Nur77 knockout mice (n = 5), real-time PCR results showed that the mRNA levels of AR and an androgen signaling target gene, Kitl, were decreased by 35% and 24%, respectively, relative to wild-type mice (n = 5), which suggested transcriptional regulation of AR by Nur77 in vivo. In cultured mouse granulosa cells and a steroidogenic human ovarian granulosa-like tumor cell line, KGN, mRNA and protein expression levels of AR were increased by overexpressing Nur77 but decreased by knocking down endogenous Nur77. Consistent with increased AR expression, chromatin immunoprecipitation showed that Nur77 bound to the NGFI-B response element (NBRE) in the AR promoter sequence. AR promoter activity was stimulated by Nur77 in HEK293T cells and attenuated in Nur77 knockout mouse granulosa cells (luciferase assay). Overexpression of Nur77 enhanced the androgenic induction of Kitl , while knockout of Nur77 attenuated this induction. These results demonstrate that AR is regulated by Nur77 in the ovaries, and they suggest that the participation of Nur77 in androgen signaling may be essential for normal follicular development.The androgen receptor (AR) is a nuclear receptor that is expressed in growing follicles and involved in folliculogenesis and follicle growth. The orphan nuclear receptor, Nur77, also has an important role in steroid signaling and follicle maturation. We hypothesized that AR levels and androgen signaling through AR are regulated by Nur77 in the ovary. In the ovaries of  Recently, the study of female reproductive physiology has been focused on the genesis and development of ovarian follicles. Follicular development and function are controlled by a plethora of intrinsic and endocrine factors, and are affected by interactions between multiple cell types within the ovaries PomcCyp17a1StARCyp21a1CRHHSD3B2The Nur77  orphan nuclear receptor is a transcription factor that activates target genes AR-deficient mouse model, there is an age-dependent progression in ovarian abnormality with a premature ovary failure (POF) phenotype that includes lower follicle numbers, impaired mammary development, fewer pups per litter, and a marked increase of atretic follicles, which indicates that AR-mediated androgen signaling is indispensable for the maintenance of folliculogenesis, and that impaired androgen signaling is a potential cause of the POF syndrome The androgenic steroid hormones are mostly produced by the adrenal glands and gonads of both sexes. Although androgens are present at much lower concentrations in the circulation of females relative to males, they are essential in maintaining female physiological functions, especially intraovarian functions, such as stimulating follicular growth on early stages Xenopus laevisin vitro, AR mediates the stimulatory effects of androgen on proliferation and follicular development Nur77 and AR are both involved in follicle maturation. Nur77 is expressed in corpora lutea and cultured ovarian granulosa cells, and it activates steroidogenic gene expression during luteinization, thus, indicating a functional relevance to stage-specific expression during follicular maturation AR and Kitl in the ovaries were examined in Nur77 knockout and wild-type mice to determine the regulation of AR by Nur77 in vivo. The mRNA and protein levels of AR and Kitl were analyzed in cultured mouse granulosa cells and a steroidogenic human ovarian granulosa-like tumor cell line, KGN, after overexpression and knockdown of Nur77 in vitro. The interactions between AR and Nur77 were also studied using chromatin immunoprecipitation and AR promoter activity. Moreover, we showed that Nur77 regulated Kitl expression through stimulation or inhibition of AR signaling using androgen or antiandrogen flutamide, thus, demonstrating an important interaction between Nur77 and androgen signaling in the ovaries.Therefore, this study aimed at investigating a novel role for Nur77 in AR regulation by testing the hypothesis that AR expression and signaling are affected by Nur77. The mRNA expression patterns of Nur77+/− mice (B6.129S2-tm1JmiNr4a1/J) were purchased from Jackson Laboratory . Three-week-old ICR mice were purchased from the Lab Animal Center of Yangzhou University . All animals were maintained in the Animal Laboratory Center of Drum Tower Hospital  on a 12∶12-h light/dark cycle (lights off at 19∶00) with food and water available ad libitum. All animal experiments were performed according to the guidelines of the Experimental Animals Management Committee .2.The KGN cell line , which was established from a human GCT and expresses typical granulosa cell markers, was cultured as previously described 2:5% CO2 and centrifuged for 5 min at 250×g. The pellets were suspended in DMEM/F12 containing 10% fetal bovine serum, 1 mM pyruvic acid (Gibco BRL/Invitrogen), 2 mM glutamine and antibiotics . The oocytes were filtered out with a 40-µm cell strainer. The mouse granulosa cells were cultured in the suspended medium at 37°C in a humidified environment with 5% CO2, and they were used within three passages in this study.Mouse granulosa cells were collected from the ovaries of immature mice (21 day old) as previously described Adenoviruses harboring the full-length Nur77 gene (Ad-Flag-Nur77) and adenoviruses harboring human Nur77 siRNA oligonucleotides  were generated using the AdMax  and pSilencer™ adeno 1.0-CMV  systems as previously described −△△CT method Total RNA was extracted from tissues and cultured cells using the TRIzol reagent . cDNA was synthesized from 1mg of purified total RNA using a PrimeScript RT reagent kit  according to the manufacturer's instructions. The specific primers used for quantitative polymerase chain reaction (PCR) analysis are listed in The proteins were prepared and separated on SDS-PAGE as previously described AR promoter sequence (−2509/−2127) was amplified by PCR from mouse granulosa cell genomic DNA with the following primers: 5′-TATAGGTACCGGCCTCAACTGCCTTACTCACAGC-3′ and 5′-TAGCAAGCTTTGGCGTTGGTGGAGTATGCAATCA-3′. Approximately 1.2 kb of the human AR promoter sequence (−2837/−1638) was amplified by PCR from human granulosa-luteinized (hGL) cell genomic DNA with the following primers: 5′-TATAGGTACCATCATTGGTTGCCTGAGGAG-3′ and 5′-TAGCAGATCTGGCAGGATGGTAGAATGGAA-3′. The PCR products were cloned into the pGL3-basic luciferase reporter plasmid  and sequenced to confirm the resulting plasmid. Preconﬂuent (75–80%) HEK293T cells in 12-well plates were transfected with pCMV-Flag-Nur77 or pCMV-empty vector (pCMV-EV), and the AR firefly luciferase report plasmid using Nanofectin . mGCs (70% confluent) from mice with different genotypes were transfected with the AR luciferase report plasmid using Lipofectamine 2000 (Invitrogen). All cells were cotransfected with the Renilla luciferase reporter plasmid  as a control for transfection efficiency. Luciferase activity was assayed 48 h after transfection using the Luciferase Assay System (Promega) and measured with a Centro XS3 LB 960 luminescence counter . At least three transfection assays were performed to obtain statistically significant data.Approximately 383 bp of the mouse 3). The crosslinks were reversed by incubation at 65°C for 5 h with a final concentration of 0.3M NaCl. DNA was purified by phenol: chloroform extraction and ethanol precipitation after the eluates were incubated with proteinase K. Finally, the purified DNA fragments were used as templates for PCR amplification and products were visualized on agarose gels. The specific primers were as follows: 5′-TATAGGTACCGGCCTCAACTGCCTTACTCACAGC-3′ and 5′-TAGCAAGCTTTGGCGTTGGTGGAGTATGCAATCA-3′ (spanning −2509/−2127 bp) for mouse AR promoter DNA fragments and 5′-TATAGGTACCATCATTGGTTGCCTGAGGAG-3′ and 5′-TAGCAGATCTGGCAGGATGGTAGAATGGAA-3′ (spanning −2837/−1638 bp) for human AR promoter DNA fragments.ChIP was performed based on the protocol provided in the kit with some modifications  as previously described 5′-AGTGCTCCAGGTGGAAGAGA-3′ and 5′-CTCTGGAAAGCCAGGATGAG-3′ (spanning −2553/−2401 bp), 5′-AGGAAAGAGTGGAGGGAGGA-3′ and 5′-AGGATGAGCTGACCTTTGGA-3′ (spanning −2584/−2413 bp) which both overlap putative Nur77-binding site of human AR promoter; 5′-TCCTTAAATGCAGGGTCCAC-3′ and 5′-AAATGCCCTGTGATTTCAGC-3′ (spanning −4713/−4526 bp), 5′-GAGTCGTTGGAGGACCTGAA-3′ and 5′-CCTGTCACTGTCTCCCCATT-3′ (spanning −4119/−3967 bp) as negative control region.To provide stronger evidence about Nur77 binding to NBRE in human AR promoter, independent quantitative ChIP experiments were performed. For these studies, chromatin was co-immunoprecipitated from KGN lysates with an antibody to Nur77 or irrelevant antibody (goat anti-rabbit IgG) as control. Lysates were sonicated to generate chromatin fragments of 200 bp which were amplified by quantitative real time PCR. The sets of primers included: Double-stranded oligonucleotides were designed for ABCD assays and biotinylated at the 3′-end of the sense strand. The following primers were designed: 5′ biotin–CACCATTTTCCAAAGGTCAGCTCATCCTGG-3′ for human AR wild-type F (from −2440 to −2411 bp); and 5′ biotin–CACCATTTTCCAgAatTCAGCTCATCCTGG-3′ for human AR mutant F. KGN cells infected with Ad-LacZ and Ad-Flag-Nur77 (10MOI) for 48 h were lysed with lysis buffer  containing protease inhibitor cocktail at 4°C. The cell extracts were incubated with 500 pmol of each double-stranded DNA immobilized on streptavidin agarose beads in binding buffer  containing protease inhibitor cocktail for 4 h at 4°C. The protein complexes on the beads were separated by SDS-PAGE, transferred onto polyvinylidine fluoride membrane and probed with an anti-Flag-HRP antibody . Immunodetection was performed using an enhanced chemiluminescence detection kit.t-test was performed for comparison between the mean values for two groups, and ANOVA was used to detect differences among more than two groups Values were determined to be significant when P<0.05.Data were expressed as the means ± SEM from at least three independent experiments. Student's in vivo, AR and Kitl mRNA expression levels were analyzed with real-time PCR in the ovaries from Nur77+/+ and Nur77−/− 20-week-old female mice. In comparison with Nur77+/+ ovaries, the expression levels of AR mRNA , we found a potential NBRE (TCAGGTCA) within mAR promoter core regions. To examine if Nur77 can bind to the AR promoter in vivo, we performed a ChIP-PCR assay. Confluent mGCs were infected with Ad-LacZ and Ad-Flag-Nur77 at an MOI of 10 for 48 h. Soluble chromatin from the mGCs was then immunoprecipitated with an anti-FLAG M2 affinity agarose gel, and the purified DNA fragments from the Flag-Nur77-Chromatin complexes were analyzed by PCR with the specific primer pairs designed for the NBRE region of the mAR promoter. AR promoter was efficiently recovered from the Flag-Nur77 immunoprecipitates but not from the control immunoprecipitates (Ad-LacZ), suggesting that Nur77 directly binds to the chromatin-associated AR promoter in mouse granulosa cells.Nur77 has been reported to regulate target gene expression through direct binding to the NBRE sequence (AAAGGTCA) as monomers (4) and/or to the palindromic NurBE sequence as homodimers (7). Based on the mouse AR promoter activity, we measured AR gene promoter activity in HEK293T cells and mGCs by transient transfection and luciferase reporter assays. As shown in AR promoter activity in a concentration-dependent manner. In mGCs from Nur77−/− mice, however, the mAR promoter activity was significantly lower than that in mGCs from Nur77+/+ mice , we first conducted a ChIP-PCR assay as described in mGCs above, and similar results were obtained . Primers used in these experiments were designed to generate a product of <200bp. It was found that chromatin fragments with potential NBRE, instead of negative control region, specifically co-precipitated with Nur77 from lysates of KGN cells  in a luciferase reporter plasmid and transiently transfected HEK293T cells to measure AR gene promoter activity. Overexpression of Nur77 activated hAR promoter activity in a concentration-dependent manner  or an androgen signaling antagonist, flutamide (FL). Real-time PCR analysis showed that Kitl expression was elevated by AD in KGN cells, which is consistent with a previous report Nur77+/+ mGCs  and a partial NBRE sequence (AGGTCA) have been observed in human and mouse −/− mice, Nurr1 expression was proved to be faint and not increased −/− mice seemed to have a fluctuation along with the mouse age (data not shown). In addition, expression levels of Nurr1 and Nor1 in human ovarian follicles and corpora lutea are much lower than Nur77 As the other two members of the NR4A family, Nurr1 and Nor1, have similar expression patterns and transcriptional roles as Nur77 KITLG promoters has been observed using a luciferase reporter assay in KGN, 293T, and HeLa cells in vivo and in vitro. To ensure this effect was androgen-dependent, we performed the Nur77 overexpression experiments in KGN cells and +/+Nur77mGCs maintained in complete medium (DMEM/F12 medium plus 10% FBS), phenol-free medium (phenol-free medium plus 10% FBS), or androgen-free medium . The results provided evidence that Kitl was regulated by Nur77 in the presence of exogenous androgen, whereas its expression was not up-regulated in androgen-free circumstance (Kit ligand (Kitl) is a growth factor that influences target cells through binding to its tyrosine kinase receptor, Kit umstance , S2. TheAR knockout (ARKO) female mice are subfertile, and they have defective folliculogenesis. Moreover, ARKO female mice ultimately develop premature ovarian failure −/− mice as a measure of reproductive function in previous studies Global Nur77 knockout mice in vivo. Our study provides new insights into Nur77 functions in the female reproductive system. Understanding the crosstalk between Nur77 and AR signaling helps us to understand the mechanisms of androgen signaling, follicle growth and oocyte maturation, which are the key pathological processes in diseases, such as premature ovarian failure (POF) and polycystic ovary syndrome .In conclusion, AR protein expression is positively regulated by Nur77 at least at the transcriptional level through the AR promoter in mouse and human granulosa cells. AR signaling is also mediated by Nur77 as shown by changes in Kitl expression. The transcriptional down-regulation is also confirmed in the ovaries from Figure S1Effects of Nur77 overexpression on mouse AR and Kitl expression.Nur77+/+ mGCs were infected with Ad-LacZ or Ad-Flag-Nur77 at the indicated MOI for 48 h. Cells were cultured in complete medium , phenol-free medium , or androgen-free medium . AR and Kitl mRNA levels were measured by real-time PCR and shown as a ratio over control (Ad-LacZ). The results are an average of three independent experiments performed in triplicate .(TIF)Click here for additional data file.Figure S2Effects of Nur77 overexpression on human AR and KITLG expression. KGN cells were infected with Ad-LacZ or Ad-Flag-Nur77 at the indicated MOI for 48 h. Cells were cultured in complete medium , phenol-free medium , or androgen-free medium . AR and KITLG mRNA levels were measured by real-time PCR and shown as a ratio over control (Ad-LacZ). The results are an average of three independent experiments performed in triplicate .(TIF)Click here for additional data file.


The arene ligand has a strong influence on the in vitro anticancer activity of this series of compounds, which correlates fairly well with cellular accumulation. The most lipophilic compound bearing a biphenyl moiety and a cyclohexylidene-protected carbohydrate is the most cytotoxic with unprecedented IC50 values for the compound class in three human cancer cell lines. This compound shows reactivity to the DNA model nucleobase 9-ethylguanine, but does not alter the secondary structure of plasmid DNA, indicating that other biological targets are responsible for its cytotoxic effect.The synthesis and  II(arene) complexes (pta)Cl2]  (en)Cl]+  (HOPO)Cl] complexes (HOPO = hydroxypyr(id)one)  Dyson, ,  ruthenium(II)], bis[dichlorido(η6-benzene)ruthenium(II)], bis[dichlorido(η6-toluene)ruthenium(II)] ruthenium(II) 1a, dichlorido(η6-p-cymene)ruthenium(II) 1 b , 125.75 (13C{1H}) and 202.44 MHz (31P{1H}) or a Bruker Avance 400 FT NMR spectrometer at 400.13 (1H), 100.63 (13C{1H}) and 161.98 MHz (31P{1H}). 2D NMR spectra were collected in a gradient-enhanced mode. Melting points were measured on a Büchi B-540 apparatus and are uncorrected. Elemental analysis was performed by the Laboratory for Elemental Analysis, Faculty of Chemistry, University of Vienna, on a Perkin–Elmer 2400 CHN Elemental Analyzer and at the Microanalytical Laboratory of the EPFL. Electrospray ionization mass spectra were recorded on a Bruker esquire3000. Na2EDTA·, NaOH (Fluka), tris(hydroxymethyl)aminoethane, glacial acetic acid , and MilliQ H2O  were used for the preparation of TAE buffer for gel electrophoresis studies. Loading buffer (6×), pBR322 DNA (0.5 μg/μL) and GeneRuler™ 1 kb DNA ladder (0.5 μg/μL) were obtained from Fermentas.All chemicals were obtained from commercial suppliers and used as received and were of analytical grade except for methanol and CH6-arene)ruthenium(II)] (1 eq.) and the phosphite ligand (2 eq.) in dry CH2Cl2 (30 ml) was stirred for 3 h at 40°C. The solvent was reduced to about 3 mL on a rotary evaporator, and diethyl ether (20 mL) was added resulting in orange or brown precipitates which were filtered, washed with diethyl ether (2 × 5 mL), and dried under vacuum.A mixture of bis[dichlorido and [(η6-benzene)RuCl(μ-Cl)]2  following the general procedure.The title compound was synthesized from 3,5,6-bicyclophosphite-1,2-18H23Cl2O6PRu·0.5CH2Cl2: C 38.25, H 4.16; found: C 38.21, H 4.19%; MS (ESI+): m/z: 560.7 [M + Na]+; 1H NMR : δ = 6.20 , 5.89 , 5.10–5.13 , 4.83 , 4.75 , 4.48-4.50 , 4.31–4.33 , 1.57–1.70  ppm. 13C{1H} NMR : δ = 113.3 (Ccyc), 105.4 (C-1), 91.2 (C-Ar), 83.4 (C-2), 79.5 (C-3), 75.7 (C-4), 75.2 (C-5), 71.5 (C-6), 36.5 (C6H10), 35.8 (C6H10), 24.8 (C6H10), 23.9 (C6H10), 23.5 (C6H10) ppm. 31P{1H} NMR : δ = 132.7 ppm.Yield: 104 mg (96%); m.p. 295–297°C (decomp); elemental analysis calcd. for CO-isopropylidene-α-D-glucofuranose  and [(η6-toluene)RuCl(μ-Cl)]2  following the general procedure.The title compound was synthesized from 3,5,6-bicyclophosphite-1,2-16H21Cl2O6PRu·0.25CH2Cl2: C 36.58, H 4.06; found: C 36.93, H 4.22%; MS (ESI+): m/z: 534.6 [M + Na]+; 1H NMR : δ = 6.19 , 5.80–5.82 , 5.58 , 5.52 , 5.09–5.12 , 4.81 , 4.74 , 4.44–4.48 , 4.31–4.33 , 2.34 , 1.52 , 1.36  ppm. 13C{1H} NMR : δ = 113.0 [C(CH3)2], 112.6 (C-Ar), 105.7 (C-1), 91.0 (C-Ar), 90.6 (C-Ar), 83.7 , 82.2 (C-Ar), 81.8 (C-Ar), 79.2 , 77.2 (C-Ar), 76.8 , 74.8 , 69.9 , 26.9 [C(CH3)2], 26.3 [C(CH3)2], 19.2 (Ar-CH3) ppm. 31P{1H} NMR : δ = 134.5 ppm.Yield: 201 mg (97%); m.p. 180–181°C (decomp); elemental analysis calcd. for CO-cyclohexylidene-α-D-glucofuranose  and [(η6-toluene)RuCl(μ-Cl)]2  following the general procedure.The title compound was synthesized from 3,5,6-bicyclophosphite-1,2-19H25Cl2O6PRu·0.15CH2Cl2: C 39.37, H 4.40; found: C 39.06, H 4.40%; MS (ESI+): m/z: 574.6 [M + Na]+; 1H NMR : δ = 6.18 , 5.80–5.82 , 5.58 , 5.48–5.51 , 5.08–5.12 , 4.82 , 4.73 , 4.44–4.48 , 4.32–4.34 , 2.34 , 1.67–1.70 , 1.56–1.58  ppm. 13C{1H} NMR : δ = 113.7 (cycC), 112.7 (C-Ar), 105.3 (C-1), 91.1 (C-Ar), 90.7 (C-Ar), 83.4 , 82.3 (C-Ar), 81.6 (C-Ar), 79.4 , 77.4 (C-Ar), 76.6 , 74.9 , 69.8 , 36.9 (C6H10), 36.2 (C6H10), 24.8 (C6H10), 24.2 (C6H10), 23.5 (C6H10), 19.2 (CH3) ppm. 31P{1H} NMR : δ = 134.4 ppm.Yield: 107 mg (95%); m.p. 281–283°C (decomp); elemental analysis calcd. for CO-isopropylidene-α-D-glucofuranose  and [(η6-biphenyl)RuCl(μ-Cl)]2  following the general procedure.The title compound was synthesized from 3,5,6-bicyclophosphite-1,2-21H23Cl2O6PRu·0.25CH2Cl2: C 42.85, H 3.98; found: C 42.97, H 3.88%; MS (ESI+): m/z: 596.6 [M + Na]+; 1H NMR : δ = 7.69–7.71 , 7.48–7.50 , 6.18–6.22 , 5.99 , 5.88 , 5.03–5.07 , 4.72 , 4.38–4.44 , 4.26 , 4.19 , 1.50 , 1.33  ppm. 13C{1H} NMR : δ = 133.7 (C-Ar), 130.1 (C-Ar), 129.0 (C-Ar), 128.8 (C-Ar), 128.7 (C-Ar), 127.3 (C-Ar), 112.5 [C(CH3)2], 108.3 (C-Ar), 105.7 (C-1), 91.2 (C-Ar), 90.8 (C-Ar), 89.9 (C-Ar), 88.9 (C-Ar), 88.2 (C-Ar), 83.5 , 79.1 , 76.8 (C-4), 74.9 , 69.6 (C-6), 26.9 [C(CH3)2], 26.3 [C(CH3)2] ppm. 31P{1H} NMR : δ = 132.3 ppm.Yield: 217 mg (91%); m.p. 178–179°C (decomp); elemental analysis calcd. for CO-cyclohexylidene-α-D-glucofuranose  and [(η6-biphenyl)RuCl(μ-Cl)]2  following the general procedure.The title compound was synthesized from 3,5,6-bicyclophosphite-1,2-24H27Cl2O6PRu·0.3CH2Cl2: C 45.61, H 4.35; found: C 45.27, H 4.38%; MS (ESI+): m/z: 636.7 [M + Na]+; 1H NMR : δ = 7.62–7.69 , 7.46–7.49 , 6.17–6.20 , 5.98 , 5.87 , 5.03–5.05 , 4.73 , 4.43–4.46 , 4.26 , 4.18 , 1.65–1.67 , 1.54–1.58  ppm. 13C{1H} NMR : δ = 133.6 (C-Ar), 130.1 (C-Ar), 129.0 (C-Ar), 128.8 (C-Ar), 128.6 (C-Ar), 127.2 (C-Ar), 113.2 (cycC), 108.5 (C-Ar), 105.4 (C-1), 91.3 (C-Ar), 90.6 (C-Ar), 90.0 (C-Ar), 89.0 (C-Ar), 88.3 (C-Ar), 83.2 , 79.3 , 76.6 (C-4), 75.0 , 70.0 (C-6), 36.5 (C6H10), 35.8 (C6H10), 24.8 (C6H10), 23.9 (C6H10), 23.5 (C6H10) ppm. 31P{1H} NMR : δ = 132.4 ppm.Yield: 119 mg (93%); m.p. 170–172°C (decomp); elemental analysis calcd. for CO-isopropylidene-α-D-glucofurano-side  was added to a solution of {(η6-phenoxyethanol)RuCl(μ-Cl)}2  in CH2Cl2 (30 mL). The reaction mixture was stirred for 12 h at room temperature. The insoluble residue was removed by filtration and diethyl ether was added to a red solution in order to precipitate the crude product. The pure product was obtained by purification on a silica gel column with CH2Cl2: MeOH (20: 1) as eluent. Yield: 268 mg (60%); m.p. 120–121°C (decomp); elemental analysis calcd. for C17H23O8RuPCl2: C 36.57, H 4.15; Found: C 36.96, H 4.35%; MS (ESI+) m/z: 522 [M - Cl]+; 1H NMR : δ = 6.25 , 6.20 , 5.65 , 5.30 , 5.24 , 4.98 , 4.89 , 4.86 , 4.57 , 4.32 , 4.3 , 3.94 , 1.51 , 1.37  ppm.13C{1H} NMR : δ = 145.3 (C-Ar), 112.6 [C(CH3)2], 105.8 (C-1), 94.3 (C-Ar), 93.7 (C-Ar), 83.6 , 79.0 , 77.2 (C-4), 74.7 (C-Ar or C-5), 73.8 (C-Ar or C-5), 72.8 , 69.0 (C-6), 60.8 (CH2-OH), 26.9 (CH3), 26.2 (CH3) ppm.31P{1H} NMR : δ = 137.1 ppm.3,5,6-Bicyclophosphite-1,2-O-cyclohexylidene-α-D-glucofuranoside  was added to a solution of {(η6-phenoxyethanol)RuCl(μ-Cl)}2  in CH2Cl2 (30 mL). The reaction mixture was stirred for 12 h at room temperature. The insoluble residue was removed by filtration and diethyl ether was added to a red solution in order to precipitate the crude product. The pure product was obtained by purification on a silica gel column with CH2Cl2: MeOH (20: 1) as eluent. Yield: 350 mg (73%); m.p. 90–91°C (decomp); elemental analysis calcd. for C20H27O8RuPCl2: C 40.14, H 4.55; Found: C 40.41, H 4.73%; MS (ESI+) m/z: 563 [M - Cl]+; 1H NMR : δ = 6.19 , 5.98 , 5.53 , 5.13 , 5.10 , 4.82 , 4.73 , 4.50 , 4.40 , 4.33 , 4.28 , 3.97 , 3.37 , 1.70–1.24  ppm. 13C{1H} NMR : δ = 145.0 (C-Ar), 113.2 (C6H10), 105.4 (C-1), 94.0 (C-Ar), 93.6 (C-Ar), 83.3 , 79.2 , 76.8 , 74.6 , 73.7 (C-Ar), 72.9 (C-Ar), 72.7 (O-CH2), 69.2 , 60.6 (CH2-OH), 36.4 (C6H10), 35.7 (C6H10), 24.8 (C6H10), 23.9 (C6H10), 23.6 (C6H10) ppm. 31P{1H} NMR : δ = 137.1 ppm.3,5,6-Bicyclophosphite-1,2-2O and the samples were analyzed by 1H and 31P{1H} NMR spectroscopy after 1, 24, 48 and 96 h. For 9-ethylguanine binding experiments, the complexes were mixed at molar ratios of 1: 1 and 1: 2 (complex: 9-EtG) in D2O and reaction progress was monitored by 1H and 31P{1H} NMR spectroscopy after 1, 24 and 96 h, while samples were kept at room temperature during this time period.For hydrolysis studies, the compounds were dissolved in D2, 4, and 6 was tested by agarose gel electrophoresis with the plasmid pBR322. TAE (1x) buffer was employed as incubation medium. Stock solutions of 1 mM of complex 2, 4, and 6 were prepared in TAE (1x) buffer, diluted with TAE (1x) buffer and stored at −20 °C. The plasmid pBR322 was diluted with TAE (1x) buffer as well. Incubation mixtures were prepared to yield rb-values corresponding to 0.01, 0.05, 0.10, 0.50, 1.00 and 5.00.The structural modification of DNA by The agarose gels consisted of 1% agarose in TAE (1×) buffer, and the incubation mixtures were subject to 35 min running time at 100 V and 70 mA in a PerfectBlue™ Mini S (PEQLAB) GE chamber. The DNA bands were stained with ethidium bromide (1 μl/mL) and were processed and analyzed with the gel documentation system GenoView UV-source and GenoSoft Version 3.08 C (VWR).p-nitrophenyl ester (CBZ = N-carbobenzoxy) as substrate. For the enzyme to be catalytically functional, the active site cysteine needs to be in a reduced form which was accomplished before dilution with an excess of dithiothreitol (DTT) for 1 h at 30°C. IC50 determinations were performed in triplicate using a fixed enzyme concentration of 500 nM and a fixed substrate concentration of 200 μM. Inhibitor concentrations ranged from 0.3 to 200 μM.Crude bovine spleen cathepsin B (cat B) was purchased from Sigma (C6286) and used without further purification. The colorimetric cat B assay was performed in 100 mM sodium phosphate, 1 mM EDTA, 0.025% polyoxyethylene (23) lauryl ether (BRIJ), pH 6.0, using Na-CBZ-L-lysine The enzyme and inhibitor were co-incubated at 30°C over a period of 24 h prior to the addition of substrate. Activity was measured over 3 min at 326 nm. Colorimetric readings were performed on a Lambda 20 Bio spectrophotometer (Perkin-Elmer).Cell lines and culture conditions. CH1 cells originate from an ascites sample of a patient with a papillary cystadenocarcinoma of the ovary and were a gift from Lloyd R. Kelland, CRC Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, UK. SW480 , and A549  cells were provided by Brigitte Marian . All cell culture media and reagents were obtained from Sigma-Aldrich Austria. Cells were grown in 75 cm2 culture flasks (Iwaki) as adherent monolayer cultures in Eagle's Minimal Essential Medium (MEM) supplemented with 10% heat-inactivated fetal calf serum, 1% v/v non-essential amino acids (from 100× ready-to-use stock), 1 mm sodium pyruvate and 4 mm L-glutamine (complete medium). Cultures were maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO2.MTT assay conditions. Cytotoxicity was determined using the colorimetric MTT -2,5-diphenyl-2H-tetrazolium bromide, Fluka) assay. CH1, SW480 and A549 cells were harvested from the culture flasks by trypsinization, and 100 μL aliquots were seeded into 96-well microculture plates  in complete medium. Densities of 1.5 × 103 (CH1), 2.5 × 103 (SW480) and 4 × 103 (A549) cells per well were chosen in order to ensure exponential growth of untreated controls throughout the experiment. Cells were allowed to settle for 24 h. Then, the test compounds were dissolved and serially diluted in complete medium, and 100 μL aliquots were added to the microcultures. Cells were exposed to the test compounds for 96 h. After exposure, all media were replaced