under flow indeed depended on sulfatides and that this interaction differed considerably from the interaction of P-selectin with P-selectin Glycoprotein Ligand-1 (PSGL-1), which underlies leukocyte-endothelium adhesion. Methods and Results: Upon platelet activation, sulfatides were translocated to the platelet surface to form focal hot-spots. Interestingly, P-selectin was observed to exclusively interact with liposomes with a sulfatide density higher than 21% ( The selectin family of lectin-based adhesion molecules consists of L-selectin (in leukocytes), E-selectin (in endothelium), and P-selectin (in platelets and endothelium) ,2. The lMerten and colleagues were the first to show that P-selectin binds to sulfatides exposed on the platelet surface upon activation, and that platelet aggregates are stabilized through sulfatide/P-selectin rather than PSGL-1/P-selectin interactions. By inducing further degranulation, thereby increasing surface expression of P-selectin, P-selectin/sulfatide interactions trigger a positive feedback mechanism that potentiates aggregation ,21,22,23EWVDV minimal motif interacted with P-selectin close to the PSGL-1 binding site on P-selectin, which was proposed to overlap with that of sulfatides -cholesteryloleate from Amersham Life Sciences . Peptides were purchased from Eurosequence , and their quality was confirmed by mass spectroscopy and HPLC. Antibody WAPS12.2 was isolated from HB-299 cells obtained from the American Type Culture Collection (ATCC).We obtained human P- and E-selectin-IgG from R&D systems , human L-selectin-IgG and monoclonal anti-P-selectin antibodies AK-4 and AC1.2 from Pharmingen , adenosine diphosphate (ADP), sulfatides , cholesterol, goat-anti-human IgG (Fc-specific), human placental collagen type III and filipin III from Sigma , fibrillar equine collagen from Hormon Chemie , prostacyclin ) and P-selectin-deficient mice (P-selectin knockout (KO)) on a C57Bl6 background were obtained from Jackson Laboratories . Mice had unlimited access to water and regular chow diet . Blood was obtained from surplus mice scheduled for sacrifice at the Leiden animal breeding facility. Animal experiments were performed at the Gorlaeus Laboratories of the Leiden Academic Center for Drug Research following the national laws and approved by the local ethics committee for animal experiments.3H]-cholesteryl oleate ) was added, and the organic solvent was evaporated by a stream of nitrogen. The resulting lipid layer was vortexed in 1 mL phosphate buffered saline (PBS) and subsequently sonicated for 40 min at 54 °C using a Soniprep 150  at 18 µm amplitude. Particle sizes were determined by photon correlation spectroscopy  at 25 °C and a 90° angle between laser and detector. Sonication resulted in monodisperse liposomes with mean particle diameters between 63–85 nm (polydispersities of 0.13–0.22). Liposomes were stored at 4 °C under argon and used for characterization and metabolic studies within 7 days following preparation, during which no physicochemical changes occurred. As the liposomes were equal in size and incorporation of the radioactive label was quantitative, the specific activity of each sulfatide-bearing liposome was calculated as the amount of [3H]-cholesteryloleate (dpm) divided by the total lipid content (ng).Liposomes were prepared by sonication as follows. Egg yolk phosphatidylcholine, cholesterol, and sulfatides, all dissolved in chloroform/methanol , were mixed at weight ratios of 4:0.8:0 (named Sf0), 4:0.8:1.3 (Sf21), 2.5:0.8:1.3 (Sf28), 1:0.8:1.3 (Sf42), and 0.2:0.8:1.3 mg/mL (Sf57). The Sf numbers indicate the number of sulfatides as a percentage of total lipid weight. A tracer amount of -CE-labeled liposomes (0–800 µg lipid per mL assay buffer containing 0.2% BSA). For competition studies, labeled liposomes (1 × 105 dpm/well) were incubated in the presence of peptides or antibodies at the indicated concentrations. After removal of unbound liposomes by washing four times with assay buffer, bound liposomes were collected in 2-propanol/butanol/water  and counted for radioactivity using a scintillation counter. Since the sulfatide liposomes were equal in size, the recovered amount of [3H]-CE could be used to calculate the binding capacity of P-selectin for the sulfatide liposomes, as well as the recovered amount of lipid (ng/well) based on the specific activity.Fc-specific goat anti-human IgG (10 μg/mL) was coated overnight at 4 °C in a high binding 96-well plate  in coating buffer . Sulfatides were immobilized on L1 sensor chips at 5 μL/min according to the manufacturer’s instructions. Binding kinetics of human fusion proteins IgG and P-, L- and E- selectins to the immobilized sulfatides were determined by injection of the fusion proteins. At the end of each injection, the sensor chips were regenerated with 100 mmol/L NaOH and equilibrated with running buffer. All curves were corrected for non-specific binding by an online baseline subtraction of ligand binding to a control flow channel. Binding kinetics were analyzed using BIAevaluation software .A Biacore 2000 instrument  was used to analyze the interaction between sulfatides and P-, L, and E-selectins, as described . Experim11 platelets/L with platelet-poor plasma prepared from the remaining blood . Washed platelets were prepared by adding 0.1 volume of anticoagulant citrate dextrose (ACD) solution  to lower the pH of the PRP to 6.5, followed by two cycles of centrifugation  and resuspension in HEPES-Tyrode buffer . Prostacyclin  10 ng/mL) was present during the final wash step, and the final resuspension was in HEPES-Tyrode buffer (pH 7.2) to a concentration of 2 × 1011 platelets/L. Experiments were performed with blood or platelets from at least 3 donors.Freshly drawn venous blood from healthy volunteers was obtained through the Mini Donor Service, a blood donation facility for research purposes, that is approved by the Medical Ethics Committee of the University Medical Center Utrecht (protocol number 07-125). All donors provided written informed consent, in accordance with the Declaration of Helsinki for blood collection through this service. All participant information is kept confidential, and only Mini Donor Service personnel have access to the information provided. The donors were free of medication for at least two weeks before blood collection. Overnight fasting before blood collection was not necessary. Blood was collected into 0.1 volume 130 mmol/L trisodium citrate and was processed the same day and within 6 h. For aggregation studies, platelet-rich plasma (PRP) was prepared by centrifugation  and adjusted to a final concentration of 2×102. Pellets were resuspended in HEPES-Tyrode buffer (pH 6.5), and the washing procedure was repeated once. The platelet pellets were subsequently resuspended in HEPES-Tyrode buffer (pH 7.2), and the platelet count was adjusted to 2 × 1011 platelets/L.For studies with murine platelets, six to eight-week-old male WT and P-selectin KO mice were anesthetized by subcutaneous injection of a mixture of xylazine (5 mg/mL), ketamine (0.04 mg/mL), and atropine (0.05 mg/mL) and exsanguinated by extraction via an intracardiac syringe. Blood was anticoagulated with 0.1 volume 130 mmol/L trisodium citrate, recalcified, and used for perfusion studies. For static adhesion experiments, washed mouse platelets were prepared by centrifugation of whole blood . Plasma and buffy coat were isolated, and PRP was concentrated by centrifugation , followed by another centrifugation step  in the presence of ACD and PGI2, pH 7.4) and incubated with blocking buffer . After washing, wells were incubated  with [3H]-CE-labeled liposomes  in the absence or presence of the monoclonal anti-sulfatide antibody SulphI (5 μg/mL). After removal of unbound liposomes by washing four times with assay buffer, bound liposomes were collected in 2-propanol/butanol/water  and counted for radioactivity using a scintillation counter.Washed mouse platelets were stimulated with vehicle or thrombin  and coated on a high-binding 96-well plate . Then, wells were washed with assay buffer  at a concentration of 100 μg/mL for 1 h at room temperature. Human placental collagen type III (solubilized overnight in 50 mmol/L acetic acid) or fibrillar equine collagen (1 mg/mL) were sprayed onto Thermonox coverslips at a density of 30 μg/mL with a retouching airbrush  under a nitrogen-operating pressure of 1 atm. Coverslips were blocked with human albumin (1% in PBS) and stored overnight at 4 °C.−1 (representative for large arteries) and to collagen type III at 800 s−1 ; adhesion of murine platelets to fibrillar equine collagen was at 150 and 300 s−1 in the absence and presence of Sulph I antibody (10 µg/mL), respectively. Higher shear rates required large volumes of murine blood, which was unfortunately not available.Prewarmed human and murine blood samples  were drawn through the perfusion chamber by an infusion pump  for 3 (human) and 5 min (mouse). Adhesion of human platelets to fibrinogen was performed at a shear rate of 300 sCoverslips with human platelets were rinsed with HEPES-buffered saline  and fixed with 1% paraformaldehyde in PBS for immunofluorescence studies. For evaluation by light microscopy, coverslips with murine platelets were fixed with 0.5% glutaraldehyde in PBS, stained with May-Grünwald-Giemsa, dried and mounted in Entellan , and analyzed with a light microscope  equipped with a JAI-CCD camera  coupled to a Matrox frame grabber  using Optimas 6.2 software . Murine platelet deposition was expressed as percentage surface coverage assessing 20 fields in a randomized manner.4Cl/PBS , washed again and blocked with PBS containing 3% BSA . To detect membrane cholesterol, coverslips were incubated with 50 μg/mL filipin III in PBS/1% BSA . For detection of sulfatides, coverslips were incubated with 1 μg/mL SulphI in PBS/3% BSA , washed and incubated with Alexa Fluor® 488 goat-anti-mouse IgG . Finally, coverslips were washed with PBS and mounted in Aqua/Poly Mount. Cells were visualized with a Nikon Eclipse E600 fluorescence microscope (60x Nikon objective) equipped with a CoolSNAP-Pro camera , using Image-Pro Plus software  for analysis.Coverslips from perfusion experiments with human blood were washed with PBS, incubated in 50 mmol/L NHAs controls, resting platelets were spun down onto microscope slides , fixed with 