Populus simonii × Populus nigra is an important cultivated forest species with resistance to cold, drought, insect and disease, and also a key model plant for forest genetic engineering. In this study, H2O2 response in P. simonii × P. nigra leaves was investigated using physiological and proteomics approaches. The seedlings of 50-day-old P. simonii × P. nigra under H2O2 stress exhibited stressful phenotypes, such as increase of in vivo H2O2 content, decrease of photosynthetic rate, elevated osmolytes, antioxidant accumulation, as well as increased activities of several ROS scavenging enzymes. Besides, 81 H2O2-responsive proteins were identified in the poplar leaves. The diverse abundant patterns of these proteins highlight the H2O2-responsive pathways in leaves, including 14-3-3 protein and nucleoside diphosphate kinase (NDPK)-mediated signaling, modulation of thylakoid membrane structure, enhancement of various ROS scavenging pathways, decrease of photosynthesis, dynamics of proteins conformation, and changes in carbohydrate and other metabolisms. This study provides valuable information for understanding H2O2-responsive mechanisms in leaves of P. simonii × P. nigra.Hydrogen peroxide (H Among them, H2O2 is the most abundant ROS in plant cells during photosynthesis, photorespiration, and respiration processes [2O2 can easily penetrate membrane through water channels, functioning as a likely long-distance signaling molecule in plant growth and stress perception [2O2 has been proven to be a regulator of many physiological processes, such as cell wall modulation, senescence, phytoalexin production, photosynthesis, stomatal movement, and cell cycle [2O2 has obvious oxidative destruction of diverse molecules  in plant cells [2O2 can improve seed germination [2O2 changes in cells showed as a bell-shaped response with an optimum, depending on the plant species, developmental stages, cell types, and environmental conditions [Various environmental stresses usually affect reactive oxygen species (ROS) homeostasis in plants, leading to the generation of excess ROS, such as singlet oxygen O2, superll cycle . Excess nt cells , which dmination  and planmination ,7, osmotmination , aluminumination , heat [6mination , paraquamination , and potmination . H2O2 ch2O2-responsive genes in various plants. For example, more than 170 non-redundant expressed sequence tags (ESTs) in Arabidopsis [2O2 responsive genes. In addition, 6156, 6875 and 3276 transcripts were differentially expressed in three wheat lines including a powdery mildew resistant (PmA) line and two susceptible (Han and Cha) lines [2O2-responsive proteins were identified in leaves from rice (Oryza sativa L. cv. 93–11) [Citrus aurantium L.) [Brachypodium distachyon [Triticum aestivum L.) [2O2 response. However, most of these proteomics studies focused on model plants and crops. Proteomic analysis of the response of forest trees to H2O2 stress has not been reported.Previous transcriptomic analyses have reported a large number of Hbidopsis , 713 ESTbidopsis , and 437bidopsis  were idea) lines . Recentltium L.) , Brachypstachyon , and wheivum L.)  using twPopulus genome, poplar has emerged as a model system for molecular and genetic studies of forest trees [Populus simonii × Populus nigra, also called as Populus xiaohei, is the hybrid of Populus simonii and Populus nigra, which widely distributes in northern China, and has been well used for afforestation and commercial forest. P. simonii × P. nigra is a fast-growing tree species with excellent properties of resistance to cold, drought, insect and disease. The early studies of P. simonii × P. nigra were mainly focused on cultivation and germplasm introduction [P. simonii × P. nigra [Tamarix androssowii late embryogenesis abundant gene)-introduced transgenic P. simonii × P. nigra dwarf mutant showed 537 genes and 99 proteins were significantly altered, respectively [P. simonii × P. nigra in response to stresses. Thus, the dynamic proteomic analysis of P. simonii × P. nigra under H2O2 stress is important for further investigation of the molecular mechanism of oxidative stress and biotechnological manipulation with the aim of enhancing poplar stress tolerance.Poplar trees are widely planted, and poplar woods are commonly used for building materials, furniture and paper . With thst trees . Populusoduction . Recentloduction , insect oduction , and disoduction . BesidesP. nigra ,27,28,29P. nigra  and SoleP. simonii × P. nigra leaves under 0, 12, 24 and 36 mM H2O2 treatments for 6 h. Our results indicate that modulation of thylakoid structure, ROS scavenging pathways, photosynthesis, and protein conformation play critical roles in P. simonii × P. nigra leaves in response to H2O2. These results provide new insights into the molecular mechanisms underlying poplar response to H2O2 stress.In this study, we performed physiological and proteomic analyses of P. simonii × P. nigra were immersed in 0, 12, 24 and 36 mM H2O2 solutions for 6 h, respectively (2O2 stress. The net photosynthetic rate (Pn) decreased from 4 μmol CO2∙m−2·s−1 in control to about 3.7 μmol CO2∙m−2·s−1 under each H2O2 treatment  increased slightly from 473.2 μmol CO2·mol−1 in control to 496.3 μmol CO2·mol−1 under 36 mM H2O2, but the transpiration rate (Tr) was not significantly altered under the H2O2 stress  content and relative electrolyte leakage (REL) in leaves were determined. MDA contents and RELs were not changed under 12 mM H2O2, but increased under 24 and 36 mM H2O2 (−1 fresh weight (FW) in control to about 21.4 nmol·g−1 FW under 24 and 36 mM H2O2  activity was increased 1.7-fold under 24 mM H2O2 (2O2 treatment (2O2 stress. The activity of monodehydroascorbate reductase (MDHAR) was inhibited, while the activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were significantly increased under H2O2 treatments  activity was induced 1.2-fold under 36 mM H2O2 treatment , dehydroascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG) were detected in leaves in response to the Hreatment G. The co mM H2O2 G. GSH co2O2 -stained gels , 877 ± 21, 838 ± 23, 768 ± 48, and 811 ± 31 protein spots from 0, 12, 24 and 36 mM H2O2-treated leaves were detected, respectively (2O2 concentrations (fold change > 1.5 and p < 0.05). All of the 114 DAP spots were subjected to in-gel digestion and protein identification using tandem mass spectrometry. A total of 83 DAPs were identified using matrix-assisted laser desorption/ ionization (MALDI) tandem time of flight (TOF-TOF) mass spectrometry (MS) and Mascot Searching with stringent criteria. Among the 83 identified DAP spots, 81 DAP spots contained a single protein each  in ectively . Among tein each , and theein each . Thus, t2O2-responsive proteins, 34 proteins were originally annotated as unknown, hypothetical proteins, or without annotation. They were all re-annotated according to the Basic Local Alignment Search Tool (BLAST) analysis (2O2-responsive proteins). Besides, carbohydrate and energy metabolism (15%), other metabolism (12%), as well as protein folding and unfolding (10%) were also over-represented.Among the 81 Hanalysis . Based oanalysis A. Intere2O2.The subcellular localization of the 81 proteins was predicted using five different tools  . In tota2O2-responsive proteins were performed, which revealed four main clusters  were categorized into Cluster IV.To better understand the abundance patterns of the coordinately regulated proteins, hierarchical clustering analysis of the 81 Hclusters . Cluster2O2-responsive proteins, the PPI networks were generated using the web-tool STRING 10 [2O2-responsive proteins, 59 unique homologous proteins were found in Arabidopsis  and potato (Solanum tuberosum) under osmotic and oxidative stresses [Brassica napus and Arabidopsis were differentially regulated by various abiotic stresses, such as drought, ozone, cold, NaCl, light, and mechanical wounding [Excessive Hf poplar A,B. Impon leaves B. PAPs, stresses ,37. Besiwounding ,39. The wounding , cold acwounding , as wellwounding ,43. All 2O2 treatment, exogenous H2O2 can permeate through cell membrane into cells, leading to the increase of intracellular H2O2 level [2O2 concentrations in leaves were increased under 12 and 24 mM H2O2 (2O2 levels were increased in leaves of grass pea (Lathyrus sativu L.) under 5 and 10 mM H2O2 for 24 h, but returned to the normal level under 20 mM H2O2, which were revealed from H2O2 content detection and the histochemical localization using DAB staining [2O2 scavenging when plants were exposed to relatively higher concentration of H2O2.Generally, when leaves are exposed to HO2 level . In this mM H2O2 A. Interestaining . These i2O2 scavenging to cope with exogenous H2O2 stress . The activities of APX and POD were increased under 5 and 10 mM H2O2, but returned to the normal level under 20 mM H2O2 [2O2 levels  systems, but also could directly reduce H2O2 as reductants in poplar leaves. Additionally, the exogenous H2O2-induced osmolytes  were suggested to protect cellular components from degeneration by scavenging ROS in poplar leaves  were Hn poplar E,F. MDHAr leaves C–E. Simireatment .2O2 stress. GSTs are mostly known as detoxifiers of electrophilic compounds by covalently linking glutathione to hydrophobic substrates for sequestration or removal, which plays an important role in improving plant stress tolerance [2O2, but the abundances of GST U30 and GST F1 were decreased , glyoxylase (Gly) and aldo/keto reductase (AKR) were altered in regulating secondary release of metabolite signals in poplar leaves to cope with Holerance . In thisecreased . It can ST1-like  and citrST1-like  were H2OST 19E50  and riceST 19E50  were H2O2O2 stresses [2O2, but decreased significantly under 36 mM H2O2 .2O2-resposive photosynthetic proteins had multi-proteoforms in poplar leaves in response to H2O2  clones, the PGM gene and soluble sugar level were all induced under drought stress [2O2-decreased abundances of cytosolic GAPDH and enolase implied that glycolysis was inhibited in poplar under H2O2 stress. In addition, the decreased abundances of alcohol dehydrogenase and cytosolic NAD-dependent malate dehydrogenase would reduce the regeneration of reducing power nicotinamide adenine dinucleotide (NADH) in H2O2-stressed poplar leaves [2O2-stressed poplar leaves.Carbohydrate and energy supply are essential for plants in response to oxidative stress . Our pro2 stress F. The int stress . This imt stress . Besidesr leaves ,56, whilO-acetylserine (thiol) lyase (OAS-TL)-involved pathway [2O2 stress -involved pathway  and O-ac pathway , respect2 stress G. The oxive days . In addi2 stress G. These 2O2 stress  and uroporphyrinogen decarboxylase (UROD), were all decreased under 36 mM H2 stress H. ALAD c2O2 stress  and two proteoforms of heat shock protein 90 (HSP90) were increased in poplar under H2 stress I. HSC80 P. simonii × P. nigra plantlets were transferred to a culture flask containing 80 mL 1/2 MS solid medium, containing 2% (w/v) sugar and 0.54% (w/v) agar. The explants were cultured in a phytotron at 26 °C/22 °C (day/night), 16 h photoperiod and 200 μmol·m−2·s−1 light intensity for 50 days. The shoots of regenerated plantlets were immersed in 0, 12, 24 and 36 mM H2O2 for 6 h, respectively. After the treatments, leaves were harvested and blotted dry on filter paper immediately. For each treatment, at least three biological replicates were performed. For each replicate, more than three whole leaves with similar size from at least three separate poplar seedlings were collected and pooled. The fresh weight was 0.2 g. The samples were either used fresh or stored at −80 °C for future analysis.The terminal buds or lateral buds excised from Pn, Gs, Ci, and Tr were measured