with 1:6 MTT/RPMI mixture (100 μL per well) containing MTT solution in phosphate-buffered saline (5 mg/ml) and MTT/RPMI1640 culture medium . After incubation for 4 h, the supernatants were removed, and the formazan crystals formed in viable cells were dissolved in 150 μL DMSO per well. Optical densities were measured at 550 nm with a microplate reader (Tecan Spectra Classic), using a reference wavelength of 690 nm to correct the unspecific absorption. The quantity of viable cells was expressed in terms of T/C values by comparison to untreated control, and 50% inhibitory concentrations (IC50) were calculated from concentration-effect curves by interpolation. Evaluation is based on means from at least three independent experiments, each comprising three replicates per concentration level.5 cells per well. Cell microcultures were incubated in a moist atmosphere at 37°C for 24 h prior to exposure to the test compounds. The cell number was determined using trypan blue staining in parallel microcultures during the 2 h exposure period. After exposure the medium was removed, cells were washed with PBS and consecutively lyzed with 0.5 mL sub-boiled HNO3 (conc.) per well for 1 h. The Ru concentration was quantified by ICP-MS in presence of 0.5 μM indium used as internal standard. Results are based on three independent experiments, each consisting of three replicates.The cellular accumulation study was conducted following a previously published protocol ruthenium(II)] precursor in CH2Cl2  compounds  compounds in Table 1b, 2, 4, 6, and 8 in CH1 cells, which are usually most sensitive to the treatment with metal compounds, are shown in Figure The cancer-inhibiting potential of 50 values in the low micromolar range. The following order of cytotoxicity was observed with regard to variation of the arene ligand in 1b, 2, 4 and 6 with the identical sugar moiety: biphenyl > cymene > toluene > benzene.Carbohydrate compounds potentially accumulate in tumors due to their high demand for glucose as a result of their upregulated glycolytic energy production. Recently, fluorescent Ru(arene) complexes bearing the pta or a sugar phosphite ligand were shown to accumulate in cells to a similar extent , and the reaction was monitored by NMR spectroscopy. Complex 3 appears to form adducts with the DNA model nucleobase 9-EtG via N7 of the guanine moiety as shown by 1H NMR spectroscopy where a low-field shift from 7.85 to 8.29 ppm was observed for H8 of guanine. Analysis by 31P{1H} NMR spectroscopy immediately after mixing 3 with 9-EtG revealed a single peak at 135.9 ppm for the unreacted compound. Within 1 h, two additional signals at 136.6 and 137.2 ppm appear due to the formation of diastereomers by exchange of one of the chlorido ligands with 9-EtG. However, after 24 h several species were formed, including signals at 93.3 and 95.1 ppm indicative of cleavage of a P-O bond, similar to the behavior observed for related analogous complexes  up to 5 within the incubation period of 2 h. Consequently, the incubation period was increased to 4 and 6 h for complexes 4 and 6, which did not lead to any observable change either, making it unlikely that the cytotoxic effect is related to DNA binding interactions.Furthermore, the reactivity to DNA and impact on its secondary structure was studied by determining the electrophoretic mobility of a dsDNA plasmid in cell-free experiments. However, no influence of complexes in vitro  compounds have been shown to be good inhibitors  complexes with carbohydrate-derived phosphorus-containing ligands. The cytotoxicity appears to be dependent on the lipophilicity, i.e., the most lipophilic compound is the most cytotoxic. Moreover, such complexes are known to exhibit certain selectivity for tumor cells over non-tumorigenic cells and are also active in cisplatin-resistant cancer cells (Berger et al., in vitro assays, but only in vivo experiments will give deeper insight into the potential of these compounds as anticancer drugs.In this paper, we report on the synthesis and biological evaluation of RuThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AR) are usually found in partial or complete androgen insensitivity syndrome (AIS).Androgens are critical in male external genital development. Alterations in the androgen sensitivity pathway have been identified in severely undermasculinized boys, and mutations of the androgen receptor gene  was performed. In silico and luciferase functional assays were performed for unreported variants.Two hundred and ninety-two Caucasian children presenting with isolated hypospadias without micropenis or cryptorchidism and 345 controls were included prospectively. Mutational analysis of the AR were identified in 9 patients with glandular or penile anterior (n = 5), penile midshaft (n = 2) and penile posterior (n = 2) hypospadias, i.e., 3%: p.Q58L (c.173A>T), 4 cases of p.P392S (c.1174C>T), 2 cases of p.A475V (c.1424C>T), p.D551H (c.1651G>C) and p.Q799E (c.2395C>G). None of these mutations was present in the control group. One mutation has never been reported to date (p.D551H). It was predicted to be damaging based on 6 in silico models, and in vitro functional studies confirmed the lowered transactivation function of the mutated protein. Three mutations have never been reported in patients with genital malformation but only in isolated infertility: p.Q58L, p.P392S, and p.A475V. It is notable that micropenis, a cardinal sign of AIS, was not present in any patient.Five missense mutations of the AR mutations may play a role in the cause of isolated hypospadias, even in the most minor forms. Identification of this underlying genetic alteration may be important for proper diagnosis and longer follow-up is necessary to find out if the mutations cause differences in sexual function and fertility later in life. Hypospadias is defined as a malformation of the penis due to an incomplete development of the ventral part of the penis. This may include (1) a defect in the developing urethra leading to the localization of the urinary meatus on the ventral aspect of the penis in a variable position from the glans to the perineum AR. These mutations are usually found in partial or complete androgen insensitivity syndrome AR sequencing is thus generally performed in selected patients with severe 46,XY DSD with normal or elevated plasma level of testosterone AR mutations are usually not considered as a cause of isolated hypospadias Androgens play a central role in male external genital development. Testosterone and its derivative 5 alpha-dihydrotestosterone are the two major androgens that mediate male sexual differentiation, and an alteration in the androgen sensitivity pathway has been identified in undermasculinized boys AR mutations and thus whether all types of hypospadias should warrant molecular analysis of the AR.It is nevertheless now emerging that many milder variants of the classic disorders exist, especially in partial androgen insensitivity syndrome. Moreover, a systematic approach to genetic analysis is providing rewards in some cases 6 ml sperm concentration, ≥40×106 total sperm count, ≥2 ml semen volume, ≥50% of a+b or ≥25% motility and a high percentage of normal forms (≥10%) according to the WHO criteria], with normal location of the urethral meatus, noncleaved prepuce and intrascrotal testis, and 50 women of known fertility . This study was approved by the Institutional Review Board of the institution  and written consent was obtained from all parents.In this study, 292 Caucasian boys presenting with isolated hypospadias  were included prospectively (newborn to 12 years). Clinical diagnosis was performed by direct clinical examination by the pediatric urologist or pediatric endocrinologist. The location of the urethral meatus ranged from glandular to perineal hypospadias . The level of division of the corpus spongiosum—which can be assessed during degloving of the penis at the time of surgical correction—was not used as a classification method here since some patients with anterior and glandular hypospadias did not undergo surgery. Three hundred and forty-five controls alleles were sequenced. They included 245 normospermic men [the definition of normospermic is ≥20×10DNA was extracted from either peripheral blood or preputial skin. When blood was used, the DNA was extracted with a QIAamp DNA blood minikit . When tissue was used, the excess skin removed at the time of hypospadias surgery and/or circumcision was frozen in liquid nitrogen. DNA was extracted from this tissue using DNAzol (Invitrogen). The manufacturer's protocol for DNA isolation was followed with minor modifications.AR using the Taq PCR Master Mix kit from Qiagen , we performed direct sequencing using the BigDye terminator v1.1 kit  and an ABI Prism310 Genetic Analyzer , as reported elsewhere AR mutations were detected, SRD5A2 and MAMLD1 genes were also sequenced to rule out another cause of hypospadias NM_000044.2 and the AR database