1% paraformaldehyde, and immunofluorescently labeled as described above.Human PRP was incubated with peptide antagonists  or vehicle and stimulated with 10 μmol/L ADP under constant stirring at 1000 rpm (37 °C). Optical aggregation was monitored in a Chrono-Log Lumi-aggregometer .2).After 15 min, aggregates were fixed in 1 volume 0.5% glutaraldehyde in PBS and applied to microscope slides. Aggregates were dehydrated with methanol and stained with May-Grünwald/Giemsa. The aggregate size was measured by light microscopy equipped with a JAI-CCD camera  coupled to a Matrox frame grabber  using the ‘watershed’ function of the Optimas 6.2 software . Results were expressed as mean aggregate size  was 33% lower than that of WT platelets . Importantly, adhesion of WT platelets was reduced by 41% by the SulphI antibody to 20.3 ± 1.8% (p = 0.0047), but with P-selectin KO platelets, the antibody had no effect   was observed for P-selectin KO platelets. In line with earlier observations -CE-labeled sulfatide liposomes to P-selectin were performed for a selection of peptide antagonists, derived from the parent peptide TM11 (CDVEWVDVSSLEWDLPC) [EWVDVS (EC50 = 12 μM), DVEWVDVA (EC50 = 8 μM), and CDVEWVDVSC (EC50 = 0.2 μM). Surprisingly, the truncated peptides EWVDV, VEWVDV, AVEWVDVS, DAEWVDVS, and DVAWVDVS, all potent inhibitors of adhesion of HL60 cells to P-selectin [AVDVS, DVEWADVS, DVEWVAVS, DVEWVDAS, and EWVKV [EWVDV (ref. [LEWDLPC) . Sf28 liKV, EWVDV, DVEWVDVS, and CDVEWVDVSC. Peptide antagonists DVEWVDVS and CDVEWVDVSC significantly reduced maximum aggregation by 28% (p < 0.05) and 43% (p < 0.001), respectively  and 20% (p < 0.05), respectively, confirming a role for sulfatide/P-selectin interactions in platelet aggregation [Since the interaction of sulfatides with P-selectin is instrumental in platelet aggregation   B–E. ThesVarious negatively charged polymers, including heparin , sulfateEWVDV-based peptide ligands enhances their avidity for P-selectin [Sulfatides are sulfated glycosphingolipids, composed of a hydrophobic ceramide and a hydrophilic sulfogalactoside unit. Their amphiphilic nature facilitates rapid and quantitative incorporation into liposomes . By multselectin . Conceivselectin .EWVDV core motif critical for P-selectin binding [EWVDV [EWVDV failed to block sulfatide liposome binding to P-selectin and platelet aggregation. As full-length peptides, such as DVEWVDV and CDVEWVDVSC, inhibited both PSGL-1 and sulfatide binding with high potency, the N-terminal extension of EWVDV must be required for inhibition of sulfatide binding as well as platelet aggregation. Intriguingly, CDVEWVDVSC was 20-fold more potent in inhibiting sulfatide than PSGL-1 binding to P-selectin.The peptide antagonists we used for mapping differences in the P-selectin binding pattern of sulfatides versus PSGL-1, all encompassed the  binding . The smag [EWVDV . RemarkaEWVDVSC and the anti-sulfatide and anti-P-selectin antibodies corresponds with this notion. Interestingly, while equally effective in inhibiting aggregate size, AK-4 appeared to be a much more potent inhibitor of platelet aggregation than SulphI. This could point to an involvement of counterligands other than sulfatides in P-selectin-dependent aggregate formation.The observation that the anti-sulfatide antibody SulphI and the anti-P-selectin antibody AK-4 were able to inhibit platelet aggregation and aggregate size underscored the relevance of the P-selectin/sulfatide dyad for aggregation. P-selectin-dependent inhibition is restricted to the later steps in the aggregation response, suggesting that sulfatides serve a vital role in aggregate stabilization. The observed marked reduction in aggregate size by CDVEWVDVSC exclusively interfered with P-selectin binding to liposomes containing intermediate sulfatide densities (28%). This is supportive of the existence of at least two separate sulfatide binding domains on P-selectin: a Ca2+-dependent binding site in the lectin domain of P-selectin for intermediate-density sulfatide liposomes, and a Ca2+-independent binding site for high-density sulfatide liposomes. The peptide antagonists were earlier shown to interact with or in proximity to the sLex binding site on P-selectin and to inhibit PSGL-1 binding to P-selectin under static and flow conditions [EWVDV), which associate with the sLex binding pocket [EWVDVS, may protrude more deeply into this sulfotyrosine binding pocket, possibly via interaction of its negatively charged N-terminal aspartic acid with the positively charged lysine residues at position 111 and 113 in P-selectin [Whereas sulfatide liposome binding could be displaced by antibody WAPS12.2 regardless of sulfatide density, blocking antibody AK-4, EDTA, and CDVensities %. This inditions ,41, sulfnditions ,43,44,45ensities %. This iselectin ,29 (Figux involved multiple binding pockets. Elongated and truncated EWVDV-based peptide antagonists appeared to be selective competitors of sulfatide and PSGL-1 binding, respectively, but only the elongated peptides interdicted P-selectin-mediated platelet aggregation. This renders the elongated EWVDV peptides attractive in therapeutic strategies that aim at reducing thrombus size and stability. Since sulfatides also contribute to platelet-leukocyte tethering [In conclusion, this study established the importance of sulfatides for P-selectin-dependent platelet-platelet interaction. It identified exposed sulfatide density as a key determinant of sulfatide binding and suggested that the interaction of P-selectin with sulfatides, PSGL-1, and sLeethering  and play


Spatholobus suberectus) on radiation-induced hematopoietic alteration and oxidative stress in the liver. Mice were exposed to a single acute γ-radiation for the whole body at the dose of 6.0 Gy, then subjected to administration of amifostine (45 mg/kg) or JXT (40 g crude drug/kg) once a day for 28 consecutive days, respectively. Bone marrow cells and hemogram including white cells, red cells, platelet counts, and hemoglobin level were examined. The protein expression levels of pJAK2/JAK2, pSTAT5a/STAT5a, pSTAT5b/STAT5b, and Bcl-2 in bone marrow tissue; levels of reactive oxygen species (ROS); and the activity of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) in serum and liver tissue were determined. At the end of the experiment, the effect of JXT on cell viability and G-CSF and G-CSFR levels in NFS-60 cells were tested by CCK-8 assay, ELISA, and flow cytometry. The results showed that the mice exposed to γ-radiation alone exhibited a typical hematopoietic syndrome. In contrast, at the end of the 28-day experiment, irradiated mice subjected to oral administration of JXT showed an obvious improvement on blood profile with reduced leucopenia, thrombocytopenia (platelet counts), RBC, and hemoglobin levels, as well as bone marrow cells. The expression of pJAK2/JAK2, pSTAT5a/STAT5a, and Bcl-2 in bone marrow tissue was increased after JXT treatment. The elevation of ROS was due to radiation-induced toxicity, but JXT significantly reduced the ROS level in serum and liver tissue, elevated endogenous SOD and GSH-PX levels, and reduced the MDA level in the liver. JXT could also increase cell viability and G-CSFR level in NFS-60 cells, which was similar to exogenous G-CSF. Our findings suggested that oral administration of JXT effectively facilitated the recovery of hematopoietic bone marrow damage and oxidative stress of the mice induced by γ-radiation.This study aims at investigating the radioprotective effect of ethanol extract from Ji-Xue-Teng (JXT,  Radiotherapy is widely used in cancer treatment. Although irradiation is targeted at malignant tissues, the surrounding normal tissues or organs are also affected –3. WholeAs a consequence, it is essential to search radioprotectors to control side effects in both planned exposure such as radiotherapy and unplanned exposure such as natural radiation . AlthougSpatholobus suberectus (S. suberectus) Dunn, recorded in Gang Mu Shi Yi as a holy drug of the blood, which can remove blood clot and produce fresh blood. Modern pharmacological researches have documented that JXT and its active components are widely used for improving blood circulation and executing anti-inflammation, antibacterial, neuroprotective, antioxidative, and anticancer effects [γ-radiation-induced myelosuppression and oxidative damage of the liver tissues in mice and the underlying hematopoietic mechanisms were explored.Ji-Xue-Teng (JXT) is a traditional herb from the dry rattan of  effects –25. Acco effects , but theJXT was purchased from Beijing Lvye Pharmaceutical Co. Ltd. , Fujian (China) origin. JXT was cut into small pieces and soaked in 75% ethanol at 8 times volume overnight and then extracted 3 times with 1 h for each time. Finally, the filtrates were mixed and concentrated by a rotary evaporator with bath temperature lower than 40°C. Ethanol extract was lyophilized to powder.The reagents and kits for determining enzyme activity were purchased from Nanjing Jiancheng Bioengineering Institute . The antibodies for evaluating G-CSFR, Bcl-2, JAK2, pJAK2, STAT5a, pSTAT5a, STAT5b and pSTAT5b were purchased from Abcam . Amifostine was purchased from Dalian Merro Pharmaceutical Factory ; RPMI-1640 culture medium and fetal bovine serum (FBS) were purchased from HyClone . G-CSF ELISA kit was purchased from Shanghai Tongwei Biotech Co. Ltd. .A total of 40 Chinese Kun Ming (KM) mice with the age of 6–8 weeks and inbred colony were purchased from the Laboratory Animal Breeding and Research Center in PLA General Hospital, Beijing, China, and maintained under controlled conditions of temperature (23 ± 2°C) and light (12L : 12D). All mice were provided with standard chow diet and water ad libitum.γ-radiation) at the dose of 6 Gy was delivered in the Department of Radiotherapy and Oncology, the Military Medical Academy of Science. The mice were randomly divided into 4 groups including nonirradiation control, irradiation control, positive treatment control, and JXT treatment groups with 10 mice in each group. The mice from the positive treatment control group were administrated with amifostine at 43.6 mg/kg by tail vein injection [Gamma radiation  level, erythrocyte (RBC) count, total leukocyte (WBC) count, and platelet (PLT) count for each mouse were measured at 0 day (before irradiation) and 21 days postirradiation using an automated hematology analyzer .The ROS level in the serum and ROS, SOD, GSH-Px, and MDA levels in the liver tissue were analyzed, respectively, according to the instructions of the kits from the manufacturer.