in fully expanded leaves of each plant using a portable photosynthesis system LICOR 6400 XT  . The meag at 4 °C for 10 min, then the supernatant was collected. Two milliliters of 0.6% (w/v) thiobarbituric acid solution was added to 2 mL of the supernatant. The reaction solution was incubated for 15 min at 100 °C followed by cooling down to ambient temperature. The absorbance was detected under 450, 532 and 600 nm using an Ultrospec 2100 pro UV/Visible spectrophotometer . The MDA content was calculated according to Li et al. [The MDA content and REL were determined using previous methods described by Wang et al. . For thei et al. .For the REL determination, 0.2 g fresh leaves were cut and completely immersed in 20 mL deionized water, then degassed for 10 min. The electrical conductivity of the solution (E1) was measured using a conductivity instrument (DDS-11A) after 20 min. Subsequently, the solution was incubated at 100 °C for 15 min and cooled to room temperature, and then the electrical conductivity of the solution (E2) was determined. In addition, the electrical conductivity of deionized water (E0) was also detected. The REL was calculated according to the equation: REL (%) = (E1 − E0)/(E2 − E0) × 100%.w/v) sulfosalicylic acid. After incubation at 100 °C for an hour and followed by cooling down to ambient temperature, the homogenate was centrifuged at 15,000× g for 10 min at 25 °C. Then, 1 mL supernatant, 2 mL glacial acetic acid, and 2 mL ninhydrin were incubated at 100 °C for an hour. After cooled to room temperature, the incubated solution with addition of 8 mL methylbenzene was allowed to stand for an hour before the detection at 520 nm using a spectrophotometer.Contents of proline and total soluble sugar were determined using ninhydrin reaction and an anthrone reagent method developed by Li et al. . For theg for 10 min at 25 °C, the supernatant was collected and diluted with deionized water to 50 mL. Then, 1 mL the extracting solution, 0.5 mL 2% (w/v) ethyl acetate solution of anthrone, and 5 mL concentrated sulfuric acid were incubated at 100 °C for 2 min. The absorbance was detected under 630 nm using a spectrophotometer after cooled down to room temperature.For the total soluble sugar assay, 0.2 g fresh leaves were ground in 5 mL deionized water. The homogenate was incubated at 100 °C for 30 min followed by cooling down to ambient temperature. After centrifugation at 15,000× w/v) reinecke salt solution. The homogenate was centrifuged at 10,000× g for 15 min at 20 °C. The supernatant was collected and filtered through a 0.45-μm-pore-size cellulose acetate filter. The filtrate was dried at 70 °C and then resuspended in 5 mL deionized water. After 5 mL reinecke salt solution was added, the reaction solution was incubated at 4 °C for 2 h, and then centrifuged at 4000× g for 15 min at 4 °C. The precipitate was collected, and then redissolved in 15 mL aether. Then, the solution was centrifuged at 4000× g for 15 min at 4 °C. The supernatant was harvested, dried, and redissolved in 70% acetone. The absorbance was determined at 525 nm using a spectrophotometer. The contents of proline, total soluble sugar, and glycine betaine content were calculated from the standard curve.Glycine betaine content was measured using reinecke salt method as described by Zhao et al. . Fresh l2O2 was determined using potassium iodide reaction as described in Suo et al. [2•− was obtained according to a method of Zhao et al. [g for 20 min at 4 °C, the supernatant was collected for enzyme activity assays, including SOD, CAT, APX, GPX, POD, MDHAR, DHAR, GR, and GST. The activities of SOD, CAT, APX, POD, GR, and GST were assayed according to our previous methods [The content of Ho et al. . Generato et al. . For ant methods . The act methods . For all methods . In addi methods .2O2 treatments were extracted using a phenol extraction method according to Wang et al. [p value smaller than 0.05 were considered to be DAP [The proteins from leaves under different concentrations of Hg et al. . About 1g et al. . For quao be DAP .p < 0.05); and (3) more than two peptides matched with nearly complete y-ion series and complementary b-ion series.The DAP spots were excised from the gels and digested with trypsin as previously described . MS/MS sThe identified proteins were searched against the NCBI database  and UniPThe subcellular localization of the identified proteins were predicted using five Internet tools according to Suo et al. : YLoc 8, LocTreeSelf-organizing tree algorithm hierarchical clustering of the protein abundance profiles was obtained from log (base 2) transformed fold change values of protein spots using Cluster software (version 3.0).The protein–protein interactions were predicted using the web tool STRING 10 . The homp value smaller than 0.05 was considered to be statistically significant.All the physiological results were presented as means ± standard deviation (SD) of at least three biological replicates. Data were analyzed by one-way ANOVA followed by Duncan's test using the statistical software SPSS 17.0 . A 2O2 homeostasis in the cells is complicated and fine-tuned. In the present study, we present a primary H2O2-responsive network in leaves of poplar (P. simonii × P. nigra) using integrative analysis of physiological and proteomic approaches. The molecular network includes 14-3-3 protein-/NDPK-mediated signaling pathway, dynamics of thylakoid membrane structure, enhancement of diverse antioxidative defense system, alteration of photosynthesis, adjustment of carbohydrate and other basic metabolisms, as well as modulation of protein synthesis and conformation (2O2-responsive mechanisms in poplar plants. Although our proteomics results highlighted some critical candidate proteins/genes in H2O2-responsive signaling and metabolic pathways, their biological functions for H2O2 response in poplar still need future characterization using molecular genetics and PTM analysis tools.In the course of poplar development and stress-response in poplars, the molecular regulation of Hormation . This st


Uridine 5′-diphosphate-glucuronosyltransferase 2B (UGT2B) genes code for enzymes that catalyze the clearance of testosterone, dihydrotestosterone (DHT), and DHT metabolites in the prostate basal and luminal tissue. The expression of the UGT2B15, UGT2B17, and UGT2B28 enzymes has not been evaluated in prostate tissue samples from hormone therapy-naïve patients.We determined the expression of UGT2B15, UGT2B17, and UGT2B28 enzymes in 190 prostate tissue samples from surgical specimens of a multiethnic cohort of patients undergoing radical prostatectomy at the Durham Veterans Affairs Medical Center. The association between each protein’s percent positive and H-score, a weighted score of staining intensity, and the risk of biochemical recurrence (BCR) was tested using separate Cox proportional hazards models. In an exploratory analysis, UGT2B17 total positive and H-score were divided at the median and we tested the association between UGT2B17 group and risk of BCR.p = 0.11), though not statistically significant. Upon further investigation, we found that patients with UGT2B17 higher levels of expression had a significant increased risk of BCR on univariable analysis , although this association was attenuated in the multivariable model .The median follow-up for all patients was 118 months (IQR: 85-144). Of 190, 83 (44%) patients developed BCR. We found no association between UGT2B15 or UGT2B28 and risk of BCR. However, there was a trend for an association between UGT2B17 and BCR (HR = 1.01, 95% CI 1.00-1.02, Our findings suggest that UGT2B17 overexpression may be associated with a significant increased risk of BCR. These results are consistent with previous reports which showed UGT2B17 significantly expressed in advanced prostate cancer including prostate tumor metastases. It is estimated that there will be approximately 161,360 new cases of prostate cancer (PC) and approximately 26,730 deaths from PC in the US population in 2017 –6. MoreoUGT2B4, UGT2B7, UGT2B11, UGT2B15, UGT2B17, and UGT2B28 along with six pseudogenes that are interspersed among the genes in the region [Uridine 5′-diphosphate-glucuronosyltransferases (UGT) are part of a superfamily of enzymes which are part of the phase II drug detoxification pathways in the hepatic tissue in mammals. The UGTs catalyze the addition of the hydrophilic moiety, glucuronide to acceptor molecules in a process called glucuronidation , 14. In e region , 17. Fure region , 19. Thee region  e region . Thus thA couple of studies have suggested that expression levels of the UGT2B17, B15, and 28 enzymes may be prognostic for clinicopathologic characteristics of prostate cancer. In one study the expression levels of UGT2B17 and B15 in prostate tumor samples from ADTh treated patients were compared to expression levels in patients that were untreated or diagnosed as benign prostatic hyperplasia (BPH) . The resTo assess whether expression levels of UGT2B enzymes would predict worse prostate cancer outcomes, we analyzed the association between expression of UGT2B15, B17 and B28 in tumor microarrays at the time of radical prostatectomy and risk of BCR. We hypothesized that higher expression of the UGT2B enzymes is associated with increased prostate cancer progression risk.A retrospective study was conducted on 196 patients undergoing radical prostatectomy at the Durham Veterans Affairs Medical Center (DVAMC) between 1993 and 2004. None of the patients had hormonal ablation or radiation therapy prior to surgery. Four core needle biopsies of cancer of the formalin fixed and paraffin embedded radical prostatectomy specimens were used to create tissue microarrays (TMAs). Slices of the TMA were stained for expression of UGT2B15, UGT2B17, and UGT2B28. We used two indicators for the staining power of the biomarkers, the average percentage of positive staining cells, and H-score. H-score was calculated for each of the three biomarkers using the formula: 3*percent staining strong +2*percent staining medium +1*percent staining weak staining.Patients were followed for a median of 118 months. Biochemical recurrence (BCR) was defined as a single PSA value of >0.2 ng/ml, two values of 0.2 ng/ml, or need for secondary treatment for elevated PSA in the post-operative period. We excluded 1 patient missing extra-capsular extension, 2 missing seminal vesicle, and 3 missing follow-up, leaving a total of 190 patients in our study cohort. Institutional Review Board approval was obtained at Duke University, North Carolina Central University and the DVAMC and all patients signed an informed consent at the DVAMC prior to enrollment.For all antibodies, TMA slides from DVAMC were baked overnight in a 60 °C oven before IHC staining. The staining protocols are as follows: UGT2B15: Sections were deparaffinized with xylene and rehydrated through graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. Heat-induced epitope retrieval (HIER) was carried out in EDTA, pH 8.0  using a vegetable steamer at 95 °C for 25 min. Mouse polyclonal anti-human UGT2B15 primary antibody  was diluted with BSA to a concentration of 1:50 and applied to the sections overnight at 4 °C. Slides were rinsed with PBS then Dakocytomation Envision System Labelled Polymer HRP anti-mouse  secondary antibody was applied for 30 min at room temperature. The antibody was then visualized using the Betazoid DAB Chromogen Kit . Finally, sections were rinsed with water, counterstained with hematoxylin, dehydrated through graded ethanol, cleared with xylene, and coverslipped.UGT2B17: Sections were deparaffinized with xylene and rehydrated through graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. Heat-induced