After polymerase chain reaction (PCR) amplification of exons 1–8 of the When a mutation that had never been reported was found, the functional consequences of amino acid changes were predicted using in silico models.AR gene and alignments were made with the ClustalW software at http://www.ebi.ac.uk/Tools/msa/clustalw2/.Regarding the homology study, ensembl.org detected the putative homologs of the human http://www.compbio.dundee.ac.uk/www-jpred/). The relative accessibility of amino acids was studied with Netsurf software http://www.cbs.dtu.dk/services/NetSurfP/). The three-dimensional structure was predicted by the Protein Homology/analogY Recognition Engine (PhyreEngine) from the Structural Bioinformatics Group, Imperial College, London, at http:www.sbg.bio.ic.ac.uk/phyrew/Regarding the structure prediction, the secondary structure for wildtype and variants was predicted using JPred software http://genetics.bwh.harvard.edu/pph/, http://www.pantherdb.org/tools/csnpScoreForm.jsp., and http://sift.jcvi.org/. These algorithms are based on the alignment of orthologous and/or paralogous protein sequences and/or structural constraints.The functional consequences of amino acid changes were predicted using three algorithms. Polyphen  . The Renilla luciferase construct phRG-TK was obtained from Promega, WI, Madison, USA.The full-length AR expression construct pSVAR0 was a kind gift of Dr. A. Brinkmann . The pAR-D551H mutant was constructed by site-directed mutagenesis in a two-step PCR using pSVAR0 as a template. In the first round, the primer pair AR-Kpn_fwd: Hela cells were maintained in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12  supplemented with 10% fetal calf serum in 5% CO2 at 37°C.Rhox5 firefly-luciferase reporter plasmid, 30 ng AR expression plasmid, 10 ng of the constitutive Renilla luciferase expression plasmid phRG-TK, and 0.72 µl Fugene HD  per well. Five hours post-transfection, cells were incubated for 18 hr with either vehicle or the indicated concentration DHT, Firefly and Renilla luciferase activities were detected using the Dual-Luciferase reporter assay kit  and a LUCY 3 Luminometer . The activity of the Renilla luciferase was used to normalize for transfection efficiency. All transfections were performed in triplicate and in at least 3 independent experiments.For transfection, HeLa cells were seeded at 50,000 cells per well in 24-well plates in DMEM medium supplemented with 10% charcoal-stripped fetal calf serum. After 24 hr, cells were transfected with 200 ng of AR were identified in 9 of the 292 patients, i.e., 3%: p.Q58L (c.173A>T), 4 cases of p.P392S (c.1174C>T), 2 cases of p.A475V (c.1424C>T), p.D551H (c.1651 G>C) and p.Q799E (c.2395C>G). None of these mutations was present in the control group. The hypospadias was not severe in 5 cases  and penile posterior in 2 cases only. Clinical data are summarized in SRD5A2 or MAMLD1 was found in these patients. The mothers were not available for sequencing.Five missense mutations of the The p.D551H (c.1651 G>C) mutation has never been described and was thus tested in silico.The secondary structure was predicted to be modified proximal to the mutation with changes in a helical domain of 11 amino acids. The relative and absolute accessibilities of the amino acid were modified from 0.55 to 0.60 and from 77.06 to 110.41, respectively. The structure prediction of the mutated protein was significantly changed (data not shown). All 3 in silico algorithms predicted affected protein function  with a conserved amino acid throughout species .The in vitro functional studies confirmed that the D551H mutation induced a reduction of the androgen receptor transactivation. The difference between the wild type protein and the mutated one was significant at DHT concentrations between 0.01 and 10 nM .AR mutation analysis, along with karyotyping and hormonal work-up, should be performed in children with (1) a phenotype of uncertain sex at birth, (2) severe hypospadias, and (3) hypospadias associated with another sign of DSD that could be a manifestation of partial androgen insensitivity syndrome, such as micropenis or cryptorchidism AR including exon 1 remain rare. Vottero et al. AR in target tissues from patients with hypospadias is more methylated than in control children, resulting in a decreased expression of the AR. However, no mutation was identified. In order to better elucidate the genotype-phenotype relation and identify which patients should be explored, we prospectively screened a large number of unselected hypospadiac subjects.The recommendations for the appropriate use of genetic testing in male children with genital malformation state that AR mutations. Two of these mutations have already been associated with severe genital malformation: p.Q799E We found that 3% of 292 boys with isolated hypospadias had The risk of infertility is known to be associated with hypospadias, but there has been no definitive and methodologically adequate study of the fertility of men born with hypospadias. In an evaluation of the social and sexual impact of this malformation, Aho et al. AR mutations even in the most minor forms of hypospadias may indicate that these mutations make up part of the common background of these two diseases. Early detection of the AR mutations known to be usually present in patients with PAIS syndromes or hypofertile men might also significantly improve (1) the hormonal and clinical follow-up of these patients during puberty, especially regarding the size of the penis. Indeed, the activities and effects of androgens during fetal life do not always predict their impact on penile growth during puberty Thus, the patients with the highest risk are mainly those with cryptorchidism and those with a severe meatus displacement AR, whereas about 85% of the mutations reported in the AR mutations database http://www.mcgill.ca/androgendb/) are localized in exons 2 to 8. In contrast, the mutations responsible for minor phenotypes, as seen in our series, are mainly present in exon 1. Of the exon 1 mutations described in the AR database, 70% induced complete androgen insensitivity syndrome (CAIS), 14% induced partial androgen insensitivity syndrome (PAIS), and 16% were implicated in infertility. Exon 1 is an important regulatory region for AR activity. It encodes the N-terminal domain of the AR protein, which contains transactivation elements, dimerization zones, several cofactor binding sites, and two regions (25FXXLF27 and 433WXXLF437) involved in N-terminal and C-terminal domain interactions. Loss of this domain results in inactivation of the AR since testosterone binding no longer leads to its conformational change and the subsequent interactions between helix H12 and helix H3 AR mutation in the database (ranging from PAIS to infertility). These results nevertheless show that missense mutations of exon 1 may slightly alter AR function, inducing minor phenotypes.It is notable that 7 out of 9 patients exhibited a mutation in exon 1 of the AR mutation. In our series, no clinical data from the medical history or physical examination were able to identify patients at risk for these mutations. Mutated subjects had no other signs of androgen insensitivity than isolated hypospadias. Size of the penis, location of testes and location of the urethral meatus were unable to identify a priori patients with a higher risk of AR abnormalities. Familial history was more frequent in patients with an AR mutation (2/7) than in the others , but this criterion is not sufficient and it misses most of the patients with a genetic alteration. The clinical data alone cannot be used as indicators of AR mutation. The hormonal work-up was of no help either. Although higher LH and testosterone concentrations are observed in patients with more severe forms of androgen insensitivity such as CAIS, measurements of LH, FSH and plasma testosterone were normal or inconclusive in our series since most patients with a minor phenotype are referred to the surgeon during infancy, far after the neonatal period of pituitary-testicular activity (mini-puberty). Conducting systematic HCG tests in these patients would be abusive.Isolated and minor hypospadias is the most frequent form of 46,XY DSD, and screening all patients with this phenotype would be very expensive and time-consuming. It would thus be helpful to identify the subgroup of these children who need to be screened for an AR mutations may play a role in the cause of isolated hypospadias, even in the most minor forms of this malformation. We found a prevalence of mutations of about 3%. These patients with AR abnormalities did not differ from the vast majority of hypospadiac boys. Identification of the underlying alteration in the AR may be important for a proper diagnosis of this frequent genital abnormality. Longer follow-up of these patients is necessary to determine whether these mutations cause differences in sexual function and fertility later in life.