γ-radiation.Bone marrow cells were collected from both femurs of each mouse at day 10 after whole-body radiation at the dose of 6 Gy. The collected bone marrow cells were rinsed with PBS, and single-cell suspension was obtained. After the lysis of red blood cells, the bone marrow cells were suspended in RPMI-1640 with 10% fetal bovine serum (FBS) and 100 U/ml antibiotics. Then, the bone marrow cells were counted in a hemacytometer under an optical microscope to determine whether JXT extract can protect the bone marrow cells from the depletion induced by β-glycerophosphate; 1 mM sodium orthovanadate; 1 μg/ml leupeptin; and 1 mM PMSF) for 20 min on the ice. After centrifugation at 12000×g for 10 min, protein was then quantitated by using a bicinchoninic acid protein assay kit . After being heated at 95°C for 8 min, the samples were loaded on 12% SDS-polyacrylamide gel for electrophoresis and transferred to PVDF membrane . The membrane was blocked for 2.5 h in TBS-T buffer  containing 3% (w/v) bovine serum albumin  at room temperature and then probed with each primary antibody (1 : 500 dilution) at 4°C overnight. After washing with TBS-T for three times, the membrane was then incubated with appropriate horseradish peroxidase-labeled secondary antibody for 2 h at room temperature. For quantitative analysis of immunoblot bands, the densities of the bands were measured by scanning densitometry . All experiments were repeated at least three times.The preparation of bone marrow cells was the same as above. For western blot analysis, total protein was extracted first, and cells were washed twice with cold PBS and lysed in the radioimmunoprecipitation assay (RIPA) buffer .NFS-60 cell line, derived from murine myeloid leukemia, is responsive to different growth factors including G-CSF. Cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 50 U/ml penicillin, 50 μl of CCK-8 solution was added to the culture medium and incubated at 37°C for 4 h. Then, the OD was spectrophotometrically measured at 450 nm using a microplate spectrophotometer . Viability was expressed as the percentage of vehicle-treated  culture that was set to 100%.NFS-60 cell viability after JXT treatment was evaluated with CCK-8 assay. Cells (3000 cells/well) were plated in 96-well plates. After 24 h culture, JXT at different final concentrations  was added into the plates and detected after 24 h culture. Briefly, 20 −6 μg/ml. Dispensed antigen standards and samples were added to each well of 96-well plates precoated with primary antibodies. After adding biotin conjugate reagent and enzyme conjugate reagent into each well, the plates were incubated at 37°C for 30 min. Then, the plates were rinsed with distilled water for 5 times and measured using a microtiter plate reader .Culture media were collected following treatments and promptly stored at −80°C until the future assay. The G-CSF level was measured using commercially available paired antibody quantitative ELISA kit according to the manufacturer's instructions. Data were expressed as 106 NFS-60 cells were plated in 6-well plates for each well. After treatment with JXT at different final concentrations  for 24 h, the cells were harvested and washed in PBS and then centrifuged at 1000×g for 5 min. The cell pellet was resuspended at the density of 5 × 106 cells/ml and incubated with G-CSFR antibody for 30 min at 37°C. Then, the cells were washed in PBS and incubated with IgG secondary antibody for 30 min at 37°C. The cells were then analyzed in a Becton Dickinson flow cytometer . Each sample was collected at the amount of ten thousand cells.The G-CSFR level in cells was measured using flow cytometry. A total of 5 × 10P < 0.05.All data were expressed as mean ± standard deviation (M ± SD). Statistical analysis was conducted by using SPSS 16.0 software. One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used for multiple group comparisons. The statistically significant difference was considered at γ-radiation. The administration with JXT significantly increased the body weight (P < 0.05), spleen index, and thymus index (P < 0.01) when compared to the radiation group. The results suggested that JXT administration played a positive role in preventing radiation-induced loss of body weight, spleen index, and thymus index of the mice.P < 0.01) when compared with those in the control group. The administration with JXT and amifostine could attenuate the decrease in WBC counts. P < 0.01) and amifostine-treated mice (P < 0.05). As shown in P < 0.01). P < 0.01 and 0.05). These results showed that the mice exposed to γ-radiation alone exhibited a typical hematopoietic syndrome. On the other hand, at the end of the 21-day experiment, irradiated mice receiving oral administration of JXT showed an improved blood profile with reduced leucopenia, thrombocytopenia (platelet counts), RBC, and hemoglobin levels.WBC counts of the mice before and after irradiation were shown in P < 0.01).Subsequently, the potential of JXT to protect irradiation-induced damage of bone marrow cells was also investigated. The effect of JXT on hematopoietic tissue in irradiated mice was evaluated by HE staining in our previous study . The marIn order to identify whether JAK/STAT signal pathway can be activated by JXT, we examined the phosphorylation of JAK2, STAT5a, and STAT5b in the bone marrow after JXT treatment. The expression of pJAK2/JAK2 and pSTAT5a/STAT5a in bone marrow tissue was decreased after radiation, and the expression of phosphor-JAK2 and phosphor-STAT5a was enhanced. But the expression of pSTAT5b/STAT5b was not changed after radiation and JXT treatment .μg/ml to 40 μg/ml), the viability of NFS-60 cells was increased in a concentration-dependent manner (P < 0.05 or P < 0.01). However, the viability of NFS-60 cells in the 200 μg/ml treatment group was suppressed (P < 0.01), but no change in the JXT treatment groups  revealed an obvious increase in the percentage of viable cells when compared to the control group. After JXT treatment at different concentrations .In order to explore the effect of JXT on the apoptosis of bone marrow cells from mice upon 60 irradiation-induced damage and significantly increase the number of peripheral blood cells , body weight, spleen index, thymus index, and bone marrow cells. Together, these results confirm the enhancement of JXT on hematopoiesis.The bone marrow is the most important hematopoietic organ with hematopoietic cells at different developmental stages. A high proliferation rate of bone marrow cells is highly susceptible to irradiation-induced injury . Bone ma60 irradiation significantly reduced WBC and PLT counts in the peripheral blood of the mice; however, JXT administration can accelerate the recovery of peripheral blood cells. In order to investigate the effect of JXT treatment on bone marrow injury, the analysis of BMC counts indicated that Co60 irradiation significantly decreased the BMC number, and JXT treatment attenuated this reduction, suggesting that JXT may accomplish the protection by improving the density of BMC and increasing the number of nucleated cells in the bone marrow of irradiated mice.Normally, the peripheral blood cell number maintains relative stability, depending on the hematopoietic function of the bone marrow . Radiatiin vivo and in vitro, and the alcohol extract of JXT can promote protein phosphorylation and the activation of JAK2-STAT5 signal pathway, thereby promoting hematopoiesis. The regulation of hematopoiesis involves multiple signal transduction pathways, in which the JAK-STAT signal pathway is an important signal pathway for the signal transduction of hematopoietic growth factors [In the present study, we confirmed the radioprotective effect of JXT extract in the hematopoietic system  factors , 35. Rec factors –38. The  factors .ΔN/ΔN mast cells have a reduced level of Bcl-2 expression. Bcl-2 is a bona fide direct target of STAT5 so that STAT5 requires the N-domain for the suppression of miR15/16, induction of Bcl-2, and induction of survival signaling in mast cells and myeloproliferative neoplasms (MPNs) [In our studies, Bcl-2 was decreased after radiation treatment and increased in JXT-treated mice. The signal transducer and activator of transcription 5 (STAT5) are a critical regulator of normal and leukemic lymphomyeloid development through activating downstream early-acting cytokines, their receptors, and Janus kinases (JAKs) –43. STATs (MPNs) , 47. Thes (MPNs) –50. Bcl-Ionizing radiation can produce free radicals in cells, such as superanion, hydroxyl, and hydrogen peroxide free radicals . The actMany natural products and compounds have been found to have radioprotective effects. A novel lignin-derived polyphenolic composition with ammonium molybdate, BP-C2 mitigates radiation-induced damage in midlethal range of radiation doses. Effects are mediated by the enhancement of extramedullar hematopoiesis in the spleen and a protective effect on the intestinal epithelium . Tea pol60 radiation. JXT treatment can improve bone marrow damage and ameliorate cellular contents of the bone marrow through stimulating the JAK2/STAT5a signal pathway, thus increasing Bcl-2 expression and blocking bone marrow cell apoptosis in Co60-irradiated mice. The antioxidant capacity of JXT in the liver can be attributed to the regulatory effect on SOD, GSH-Px, and MDA. Therefore, JXT may be a promising radioprotective agent for the suppression of hematopoiesis.In our study, JXT has a protective function against hematopoietic dysfunction induced by Co


Scientific Reports 10.1038/s41598-019-53866-z, published online 02 December 2019Correction to: This Article contains a typographical error in the Introduction section.“Where this postulate holds true, the fractional base compositions  for a base in SSM-mediated SSR length variants are necessarily nearly identical and essentially independent of the total length of the SSR, given the SSR > RU length.”should read:“Where this postulate holds true, the fractional base compositions  for a base in SSM-mediated SSR length variants are necessarily nearly identical and essentially independent of the total length of the SSR, given the SSR > > RU length.”