epitope retrieval (HIER) was carried out in EDTA, pH 8.0  using a vegetable steamer at 95 °C for 25 min. Rabbit polyclonal anti-human UGT2B17 primary antibody  was diluted with BSA to a concentration of 1:50 and applied to the sections overnight at 4 °C. Slides were rinsed with PBS then Dakocytomation Envision System Labelled Polymer HRP anti-rabbit  secondary antibody was applied for 30 min at room temperature. The antibody was then visualized using the Betazoid DAB Chromogen Kit . DAB was rinsed away with distilled water, cupric sulfate was applied for 5 min, and the tissue was rinsed again with distilled water. Finally, sections were rinsed with water, counterstained with hematoxylin, dehydrated through graded ethanol, cleared with xylene, and coverslipped.UGT2B28: Sections were deparaffinized with xylene and rehydrated through graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. Heat-induced epitope retrieval (HIER) was carried out in 0.01 M citrate buffer, pH 6.0 using a vegetable steamer at 95 °C for 25 min. Rabbit polycloncal anti-human UGT2B28 primary antibody  was diluted with BSA to a concentration of 1:50 and applied to the sections for 45 min at room temperature. Slides were rinsed with PBS then Dakocytomation Envision System Labelled Polymer HRP anti-rabbit  secondary antibody was applied for 30 min at room temperature. The antibody was then visualized using the Betazoid DAB Chromogen Kit . Finally, sections were rinsed with water, counterstained with hematoxylin, dehydrated through graded ethanol, cleared with xylene, and coverslipped.Definiens’ Tissue Studio  to determine the percentage of UGT2B15, UGTB17, UGTB28 and EGFR positive cells in a non-biased method. Briefly, stain specific algorithims were created using the pre-defined cytoplasmic detection module and classification tool, positive and negative stained cells within each tissue region were identified. Thresholds were set to classify hematoxylin stain for nuclei and DAB stain for positive cytoplasmic staining. The data were exported to Excel for further statistical analysis. Scanning and analyses were performed through the Translational Pathology Core Laboratory, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA.Slides were digitized on a ScanScope AT  and morphimetric analysis performed with Patient characteristics were summarized using median, 25th percentile, and 75th percentile for continuous variables and count and percentage for categorical variables. We tested the association between each biomarker’s  percent positive and H-score and the risk of BCR using separate Cox proportional hazards models. Both crude models and models adjusted for PSA , age (continuous), pathological Gleason score (2-6 vs. 3 + 4 vs. ≥4 + 3), race (black vs. non-black), positive surgical margins (yes vs. no), extracapsular extension (yes vs. no), and seminal vesicle invasion (yes vs. no) were fit. In these models, UGT2B15, UGT2B17, and UGT2B28 percent positive and H-score were modeled per 10-unit increase. In an exploratory analysis, we split UGT2B17 total positive and H-score at the median and tested the association between UGT2B17 group and risk of BCR.We also tested whether UGT2B15, UGT2B17, or UGT2B28 H-scores were correlated with pathological Gleason score using the Kruskal-Wallis test or chi-square test. For each level of Gleason score (2-6 vs. 3 + 4 vs. ≥4 + 3), we reported median, 25th percentile, 75th percentile of the biomarker H-score, <median (low expression), and ≥median scores (high expression).The demographic characteristics of the study population and their clinical and pathologic phenotypes are summarized in Table p > 0.5; Table p > 0.2; 1.03, p > 0.4). However, there was a trend between higher UGT2B17 percent positive and increased risk of BCR  on univariable analysis, although this did not reach statistical significance. This trend was weakened on multivariable analysis .There was no association between UGT2B15 or UGT2B28 percent positive or H-score on univariable or multivariable analysis . There was no association between UGT2B17 H-score groups and BCR on univariable (p = 0.20) or multivariable (p = 0.32) analyses. Upon further inspection, pathological Gleason score and seminal vesicle invasion were the greatest mediators between UGT2B17 and BCR. Specifically, after adjusting for these two variables, the HR is 1.28 .Notably in secondary analysis, patients with UGT2B17 total positive above the median had a significant increased risk of BCR on univariable analysis .Finally, we found no association between H-scores of UGT2B15, UGT2B17, or UGT2B28 and pathological Gleason score  have been significantly associated with decreased survival following BCR [UGT1A risk alleles did not significantly change androgen glucuronide levels. UGT1A class expression was shown to be decreased in prostate cancer stem cells and in metastatic CRPC cells while in vitro experiments with LNCaP prostate cancer cell lines suggests this decreased expression is associated with cell survival [UGT2B genes in the prostate are critical to the hormonal microenvironment.Genetic studies have demonstrated a relationship between UGT2B genes and clinical and pathologic characteristics of prostate cancer. For example, a study has shown that deletions of n Asians . While se levels , 23. In wing BCR . In thatsurvival . This maOur study has some limitations. The small sample size may have limited the power to detect significant associations between UGT2B17 expression and BCR on multivariable analysis. In addition we had a few number of events for CRPC and metastases, thus we could not evaluate the associations between UGT2B expression levels and those long-term prostate cancer outcomes. UGT2B17 expression has been found to be significantly expressed in prostate cancer metastases and CRPC tumors, while UGT2B15 is negatively regulated in those tissues . ElevateUGT2B gene variants are needed, as those variants continue to show impact on the risk for prostate cancer [In conclusion, the results from this study are suggestive that UGT2B17 overexpression may be associated with an increased risk of BCR. This study contributes to an emerging field of investigation that seeks to uncover the novel predictors of intraprostatic levels of androgens that persist in CRPC and the subsequent development of prostate tumor metastases . Future e cancer . The UGT


Sulfolobus solfataricus that belong to the Prx family have recently been characterized. One of these proteins, Bcp1, was chosen to determine its antioxidant effects in H9c2 rat cardiomyoblast cells. Bcp1 activity was measured in vitro under physiological temperature and pH conditions that are typical of mammalian cells; the yeast thioredoxin reductase (yTrxR)/thioredoxin (yTrx) reducing system was used to evaluate enzyme activity. A TAT-Bcp1 fusion protein was constructed to allow its internalization and verify the effect of Bcp1 on H9c2 rat cardiomyoblasts subjected to oxidative stress. The results reveal that TAT-Bcp1 is not cytotoxic and inhibits H2O2-induced apoptosis in H9c2 cells by reducing the H2O2 content inside these cells.Peroxiredoxins (Prxs) are ubiquitous thiol peroxidases that are involved in the reduction of peroxides. It has been reported that prokaryotic Prxs generally show greater structural robustness than their eukaryotic counterparts, making them less prone to inactivation by overoxidation. This difference has inspired the search for new antioxidants from prokaryotic sources that can be used as possible therapeutic biodrugs. Bacterioferritin comigratory proteins (Bcps) of the hyperthermophilic archaeon At high concentrations, ROS can damage all major classes of biological macromolecules, leading to protein oxidation, lipid peroxidation, DNA base modifications, and strand breaks. In addition, H2O2 plays a key role in cellular metabolism because it functions as a signalling molecule that regulates cell growth, cell adhesion, cell differentiation, and apoptosis. For these reasons, the ROS concentrations must be strictly controlled. However, the regulation of the ROS concentrations within cells is not clearly understood [Reactive oxygen species (ROS), notably, superoxide , and peroxiredoxin (Prx). Recently, attention has been given to Prxs, which are ubiquitous thiol peroxidases in archaea and eukaryotes, including humans. All Prxs share a common structure characterized by the thioredoxin (Trx) fold [P), whereas the second cysteine is called the resolving cysteine (CR). The CR is situated at the C-terminus or in different central positions downstream of CP [2O2 oxidizes CP to sulfenic acid (CP-SOH), which in turn condenses with CR to form a disulfide bond. A disulfide reducing system, generally composed of thioredoxin reductase (TrxR)/thioredoxin (Trx), is coupled to Prx for recycling [2O2 can further oxidize CP-SOH to its inactive forms, the sulfinic (CP-SO2H) or sulfonic (CP-SO3H) acids, thereby preventing disulfide bond formation and inactivating the enzyme. The reason for this sensitivity is due to specific structural motifs. Specifically, a “GGLG” sequence and a YF C-terminal extension stiffen and stabilize the fully folded (FF) active site, making the enzyme more susceptible to overoxidation [ sensitive in contrast to the large majority of prokaryotic Prxs, which are considered robust because they lack these motifs [Living organisms have evolved different antioxidant defence systems to protect themselves against ROS toxicity. Among the antioxidant enzymes, superoxide dismutase (SOD) is involved in the dismutation reaction of Oam of CP , 4. H2O2ecycling . In somexidation –8. For te motifs . This ste motifs . Sulfolobus solfataricus [ E. coli and characterized [ SsTrxR (Sso2416) and Protein Disulfide Oxidoreductase (SsPDO) (Sso0192) [P45 forms an intramolecular disulfide bond with CR50. The X-ray structure of the C45S/C50S double mutant, which represents the fully reduced enzyme state, was determined to a resolution of 2.15 Å and showed a Trx fold that was similar to other Prxs [An array of bacterioferritin comigratory proteins (Bcps) belonging to Prx family, named Bcp1, Bcp2, Bcp3, and Bcp4, have been recently characterized from the hyperthermophilic archaeonataricus –13. ArchSso0192) –16 repla Sulfolobus) is used [2O2 levels inside the cells were also evaluated. Our results indicate that in an in vitro cellular system , the archaeal enzyme can (1) function as an antioxidant to reduce the endogenous peroxide levels and (2) decrease cellular apoptosis following H2O2-induced stress.This paper shows that archaeal Prx can function as an antioxidant in eukaryotic cells. Specifically, our data show Bcp1 peroxidase activity under pH and temperature conditions that are typical of mammalian cells. Furthermore, we show that the archaeal Prx is active, although eukaryotic recycling system (different from that of is used . A fusio is used  and Bcp1Escherichia coli BL21-CodonPlus (DE3)RIL/pETBcp1 [600 nm of 0.8 in Luria-Bertani (LB) medium supplemented with kanamycin (10 μg mL−1) and chloramphenicol (34 μg mL−1) at 37°C. Bcp1 expression was induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG)  for 6 h at 37°C. E. coli cells containing the recombinant protein were harvested by centrifugation, and the pellet from a 1 L culture was suspended in 20 mL of ice-cold 20 mM Tris-HCl, pH 8.0, containing a complete EDTA-free protease inhibitor cocktail . The protein was purified using a previously described method [/pETBcp1  was growd method .E. coli BL21-CodonPlus (DE3)RIL was transformed with pET17yTrx or pET17yTrxR [μg mL−1) and chloramphenicol (34 μg mL−1) at 37°C. The expression of both yTrx and yTrxR was induced with 0.5 mM IPTG for 16 h at 37°C to an OD600 nm of 