Pinus massoniana (AR-sensitive) and Taxus wallichiana var. mairei (AR-resistant) are widely distributed in southern China. Under AR stress, significant necrosis and collapsed lesions were found in P. massoniana needles with remarkable yellowing and wilting tips, whereas T. wallichiana var. mairei did not exhibit chlorosis and visible damage. Due to the activation of a large number of stress-related genes and the synthesis of various functional proteins to counteract AR stress, it is important to study the differences in AR-tolerance mechanisms by comparative proteomic analysis of tolerant and sensitive species. This study revealed a total of 65 and 26 differentially expressed proteins that were identified in P. massoniana and T. wallichiana var. mairei, respectively. Among them, proteins involved in metabolism, photosynthesis, signal transduction and transcription were drastically down-regulated in P. massoniana, whereas most of the proteins participating in metabolism, cell structure, photosynthesis and transcription were increased in T. wallichiana var. mairei. These results suggest the distinct patterns of protein expression in the two woody species in response to AR, allowing a deeper understanding of diversity on AR tolerance in forest tree species.Acid rain (AR), a serious environmental issue, severely affects plant growth and development. As the gymnosperms of conifer woody plants,  To daPinus massoniana, a widely distributed coniferous species in southern China, has suffered seriously from AR in recent decades [P. massoniana in several AR-affected regions of southern China was revealed in previous studies and the growth was significantly reduced under AR treatment [Taxus wallichiana var. mairei, another conifer tree species, is distributed in adjacent regions that are also suffering from serious AR [T. wallichiana var. mairei. The results suggest that T. wallichiana var. mairei is a resistant species to AR stress [ decades . Severe reatment ,10. Taxurious AR . PhysiolR stress ,13; howeetc., have been successfully identified and characterized, and may play important roles in mediating plant responses to AR for P. massoniana and Arabidopsis [P. massoniana and T. wallichiana var. mairei, which respectively represent AR-sensitive and AR-tolerant species. Furthermore, we also characterized the AR-responsive proteins, analyzed the functions of these differentially expressed proteins, and established the different responsive networks of metabolism in P. massoniana and T. wallichiana var. mairei under AR stress.Recently, by using a 2-DE-based proteomic approach, a set of proteins associated with: primary metabolism, secondary metabolism, protein stability and defense, photosynthesis and energy metabolism, bidopsis –16. In tThe results of this study, providing different evidence from that evaluating the effects of AR stress on single tree species, could further enhance the current understanding of the protein changes underlying AR stress-related cellular and physiological responses between AR-sensitive and AR-resistant woody plants, and further dissect the different tolerance mechanisms of forest trees to AR stress.2.2.1.2.1.1.P. massoniana and T. wallichiana var. mairei, an AR-sensitive and AR-tolerant tree species respectively, were treated with simulated AR (pH 3.0) for two months. First we investigated the detrimental phenotypical and physiological changes of these two species under AR stress. Morphological changes of both species are shown in Pn) of the saplings were also measured , the following groups were photosynthesis and energy production (16.9%), stress and defense (16.9%), protein synthesis and modification (12.3%), signal transduction (7.7%), transcription (7.7%), hormone response (4.7%), cell structure (1.5%), and function unknown and hypothetical proteins (12.3%) , metabolism (11.5%), cell structure (11.5%), signal transduction (11.5%), transcription (11.5%), stress and defense (3.9%), hormone response (7.7%), and function unknown and hypothetical proteins (7.7%)  . In T. ws (7.7%) . This reP. massoniana compared to those in T. wallichiana var. mairei under AR stress. In AR-treated P. massoniana, 15 proteins were increased and 50 proteins were decreased -NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin) to redox-based metabolisms in plastids and mitochondria [T. wallichiana var. mairei reflects the role of this protein in basal metabolism under stress conditions. ABC transporters constitute one of the largest protein families with diverse functions in membrane transport [P. massoniana under AR treatment. Earlier studies have detected a decrease in ABC transporter substrate binding protein in response to copper stress in Cannabis sativa roots, suggesting that the ABC transporter may play a very important role in the tolerance response of plant to environmental stresses [P. massoniana under AR stress. The down-regulation of the protein related to transmembrane transport of molecules  and down-regulation of metal ion related proteins, is propitious for re-establishing cellular homeostasis in AR-treated P. massoniana. Based on the above results, we speculate that AR stress might affect more metabolic processes in P. massoniana than those in T. wallichiana var. mairei.Donation of electrons by ferredoxin (Fd) has been demonstrated in many other plastid enzymes, which is essential for multiple cellular processes, including nitrogen and sulfur assimilation, amino acid and fatty acid synthesis ,22. In tchondria . It is nchondria . The up-ransport . Comparestresses . Furtherstresses . In planstresses . In our 2.2.2.T. wallichiana var. mairei under AR stress. Previous studies reported that the transverse orientation of cortical microtubule arrays in tobacco BY-2 cells was remodelled to a more random arrangement and the tubulin a-6 chain was induced in Arabidopsis roots after NaCl treatment [T. wallichiana var. mairei. These universal cytoskeletal proteins may call into question their stronger tolerance in T. wallichiana var. mairei in response to AR than that in P. massoniana. Cell wall proteins are essential constituents of the plant cell wall, which are involved in modifications of cell wall components and structure, and signaling and interactions with plasma membrane proteins. Beta-fructofuranosidase is an enzyme involved in cell wall biosynthesis. In this study, the abundance of beta-fructofuranosidase  could play an important role in plant adaptation to environmental stresses in addition to its role in peptide elongation [P. massoniana after AR treatment, indicating that protein synthesizing machinery plays an important role in AR adaptation in this plant. Glycogen synthase kinase 3 (GSK-3) was originally identified as a regulator of glycogen synthesis in mammals. In plants, GSKs are reported to be involved in diverse important processes including hormone signaling, development, pathogenic stimuli and stress responses [P. massoniana under AR treatment. Indeed, our result provides extra evidence that GSKs play an important regulatory function under AR stress. Moreover, it is known that chaperonin is down-regulated under oxidative stress in rice [P. massoniana. This finding suggests that chaperonin may be a general AR stress response element in P. massoniana. As a whole, these results suggest that AR treatment affected the biosynthesis and refolding of proteins and led to protein degradation, which is more pronounced in P. massoniana than in T. wallichiana var. mairei.It is not surprising that AR stress also damaged the homeostasis of protein metabolism between biosynthesis and degradation. Singh ongation . Indeed,esponses . In this in rice . Consist2.2.4.P. massoniana leaves, and similar patterns were also observed for most photosynthesis-related proteins  . These results, together with the complex expression patterns of stress related proteins in both woody plants provide new insights into the relationships between the impacts of AR stress and defense and stress responses.Reactive oxygen species (ROS), which play a critical role in plant cellular signaling and stress responses, are readily produced by abiotic stresses . Plants spot 51, . Thus, tP. massoniana under AR stress, which is consistent with our previous results that AR stress led to a decrease in the abundance of gibberellin-responsive protein in Arabidopsis [P. massoniana. Moreover, the abundance of auxin-responsive protein  is required to synthesize salicylic acid for plant defense [T. wallichiana var. mairei. Its presence may provide protection against AR stress and possibly endow T. wallichiana var. mairei with greater AR tolerance.Plant hormones are not only involved in plant growth and development, but also important in response to abiotic and biotic stresses. Recent studies suggest that environmental stimuli can regulate endogenous gibberellin (GA) level through the changes of enzymes involved in GA biosynthesis and inactivation . In thisbidopsis , suggest spot 47  decrease defense . Further2.2.6.P. massoniana under simulated AR, whereas the abundance of light-mediated development protein DET1-like isoform 2  are implicated as major primary Ca2+ sensors in plants, and CDPK-controlled signaling pathways regulate specific responses to biotic and abiotic stresses. Here, the abundances of CDPK , RNA polymerase II C-terminal domain phosphatase-like 1 (spot 24) in T. wallichiana var. mairei were found to change their expression under AR stress , relative humidity of 60%–70%, a light intensity of 390 μmol·m−2·s−1 photosynthetically active radiation (PAR). The seedlings were sprayed once each day with 200 mL·pot−1 distilled water as control  or simulated AR solution . The ion compositions of the CK solution were adopted from Liu et al., while AR solution was made from CK solution and the pH was adjusted with a mixture of H2SO4 and HNO3 in the ratio of 5 to 1 by chemical equivalents, which represents the average ion compositions of rainfall in southern China [42− and NO3− were 0.45 and 0.09 mM, respectively. After 2-month simulated AR treatment, the sapling needles were collected for physiological measurements and proteomics research.The seedlings of rn China . The fin3.2.v/v acetone. Absorption of the extract was measured at 663 and 646 nm with a spectrometer  and the chlorophyll content was calculated as described by Wellburn [Plant leaves (0.1 g of fresh weight (FW)) were prepared, and chlorophyll was extracted with ice-cold 80% Wellburn .Pn) was measured with a portable photosynthesis system . Air temperature, CO2 concentration, light intensity, and air relative humidity were maintained at 25 °C, 380 μ·L−1, 800 μmol·m−2·s−1 PAR, and 80%, respectively. At least ten saplings were randomly selected from the CK or AR treatment group for Pn measurement.Leaf net photosynthetic rate  was performed according to Hu et al. . The sam [et al. . The pro3.4.et al. [m/z 842.5, and m/z 2211.10). The spectra were analyzed with the flex analysis software . Since the P. massoniana and T. wallichiana var. mairei genome are still unsequenced, a homology-based search was performed. The measured tryptic peptide masses were searched against the National Center for Biotechnology Information non-redundant  database (release date: 16 July 2012), and selecting the taxonomy of green plants using MASCOT interface . The following parameters were used for database search: MH+ monoisotopic mass values, a fragment ion mass tolerance of ±0.3 Da, permitting one missed cleavage, alkylation of cysteine by carba-midomethylation as a fixed modification, and oxidation of methionine as a variable modification. MASCOT Peptide Mass Fingerprint (http://www.matrixscience.com) protein scores greater than 73 with the NCBInr database were considered significant (p < 0.05). A number of other criteria were further evaluated in the final assignment of protein and peptide identifications: the number of matching peptides (at least four), the coverage (a minimum of 9%), and the molecular weight (Mr) and isoelectric point (pI) of the protein. Furthermore, to avoid false positives, an additional in-house Basic Local Alignment Search Tool (BLAST) search against the NCBI protein database (http://www.ncbi.nlm.nih.gov) was done to reconfirm all the matches.In-gel protein digestion and protein identification was followed as described by Liu et al. . Matrix-http://www.uniprot.org) and TAIR database  to find out if their functions are known, they were then further classified using Functional Catalogue software .The identified proteins were used to search for over/under-representation of the searched proteins adopting the AGI codes as input and then searched within the UniProt  .Values in figures and tables were expressed as means ± SE. The statistical significance of the data was analyzed using a univariate analysis of variance (4.P. massoniana than in T. wallichiana var. mairei in response to simulated AR treatment. Taken together, metabolism, photosynthesis, signal transduction and transcription related proteins in P. massoniana, a sensitive species, were depressed by AR stress. However, the abundances of proteins participating in photosynthesis as well as in signal transduction and transcription were increased in T. wallichiana var. mairei, a tolerant species, under simulated AR. We presume that AR was a strongly oxidative situation experienced by P. massoniana but was of weak oxidative intensity in T. wallichiana var. mairei. On the other hand, AR stress influenced only part of the photosynthetic network and modified gene expression in such a way as to enhance metabolism systems and strengthen plant defense responses to maintain physiological and biochemical homeostasis in T. wallichiana var. mairei. This is likely one of the reasons P. massoniana is more sensitive to AR stress than T. wallichiana var. mairei. The proteins identified in this study might be useful in investigating the different defense mechanism of woody plants to AR stress.In this study, a comparative proteomics analysis was carried out to clarify the differentially expressed protein profiles of two tree species under AR treatment. Our data revealed that many more proteins altered their expression level in P. massoniana and T. wallichiana var. mairei, together with previous reports, we outlined a schematic overview model associated with the different systematic response of P. massoniana and T. wallichiana var. mairei to AR stress (P. massoniana and T. wallichiana var. mairei under AR challenge and deepen our understanding in AR tolerance in woody plants.Based on the putative functions and expression changes of the identified proteins in R stress . These r


The casual and severity distribution of allergic rhinitis (AR) in Hungary is unknown.The aim of this survey was to evaluate symptom perception, disease severity, concomitant asthma frequency and the impact of AR on everyday life activities in a cross-sectional, multicenter study in Hungary under the supervision of Hungarian Respiratory Society.Data were recorded by 933 AR patients (65.93% women) and their treating specialists. The perceptions of patients regarding the symptoms  of AR and its severity, together with its impact on everyday life were assessed. Physicians recorded data regarding the diagnosis and severity of AR, and comorbidities.52.5% of patients suffered from seasonal AR, 35.1% from perennial AR. A large proportion of patients had moderate to severe disease (MS-AR) (57.34%), persistent disease (98.0%) and concomitant asthma . MS-AR was more frequent among women. Despite the treatment used, in MS-AR the proportions of patients reporting moderate to severe rhinorrhoea, nasal obstruction, ocular itching/redness, watering, itchy throat and sneezing were as high as 52.0%, 54.0%, 33.8%, 26.5%, 44.0% and 31.2%, respectively. Overall, there was a poor agreement between disease severity reported by patients and specialists. The adherence to oral antihistamines and intranasal corticosteroids was found to be between 50 and 65%; mostly depending on the dosage form.AR remains a significant health problem in Hungary because of the burden of symptoms, high rate of concomitant asthma and the significant proportion of MS-AR affecting general well being. The incidence and prevalence of allergic rhinitis (AR) has increased worldwide over the past decades. Presently, AR is the most common allergic respiratory disorder, affecting about 10-30% of the general population worldwide, depending on the area and on the age of patients. About 19% of the general population in Europe, while 8.8% to 16% in the United States of America suffers from mild or moderate-severe AR. The disAlthough the prevalence of AR is increasing in Hungary according to some national epidemiological data, there aThe present multicenter cross-sectional study was initiated and supervised by the Hungarian Respiratory Society and was conducted with Hungarian National Ethical Board approval. All the counties in Hungary were represented with sample sizes proportional to respective general population sizes. The perceptions of patients and physicians regarding the symptoms of AR and its severity and impact on general well being and everyday life were assessed. All the attending doctors were specialists, including allergy specialists, ear, nose and throat doctors and respiratory physicians, and they enrolled the AR patients consecutively on the occasion of patients’ regular visits. AR was diagnosed by the physicians, and adult patients were targeted. Physicians recorded data relating to patient characteristics, diagnosis, AR severity, common triggers, comorbidities, current and past drug treatments and smoking history.st, 2009 and October 1st, 2009. It must be noted that a patient number above 1,000 was targeted; however 74 surveys were either not completely filled out or were not filled out at all, so these surveys were not used in the data analysis. 65.93% women (N=615) and 34.07% men (N=318) were enrolled . Diagnostic skin prick tests to confirm AR had been performed on 97.18% of patients at least since the diagnosis. Allergen specific IgE antibody tests were carried out in 22.86% of patients. 