To test two related hypotheses that elevated blood pressure (BP) is a risk factor for aortic valve stenosis (AS) or regurgitation (AR).In this cohort study of 5.4 million UK patients with no known cardiovascular disease or aortic valve disease at baseline, we investigated the relationship between BP and risk of incident AS and AR using multivariable-adjusted Cox regression models. Over a median follow-up of 9.2 years, 20 680 patients (0.38%) were diagnosed with AS and 6440 (0.12%) patients with AR. Systolic BP (SBP) was continuously related to the risk of AS and AR with no evidence of a nadir down to 115 mmHg. Each 20 mmHg increment in SBP was associated with a 41% higher risk of AS  and a 38% higher risk of AR . Associations were stronger in younger patients but with no strong evidence for interaction by gender or body mass index. Each 10 mmHg increment in diastolic BP was associated with a 24% higher risk of AS  but not AR . Each 15 mmHg increment in pulse pressure was associated with a 46% greater risk of AS  and a 53% higher risk of AR .Long-term exposure to elevated BP across its whole spectrum was associated with increased risk of AS and AR. The possible causal nature of the observed associations warrants further investigation. The CPRD database contains retrospective anonymised patient data from 674 general practices in the UK.,A total of 6 613 644 patients between 30 and 90 years with at least one BP measurement were identified. Patients entered the study at the date of their earliest blood pressure measurement (baseline) and exited the study at the earliest date of transfer out of the general practice as recorded in the database, death, end of the study period, or a record of aortic valve disease (whatever came first). To reduce the risk of reverse causation, we further excluded all individuals who, at baseline, had a prior diagnosis of cardiovascular disease (414 250), AS or AR (9417), or those prescribed lipid-lowering or anti-hypertensive medication (196 451). Cardiovascular disease was defined, as previously reportedTable S1. We excluded diagnostic codes that clearly classified aortic valve disease as congenital, or when mixed aortic valve disease was reported with no clear indication of the dominant condition. However, we included reports of rheumatic heart disease in our main analysis due to the clinical uncertainty in distinguishing between rheumatic and other non-congenital types of aortic valve disease diagnosed at middle and old age . Previous validation studies based on electronic health records have shown that the majority of clinically recorded valve disease codes are based on echocardiographic assessments and recorded cases typically represent moderate to severe severity rating.Our primary outcomes were incident reports of AS or AR, which were identified from hospital discharge reports, death registers, or primary care records, using the diagnostic codes shown in ,mmHg − DBPmmHg) as alternative exposure variables to investigate any differential associations between BP indices on risk and to enable comparison of our findings with epidemiological studies of BP associations with other outcomes. Blood pressure measurements were taken from recordings in primary care records . To take account of measurement errors resulting from variability in BP measurements over longer time, we used multiple repeated BP measurements (mean of 6.7 measurements per patient) to calculate ‘regression dilution-corrected’ or ‘usual’ BP values.,,,We defined SBP as our primary exposure because SBP has been shown to have the strongest predictive ability among most other measures of BP for most cardiovascular outcomes.2), smoking status , cholesterol ,We determined the association between SBP (and other BP indices) and each outcome using multivariable Cox regression and displayed hazard ratios (HRs) with floating absolute risks.To investigate the validity of our outcome variables and potential effect of valve severity on outcomes, we performed the following three sensitivity analyses. First, we extracted information on echocardiograms within 14 days prior to the recorded diagnosis of valve disease and stratified analyses by echocardiographically supported diagnoses of aortic valve disease vs. other reports with no recorded information on diagnostic tools during this 14-day interval. Second, we stratified our analyses according to whether or not there was a definitive report of valve replacement to assess whether observations might have been diluted due to inclusion of mild or asymptomatic cases. Third, we stratified our analyses by initial source of case reporting , assuming hospital diagnoses cases to be more severe and more likely to be verified by specialists. In addition, we performed the following analyses to test the validity of our findings. First, to test the validity of methods for measuring and modelling exposure variables and co-variates, as well as the possibility of informed presence bias , we chose incident stroke as a positive control outcome and report BP associations with stroke. Second, we excluded total cholesterol, LDL and HDL as covariates from the primary analyses due to their high proportion of missing values. Third, we assessed the impact of progressive covariate adjustment to assess the robustness of our estimates to further adjustments.P-value <0.05. Study findings are reported in accordance with the REporting of studies Conducted using Observational Routinely-collected health Data (RECORD) recommendations.Statistical significance threshold was defined as a two-sided Table 1. Overall, 1.1% of patients had diabetes at baseline and about a third were smokers. The incidence of several vascular conditions during follow-up was higher among those with higher SBP.In total, 5 392 183 individuals with a median follow-up of 9.2 [interquartile interval (IQI) 4.4–16] years and median age of 39 (IQI 32–53) years were included in the study. Of these individuals, 27 977 (0.52%) had a diagnosis of aortic valve disease (AS or AR). A total of 20 680 (0.38%) were diagnosed with only AS, and 6440 (0.12%) were diagnosed with only AR and were included in the analyses. The mean (standard deviation) age at the time of diagnosis were 64.2 (12.1) years and 57.0 (16.5) years, for AS and AR, respectively. Patient characteristics by SBP categories are shown in Figure ), and a 1.38 times higher risk of AR   with no evidence of a threshold below or above which the associations were different. Compared with the reference category of SBP ≤120 mmHg, patients with SBP ≥161 mmHg were more than twice as likely to be diagnosed with AS  and about twice more likely to be diagnosed with AR .When SBP was analysed as a continuous variable across all age groups, each 20 mmHg increment in SBP was associated with a 1.41 times higher risk of AS  1.38–1.45) ; whereas in patients aged 71–90 the risk was 1.23 times higher  . The corresponding hazard ratios for AR were 1.86 (CI 1.66–2.07) in the youngest age group and 1.22 (CI 1.10–1.35) in the oldest age group . Proportional differences did not differ between men and women or by BMI categories for both AS and AR .Adjusted hazard ratios among subgroups are shown in P for heterogeneity 0.51 for AS and 0.07 for AR); when hospital discharge reports were compared with outpatient diagnoses (P for heterogeneity 0.58 for AS and 0.13 for AR); or when patients with a definitive report of valve replacement were compared with those with no clear reports of valve replacement (P for heterogeneity 0.24 for AS and 0.26 for AR) .Associations were similar when echocardiographically-supported reports were compared with reports without information on imaging , and a 1.53 times higher risk of AR . Each 10 mmHg higher DBP was associated with a 1.24 times greater risk of AS  but not significantly with AR . However, associations differed by age group . In addition, when PP and SBP were simultaneously entered into the models, the overall HR for SBP was substantially attenuated for AS  and was no longer significant for AR . In these SBP adjusted models, for a given SBP, DBP was negatively associated with AR or AS .The results of additional sensitivity analyses are reported in In a population of adults, there was a significant association between higher SBP and increased risk of AS and AR. Proportional increases in risk for a given difference in SBP were similar throughout the range of SBP with no evidence of a threshold below or above which the relationship changed. Overall, a 20 mmHg higher SBP was associated with an approximately 40% higher risk of AS or AR, with stronger associations observed in younger groups.A few cross-sectional studies have previously reported that patients with hypertension also have a higher chance of concomitant AS.,,,,,To our knowledge, our study is the first longitudinal study with enough power to reliably report on age-specific associations across the whole spectrum of typical BP levels for both AS and AR. The continuous log-linear relationships between SBP and risk of aortic valve disease observed in our study are consistent with other large-scale epidemiological studies that have examined associations with other vascular outcomes.Previous clinical and experimental studies have suggested that mechanical stress related to high BP plays an important role in initiating the process of aortic valve sclerosis.absolute risk of AR increased less strongly with age. Thus, one could speculate that younger patients, who develop AR, have a higher predisposition for aortopathy or a connective tissue disorder trait, and hence respond to elevated BP and PP faster, leading to aortic root dilatation and then AR. In contrast, people without such traits may predominantly respond with arterial stiffening and aortic valve calcification over several years that eventually manifests as AS. Future mechanistic studies could explore the differences in BP pathways on AS and AR.Whilst these interdependent processes have been mainly postulated to explain the pathophysiology of AS, it is not entirely clear why the same exposure, namely elevated BP, is more likely to lead to AR in some patients and to AS in others. The age-specific associations in the present study show that compared with AS, the http://www.ukbiobank.ac.uk/2017/02/causes-and-consequences-of-valvular-heart-disease/) that are less prone to reverse causality and confounding are ideally suited to investigate the causal relationship of the observed associations in this study. A further limitation of our study is that our analyses were based on routinely collected data from linked electronic health records, which might be more prone to measurement errors of exposure and outcome variables. However, several steps were undertaken to reduce the effect of such possible measurement errors (e.g. regression dilution correction for our exposure variable) and to validate our findings . Finally, studies based on clinically reported events are more likely to only capture functionally relevant and symptomatic disease states and are likely to underestimate rates compared to epidemiological studies that screen the entire population for subclinical and clinical disease. Thus, the observed associations may not be generalizable to less severe and subclinical cases of aortic valve disease.As an observational study, we are unable to entirely rule out uncontrolled confounding or reverse causality. Indeed, given the potential bidirectional relationship between aortic stiffness and elevated BP,In conclusion, we found significant associations between higher BP and pulse pressure and increased risk of AS and AR. These findings suggest that risk of aortic valve disease might be modifiable but further research, ideally from meta-analyses of BP lowering trials or Mendelian randomization studies are needed to assess the causal nature of the observed associations.This project was supported by the ‘Oxford Martin School grant for the Deep Medicine programme’. K.R., H.M., and F.R. are supported by the ‘National Institute of Health Research (NIHR) Oxford Biomedical Research Centre’. J.T. is funded by ‘The Rhodes Trust’ and ‘The Clarendon Council’. M.W. is supported by a Principal Research Fellowship from the ‘Australian Health and Medical Research Council’.Conflict of interest: none declared.Supplementary DataClick here for additional data file.