0.8. The E. coli cells were harvested by centrifugation and each pellet was suspended in 20 mL of ice-cold 20 mM Tris-HCl, pH 7.5, 10 mM NaCl, 5 mM DTT, 1 mM EDTA, and a complete EDTA-free protease inhibitor cocktail and disrupted by sonication with 5 min pulses at 20 Hz (30′′ on and 30′′ off). The soluble fractions were obtained after ultracentrifugation at 160,000 ×g for 30 min at 4°C.T17yTrxR   that had been equilibrated with 50 mM Tris-HCl, pH 7.5, and 0.2 M KCl and was connected to an AKTA system . The elution was performed at a flow rate of 0.4 mL min−1.The cell extract was heated at 75°C for 30 min, centrifuged at 15,000 ×g at 4°C for 30 min, and then concentrated  to purify−1.The sample was loaded onto a Hi-Trap DEAE FF column  in 20 mM Tris-HCl, pH 7.5, and 1 mM EDTA connected to AKTA system  to purify yTrxR. The proteins were eluted with a linear gradient of 0–0.5 M NaCl in 30 min at a flow rate of 0.5 mL minFor both proteins, the fractions were pooled, analysed by SDS-PAGE, and extensively dialysed against 20 mM Tris-HCl, pH 7.0.yTrx activity was detected using the insulin precipitation assay [−1 insulin, 1 mM DTT, and 5 μM yTrx. The increase in the absorbance due to the precipitation of the insulin B chain was monitored at 650 nm in a Varian Cary 50 Bio UV-Vis spectrophotometer.on assay , in a reyTrxR activity was evaluated using the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) reduction method [A412 nm at 30°C. The reaction mixture contained 0.1 M potassium phosphate, pH 7.0, 2 mM EDTA, 0.2 mM NADPH, 0.05 mM FAD, 0.5 mM DTNB, and 0.2 μM yTrxR.n method . The for2O2, and 10 μM Bcp1. After incubation for 5 min at 37°C, the reaction was stopped adding a trichloroacetic acid solution (10% w/v). The amount of H2O2 remaining was detected by measuring the A490 nm of the purple-coloured ferrothiocyanate complex that developed after the addition of 0.2 mL of 10 mM Fe(NH4)2(SO4)2 and 0.1 mL of 2.5 M KSCN.Bcp1 peroxidase activity was tested in an assay using DTT . The reaThe effect of pH on Bcp1 activity was analysed using 0.1 M citrate phosphate (pH 5.5–6.5) and 0.1 M Hepes (pH 7.0–7.5) buffers and the assay conditions reported above. The pH stability of Bcp1 was also evaluated after the enzyme was incubated in the buffers (pH 5.5–7.0) for 30 min, 1 h, 2 h, 4 h, and 16 h, and the residual peroxidase activity was determined under standard conditions.μM yTrx, 0.1 μM yTrxR, and 3.5 μM Bcp1. After preincubation at 30°C for 2 min, the reaction was started by adding 0.2 mM H2O2. The NADPH oxidation was monitored spectrophotometrically at 340 nm.The peroxidase activity was also evaluated using NADPH/yTrxR/yTrx as the electron donor system to reduce Bcp1. The assay mixture contained 50 mM Hepes, pH 7.0, 1 mM EDTA, 0.2 mM NADPH, 5–100  bcp1 coding sequence was isolated from the pET30Bcp1 vector [ NdeI and XhoI restriction endonucleases. The bcp1 fragment was purified by agarose gel electrophoresis using the QIAquick Gel Extraction Kit  and cloned into the pTAT2.2 expression vector . E. coli BL21-CodonPlus (DE3)RIL competent cells were transformed with the obtained vector, pTAT2.2-Bcp1, and selected on LB agar plates with kanamycin (10 μg mL−1) and chloramphenicol (34 μg mL−1). The selected colony was cultured in LB medium with the same antibiotics at 37°C. The cells were grown up to an OD600 nm of 0.8 and the expression of TAT-Bcp1 was induced with 1 mM IPTG for 3 h at 37°C. TAT-Bcp1 was purified using the same conditions described for Bcp1. Peroxidase activity was evaluated using the DTT assay at 37°C, as reported for Bcp1.The1 vector  after di−1), and 1 mM sodium pyruvate  at 37°C in a 5% CO2 incubator. A freshly prepared H2O2  stock aqueous solution was added to the cells at a final concentration of 0.3 mM and incubated for 1 h or 3 h to induce oxidative stress.H9c2 cells  were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 4 mM L-glutamine, a 1% v/v penicillin/streptomycin solution  was added to the cells and incubated for different times . As a control, the cells were incubated with buffer diluted in medium. The cells were then detached with trypsin-EDTA, harvested by centrifugation, and washed with PBS. The cells were lysed on ice for 20 min in 0.5% Triton X-100 in PBS containing a complete EDTA-free protease inhibitor cocktail. After centrifugation at 1,500 ×g for 10 min at 4°C, the supernatant representing the cytoplasmic fraction was collected and quantified by the Bradford method [H9c2 cells were seeded in 6-well plates at a density of 2 × 10d method  using Bi3 cells/well in 0.1 mL of DMEM. After 24 h, the cells were incubated with 10 μM TAT-Bcp1 for different times . As a control, the cells were incubated with buffer diluted in medium. The MTT solution  was added at 0.5 mg mL−1 in DMEM without phenol red (0.1 mL/well). After a 4 h incubation at 37°C, the formazan salts were solubilized in 0.04 M HCl in anhydrous isopropanol and cell viability was evaluated by measuring the A570 nm using an automatic plate reader spectrophotometer . Cell survival was expressed as the means of the percentage values compared to the control. Each sample was analysed in triplicate and the assay has been repeated at least three times.Cell viability was evaluated using the 3--2,5-diphenyltetrazolium bromide (MTT) assay . H9c2 ce2O2 levels were determined using a Fluorescent Peroxide Sensor (MAK164 Sigma-Aldrich), which generates a fluorescent product proportional to the H2O2 content following a reaction with H2O2.The H2O2 levels were measured under (1) physiological and (2) stressed conditions.μM) for 60 min at 37°C. Next, the cells were washed twice in PBS and lysed as reported above. Then, the samples were incubated with the Fluorescent Peroxide Sensor diluted in working solution according to the manufacturer's instructions and incubated for 30 min at 37°C. The fluorescence intensity was measured using a multimode reader synergy HT calibrated for excitation at 490 nm and emission at 525 nm.H9c2 cells were pretreated or with or without increasing concentrations of TAT-Bcp1 (1–20 μM for 16 h) and then stressed with 0.3 mM H2O2 for 30 or 60 min at 37°C. The cells were then washed and lysed, and finally the H2O2 levels were measured using the fluorescence assay described above. Every experiment included four replicates.H9c2 cells were pretreated with TAT-Bcp1  before treatment with H2O2 (0.3 mM for 1 and 3 h).H9c2 cells were seeded in 6-well plates at a density of 1.5 × 10Apoptotic cells were detected by ethidium bromide (EB) and Acridine Orange (AO) staining of the nuclei . AO permμL of PBS containing a 5 μg mL−1 EB/AO dye mixture, and incubated at 37°C for 20 min. The stained cells were visualized under a fluorescence microscope (Nikon Eclipse E-100). Images were taken at 100x magnification. Both the apoptotic (red) and live (green) cells in each sample were counted in five microscopic fields.The cells were detached with trypsin-EDTA, washed with ice-cold PBS, resuspended in 50 2, 0.5 mM DTT, and a complete EDTA-free protease inhibitor cocktail (Roche), lysing them by adding 0.1% NP-40, and centrifuging the lysates at 1,500 ×g for 10 min at 4°C. The pellet was dissolved in RIPA buffer  containing a protease inhibitor cocktail and incubated on ice for 30 min. After centrifugation at 1,500 ×g for 30 min at 4°C, the nuclear protein concentration was measured using the Bradford method [μg of the protein extracts was separated by 10% SDS-PAGE. Western blotting analysis was performed using the chemiluminescent method and the following antibodies: poly(ADP-ribose) polymerase-1 (PARP-1) mAb  diluted 1 : 2,000 in blocking buffer (5% BSA in TBS buffer containing 0.1% v/v Tween 20) or the B23 mAb  as loading control for the nuclear proteins. An HRP-conjugated goat anti-mouse IgG was used for detection .Nuclear extracts were prepared by detaching the cells with trypsin-EDTA, incubating them on ice for 10 min in 10 mM Hepes, pH 8.0, containing 10 mM KCl, 1.5 mM MgCld method , and 30  t-test. A p value < 0.05 was defined as indicating a statistically significant difference.The data for the cell viability and cell apoptosis assays are presented as the means ± SD of at least three repeats. Statistical analysis was performed using a two-tailed Student'shPrx1 and hPrx2) [ Saccharomyces cerevisiae [ Vibrio vulnificus (VvPrx1 and VvPrx2) [ hPrxI of 35% and 33%, respectively. The first similarly to Bcp1 is the atypical 2-Cys Prxs, and the two enzymes are characterized by a CxxxxxC motif, in which the two cysteine residues are the CP and CR, respectively [ VvPrx1 and VvPrx2, which belong to the prokaryotic domain, show lower identity with Bcp1 , and only the first presents the overoxidation motif. This analysis suggests that prokaryotic Bcp1 could be a robust peroxiredoxin that also functions in all eukaryotic environments.The CLUSTAL multiple alignment of Bcp1 versus human Prxs (d hPrx2) , Prx   was perfectively , 25, but E. coli, purified [2O2 at 37°C (data not shown) and that the optimal peroxidase activity was achieved at pH 7.0  and NADPH as an electron source to verify that Bcp1 activity was supported by eukaryotic disulfide reductase system. This system was successfully used to measure the peroxidase activity of hPrx1 and hPrx2 [ yTrxR and yTrx from E. coli and determined their reducing activities, as reported in the MM (data not shown). Initially, Bcp1 peroxidase activity was determined by measuring the consumption of NADPH, which was revealed by a decrease in absorbance at 340 nm  domain, a short basic polypeptide of 11 amino acids residues, was fused with a target protein it confers the ability to penetrate across the lipid bilayer of the cells due to the strong binding of the positively charged TAT domain to the negative charges on the cell surface . bcp1 into pTAT2.2 vector to allow Bcp1 internalization into the cell. The recombinant plasmid, pTAT2.2-Bcp1 , was used to produce a soluble fusion protein with the TAT domain at the N-terminus and a His tag at the C-terminus. TAT-Bcp1 was purified by heat-treatment followed by IMAC chromatography using the conditions described for Bcp1 [We generated the TAT-Bcp1 fusion protein by cloningfor Bcp1 . The SDS2O2 under the same pH and temperature conditions (data not shown).The purified enzyme was functionally characterized. The DTT assay was performed and the results revealed that, similar to Bcp1, TAT-Bcp1 was able to reduce all of the H2O2 contributes to the initiation and the progression of cardiovascular diseases, including atherosclerosis [μM) of the fusion protein for 1 h. As shown in μM TAT-Bcp1 was the optimal concentration for internalization into H9c2 cells. Afterwards, TAT-Bcp1 internalization was analysed at 1 h, 4 h, 16 h, and 24 h (http://dx.doi.org/10.1155/2016/7424870)). No band was detected when buffer alone was diluted into the culture medium , showing that TAT-Bcp1 was not cytotoxic to H9c2 cells. The incubation was also extended to 72 h , and the results showed that 95% and 91% of the cells survived at 48 h and 72 h, respectively, confirming the data described above.The MTT assay was performed to evaluate whether TAT-Bcp1 had any cytotoxic effect on the H9c2 cells. The cells were cultured in growth medium and treated with 10 and 24 h . After a2O2 content was measured in lysates of H9c2 cells that had been treated with increasing concentrations of TAT-Bcp1 and were or were not subjected to oxidative stress to establish the effect of TAT-Bcp1 on the H2O2 content in H9c2 cells.The endogenous H2O2 levels in H9c2 cell lysates were determined after incubation with increasing amounts (1 to 20 μM) of TAT-Bcp1 for 1 h at 37°C. The H2O2 levels in the lysates were determined using the Fluorescent Peroxide Sensor  