Coenzyme Q10 was also found to bind and activate both PPARs in a similar fashion, suggesting an endogenous role in relaying the states of mitochondria, peroxisomes and cellular redox to the two receptors. Testing idebenone in a mouse model of type 2 diabetes revealed the ability to reverse fatty liver development. These findings indicate new mechanisms of action for both PPARα and PPARγ, and new potential treatment options for nonalcoholic fatty liver disease (NAFLD) and steatosis.Current peroxisome proliferator-activated receptor (PPAR)-targeted drugs, such as the PPARγ-directed diabetes drug rosiglitazone, are associated with undesirable side effects due to robust agonist activity in non-target tissues. To find new PPAR ligands with fewer toxic effects, we generated transgenic zebrafish that can be screened in high throughput for new tissue-selective PPAR partial agonists. A structural analog of coenzyme QThis article has an associated First Person interview with the first author of the paper. Summary: A zebrafish screen identifies a novel PPARα/γ ligand, idebenone, with potential for treatment of fatty liver diseases, as seen by testing it in a mouse model of type 2 diabetes. Obesity and associated metabolic disorders have become an epidemic worldwide with some of the most costly and deadly outcomes . Obese iPPARA, PPARD and PPARG  as an animal model to study development and disease has revealed many advantages that make it ideal for in vivo drug testing and screening (10 (CoQ10), suggested that CoQ10 may serve as an endogenous ligand for PPARs. Indeed, we found that CoQ10 and many of its derivatives serve as direct partial agonists, not only for PPARγ but also for PPARα. While idebenone is able to regulate the expression of a subset of known PPARα and PPARγ target genes, unlike TZDs, it does not induce lipogenesis. In obese mice, treatment with idebenone resulted in reversal of fatty liver development, whereas treatment with rosiglitazone exacerbated the problem.The use of zebrafish  reporter (We have expanded our previously described ‘ligand trap’ (LT) system for human NR studies  to inclureporter .Fig. 1.Fig. S1; Fig. S1; Fig. S1. Note that the eGFP reporter protein, which includes a nuclear localization signal, results in dot-like expression patterns .The three PPAR fish lines generated here respond to endogenous zebrafish small molecules (i.e. metabolites) and cofactors in a tissue-specific manner B. At 2 din vivo drug discovery, we conducted a screen to identify PPARγ-selective partial agonists (Table S1). Among these were the well-known PPARγ TZD drugs rosiglitazone, troglitazone and pioglitazone, as well as several nonsteroidal anti-inflammatory drugs and capsaicin , a natural component of hot peppers. Fenofibrate, a PPARα selective agonist known to activate PPARγ at micromolar concentrations, was also identified . The PPARγ antagonist GW9662 was able to block this activation, consistent with direct competition within the LBD pocket  core with variable numbers of isoprenoid subunits in their side chains, as indicated by the subscript number , with similar results to those induced by LipoQ . These co-purifications confirmed strong binding of idebenone and CoQ10 to both the PPARα and PPARγ LBDs, but not to PPARδ . Control nano-electrospray ionization (nanoESI) MS spectra obtained from LBD incubated in the absence of idebenone show no presence of a complex .To test whether idebenone and CoQto PPARδ A. As an to PPARδ B. Two madb/db) mice fed on a normal diet supplemented with either vehicle (DMSO), rosiglitazone (10 mg/kg body weight) or idebenone (900 mg/kg or 1800 mg/kg). Although these doses may seem relatively high, they are only two to three times higher  were ordered from Jackson Laboratories (#000697) at 7 weeks of age. Mice were housed individually with free access to food  and water on a 14-h light/dark cycle (6 a.m. to 8 p.m. light). 10% sucrose and 100 µl Tween-80 was added to 300 g food that was supplemented with either vehicle (DMSO), 10 mg/kg body weight rosiglitazone, 900 mg/kg idebenone or 1800 mg/kg idebenone. Food was exchanged daily. Mice were randomized by body weight into four treatment groups (n=8). Dosing occurred for 21 days. Mice were euthanized by exsanguination under anesthesia with isoflurane and livers were fixed in neutral buffered formalin for 24 h. The next day, samples were transferred to 10% sucrose (for another 24 h) and then 30% sucrose. Oil-O-Red staining was performed through the Pathology Research Program at the University Health Network in Toronto.Male C57BL/6 db/db livers were homogenized in RIPA buffer, and protein concentration was quantified using a BCA protein assay kit  followed by measurement using a TBARS assay kit according to the manufacturer's instructions .Thiobarbituric acid reactive substances (TBARS) were measured as a surrogate marker of oxidative stress. 100 mg of 2/methanol. The organic phase was separated and brought up to 5 ml with chloroform. Dried aliquots of standards and samples were re-dissolved in 10 µl of 1:1 cholorform:Triton X-100 and evaporated overnight. Samples were assayed for cholesterol and triglycerides using commercial colorimetric assays (Thermo).Liver lipids were extracted using the Folch method as previously described . Briefly4 (2%) in a 0.1 M cacodylate buffer. After washing in 0.1 M cacodylate buffer, the cells were dehydrated through an ethanol series. The material was then penetrated by an increasing series of Spurr resin diluted in acetone. Finally, the tissues were embedded in Spurr resin. Both the thick and ultrathin sections were sectioned with an Ultracut Leica UC6 ultramicrotome  using a diamond knife. Thick sections (0.5 μm) were placed on the slides, stained with Toluidine Blue, and imaged with a Leica DMRA2 fluorescence microscope using a Q-Imaging Retiga EX camera and Openlab 3.1.7 software. Ultrathin sections (80 nm) were placed onto copper grids coated with Formvar film, stained with uranyl acetate and lead citrate solutions, and examined with a Zeiss Libra 120 transmission electron microscope .Tissues were fixed in 2.5% glutaraldehyde in a 0.1 M cacodylate buffer and post-fixed in OsOdb/db livers using RNA STAT-60 , treated with DNase I  and reverse transcribed into cDNA with random hexamers using the High Capacity Reverse transcription system . Primer sequences are listed in Total RNA was extracted from 100 mg of


AR) gain identifies metastatic castration-resistant prostate cancer (mCRPC) patients with worse outcome on abiraterone/enzalutamide, but its relevance in the context of taxane chemotherapy is unknown. We aimed to evaluate whether docetaxel is active regardless of plasma AR and to perform an exploratory analysis to compare docetaxel with abiraterone/enzalutamide. This multi-institutional study was a pooled analysis of AR status, determined by droplet digital polymerase chain reaction, on pretreatment plasma samples. We evaluated associations between plasma AR and overall/progression-free survival (OS/PFS) and prostate-specific antigen (PSA) response rate in 163 docetaxel-treated patients. OS was significantly shorter in case of AR gain , but not PFS  or PSA response . We investigated the interaction between plasma AR and treatment type after incorporating updated data from our prior study of 73 chemotherapy-naïve, abiraterone/enzalutamide-treated patients, with data from 115 first-line docetaxel patients. In an exploratory analysis of mCRPC patients receiving first-line therapies, a significant interaction was observed between plasma AR and docetaxel versus abiraterone/enzalutamide for OS  and PFS . Specifically, we reported a significant difference for OS favoring abiraterone/enzalutamide for AR-normal patients  and a suggestion favoring docetaxel for AR-gained patients . These data suggest that AR-normal patients should receive abiraterone/enzalutamide and AR-gained could benefit from docetaxel. This treatment selection merits prospective evaluation in a randomized trial.Plasma androgen receptor (AR) predicted outcome in metastatic castration-resistant prostate cancer (mCRPC) patients treated with docetaxel, and we performed an exploratory analysis in patients treated with docetaxel or AR-directed drugs as first-line mCRPC therapy. We showed that plasma AR normal favored hormonal treatment, whilst plasma AR-gained patients may have had a longer response to docetaxel, suggesting that plasma AR status could be a useful treatment selection biomarker.We investigated whether plasma androgen receptor ( AR)-normal status favored treatment with abiraterone or enzalutamide, whilst docetaxel benefit was unrelated to plasma AR. AR testing in plasma may have clinical utility for treatment selection in mCRPC.Analysis of metastatic castration-resistant prostate cancer (mCRPC) patients receiving first-line therapy showed that plasma androgen receptor ( AR aberrations and worse outcome with AR-directed drugs AR status in the context of taxanes is unknown.There are currently several approved life-prolonging therapies for the treatment of metastatic castration-resistant prostate cancer (mCRPC), including androgen receptor (AR)-directed drugs and taxanes. Plasma DNA analysis from mCRPC patients has suggested potential clinical applicability with an association between plasma AR status with outcomes in mCRPC patients treated with docetaxel. Additionally, we aimed to perform an exploratory analysis to compare the difference in outcome by plasma AR status for patients treated either with first-line docetaxel or AR-directed therapy.We here aimed to evaluate the association of plasma 2 every 3 wk with prednisone 5 mg twice daily for a maximum of 10 cycles for mCRPC AR copy number (CN) droplet digital polymerase chain reaction assay are described in the Supplementary material.Plasma samples were collected, with the primary aim of biomarker evaluation, from mCRPC patients, treated with standard-dose intravenous docetaxel 75 mg/mAR status in plasma collected from 166 docetaxel-treated mCRPC patients prior to first- or second-line mCRPC therapy . The median number of docetaxel cycles was the same in AR-normal and AR-gained patients . The median follow-up period of alive patients was 24 mo. As 98% of the deaths were prostate cancer related, only overall survival (OS), and not cancer-specific survival, was analyzed. The median OS was 14 mo  for AR-gained patients and 22 mo (95% CI 20–29) for AR-normal patients. Median progression-free survival (PFS) was 7 mo (95% CI 5–8) in AR-gained patients and 7 mo (95% CI 6–8) in AR-normal patients. OS was significantly shorter in AR-gained versus AR-normal patients , but no significant difference was observed for PFS  or prostate-specific antigen (PSA) decline ≥50% . When comparing AR-normal with AR-gained patients in each treatment group, serum LDH and PSA were significantly higher in AR-gained patients treated with abiraterone/enzalutamide and serum alkaline phosphatase was significantly higher in plasma AR-gained patients treated with docetaxel (Supplementary Table 2).Next, we selected the 115 patients treated with docetaxel as first-line therapy and in an exploratory, analysis compared them with 73 previously described patients treated with first-line abiraterone/enzalutamide A [5]. A AR status was investigated using a multivariable Cox proportional hazard model, which showed a significant treatment interaction with AR status for both OS  and PFS . The median follow-up period for alive patients of the abiraterone/enzalutamide cohort was 32 mo.The interaction of docetaxel or abiraterone/enzalutamide therapy and AR CN status are depicted in AR-normal patients treated with abiraterone/enzalutamide had a significantly lower risk of death  and progressive disease  when compared with those treated with docetaxel, and in AR-gained patients there was a suggestion toward a lower risk of death  and PFS .The estimated median OS and PFS as a function of treatment and AR aberrations is associated with worse outcome on abiraterone/enzalutamide AR gain in docetaxel-treated patients was associated with significantly shorter OS. This emphasizes the urgent clinical need of alternative treatments for AR-gained patients AR gain status is associated with resistance to taxanes in an abiraterone/enzalutamide-naïve population and, to avoid the influence of possible cross-resistance events on the interpretation of survival data, we compared it with the effect seen in taxane-naïve abiraterone/enzalutamide-treated patients AR status in treatment-naïve docetaxel-treated patients introduces the hypothesis that AR-gained patients would derive greater benefit from treatment with taxanes in preference to abiraterone/enzalutamide. However, we recognize some limitations of our study, including the significantly different durations of median follow-up of alive patients between the docetaxel and the abiraterone/enzalutamide cohort ; the relatively modest sample size of the cohorts, especially of AR-gained patients treated with abiraterone/enzalutamide (n = 10); and the retrospective, nonrandomized