were significantly reduced compared to the untreated samples. Taken together, these results indicate that TAT-Bcp1 reduces the H2O2 content in H9c2 cells under both physiological and oxidative stress conditions.In the latter case, we investigated the antioxidant activity of TAT-Bcp1 by measuring the HμM TAT-Bcp1 for different incubation times (1–24 h) and then exposed to 0.3 mM H2O2 for 1 h to study the effect of TAT-Bcp1 on H2O2-induced apoptosis. Cell apoptosis was analysed by EB and AO staining (The H9c2 cells were pretreated with 10 staining .2O2-treated apoptotic cells was approximately 3-fold higher (45.3 ± 2.3%) . Furthermore, the administration of TAT-Bcp1 to the unstressed cells did not induce any increase in cell apoptosis  [ S. solfataricus, in which four enzymes have been extensively characterized, to man, in which six isoenzymes with different roles and subcellular localizations have been identified. The ubiquity of Prxs and their homology conserved among the different phylogenetic domains show the important roles of these enzymes in addition to their peroxidase activities [2O2 levels, which are responsible for cell proliferation, angiogenesis, and apoptosis [Prxs have recently received more attention because they are very efficient at removing low concentrations of H~20 μM) , 29. Prxtivities . Althoug0 μM , 29poptosis –33. Furtpoptosis . S. solfataricus that lacks the sensitivity motif and has a higher robustness compared to its eukaryotic counterparts. Starting from these differences, we first assessed whether Bcp1 exhibited peroxidase activity under conditions that are typical of mammalian cells and in the presence of the eukaryotic recycling system yTrxR/yTrx. Our results showed that the enzyme can function at 37°C and pH 7.0 and is still stable after 16 h incubation under these conditions, highlighting the potential use of this enzyme in biotechnological applications [2O2 levels in the presence of the yTrxR/yTrx system but also showed that its affinity with yTrx was comparable to that reported for human Prxs [ in vitro characterization, we analysed the effect of Bcp1 on H2O2-induced apoptosis into H9c2 cells, with the aim of analysing the possible use of the archaeal Prx as an antioxidant.Our studies have focused on the antioxidant activity of a robust Prx to develop new antioxidant drugs; we chose prokaryotic Prx, Bcp1, fromications . In contications  instead man Prxs , suggest2O2 levels in H9c2 cells under both physiological and stressed conditions, showing that the enzyme can use the endogenous recycling system, but was also able to reduce apoptosis, as shown in the EB/AO staining assay in We designed the fusion protein TAT-Bcp1 to mediate its internalization into H9c2 cells and to analyse the effect of Bcp1 on the cell stress response. TAT-Bcp1 was effectively detected in the cells at 1 h after administration and gradually decreased to 70% after 24 h, in agreement with the protein turnover rates. Furthermore, the protein did not induce any cytotoxic effects, but it could have a cytoprotective effect, as shown by the slight but significant increase in cell viability at 16 h and 24 h. The results obtained in this study indicate that TAT-Bcp1 not only efficiently decreased the HThis study is the first report of an archaeal enzyme delivered into mammalian cultured cells that is able to protect cells from oxidative stress by reducing both the peroxide levels inside the cells and the resulting apoptosis. The intrinsic stability of Bcp1 and its modification, which provides the ability to penetrate cells, makes TAT-Bcp1 a good candidate to study the effectiveness of scavenging ROS in the cell. Growing evidence is highlighting the roles of Prxs in different diseases, ranging from diabetes to neurological disorders to cardiovascular diseases; therefore, this work aims to provide new insights into the possibility of using alternative sources of antioxidant enzymes, such as those from thermophilic microorganisms.E. coli BL21-CodonPlus (DE3)-RIL cellular extract; lane 3: E. coli BL21-CodonPlus (DE3)-RIL cellular extract induced with IPTG; lane 4: heat-treated cellular extracts; lane 5: IMAC chromatography.Figure S1: Cloning and expression of the TAT-Bcp1 fusion protein. (A) pTAT2.2-Bcp1 map. The T7 promoter region and TAT sequence are indicated by the black lines upstream of the bcp1coding region (grey). The His-tag is indicated by a dashed line downstream of bcp1. NdeI and XhoI are the restriction sites used for cloning. (B) SDS-PAGE analysis of the different purification steps for TAT-Bcp1. Lane 1: molecular weight markers (Protein Marker VI-Applichem); lane 2: Figure S2: Detection of extracellular TAT-Bcp1. A western blot analysis was performed to detect TAT-Bcp1 in the medium of cultured H9c2 cells; the same amounts of conditioned medium were analysed with the penta-His HRP conjugated mAb. H9c2 cells were cultured in growth medium (lane 5) and incubated with TAT-Bcp1 (10 µM) for 1 h (lane 6), 4 h (lane 7), 16 h (lane 8) and 24 h (lane 9). Decreasing amounts of purified TAT-Bcp1 were loaded to construct a calibration curve: 100 ng (lane 1), 75 ng (lane 2), 50 ng (lane 3), and 25 ng (lane 4).p < 0.001; the results were derived from four replicates of a representative experiment.Figure S3: Effect of TAT-Bcp1 on H9c2 cell viability at different incubation times. Cell viability was determined using the MTT assay. The cells were cultured in normal growth medium and then treated with TAT-Bcp1 (10 µM) for 24 h, 48 h and 72 h. Cell viability was evaluated by measuring the A570nm; the data are expressed as the mean percentages ± S.D. compared to the control. ∗


The mitochondrial protein p32 is a validated therapeutic target of cancer overexpressed in glioma. Therapeutic targeting of p32 with monoclonal antibody or p32-binding LyP-1 tumor-homing peptide can limit tumor growth. However, these agents do not specifically target mitochondrial-localized p32 and would not readily cross the blood–brain barrier to target p32-overexpressing gliomas. Identifying small molecule inhibitors of p32 overexpressed in cancer is a more rational therapeutic strategy. Thus, in this study we employed a pharmacophore modeling strategy to identify small molecules that could bind and inhibit mitochondrial p32.A pharmacophore model of C1q and LyP-1 peptide association with p32 was used to screen a virtual compound library. A primary screening assay for inhibitors of p32 was developed to identify compounds that could rescue p32-dependent glutamine-addicted glioma cells from glutamine withdrawal. Inhibitors from this screen were analyzed for direct binding to p32 by fluorescence polarization assay and protein thermal shift. Affect of the p32 inhibitor on glioma cell proliferation was assessed by Alamar Blue assay, and affect on metabolism was examined by measuring lactate secretion.Identification of a hit compound (M36) validates the pharmacophore model. M36 binds directly to p32 and inhibits LyP-1 tumor homing peptide association with p32 in vitro. M36 effectively inhibits the growth of p32 overexpressing glioma cells, and sensitizes the cells to glucose depletion.This study demonstrates a novel screening strategy to identify potential inhibitors of mitochondrial p32 protein overexpressed in glioma. High throughput screening employing this strategy has potential to identify highly selective, potent, brain-penetrant small molecules amenable for further drug development. C1qBP) is a direct transcriptional target of the cMyc oncogene  G [C] GNKRTRG [C] [COOH] was synthesized by ThermoFisher Scientific. The peptide was made cyclic by disulfide bond between C2 and C10. Unlabeled linear LyP-1 peptide (H-CGNKRTRGC-OH) (synthesized by AnaSpec) was used for competition experiment. The labeled peptide was used at a final concentration of 0.5 nM. Fluorescence polarization assay was performed at final buffer concentrations of 10 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgClData are expressed as means ± standard deviations (SD) for all experiments. GraphPad Prism was used for statistical calculations and for graph generation.We developed a 3D configuration of the peptide LyP-1. To develop a pharmacophore model of LyP-1, we need to find its possible binding site to p32 and develop a pharmacophore. Since the crystal structure of Lyp-1 has not been solved, we built a model based on its nine-residue sequence, and later used its similarity to a fragment of C1q, which binds to p32. The P32 crystal structure (PDB ID: 1p32) has gaps in its chains. To make docking more accurate, we developed loops to fill these gaps, using p32 sequence and MOE Loop Modeler module. Docking with C1q showed a binding fragment of C1q containing 13 residues (152–164: SIVSSSRGQVRRS) that was separated  with 63 members, one (1B) with eight members, four with three members, and five with two members], and 16 clusters with one member. Cluster 1B was the best: six of eight compounds have a property to penetrate the blood–brain barrier (range of logBB = + 0.35 to + 0.65), and in the cluster 1C all four compounds (range logBB = − 0.31 to − 0.83). Since cluster 1A was too general, we performed a second clustering run using the GpiDAPH2 fingerprint and an SO of 45%. As a result, we obtained 15 clusters, 1 (2A) cluster with 33 members, 1 cluster (2B) with 11 members, 1 cluster (2C) with 5 members, 2 clusters with 2 members, and 10 clusters with 1 member. The compounds in cluster 2B belong to the same family and their logBB exceed − 1.6. The logBB range of compounds in cluster 2B is − 0.76 to − 1.41 and in cluster 2C is + 1.59 to − 1.41. The third clustering run included the cluster 2A and was provided with GpiDAPH3 fingerprint and an SO of 53%. This yielded one cluster (3A) with eleven compounds, one cluster (3B) with two compounds, and 10 clusters with one compound. A list of 19 compounds from clusters 1B, 2C, and 3A was selected.To reduce experimental work, we clustered 109 resulting compounds with MOE Compute/Fingerprint/Clusters module, using the GpiDAPH3 fingerprint and similarity—overlap parameter Myc-overexpressing cells have been shown to be addicted to glutamine –31. We pIn order to confirm the hypothesis of the pharmacophore model, we established a fluorescence polarization (FP) assay to validate M36 as a molecule interfering with p32 and LyP-1 peptide association. Cyclic LyP-1 peptide  was fluorescently labeled at the amino terminus with 5-FAM (5-Carboxyfluorescein) and used in p32-binding assays. Binding of human recombinant p32 to the fluorescent-tagged LyP-1 peptide increases the size and enhances fluorescence polarization  Fig. a. This sThe mitochondrial protein p32 is a validated cancer target overexpressed in glioblastoma and several other cancers . Geneticc release [We previously reported that mitochondrial p32 has a role in metabolism of Myc overexpressing cells and showed loss of p32 attenuates Myc-mediated glutamine addiction . Within  release , 33, 34. release . Accumul release , 36. In  release . GSCs ca release , 39. DirM36 effectively antagonized the interaction between p32 and LyP-1 peptide. In addition, direct binding of M36 to p32 was observed by thermal shift thus validating the pharmacophore model. LyP-1 can direct nanoparticles to p32-expressing tumors . SimilarPharmacophore modeling of p32 association with the tumor homing peptide LyP-1 and C1q identifies potential p32 binding molecules. One of these molecules is shown to inhibit p32 function and inhibit proliferation of glioma lines overexpressing p32. In conclusion, the validation of this screening strategy offers potential to identify potent and selective inhibitors of mitochondrial p32 amenable for further drug development.


ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1  •The structure of the KREMEN 1 ectodomain is solved from three crystal forms•Kringle, WSC, and CUB subdomains interact tightly to form a single structural unit•The interface to DKKs is formed from the Kringle and WSC domains•PE1PE2The CUB domain is found to interact directly with LRP6 Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family. Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling. Xenopus embryogenesis to regulate anterior-posterior patterning  is followed by a WSC domain of unknown fold . The thiPE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.We sought to provide structural insights into the multi-functionality of this cell-surface receptor. The structures presented here reveal the unknown fold of the WSC domain and the tight interactions of all three domains. We further succeeded in determination of a low-resolution LRP6ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown). Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein.The recombinant production of the extracellular domain of Krm for structural studies proved challenging see . We succECD in three crystal forms at 1.9, 2.8, and 3.2 Å resolution  and, like other Kringle domains, is low in secondary structure elements. The structurally most similar Kringle domain is that of human plasminogen  affinity. In contrast, a SUMO fusion of DKK1CRD1-L did not display binding for concentrations tested up to 325 μM  and WSC (189VCF191→NCS) domains pointing toward DKK (309NQA311→NQS), was wild-type-like in DKK1 binding  similar to PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment -R/K; CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment. Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2  of LRP6 is available for Krm binding.For this crude approximation of a true ternary complex, we noted very close proximity between the CaP6PE1PE2 C. In con binding C, while  binding D. We con surface . FurtherCUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2  with a C-terminal His10 tag that contained a TEV protease cleavage site after E324. The expected sequence of the secreted protein is ETG-23APSPGLGPGPE31 … 320AVKEE324-GSENLYFQGGS-325LPQ … VPG373-THHHHHHHHHH (the isoform-2-specific PG insertion and the TEV site are underlined). This construct was well secreted and could be processed using TEV protease. However, 80%–90% of the protein eluted as aggregates from a size-exclusion column even before TEV treatment. The same applied to analog constructs for Krm1 from zebrafish, frog, and mouse. No monomeric protein at all could be obtained for several Krm2 constructs from multiple species. A KRM1ECD-TEV expressing stable GntI-deficient HEK293S cell line was generated by excision of an EcoRI-XhoI fragment, sub-cloning into pNeo-Sec-1, and selection of neomycin-resistant cells transfected the stable cell line with DKK and LRP6PE3PE4 constructs described earlier  iso-propanol. For cryoprotection, crystals were transferred to mother liquor mixed 1:1 with 3.4 M sodium malonate (pH 7.0). The slightly less well-ordered crystal of crystal form I and crystals of form II were obtained from a KRM1ECD-DKK1CRD2 complex using the SUMO-DKK1Linker-TEV-CRD2 construct and releasing SUMO and the DKK linker region by TEV and 3C protease treatment. Crystals of form I (2.1 Å) appeared from protein at 12 mg/mL in 1.0 M (NH4)H2PO4, 0.100 M sodium citrate (pH 5.6) and were cryoprotected by transfer to 2.9 M sodium malonate (pH 5.0). Crystals of form II grew from protein concentrated to 17 mg/mL in 1.0 M MgSO4, 0.1 M trisodium citrate . For cryoprotection, crystals were transferred to mother liquor mixed 1:3 with 3.0 M ammonium sulfate, 18% glycerol. Crystal form III appeared after 11 months in a dried-out drop of condition H5 of the Morpheus screen. The protein concentration had been 9 mg/mL. For cryoprotection, fresh liquid from Morpheus/H5 was added. The ternary complex structure was obtained from an LRP6PE3PE4-DKK1fl-KRM1ECD complex at 9 mg/mL that grew in condition E10 of the PACTpremier screen (pH approximately 6.8) over the course of 2–11 months. For cryoprotection, 10% PEG200 was added. By mistake, the crystals were incubated for 1 hr with 1 mM platinum compound in this cryosolution before cryocooling.All samples subjected to crystallization were purified from CM by affinity and size-exclusion chromatography . After t2WNO, CUB_C domain of Tsg-6 . All data to 3.5 Å were used during structure determination by MR. LRP6PE3PE4  could be placed independently by PHASER, giving Z scores of >10 and log likelihood gains (LLG) of >200. The combined LLG was 673, increasing to 901 after rigid-body refinement. Strong electron density became apparent at glycosylation sites and close to methionines (see platinum soak above), further supporting the MR solution. Additional strong density was evident between LRP6 and KRM1, suggesting the presence of DKK1. A model of the DKK1CRD2  could tRwork/Rfree errors of 32.5%/36.1% against the anisotropy-corrected data and 32.1%/35.5% against the unmodified but ellipsoidally truncated (We then performed restrained refinement of the coordinates against the ellipsoidally truncated and anisotropically scaled  diffractruncated  diffract2 for experiments investigating the direct LRP6PE1PE2-KRM1ECD interaction.Equilibrium experiments were performed as described before  with theM.Z. and V.A.J. performed experiments with support from Y.Z., who generated the stable cell line. M.Z. and E.Y.J. designed the research. M.Z. wrote the paper with input from all other authors.


PEP02  is a novel nanoparticle formulation of irinotecan encapsulated in liposomes. The aims of this study were to investigate the dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and pharmacokinetics (PK) of PEP02 in combination with 5-FU and LV, in patients with advanced refractory solid tumors.2  as a 90-min intravenous infusion on day 1, followed by 24 h infusion of 5-FU 2,000 mg/m2 and LV 200 mg/m2 on days 1 and 8, every 3 weeks.Patients were enrolled in cohorts to receive PEP02 from 60 to 120 mg/m2 (two patients). DLT was observed in four patients, two at the 100 mg/m2 dose level , and two at the 120 mg/m2 dose level . The MTD of PEP02 was determined as 80 mg/m2. The most common treatment-related adverse events were nausea (81%), diarrhea (75%) and vomiting (69%). Among the six patients who received the MTD, one patient exhibited partial response, four patients had stable disease and one showed progressive disease. Pharmacokinetic data showed that PEP02 had a lower peak plasma concentration, longer half-life, and increased area under the plasma concentration-time curve from zero to time t of SN-38 than irinotecan at similar dose level.A total of 16 patients were assigned to four dose levels, 60 (three patients), 80 (six patients), 100 (five patients) and 120 mg/m2, which will be the recommended dose for future studies.The MTD of PEP02 on day 1 in combination with 24-h infusion of 5-FU and LV on days 1 and 8, every 3 weeks was 80 mg/mNCT02884128) with date of registration: August 12, 2016.The trial was retrospectively registered (The online version of this article (doi:10.1186/s12885-016-2933-6) contains supplementary material, which is available to authorized users. PEP02  is an encapsulated nanoliposomal formulation of irinotecan hydrochloride CPT-11) [1 [1]. Ir2 at 3-week interval with favorable pharmacokinetic (PK) parameters of the active metabolite, SN-38 [In our previous study, the maximum tolerated dose (MTD) of PEP02 monotherapy was found to be 120 mg/me, SN-38 . A synere, SN-38 , 6. On tUGT1A family members. Individuals with genetic mutations of UGT1A exhibit reduced glucuronidation of SN-38 and an elevated risk of neutropenia and diarrhea compared with patients with wild-type alleles [UGT1A polymorphisms and toxicities is discussed.Irinotecan is converted by carboxylesterases to its potent metabolite, SN-38, which is detoxified in part by converting to inactive SN-38 glucuronide (SN-38G) through UDP-glucuronosyl transferase 1A isoforms (UGT1A) . The act3, absolute neutrophil count ≥ 1,500/mm3, platelets ≥ 100,000/mm3, hemoglobin ≥ 10 g/dL, serum total bilirubin within normal range, AST and ALT ≤ 3× upper limit of normal range, serum creatinine ≤ 1.5 mg/dL and blood urea nitrogen ≤ 25 mg/dL; (6) no prior treatment for at least 4 weeks before study initiation, including major surgery, chemotherapy, any investigational products or radiotherapy (6 weeks for nitrosoureas and mitomycin C); (7) recovered from all treatment-related toxicities or resolved to no greater than grade 1 before enrollment; and (8) written informed consent.This trial was a multi-center, open-label, Phase I, dose escalation study of PEP02  in combination with 5-FU and LV in patients with advanced solid tumors. The inclusion criteria were as follows: (1) histologically or cytologically confirmed advanced solid tumor refractory to standard systemic chemotherapy; (2) aged between 20 and 70 years; (3) Eastern Cooperative Oncology Group performance score (ECOG PS) of 0 or 1; (4) life expectancy ≥ 2 months; (5) adequate bone marrow, hepatic and renal functions: white blood cells ≥ 3,000/mmThe exclusion criteria were as follows: (1) known or suspicious primary or secondary brain tumors; (2) HBsAg-positive or anti-HCV antibody-positive with splenomegaly (defined as spleen size > 11 cm measured in longest diameter by CT scan); (3) uncontrolled active infection or other concomitant serious disease; (4) pregnancy or breast-feeding; (5) previous exposure to irinotecan; (6) history of allergic reactions to compounds of similar chemical or biologic composition as PEP02, 5-FU, or LV. This trial was approved by the independent ethics committee of each participating institute and the Department of Health, Executive Yuan, Taiwan, and was performed in accordance with International Conference on Harmonization Good Clinical Practice guidelines and Good Clinical Laboratory Practice.2 with dose expressing as the irinotecan hydrochloride trihydrate salt, which was escalated by increments of 20 mg/m2 between dose levels. Each patient was assigned to a dose level, and no intra-patient dose escalation was allowed. 5-FU and LV were administered at a fixed dose of 2000 and 200 mg/m2, respectively. PEP02 was administered by intravenous infusion over 90 min on Day 1, followed by 24-h intravenous infusion of 5-FU and LV on days 1 and 8 every 3 weeks. Pre-medication included dexamethasone and a serotonin-antagonist. Prophylactic anti-cholinergic agent was not administered unless acute cholinergic reaction was observed in prior cycles of treatment. Anti-diarrhea agents were started according to the guideline of American Society of Clinical Oncology. Treatment was continued to a maximum of 6 cycles or until disease progression, unacceptable toxicity, treatment delay > 2 weeks, or patient’s refusal or death.The study had a traditional 3 + 3 design with three-patient cohorts for each dose level. Dose escalation was only performed after the successful completion of at least 1 full 3-week cycle by each patient in the dosing cohort. If none of the first three patients experienced DLT, dose escalation was carried out for the next cohort of patients. If one of three patients developed DLT, the cohort was expanded to six patients. If two or more patients experienced any DLT, no more patients were to be entered at the current dose level and the lower dose level was to be declared the MTD. The MTD was the highest dose level with no more than 1 DLT among the accruals. A minimum of six patients were required to be tested at the dose level defined as the MTD. The starting dose of PEP02 was 60 mg/mDose modification on day 1 of subsequent cycles was only applied to PEP02, while the dosage of 5-FU/LV remained unchanged. All dose modifications were to be based on the worst proceeding toxicity. For patients who experienced ≥ grade 3 hematologic or non-hematologic toxicities, the dose of PEP02 was reduced by one dose level. In addition, the dose of 5-FU on day 8 of each cycle could be adjusted according to the laboratory data before the dosing. If the absolute neutrophil count (ANC) is between 1,000 and 1,499/μL, platelet count is between 50,000 and 99,999/μL, or diarrhea of grade 2 severity is observed, the dose of 5-FU could be decreased by 25%. 5-FU was withheld when ANC < 999/μL, platelet count < 50,000/μL or grade 3 diarrhea was observed. The conditions for the administration of the next cycle of treatment were ANC ≥ 1,500/μL, platelet counts ≥ 100,000/μL, serum creatinine ≤ 1.5 mg/dL, and full resolution of gastrointestinal toxicities.Toxicities were assessed according to the National Cancer Institute’s CTCAE version 3.0 . DLT was defined as occurrence of 1 or more of the following events attributable to the study drugs during the first cycle: (1) grade III or IV non-hematological toxicity, except grade III nausea, vomiting, or anorexia; (2) grade IV hematologic toxicity lasting for ≥3 days; (3) grade III hematologic toxicity associated with complications (e.g. neutropenic fever or bleeding); (4) dose delay of more than 2 weeks owing to drug-related toxicity. In addition, hematological assessment was performed daily whenever grade IV hematological toxicity occurred.Pretreatment evaluations included medical history, physical examination, performance status, complete blood count, hepatic and renal functions and serology of HBsAg and anti-HCV antibody. Patients were evaluated weekly with complete blood count and biochemistry analysis. Radiologic studies to assess response were performed at baseline and then every 2 cycles of therapy according to the guidelines of Responses Evaluation Criteria in Solid Tumors criteria version 1.0. All complete and partial responses required confirmation by two consecutive observations at least 4 weeks apart.max), time at which Cmax occurred (Tmax), elimination half-life (t1/2), area under the plasma concentration-time curve from zero to time t (AUC0→t), AUC through infinite time (AUC0→∞), and clearance (CL) were calculated. Pharmacokinetic parameters of individual data set were analyzed by a non-compartmental model by using WinNonlin™ .During the first cycle of treatment, blood samples were collected before treatment, during the infusion at 30 and 60 min, at the end of infusion, at1, 3, 9, 24, 48, 72 and 168 h after the end of infusion, and before the second cycle. Plasma levels of irinotecan and SN-38 were measured by validated LC/MS/MS analytical methods. The peak plasma concentration , minimum, and maximum were tabulated.Between March 2006 and August 2008, a total of 16 patients (seven men and nine women) were enrolled. The demographics and baseline characteristics of all patients are summarized in Table 2. Because both of the initial two patients at 120 mg/m2 level experienced DLT during the first cycle of treatment , three additional patients were recruited at the prior dose level, 100 mg/m2. However, both of the two newly accrued patients at 100 mg/m2 level experienced DLTs , resulting in 2 episodes of DLT among the five patients at this dose level. Therefore, the tested dose level was further de-escalated to 80 mg/m2. Since none of the patients experienced any DLT, 80 mg/m2 of PEP02 by 90-min intravenous infusion was determined as the MTD in combination with weekly infusion of 5-FU/LV on days 1 and 8 of a 21-day cycle.The dose escalation schedule is outlined in Table All 16 patients were assessed for toxicity. Table 2, the MTD dose of PEP02 experienced less treatment-related AEs (51.1% versus 57.6%), as well as grade III or above AEs (10.6% versus 18.4%).The most common treatment-related AEs included nausea (81.3% in incidence), followed by diarrhea (75.0%), vomiting (68.8%), fatigue (43.8%), mucositis , leucopenia (37.5%), neutropenia (37.5%), weight loss (37.5%), anemia (31.3%), and alopecia (31.3%). Acute cholinergic reaction was rarely observed. Compared with the entire safety population, patients who received 80 mg/m2 by 90-min intravenous infusion. Changes in the plasma concentration of CPT-11 showed almost the same pattern at all levels. All concentration curves of plasma CPT-11 peaked quickly and reached the maximum around 1 h after the end of PEP02 infusion and gradually dropped in a mono-exponential pattern until the last sampling point, which was similar to that observed for PEP02 monotherapy in a previous study [2 (26.3 ± 11.9 ng/ml), whereas the AUC of SN-38 was higher than that of irinotecan (AUC0 → t: 343.36 ± 133.24 ng/ml*h vs. 229 ± 108 ng/mL*h). The t1/2 of SN-38 at the MTD of PEP02 was 57.54 ± 17.81 h, which was relatively longer than that of the conventional formulation (10.4 ± 3.1 h). No statistically significant difference was observed in the mean values of all pharmacokinetic parameters of SN-38 among the 4 dose levels.The PK of PEP02 is shown in Table us study . At the UGT1A1*28 (TA6TA6: 88%) and UGT1A1*6 (GG: 69%). No subject harbored homozygous mutation in UGT1A1*28 or UGT1A1*6 allele. Two and five patients had heterozygous UGT1A1*28 and UGT1A1*6, respectively. Of which, one patient with heterozygous UGT1A1*28 and UGT1A1*6 experienced grade IV neutropenia and grade III diarrhea, and had the largest dose-normalized AUC of SN-38. Four out of the 5 subjects with heterozygous UGT1A1*6 possessed relatively higher dose-normalized AUC of SN-38 comparing to other subjects; of which 3 patients experienced grade III toxicities.The majority of subjects showed wild type alleles for 2, 1 PR and 4 SD were observed among six patients. The tumor response rate and the disease control rate were 16.7 and 83.3%, respectively. PR was observed in one gastric cancer patient (at the 80 mg/m2 dose level) and one breast cancer patient (at the 100 mg/m2 dose level).One patient at dose level III, who suffered from DLT did not complete at least one post-treatment tumor assessment. Among the 15 efficacy evaluable patients, two (13.3%) had confirmed partial response (PR) and nine (60%) had stable disease (SD), leading to the overall disease control rate (DCR) of 73.3%. At the MTD of 80 mg/m2) of PEP02, in combination with infusion of 5-FU and LV on days 1 and 8 of every-3-week schedule is recommended for the future studies. In a previous study, the MTD of PEP02 monotherapy with a 3-week interval was 120 mg/m2 [2, which mimicked the AIO regimen commonly used in Europe and Asia [The current study evaluated the safety profile and preliminary efficacy of PEP02 in combination with 5-FU and LV, in patients with refractory advanced malignancy. Gastrointestinal toxicities and myelosuppression were the major DLTs, which were comparable to those of free irinotecan and PEP02 monotherapy , 9. The and Asia , 11. The2 free-form irinotecan, 80 mg/m2 of PEP02 showed lower Cmax (8.0 ± 4.4 ng/mL vs. 26.3 ± 11.9 ng/mL), longer terminal t1/2 (57.5 ± 17.8 h vs. 10.4 ± 3.1 h) and higher AUC (343 ± 133 ng/mL*hr vs. 229 ± 108 ng/mL*hr) of SN-38 [2, but no statistically significant difference was observed in the mean values of pharmacokinetic parameters of CPT-11 and SN-38, including dose-normalized Cmax, AUC parameters, t1/2, CL, and Vss, possibly owning to narrow dose increments, small sample size and high inter-individual variability.PEP02 affected the PK characteristics of irinotecan. Compared to the data of 125 mg/mUGT1A1 gene encoded a varied spectrum of active enzymes that are responsible for drug metabolism, including UGT. The UGT1A1*28 allele is characterized by the presence of a 7th dinucleotide repeat in the TATA box of the promoter region, compared to the UGT1A1*1 allele with 6 repeats. This increased number of repeats results in the reduction in the expression of UGT, leading to decreased SN-38 detoxification and prolonged exposure time of SN-38 in the intestines. Thus, patients with homozygous or heterozygous UGT1A1*28 and treated with irinotecan commonly developed dose limiting neutropenia and late diarrhea [UGT1A1*28 polymorphism, the UGT1A1*6 allele also can decrease the activity of the enzyme in the heterozygous or homozygous genotype. It has been reported that patients with both UGT1A1*28 and UGT1A1*6 heterozygosity were at high risk to develop irinotecan-related toxicities [UGT1A family genes and pharmacokinetic parameters or toxicity of PEP02. However, one subject with heterozygous mutation in both UGT1A1*6 and UGT1A1*28 had the highest dose-normalized AUC of SN-38 and experienced grade IV neutropenia and grade III diarrhea. To draw any firm conclusions, a PK/PD study according to polymorphism of UGT1A family genes should be performed [The diarrhea . Similarxicities , 16. In erformed .With the limitation of being a very small sample size study of 15 efficacy evaluable population, two subjects had confirmed PR and nine subjects had SD as their best-ever responses during this study period. The tumor response rate and disease control rate were 13 and 73%, respectively. In a Phase I trial, clinical efficacy cannot be defined accurately because of heterogeneous tumor types and different dose levels. Of the evaluable patients, PR was noted in a heavily treated breast cancer patient and a gastric cancer patient, and four out of five patients with pancreatic cancer had SD, implying that this combination regimen is worthy of further investigation. Indeed, PEP02 either alone or in combination with 5-FU/LV was investigated in a phase II PEP0208 study  and a phmax, longer t1/2 and increased AUC0→t of SN-38 compared to irinotecan; similar results were observed in another study on PEP02 infusion alone. The dose of 80 mg/m2 of PEP02 in combination with D1 and D8 infusion of 5-FU/LV with every-3-week schedule is recommended for future studies.This is the first trial to apply PEP02 in combination with 5-FU and LV in patients with solid tumors, and major treatment-related DLTs were myelosuppression and diarrhea. PEP02 had a lower C


BdOctβR1) from the oriental fruit fly, Bactrocera dorsalis (Hendel), a destructive agricultural pest that occurs in North America and the Asia-Pacific region. As indicated by RT-qPCR, BdOctβR1 was highly expressed in the central nervous system (CNS) and Malpighian tubules (MT) in the adult flies, suggesting it may undertake important roles in neural signaling in the CNS as well as physiological functions in the MT of this fly. Furthermore, its ligand specificities were tested in a heterologous expression system where BdOctβR1 was expressed in HEK-293 cells. Based on cyclic AMP response assays, we found that BdOctβR1 could be activated by octopamine in a concentration-dependent manner, confirming that this receptor was functional, while tyramine and dopamine had much less potency than octopamine. Naphazoline possessed the highest agonistic activity among the tested agonists. In antagonistic assays, mianserin had the strongest activity and was followed by phentolamine and chlorpromazine. Furthermore, when the flies were kept under starvation, there was a corresponding increase in the transcript level of BdOctβR1, while high or low temperature stress could not induce significant expression changes. The above results suggest that BdOctβR1 may be involved in the regulation of feeding processes in Bactrocera dorsalis and may provide new potential insecticide leads targeting octopamine receptors.The biogenic amine octopamine plays a critical role in the regulation of many physiological processes in insects. Octopamine transmits its action through a set of specific G-protein coupled receptors (GPCRs), namely octopamine receptors. Here, we report on a β-adrenergic-like octopamine receptor gene ( Octopamine, a biogenic amine, affects many diverse processes in invertebrate physiology, and has significant similarities between the octopaminergic signaling system in invertebrates and the adrenergic system of vertebrates . It play2+ concentration ([Ca2+]i) and a small increase in the level of intracellular cAMP ([cAMP]i), whereas the activation of OctβRs causes a moderate increase in [cAMP]i but no increase in [Ca2+]i [i and the generation of Ca2+ signals. Structural and pharmacological similarities of the TyrRs have been shown with vertebrate α2-adrenergic receptors. The TyrRs was preferent to be activated by tyramine to induce a decrease in [cAMP]i i ,4, and t [cAMP]i ,5.Drosophila melanogaster, many additional receptors have been characterized in insects. In D. melanogaster, three subtypes of OctβRs have been identified. Among them, β3-octopamine receptor (OctβR3) plays a role in metamorphosis by regulating ecdysone synthesis [Bombyx mori [Schistocerca gregaria (SgOctβR) has also been cloned [Chilo suppressalis) was characterized in a work that also provided evidence that CsOctβR2 is a likely mediator of locomotion [Nicrophorusvespilloides [Identifying the properties of octopamine receptors could contribute to the understanding of the specific functions of the octopaminergic system in insects. Since the isolation of the first octopamine receptor in the fruitfly ynthesis , and Octynthesis ,8. Two obyx mori  and a pan cloned . In addiilloides .Bactrocera dorsalis (Hendel), is one of the most economically significant and widespread pests in the world, causing damage to