design. The majority of patients were treated with taxanes in centers when abiraterone or enzalutamide were not widely available prior to chemotherapy. Nonetheless, there could be a bias due to patient selection, given the different toxicity profiles of taxanes compared with AR-targeting drugs. Additionally, detection of an AR-gained clone may be more likely at higher circulating tumor fraction that in itself is prognostic; this could bias the ability to ascertain the predictive value of plasma AR with AR-targeting drugs but would not change the interpretation of the absence of difference in our treatment-naive taxane-treated cohort. Lastly, we only considered AR gain, but other concurrently assessed AR aberrations (somatic point mutations or splice variants) could provide additional or overlapping information. Our findings suggest that AR gain detected in plasma is associated with resistance to abiraterone/enzalutamide but not with taxanes when used in the first-line setting. In conclusion, prospective randomized trials are warranted to validate the utility of plasma AR status for treatment selection in mCRPC patients.Metastatic CRPC patients can be treated with docetaxel or AR-targeting therapies as first-line therapy. We and others have reported that detection of circulating Author contributions: Gerhardt Attard had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Study concept and design: Conteduca, Jayaram, Wetterskog, Castro, Gonzalez-Billalabeitia, Olmos, Attard, De Giorgi.Acquisition of data: Conteduca, Jayaram, Romero-Laorden, Wetterskog, Salvi, Gurioli, Castro, Wingate, Casadio, Gonzalez-Billalabeitia, Olmos, Attard, De Giorgi.Analysis and interpretation of data: Conteduca, Jayaram, Romero-Laorden, Wetterskog, Castro, Gonzalez-Billalabeitia, Olmos, Attard, De Giorgi.Drafting of the manuscript: Conteduca, Wetterskog, Attard.Critical revision of the manuscript for important intellectual content: All authors.Statistical analysis: Scarpi.Obtaining funding: Gonzalez-Billalabeitia, Olmos, Attard, De Giorgi.Administrative, technical, or material support: Maugeri.Supervision: Attard.Other: Provided patients—Conteduca, Jayaram, Romero-Laorden, Castro, Marin-Aguilera, Lolli, Schepisi, Farolfi, Maugeri, Medina, Puente, Vidal, Morales-Barrera, Villa-Guzmán, Hernando, Rodriguez-Vida, González-del-Alba, Mellado, Gonzalez-Billalabeitia, Olmos, Attard, De Giorgi.Financial disclosures: Gerhardt Attard certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript , are the following: G. Attard reports receiving commercial research grants from Janssen, Arno Therapeutics, and Innocrin Pharma; has received honoraria and/or travel support from the speakers’ bureaus of Janssen, Astellas, Sanofi-Aventis, and Roche/Ventana; has received travel support from Pfizer, Abbott Laboratories, Bayer Healthcare, and Essa Pharmaceuticals; has ownership interest (including patents) in The Institute of Cancer Research Rewards to Inventors; and is a consultant for/advisory board member of Janssen-Cilag, Veridex, Bayer Healthcare, Roche/Ventana, Astellas, Medivation, Pfizer, Novartis, Millennium Pharma, Abbott Laboratories, and Essa Pharma. V. Conteduca, E. Gonzalez-Billalabeitia, and U. De Giorgi received speaker honoraria or travel support from Astellas, Janssen-Cilag, and Sanofi-Aventis. V. Conteduca received consulting fee from Bayer. D. Olmos received research funding from Janssen and Bayer. J Puente reports receiving commercial research grants from Pfizer and Astellas; has received honoraria and/or travel support from the speakers’ bureaus of Pfizer, Astellas, Janssen, MSD, Roche, Bristol, AstraZeneca, Boehringer, Pierre Fabre, Kyowa, Celgene, Lilly, Merck, Ipsen and Eisai; and is a consultant for Pfizer, Astellas, Janssen, MSD, Bayer, Roche, Bristol, AstraZeneca, Boehringer, Novartis, Clovis, Ipsen, EssaPharma, Eisai and Sanofi. B. Mellado reports receiving commercial research grants from Roche and Bayer; has received travel support from Pfizer and Janssen; and is a consultant for/advisory board member of Roche, Sanofi, Janssen, Astellas Oncology, Pfizer, Novartis, Bristol-Myers Squibb and Ipsen. M. Marín-Aguilera has received travel support from Bristol-Myers Squibb. A. Rodriguez-Vida reports receiving commercial research grants from Takeda, MSD and Pfizer; has received honoraria and/or travel support from the speakers’ bureaus of Janssen, Astellas, Sanofi-Aventis, Bayer, BMS and Roche. N. Romero Laorden has received honoraria and/or travel support from Bayer, Astellas, Janssen-Cilag, and Sanofi-Aventis. M.J. Mendez Vidal has received honoraria and /or travel support from Janssen-Cilag, Bayer Healthcare, Sanofi Aventis, Astellas Medivation, Roche, Novartis and Pfizer. R Morales-Barrera has received honoraria and/or travel support from Bayer, Roche, Astellas, Janssen-Cilag, MSD and Sanofi-Aventis. A. González del Alba has received honoraria and/or travel support from Sanofi Aventis, Astellas Medivation, Janssen-Cilag, Bayer, BMS, Pfizer, Novartis, MSD, Roche, EUSA Pharma and Eisai. E. Castro reports receiving commercial research grants from Astra Zeneca, Bayer, Janssen; has received honoraria and/or travel support from the speakers’ bureaus of Astra Zeneca, Astellas, Bayer, Janssen, Pfizer, Bristol-Myers and Roche; and is an advisory board member of Astellas, Bayer and Janssen. S. Hernando Polo has received honoraria and/or travel support from Sanofi Aventis, Astellas Medivation, Janssen-Cilag, Bayer, BMS, Pfizer, Novartis, MSD and Roche. A. Medina has received honoraria and/or travel support from BMS, Janssen-Cilag, Bayer healthcare, Sanofi Aventis, Astellas Medivation, Roche, Novartis and Pfizer. D. Olmos has a compensated advisory role for Astellas, Astra-Zeneca, Bayer, Clovis, Genetech/Roche, Janssen, and uncompensated advisory role for BioOncotech, Tokai; has received a speaker fee from Astellas, Bayer, Janssen, Sanofi, and travel support from Astellas, Bayer, Janssen, Roche; has received research funding (to the institution): Astra-Zeneca, BioOncotech, Bayer, Janssen. No potential conflicts of interest were disclosed by the other authors.Funding/Support and role of the sponsor: V. Conteduca was funded by a European Society of Medical Oncology Translational Clinical Research Fellowship. A. Jayaram is supported by a grant from the Medical Research Council (MR/P002072/1). G. Attard is supported by a Cancer Research UK Advanced Clinician Scientist Grant (A22744). This work was funded in part by Prostate Cancer UK (PG12-49), the “Instituto de Salud Carlos III” (ISCII) PI16/01565 grant. E. Gonzalez-Billalabeitia was funded by a grant from the “Instituto de Salud Carlos III” (ISCIII) PI15/01499. N. Romero-Laorden was funded by a grant from the “Instituto de Salud Carlos III” (CM14-00200). E. Castro is supported by a Prostate Cancer Foundation Young Investigator Award (2017). E. Castro and D. Olmos are supported by grants from the Ministerio de Economía, Industria y Competitividad . B. Mellado and M. Marin-Aguilera work were supported by the Instituto de Salud Carlos III-Subdirección General de Evaluación y Fomento de la Investigación (PI12/01226 and PI15/676) and co-funded by the European Regional Development Fund. Funding from CERCA Programme/Generalitat de Catalunya is gratefully acknowledged. During the conduct of the study, E. Castro was supported by a grant from the Ministerio de Educación, Cultura y Deportes (CAS17/00182). The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding authors had full access to all data and had the final responsibility for the decision to submit for publication.Acknowledgments: We thank the participating men and their families who suffered from metastatic prostate cancer and nonetheless gave the gift of participation so that others might benefit.


AR aberrations with clinical outcome in CRPC patients treated with AR-directed therapies, whereas no association was observed in patients treated with taxanes. This suggests the potential for using plasma AR as a biomarker for selecting treatment, i.e., hormone therapy or chemotherapy, and the possibility of modulating taxane dose. In recent years, plasma AR status has also been investigated in association with novel agents, such as 177Lu-PSMA radioligand therapy and PARP inhibitors. This review will focus on AR testing in plasma that may have clinical utility for treatment selection in advanced prostate cancer.The therapeutic landscape of prostate cancer has expanded rapidly over the past 10 years, and there is now an even greater need to understand the biological mechanisms of resistance and to develop noninvasive biomarkers to guide treatment. The androgen receptor (AR) is known to be involved in the pathogenesis and progression of prostate cancer. Recently, highly sensitive next-generation sequencing and PCR-based methods for analyzing androgen receptor gene (AR) copy numbers (CN) and mutations in plasma were established in cell-free DNA (cfDNA) of patients with castration-resistant prostate cancer (CRPC) treated with different drugs. The study of cfDNA holds great promise for improving treatment in CRPC, especially in the advanced stage of the disease. Recent findings showed the significant association of plasma  Prostate cancer is the most frequent cancer in men and the second most common cause of cancer-related death in Western countries . The disThe majority of hormone-naïve prostate cancer (HNPC) patients characterized by non-castrate testosterone levels who undergo androgen deprivation treatment (ADT) may become castration-resistant (CRPC). CRPC is defined as radiographic progression and/or increase in serum prostate-specific antigen (PSA) levels despite a low testosterone concentration <50 ng/mL) ,4 Despit0 ng/mL ,There are several therapeutic options available for prostate cancer, including radiotherapy, ADT, second generation AR-signaling inhibitors, chemotherapy, and bone-targeted agents. Numerous trials are also ongoing for the approval of novel targeted drugs .In this paper, we review the literature on the role of AR detected in the plasma of patients with prostate cancer as a novel prognostic and predictive biomarker in different settings of prostate cancer.Currently, prostate cancer treatment decisions (curative treatments or active surveillance) in the setting of localized disease are still based almost exclusively on histological architecture (Gleason score) , prostatDuring disease evolution, further clinical factors are used for prognostication and risk stratification. In patients diagnosed with nonmetastatic disease, baseline PSA levels, PSA velocity, and PSA doubling time are associated with clinical outcome and can be used to select patients whose follow-up requires imaging studies .Although there is no universally accepted prognostic classification for patients with metastatic HNPC (mHNPC), a recent study  identifiSeveral prognostic nomograms, especially in CRPC, have been developed to predict overall survival (OS) and to assess risk stratification in clinical trials ,18,19,20One important aspect to consider in the evaluation of treatment outcome is the biological heterogeneity of prostate malignancy. Thus, disease stratification based on molecular signatures is warranted in prostate cancer, as it has also been shown to aid the prognostic evaluation and management of other epithelial cancers such as breast cancer .Over the years, many studies have consistently shown that the majority of CRPCs are still dependent on AR signaling, which may also play a role in the transition from early to advanced disease, even at low concentrations of androgens. One of the most important objectives of recent molecular studies has been to identify synergistic factors which, together with AR, are involved in prostate cancer progression.TMPRSS2–ERG fusion (61.5%), TP53 mutation or deletion (21.1%), PTEN deletion (11.5%), FOXA1 (11.5%), and SPOP (7.7%) mutation, with no significant enrichment in post-treated specimens.The phase III CALGB 90203 trial  aimed toAR aberration was observed in the neoadjuvant setting, probably because AR amplification and mutations are typically later events in the process of prostate cancer progression (present in 60% of metastatic disease)  or malignant prostate diseases. Nevertheless, ctDNA, which represents less than 3.0% of total cfDNA, is about three to four times higher in cancer patients than in healthy individuals  Figure .PTEN-deleted patients [Plasma ctDNA analysis is particularly useful for detecting the evolving therapeutic landscape of mCRPC and for monitoring unpredictable cross-resistance phenomena among agents targeting different pathways such as AR signaling  ,22,68,69patients . ConsequThe estimation of