over 250 different types of fruits and vegetables [B. dorsalis has greatly expanded its geographical distribution adaptability also due to a powerful reproductive ability [B. dorsalis has evolved high levels of resistance against most commonly used insecticides [BdOctβR1) from B. dorsalis. Quantitative Reverse Transcription-PCR (RT-qPCR) was used to investigate its expression profile over different developmental stages and tissues. Subsequently, with the use of a heterologous expression system, the pharmacological properties of BdOctβR1 were determined for agonistic and antagonistic activity with typical ligands. Additionally, we report on the association between BdOctβR1 and unfavorable (high and low) temperatures or starvation. We believe our works on the characterization of octopamine receptors in B. dorsalis may offer insights into the development of novel leads to control pest insects.The oriental fruit fly, getables . B. dors ability . B. dorscticides . TherefoBdOctβR1 was obtained by RT-PCR. The open reading frame (ORF) consists of 1365 bp encoding a 454 amino acid protein (GenBank accession number: XP_011212557). The putative amino acid sequence of BdOctβR1 contains seven transmembrane domains, which is a signature of GPCRs. Multiple sequence alignment with DmOctβR1 and DmOctβR2 showed that BdOctβR1 has highly conserved cysteine residues in the ECLs II and III, which form a disulfide bridge that is important in stabilizing the functional receptor structure. BdOctβR1 also has the highly conserved DRY sequence at the cytoplasmic end of transmembrane domain III, which is considered important in G-protein coupling. In addition, it contains the NP(L/I)IY motif located within TM7 that is required for ligand-induced internalization and is conserved in all adrenergic receptors. Compared with the other two receptors, BdOctβR1 has a relatively shorter N-terminal region than that in D. melanogaster  and Malpighian tubules (MT), and the receptor was nearby absent in the ovaries.The RT-qPCR results showed that t stages . The hige adults , the higBdOctβR1 was successfully carried out in HEK-293 cells transfected with the expression plasmid pcDNA3.1-BdOctβR1. In this system, ligand-mediated GPCR activation initiates cAMP accumulation that is measured by an increased luminescence of Glosensor. In our assay, we tested the concentration effects of five ligands: octopamine, tyramine, dopamine, phentolamine, and naphazoline. Octopamine and tyramine could significantly induce the increase of cAMP production in BdOctβR1-expressioning cells, but not in empty pcDNA3.1 vector-transfected cells (50) of 9.11 × 10−10, 6.35 × 10−10, and 1.97 × 10−8 M (50 = 3.32 × 10−6 M) compared with the other ligands. Phentolamine, an adrenoceptor antagonist, showed only very weak agonistic effects: about 20% at a very high concentration of 1.00 × 10−5 M.Transient expression of ed cells . The res× 10−8 M . DopaminB. dorsalis, including phentolamine, mianserin, and chlorpromazine. The antagonistic activity was quantified as described . These data were used to construct concentration response curves. As shown in 50) of 4.84 × 10−7 M. The two other compounds, phentolamine and chlorpromazine, had a respective IC50 of 3.80 × 10−6 M and 2.66 × 10−6 M (−5 M). In the negative controls with empty pcDNA3.1 vector-transfected HEK-293 cells, all tested ligands at 1.00 × 10−6 M did not affect the cAMP luminescence.For the pharmacological characterization of BdOctβR1, we examined the effects of various potential antagonists on the octopamine receptor of × 10−6 M . HoweverB. dorsalis adults, BdOctβR1 exhibited no significant differences in expression. Compared with the control (27 °C), the transcript level of BdOctβR1 showed a slight increase of 20% at 42 °C and a slight decrease of 12% at 4 °C (BdOctβR1 showed a significant (p < 0.05) increase of 44% over the controls  [A. mellifera, the EC50 of octopamine for AmOctβR1 was 4.39 × 10−8 M [The result of the agonist assays was very similar to OctβR1 in nogaster . General 10−9 M) ; in A. m× 10−8 M . This suB. dorsalis. This is consistent with a previous study [Drosophila. In addition, mianserin also had antagonistic activity on OctβR2 [Drosophila. Moreover, mianserin has been identified as a very efficient inhibitor of AmOctβRs at low concentrations (IC50S: 5 × 10−9–3 × 10−9 M) [D. melanogaster [B. mori [C. suppressalis [−5 M. This is consistent with the results in Drosophila that chlorpromazine is ineffective on DmOctβR1 [C. suppressalis [B. mori [Additionally, we examined the effects of several antagonist candidates which have been proven to be effective antagonists for insect octopamine receptors ,9,19. Amus study , in whicn OctβR2 , OctβR3  10−9 M) . TherefoAedesaegypti [To date, different studies on the agonist or antagonist profile of octopamine receptors have been performed. These results had attracted the attention of many scientists to elucidate the pathways of formamidines with regard to an insecticidal action ,19. Furtaegypti . Meanwhiaegypti . These sD. melanogaster, feeding behavior in Phormia regina [A. mellifera [BdOctβR1. Similarly, various stress factors such as vibration and those that are optical, thermal and chemical could increase the octopamine level in P. americana [BdOctβR1 showed a similar response to stress as food deprivation. We believe that there may exist an association between the increase of the BdOctβR1 transcript level and inhibition of food uptake. Therefore, although the function of BdOctβR1 has not been identified yet, we believe it could be involved in the regulation of feeding in B. dorsalis.Octopamine has various roles in insects, such as inducing different effects on regulating larval locomotion and grooming , conditia regina , and olfellifera ,32,33. Imericana ,35. As tB. dorsalis was established from pupae obtained from Haikou, China, in 2008. The insects were reared as previously described in a temperature controlled room at 27 ± 0.5 °C, 70% ± 5% relative humidity on a 14:10 h photoperiod (light:dark) in the Key Laboratory of Entomology and Pest Control Engineering, Southwest University, Chongqing, China [The laboratory colony of the oriental fruit fly g, China . Larvae g, China . The 3-dTyramine hydrochloride, (±)-octopamine hydrochloride, dopamine hydrochloride, naphazoline hydrochloride, forskolin, chlorpromazine hydrochloride, phentolamine hydrochloride, and mianserin hydrochloride were all purchased from Sigma-Aldrich . To culture HEK-293 cells, DMEM/F12 medium, fetal bovine serum (FBS), fungizone, penicillin, and streptomycin were purchased from Gibco Cell Culture at Life Technologies . TransIT-LT1 Transfection Reagent was obtained from Mirus Bio LLC .260/280) and OD260/230.RNA was extracted from different developmental stages  and specific adult tissues  from treated and control adults using Trizol reagent  according to the manufacturer’s protocol. The single-strand cDNA was synthesized by the PrimeScriptTM first-strand synthesis system . RNA/DNA was quantified with a Nanovue UV-Vis spectrophotometer , and the quality was ascertained by the absorbance ratio of optical density  were designed to obtain the full-length cDNA sequence. PCR was carried out using the high fidelity DNA polymerase with the following procedure: initial denaturation at 98 °C for 2 min, followed by 35 cycles of 98 °C for 15 s, 58 °C for 15 s, and 72 °C for 2 min, and final extension at 72 °C for 10 min. PCR products were separated by electrophoresis on an agarose gel (1.0%). The purified amplicons were cloned into the pGEM-T Easy Vector  and transformed into Trans5α chemically competent cell . The transformants were selected with Luria-Bertani agar plates containing 0.1% ampicillin, and positive clones were sequenced .Specific primers  for nesthttp://www.ncbi.nlm.nih.gov). Multiple sequence alignments were performed with DNAMAN v. 6.03 . Transmembrane helices were predicted using the TMHMM server (http://www.cbs.dtu.dk). The amino acids and locate motifs were predicted on the position identifiers proposed by Ballesteros and Weinstein [http://www.ebi.ac.uk) and the neighbor-joining method with 1000 bootstrap tests. The resulting tree was displayed graphically using MEGA 5.0 [D. melanogaster served as an out-group.Similar sequences were obtained by a BlastP search against the nonredundant protein database on NCBI (einstein . A phyloMEGA 5.0 . The FMRα-tubulin (GU269902) was used as an internal reference based on previous evaluations [Ct−∆∆ method [RT-qPCR was performed on a Stratagene Mx3000P system  using iQ-SYBR Green Supermix . The 20-μL reaction system contained 10 μL of SYBR Green Supermix, 7 μL of nuclease free water, 10 μM of each primer, and 500 ng of cDNA. The PCR conditions were 95 °C for 2 min, and 40 cycles of 95 °C for 15 s and 60 °C for 30 s. A melting curve analysis (60–95 °C) was carried out at the end of procedure to ensure the specificity of each pair of primers. In addition, luations . For theBdOctβR1 in HEK-293 cells and procedures for the assays were followed as previously described [Constructs for transient expression in HEK-293 cells were made by conducting a NotI digestion for both pGEM-T-BdOctβR1 and pcDNA3.1(+) . The NotI digested BdOctβR1 and pcDNA3.1(+), and they were then ligated by T4 DNA ligase (Promega) yielding pcDNA3.1-BdOctβR1. The correct insertion was confirmed by DNA sequencing. For the cAMP response assays, we used the non-lytic GloSensor cyclic AMP assay kit (Promega) for monitoring intracellular cAMP in live cells. At the same time, the empty pcDNA3.1 vector was transfected in HEK-293 cells as a negative control. The methods for transient expression of escribed ,42,43,44escribed .−5 M) was used as a positive control. The elevated luminescence caused by cAMP accumulation was measured over a period of 15 min in 30-s intervals using an Orion microplate luminometer . The authentic ligand octopamine was selected as the model ligand for normalization of the luminescence. A concentration–response curve was generated for each receptor by logistic fitting in Origin 8.6  as previously reported [A 10-fold serial dilution of the ligands octopamine, tyramine, dopamine, naphazoline, and phentolamine was applied to the cells. Forskolin  after background subtractions [The antagonist assay was examined with serial dilutions of antagonists in the transfected cells on a luminescence detection systems (Berthold Technologies). Basically, the antagonistic activity assay was conducted in 2 sequential treatments. In the 1st treatment, various concentrations of ligands (50 μL in volume) were plated in each well, including a solvent  as control. Following the injection of the cells into the well, a 30-min incubation with the tested ligands was conducted. Subsequently, the 2nd treatment was conducted. The cells were treated with 1.00 × 10ractions . The valBdOctβR1 expression. Three biological replicates were maintained for each temperature treatment.For thermal treatments, 90 five-day-old flies were placed in testing chambers  at 42 °C for 1 h (high temperature) or 4 °C for 30 min (low temperature) to ensure test flies could recover. Flies kept at 27 °C served as controls. Artificial diet was supplied by inserting into the fly chambers a glass vial containing adult diet and a wet sponge for water supply. Subsequently, one live female and one live male were collected randomly, then frozen together in liquid nitrogen and stored at −80 °C until RNA extraction. RT-qPCR was performed as described above using the same primers that were used to study In the starvation experiments, we investigated the responsiveness after 24 h of food deprivation. In essence, 5-day-old flies were collected and fed after 12 h of starvation to ensure fly satiety, and the treatments were then deprived of food for a period of 24 h. In the control groups, the flies were provided with an artificial diet. After the starvation period of 24 h, the live flies were collected individually for sample preparation as stated above.BdOctβR1, which is a necessary step in elucidating the functions of octopamine receptors in B. dorsalis. Our study suggests that BdOctβR1 has potential as a target for the development of novel insecticides and is in relation to the regulation of feeding behavior, although the function of the receptor has not yet been fully determined. Therefore, further studies need to identify the functions of octopamine receptors in insect physiological processes, and this will provide more opportunities for discovering novel insect control agents.In summary, we characterized the molecular and pharmacological properties of