ctDNA fraction has also been acknowledged as a predictor of prognosis and treatment resistance in mCRPC, whereas there is only preliminary evidence of this in HNPC. Recently, Chi et al. did not observe an increase in the acquisition of AR aberrations  from cell-free DNA and cfRNA using next-generation sequencing and PCR, respectively, in mHNPC patients treated with apalutamide plus ADT .Moreover, ctDNA tumor fraction may be underestimated in some cases because of the emergence of lesions not included in the targeted gene custom panel used for estimating tumor content.Despite the fact that ctDNA fraction can also be considered as a useful prognostic biomarker in castration-resistant disease, it should be borne in mind that plasma DNA sequencing is characterized by several limitations involving costs, genome coverage and sensitivity to low ctDNA fraction. Nevertheless, the main limitation of these recent studies on cfDNA concentration remains its inability to specifically quantitate tumor-derived cfDNA fraction in plasma DNA.However, in general, leveraging plasma specimens collected from different stages of prostate cancer may improve the characterization of key targets for prostate cancer, such as AR, in the real-time and genomic sub-classification of CRPC.The persistent activation of the AR pathway, including AR gene amplification and mutations, and the generation of truncated splice variants lacking the ligand-binding domain (LBD) , represeAR aberrations have been studied using targeted-next generation sequencing (NGS). In several studies a customized AmpliSeq targeted gene panel including AR and other gene targets was sequenced on an Ion Torrent Personal Genome Machine or Proton [AR copy number variation and point mutation detection (with a sensitivity of 98–99%). Recently, our group identified a multiplex digital droplet PCR (ddPCR) to assess AR copy number and mutations in plasma cfDNA [p < 0.001, 95% CI, −0.015 to 0.016, respectively) [Plasma r Proton ,64. SubsAR status are summarized in Other more common methods available for detecting ctDNA and analyzing plasma AR copy number in advanced disease, mainly in a post-docetaxel setting. Romanel et al. [AR gain and point mutations were mutually exclusive, and that AR copy number was unchanged during abiraterone treatment. Moreover, the onset of T878A or L702H AR amino acid changes was reported in 13% of samples at progression on abiraterone. The association between plasma AR aberrations and outcome was very interesting, revealing a letter reduction in PSA in AR-aberrant patients than in AR-normal patients, and significantly shorter OS [hazard ratio (HR) 7.33, 95% CI 3.51–15.34, p = 1.3 × 10−9) and progression-free survival (PFS) . Subsequently, Conteduca et al. assessed the role of plasma AR not only in a post-docetaxel group, but also in a chemotherapy-naïve setting [AR gain. Eight (11%) post-docetaxel but no chemotherapy-naïve abiraterone-treated patients were AR mutant. Chemotherapy-naïve and post-docetaxel AR-gained patients had a worse outcome . Patients with AR mutations also had a significantly shorter OS . These data were validated in a secondary cohort of 94 chemotherapy-naïve patients treated with enzalutamide in 16 institutions in the PREMIERE trial (NCT02288936). Eleven (12%) had AR gain. AR-gained patients had worse biochemical PFS , radiographic PFS , OS . Subsequently, plasma AR was associated with different prognostic/predictive factors to identify additional mechanisms of resistance to AR-directed therapies.AR gene aberrations have been shown to be uncommon in the early stages of prostate cancer, but very frequent in CRPC. In fact, NGS- and PCR-based studies have focused in particular on l et al.  sequence setting . A primaAR gain in CRPC patients receiving AR signaling inhibitors, Conteduca et al. evaluated its association with other biomarkers, such as choline uptake in 18F-fluorocholine positron emission tomography/computed tomography (FCH–PET/CT) [AR gain and some FCH–PET/CT parameters (tumor lesion activity and metabolic tumor volume), both of which can be considered as independent factors of PFS and OS in multivariate analysis.After establishing the prognostic/predictive role of plasma –PET/CT) ,79. EighAR copy number by ddPCR on the outcome of 256 CRPC patients treated with AR-directed therapies [AR gain and high pre-treatment levels of serum and LDH were viewed as independent predictors of PFS and OS, suggesting the utility of assessing serum CGA before starting abiraterone or enzalutamide treatment together with plasma AR status to identify a subgroup of CRPC patients who are not likely to respond to AR-directed agents but have no AR aberrations.An additional association was observed between plasma AR and serum concentration of chromogranin A (CGA) ,81, whicherapies . Plasma AR gain in patients treated with taxanes was explored in two multi-institutional studies [AR and OS/PFS and PSA response rate in 163 docetaxel-treated patients, reporting only a significant shorter OS in AR-gained patients . In addition, the same authors studied the interaction between plasma AR and treatment type in 73 patients of the abiraterone/enzalutamide-treated group . In addition, Conteduca et al. performed an exploratory analysis of an AR-gained cabaxitaxel cohort, revealing that these patients, treated with an initially reduced dose of cabazitaxel, had a significantly shorter OS/PFS and would probably need a standard initial dose of cabazitaxel. Plasma AR status was shown to have a potential clinical utility in patients being considered for treatment with taxanes.Docetaxel and cabazitaxel represent the most widely used chemotherapies for patients with mCRPC. They have a similar function, preventing mitosis and microtubule-dependent trafficking and eliciting apoptosis. In addition, they may block nuclear translocation and AR activity . The rol studies ,87 that or study ) and 115177Lu–PSMA–617, plasma AR gene status was also assessed in association with clinical outcome [AR gene gain and 5 of 25 (20%) with AR normal (p = 0.0002)). The OR for patients with early disease progression and AR gain was 16.00, 95% CI 3.23–79.27, p = 0.0007. These preliminary data suggest that plasma AR status determined by ddPCR could be useful to identify mCRPC patients resistant to 177Lu–PSMA–617.In a phase 2 clinical trial (NCT03454750) of mCRPC patients treated with  outcome . Early pAR status could thus help to increase clinical benefit in a broader population of mCRPC.Recently, preclinical data indicated a synergy between olaparib and androgen pathway inhibitors. Specifically, a randomized, double-blind, placebo-controlled, phase 2 trial assessed the efficacy of olaparib plus the androgen pathway inhibitor abiraterone in patients with mCRPC regardless of HRR mutation status, results showing the clinical benefit of the combination compared with abiraterone alone . Plasma AR mutations was very low, underlining that they could be not considered as a helpful biomarker to identify acquired resistance to apalutamide.In addition, recent studies assessed the role of AR mutations, e.g., F877L and T878A, in the resistance to next-generation AR-directed therapies, such as apalutamide  and daroRecent preclinical evidence  has alsoAR status, given its role in patients treated with different drugs, could become an important predictive biomarker to guide treatment selection in mCRPC, along with other factors such as PSA level. Indeed, plasma AR status has also recently been assessed in the context of a novel potential prognostic index model for survival including clinical, imaging, and molecular factors [The current therapeutic and molecular landscape of prostate cancer is rapidly changing and has led to novel findings not only in CRPC, but also in earlier stages of the disease. In this context, the identification of predictive and prognostic biomarkers is essential, and it is possible thanks to the use of plasma DNA to monitor tumor dynamics and treatment outcome. In prostate cancer, plasma  factors .AR status in prostate cancer, could substantially improve prognostication and stratification of these patients. A prospective evaluation of plasma AR as a biomarker of treatment selection is warranted with a possible validation cohort, and there is an urgent need to assess the utility of plasma AR not only in advanced patients, but also in those with earlier stages of the disease.In conclusion, the integration of different biomarker strategies, including genomics, with plasma


The results suggested that the total ARs content on the seed surface can be used to predict their content in whole-grain flour. Without affecting the seed germination rate and damaging seed structure, we demonstrate that a non-destructive extraction approach is an appropriate and useful method, facilitating the development of rapid screening methods suitable for use in wheat breeding.Alkylresorcinols (ARs) are beneficial for human health and can be used as biomarkers for whole-grain cereal intake. In previous studies, ARs content in whole-grain flour was determined by milling the seeds into powder, thus destroying their structure. In this paper, we adopted a non-destructive extraction approach. Chloroform and ethyl acetate extraction methods were carried out to extract lipids from the seed surface and whole-grain flour of 36 wheat varieties in China, respectively. GC-MS analysis identified chemical compounds in which ARs were the major compounds for all the samples. The average total content of ARs (624.223 µg/g) in whole grains was much higher than that on seed surfaces (4.934 µg/g), with a positive correlation (r = 0.863,  The numbers of homologues in these series was also much lower than that in SSL b. All thIn our study, ARs were found in all 36 wheat samples. Total ARs and AR homologues content on seed surface and in whole grains from 36 samples grown in China during the 2017 crop year, are shown in On the other hand, the relative abundances of AR homologues in whole grains and seed surfaces were different. AR 23:0 was the predominant homologue on seed surfaces of all wheat samples , with lap < 0.001) between the major homologue (AR 21:0) and total ARs was observed in whole grains. Furthermore, contents of all the homologues were well correlated, except for AR 15:0 (p < 0.001) between the major homologue (AR 23:0) and total ARs was also observed. As expected, all the homologue contents correlated well with each other, except for AR 19:0 and AR 25:0; however, the reason for this correlation is unknown  between these data, and a good correlation  between total AR content in whole grains and seed surface was confirmed (p < 0.001). To determine the relationship between the AR content in whole grains and that on seed surfaces, we also performed correlations analysis between the two data sets. As shown in onfirmed . At the 2 = 0.683, p < 0.001) relationship between total ARs on seed surfaces and that in whole grains. We could predict total ARs concentrations in whole grains based on linear regression equation. There were very good linear relationships between AR 23:0 on seed surfaces and AR 21:0 in whole grains   a. The abTo validate which linear regression equation (predictive model) was feasible for predicting AR content in whole grain flour, agreements between ARs (AR 21:0) content of whole grain samples determined by GC and predictive models were found . Two met2 = 0.826, p < 0.001). At the same time, the Bland–Altman analysis showed that more than 92% of variance points fell in the 95% confidence intervals; thus, the two methods were said to agree. Although there was a weak trend  showing increased difference between the two methods . The results showed that there were statistically nonsignificant differences in the germination rates of the normal seeds compared to the chloroform-soaked seeds . TherefoThis is the first time quantitative analysis of the lipids in whole grains and seed surfaces is reported. Furthermore, correlation analyses were conducted to determine the relationship between ARs on seed surfaces and in whole grains.Both content of total ARs and AR homologues on seed surfaces and that in whole grains varied dramatically among the 36 wheat cultivars, while the relative composition of AR homologues in whole grains showed smaller variations. In whole grains, the average AR 17:0/AR 21:0 ratio was 0.1. Ratios of 0.1, 0.01 and 1.0 were reported for common wheat, durum wheat, and rye, respectively. These ratios have been suggested as an index for determining the source of cereal products ,48. Our A low AR content was present on seed surfaces, and it showed a different homologue composition, indicating that AR compositions were different in different parts of wheat seed. Dayan et al. suggested that the formation of ARs with different chain lengths is highly dependent on the specificity of the appropriate enzyme for the substrates ,50. We sOn the other hand, the chloroform extraction procedure is a time-saving and simple extraction method, which can meet the need to efficiently screening large numbers of lines. Without affecting the seed germination rate and damaging seed structure, this method is suitable for breeders and nutritionists.p < 0.001) between total AR concentrations in whole grains and that on seed surfaces. Along with a substantially shorter sample extraction time, the major advantage of this method was that using chloroform allowed easy and non-destructive extraction of the ARs from seed surfaces. Therefore, results from this study could provide a non-destructive extraction method for determining ARs contents on seed surfaces, so as to predict the contents in whole grain flour. Using the methods developed in this paper can therefore help breeders select appropriate recombination events to accelerate the selection process without destroying seed structure or affecting seed germination rates.In summary, in this work, we analyzed the composition of SSL and WGL. Although ARs were the major compounds in both SSL and WGL, there were many differences between their compositions. Moreover, there was a highly significant correlation - trifluoroacetamide (BSTFA) were purchased from Sigma-Aldrich . GC-MS , and GC-FID  were applied for identifying wheat lipid components.Chloroform and ethyl acetate were purchased from Xilong Scientific  and used for lipid extraction from wheat samples. Pyridine, n-tetracosane (C24) and Surface lipids were operationally defined as the lipid material extracted by quickly immersing these organs in chloroform ,37,39. IThe sample extraction procedure followed a previously established method for grains ,46,47. RFor GC analysis, each lipid sample was derivatized with 40 μL pyridine and 40 μL BSTFA for 1 h at 70 °C, and each lipid sample was vortexed every 20 min ,37,39. Tm/z. Compounds were identified by comparing their retention times and mass spectra with authentic standards and literature data [After derivatization, lipid composition was analyzed on a capillary GC column  and attached to an MS; helium (He) was used as carrier gas at a rate of 1.5 mL/min, programmed to temperature as follows: Set at 50 °C for 2 min, ramp 20 °C/min to 220 °C, hold for 2 min, ramp 1.6 °C/min to 310 °C, and hold for 18 min at 310 °C. The MS operated in full-scan mode applying a mass in the range of 35 to 700 ure data ,38,39,402) carrier gas inlet pressure regulated for a constant flow of 2.0 mL/min. Individual lipid compounds were quantified against the C24  by automatically integrating peak areas. All values were reported on a dry matter (DM) basis. DM was determined by drying samples in an oven at 105 °C overnight, cooling in a desiccator, and then weighing. All wheat samples were analyzed in triplicate; the coefficient of variance (C.V.) was less than 10% for the three samples.The GC equipped with a flame ionization detector  was used for composition quantitative analysis. GC-FID was carried out on the same GC conditions as above, but with Nitrogen  seeds were in the same germination conditions. The germination test was performed in a petri dish with two layers of filter papers moistened with distilled water, at 25 °C in the dark. Each petri dish contained 100 seeds, 5 mL of sterilized water was added to meet the conditions of germination humidity, and the number of statistical seed germinations was observed every day. A seed was regarded as germinated when the radicle had pierced the seed coat. The germination rate was counted on the 7th day. The results presented here are the means of the germination rates obtained from four replicates; the coefficient of variance (C.V.) was less than 10% for the four samples.p-value less than 0.05 was considered to be statistically significant. All results in our study denote the means ± SD. Pictures were drawn using Sigma plot 14.0 software, except for chemical structures, which were drawn using ChemDraw Professional 15.1 software. Correlation and linear analyses were performed using SPSS 19.0 software. Method agreement was assessed by a Bland–Altman plot . A p-val


AR expression in Japanese patients.Non-muscular invasive bladder cancer (NMIBC) has a high risk of recurrence. As androgen receptor (AR) reportedly affects bladder cancer, we assessed the correlation between NMIBC recurrence and tumor AR mRNA expression. Kaplan–Meier product-limit estimators were used to assess RFS distribution, log-rank tests to analyze differences in RFS between high- and low-risk groups; and multivariate analyses of AR mRNA expression and other clinicopathological factors to predict independent factors for RFS.We retrospectively reviewed 53 specimens of non-metastatic NMIBC, with recurrence-free survival (RFS) as the primary endpoint. We used real-time quantitative polymerase chain reaction to quantify AR mRNA-expressing group (n = 43) tended to have a longer median RFS (not reached) than did the low-AR group . Multivariate analysis showed female sex , tumor size ≥3 cm  and low AR mRNA expression  to be independent predictors of shorter RFS.The high AR mRNA expression level is an independent risk factor for RFS in Japanese patients with NMIBC. Further studies are necessary but AR expression might be a new indicator of recurrence of NMIBC.Our study showed that low  Bladder cancer (BC) is a common malignancy, with an estimated 429,800 new cases and 165,100 deaths in 2012, worldwide [Men tend to be diagnosed about four times more often than women , which iAR mRNA expression with real-time quantitative polymerase chain reaction (RT-qPCR).Thus, in this study, we assessed the correlation between NMIBC recurrence and tumor AR expression in Japanese patients, by quantifying We retrospectively reviewed 53 specimens of TUR-BT among 51 patients with non-metastatic NMIBC treated at Yokohama City University Medical Center and Yokohama City University Graduate School of Medicine from 2008 to 2015. In the two patients, specimens were obtained two times at the time of initial TUR-BT and at the time of recurrence. Samples for RT-qPCR came from biopsies performed during TUR-BT procedures. Both primary and recurrent NMIBC was diagnosed by TURBT. The experimental procedures were conducted in accordance with the ethical standards of the Helsinki Declaration. This study was approved by the institutional review board of Yokohama City University Medical Center. Informed consent to participate in the study were obtained from all subjects.The primary endpoint of this study was recurrence-free survival (RFS), which was defined as the time between the date of the initial TUR-BT and the date when the TUR for the recurrent BC was performed. Cancers detected in second-look TUR were not defined as recurrence, and RFS was counted from the first TUR-BT. Histopathological factors were assessed by pathologists specialized in uropathology.AR mRNA level was assessed by quantitative RT-qPCR method. We obtained 53 BC tissue specimens by TUR-BT to analyze gene expression. Total RNA (0.5 μg), which was isolated from the bladder tissue specimens, using Isogen , was reverse transcribed using 1 μM oligo (dT) primers  and 4 units of Omniscript reverse transcriptase (Qiagen) in a total volume of 20 μL. Gene expression for AR and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined by real-time qPCR, using AR primers . Gene expressions were determined using TaqMan® Gene Expression Assays . All data were normalized to GAPDH; expression of one tumor was assumed to be 1 and used as reference. The 2−ΔΔCT method was used to calculate relative amounts of target genes.AR mRNA level was determined by a web-based calculator cut-off finder (http://molpath.charite.de/cutoff.) and median values were used as the cut-off points for other factors to categorize continuous measurements [AR mRNA expression level and clinical factors. A Kaplan–Meier product-limit estimator was used to assess RFS distribution. Log-rank tests were used to analyze differences in RFS between the high- and low-risk groups. Univariate and multivariate COX models were used to analyze factors for predicting RFS. We derived relative risks and 95% confidence intervals (95% CIs). All tests were two-sided; alpha = 0.05 was considered significant. All analyses were conducted using IBM SPSS Statistics software for Windows, version 22 .The cut-off point for the urements . SpearmaPatients’ characteristics are shown in Table AR mRNA expression level and other clinicopathological factors  than did the low-AR group  and tumor size ≥3 cm  were significantly related to shorter RFS. Multivariate analysis showed that female sex , tumor size ≥3 cm  and low AR mRNA expression  were independent predictors of shorter RFS.Results of univariate and multivariate analyses to identify risk factors for RFS are shown in Table Although the function of AR signaling in BC is unclear, it is known to play important roles in prostate cancer occurrence and progression, and reportedly affects kidney, lung, breast and liver cancers .AR is a nuclear steroid hormone receptor, composed of several domains, N-terminal transactivation domain, DNA binding domain, a hinge region and a ligand-binding domain (LBD). In the cytoplasm, heat shock protein-90 (HSP90) binds with the LBD. As 5α-dihydrotestosterone binds to AR, AR releases HSP90 and translocates into the nucleus, binds to the androgen-response element, and promotes gene transcription –6, 21.AR signaling and the urinary tract may be critically associated. Studies in animal models have shown AR to be present in several tissues in lower urinary tract and may affect growth, differentiation and maintenance of urinary bladder tissue , 22, 23.Reportedly, AR signaling has a pivotal role in BC occurrence and progression. Testosterone increased the risk of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder tumors in female rats, whereas diethylstilbestrol, a synthetic form of female hormone estrogen, inhibited incidence in male rats . Also, iAR-knockout mice (ARKO) did not develop BBN-induced tumors, which suggests that AR has a crucial activity in bladder carcinogenesis [Studies with animal models have indicated that AR has an important role in BC. Miyamoto et al. found that both male and female ogenesis . That stogenesis , 24. Anoogenesis .+ BC specimens [AR mRNA expression in pT1 NMIBC showed that high AR mRNA expression was an independent predictor for longer RFS and cancer-specific survival [AR mRNA level was an independent predictor for RFS in NMIBC (including pTa and pT1).Therefore, the association between BC and AR has been the focus of several recent studies. Expression of AR have indicated to have high risk in bladder cancer occasion, but as for recurrence, expression of AR have shown to contribute to longer RFS. An immunohistochemical study of 169 patients indicated that recurrence was less likely for patients with ARpecimens ; anotherpecimens . A studysurvival , which w+ BC [+ BC cell growth in vivo [Reportedly, AR pathway inhibitors have been shown to be effective in preventing BC recurrence. In patients with double primary cancers of the prostate and the bladder, androgen-deprivation therapy (ADT) to treat their prostate cancers reduced the risk of recurrence of AR+ BC . Enzalut in vivo . BCG IVI in vivo .As described above, previous clinical studies demonstrated that higher AR expression in both mRNA levels and protein levels were correlated with favorable outcome in NMIBC –15. MoreThe mechanism of AR signaling in BC is still unclear, therefore, further studies of the AR pathway in NMIBC are needed.Our study had several limitations. First, it was a retrospective study with relatively few patients and a short follow-up period. Second, CIS was not analyzed in this study. CIS is an essential risk factor of recurrence and progression in NMIBC, but our study cohort had only two BCs with coexisting CIS. Finally, for verify the role of AR pathway in NMIBC, not only AR mRNA expression levels, but also protein expression levels should be investigated in current study. Future clinical studies would need to estimate an appropriate sample size to include enough of these patients and investigate mRNA and protein expression levels simultaneously.AR mRNA expression is an independent predictor for RFS in Japanese patients with NMIBC. The AR pathway is now attracting attention in BC research, and may provide new methods to treat bladder cancer and prevent its recurrence.Our study show that