14.6% of the patients were smokers, 19.42% ex-smokers, while 65.93% non-smokers. Patients’ characteristics are summarized in TableData were recorded for 933 patients suffering from AR between June 1= no symptoms, 1 = mild symptoms, 2 = moderate symptoms and 3 = severe symptoms. Thus, the total of nasal, ocular and other scores could have been between 0 and 6, where the higher was the total score the most severe was the symptom. Besides the physicians’ evaluation of the severity of AR (whether it was mild or moderate to severe according to ARIA guideline)[Data regarding the presence and severity of AR symptoms as well as the impact of AR on everyday life activities were reported by the patients using a questionnaire which was developed by the authors based strictly on Allergic Rhinitis and its Impact on Asthma (ARIA) guideline for thisideline), the dis2) method.In the present study Student’s-t test was used and a p value of < 0.05 was considered significant. To assess any association between AR severity and sex we used the chi-square . Aeroallergens most frequently involved in seasonal atopic sensitization were ragweed, mugwort, and birch, while the most common cause of perennial disease was house dust mite. Overall, 98.0% of the patients surveyed had persistent disease (defined as symptoms experienced on > 4 days/week and for > 4 consecutive weeks).Physicians reported on the cause of visits. Most patients (49.5%) were consulting for repeat prescriptions, 30.9% for routine follow up, and 14.1% because of worsening symptoms (5.5% of patients did not answer the question regarding the cause of doctor visit). Comparing the groups of mild AR and moderate-severe AR, the causes of physician visits were markedly different. In case of mild AR only 1% of patients were consulting due to symptom worsening and 34% for routine follow up. On the contrary, regarding moderate-severe AR patients 22.4% visited their specialist because of disease deterioration and only 22% for routine follow up.2< 0.05). The mean ages were not significantly different between the two severity groups . The frequency of food cross-allergy was found to be higher among moderate to severe AR patients (23%) than in the subcategory of mild AR (16%). AR had been diagnosed 5.2 years before in mild AR, and 7.0 years before for moderate-severe patients at the visit.Severity of AR was gender related. While among mild AR patients only 61.23% were women, in case of moderate-severe AR 69.23% of the patients were woman, pointing on higher prevalence of moderate-severe AR among women  and patients with moderate to severe disease were on both oral antihistamine and intranasal steroid  therapy in most cases. The optimal adherence regarding antihistamine tablets was reported to be as low as 64.32% for mild and 60.19% for moderate-severe AR patients, while regarding intranasal corticosteroids 50.15% and 50.22% for mild and severe AR group, respectively. Evaluating the possible causes of non-adherence the following were identified: 39.68% of mild AR and 31.49% of moderate-severe AR patients were without symptoms, thus they did not take the tablets. The respective values for nasal corticosteroids were 88.66% and 64.29%. The second most frequent reason of non-adherence with oral treatment was forgetting to take it: this was recorded in 40.48% and 35.91% of mild and moderate/severe AR patients, respectively. Of note, regarding intranasal steroids, this reason was rarely indicated. Side effects were the causes of non-adherence only in 2-4%.This cross-sectional, multicenter survey was the first to evaluate the severity, impact on everyday life activities, symptom perception, and concomitant asthma frequency in allergic rhinitis patients in all counties of Hungary. Conducted among 933 patients with AR presenting to their specialist, the study found that AR was mostly confirmed with diagnostic tests in Hungary. A marked proportion of patients had moderate or severe disease (57.34%), persistent symptoms (98.0%) and comorbidities such as asthma . Underrepresentation of patients with intermittent disease might be the consequence of over-the-counter freely available antihistamine tablets available for intermittent AR patients for 4 weeks without medical prescription.As reflected in this and other surveys, AR imposes a substantial burden on patients regarding everyday life limitations and work performance,13. NearOur results confirm higher rate of seasonal AR and are similar to those of AILA study (Allergies in Latin America), revealing that 62% of Latin American AR patients are seasonally affected, with the remaining 38% suffering year-long symptoms. In the Overall, there was only a poor correlation between the AR severity evaluated by physicians and that based on patients’ opinion. In good agreement with previous results of Canonica et al. it can bSeveral studies have confirmed the importance of rhinitis symptoms in the future development of asthma. The sevThis is the only multicenter study ever conducted in Hungary to describe the symptoms, impact on everyday life and concomitant asthma frequency of AR; however as a real-life investigation it has many limitations. The bias on symptom severity caused by a possible not proper AR diagnosis or treatment non-adherence cannot be excluded in some cases. There were 74 surveys which were not included in the analysis due to missing data. Furthermore, some local and oral antihistamines, nasal decongestants may be purchased over-the-counter in pharmacies, therefore their use is completely unknown in this survey.In conclusion, this survey, enrolling 933 patients with AR presenting to their specialist and lead by the Hungarian Respiratory Society, found that a significant proportion of patients have moderate or severe disease, persistent symptoms and comorbidities such as asthma in Hungary. A more severe disease was shown to be more frequent among women. Furthermore, our results highlighted the poor symptom control of patients who presented with moderate or severe and persistent disease.The authors declare that they have no competing interests.


The androgen receptor (AR) expression and the CAG repeat length within the AR gene appear to be involved in the carcinogenesis of male breast carcinoma (MBC). Although phenotypic differences have been observed between MBC and normal control group in AR gene, there is lack of correlation analysis between AR expression and CAG repeat length in MBC. The purpose of the study was to investigate the prognostic value of CAG repeat lengths and AR protein expression.81 tumor tissues were used for immunostaining for AR expression and CAG repeat length determination and 80 normal controls were analyzed with CAG repeat length in AR gene. The CAG repeat length and AR expression were analyzed in relation to clinicopathological factors and prognostic indicators.P = 0.001) and controls are more likely to exhibit short CAG repeat sequence than MBCs. There was statistically significant difference in long CAG repeat sequence between AR status for MBC patients (P = 0.004). The presence of long CAG repeat sequence and AR-positive expression were associated with shorter survival of MBC patients .AR gene in many MBCs has long CAG repeat sequence compared with that in control group (The CAG repeat length within the AR gene might be one useful molecular biomarker to identify males at increased risk of breast cancer development. The presence of long CAG repeat sequence and AR protein expression were in relation to survival of MBC patients. The CAG repeat length and AR expression were two independent prognostic indicators in MBC patients. Male breast carcinoma (MBC) is an uncommon disease, accounting for less than 1% of all cases of breast carcinoma. Hormonal factors are specifically important in carcinogenesis of male breast. Androgens, like dihydrotestosterone, restrain tissue growth in the breast thereby exerting anti-mitogenic effects Germline mutations in the AR gene have also been suggested to predispose to MBC The AR has biological and therapeutic significance in prostate cancer The aims of the present study were to: (a) investigate whether increased length of the CAG repeat sequence in the AR gene is associated with the development of MBC. (b) compare CAG repeat lengths with clinicopathological features and hormone receptor expression; (c) evaluate prognostic significance of AR (CAG)n repeats length and AR expression; and (d) present a new opinion of endocrine therapy (anti androgenic therapy) for MBC patients.Controls and patients in this study came from two hospitals, The Third Affiliated Hospital of Harbin Medical University (the 3rd AHMU) and the JingWei Affiliated Hospital of Harbin Medical University (the JW AHMU) in Heilongjiang Province northeast China.Eighty blood samples were collected from blood donors at the JW AHMU from January to July of 2012. Samples were from residual whole blood donations of men who did not have a personal history of breast or prostate cancer or a family history of MBC. The study was approved by the Institutional Review Boards of Harbin Medical University and the Heilongjiang Province Cancer Society.MBC patient records were obtained from 175 patients at the 3rd AHMU and 33 patients at the JW AHMU; these 208 subjects represented all male patients with breast cancer seen at the two institutions during the period of 1990–2007. Patients’ medical records were retrieved and the following information was abstracted from them: histopathologic and treatment characteristics, such as tumor size, histological grade, hormone receptor status, and lymph node involvement. Of the 208 subjects, tissues were obtained from 85 patients, 76 from the 3rd AHMU, and 9 from the JW AHMU; AR (CAG)n repeats length was determined for 77 and 8, respectively. No patient received any therapy before surgery. Ethical approval for the study was obtained through the Heilongjiang Regional Ethics Committee.Formalin-fixed paraffin-embedded (FFPE) tumor tissues were obtained from 85 Chinese men with breast cancer. Hematoxylin and eosin staining and analysis by tow pathologists allowed for differentiation of normal tissue from tumor tissue for 81 cases. 31 blood samples were obtained from our tissue bank in these 81 cases. Microdissection of normal tissue from the tumor blocks allowed for working samples to represent the true genome in MBC patients. DNA was isolated according to a protocol specialized for FFPE tissues provided by OMEGA . For the control series, DNA extraction was from whole blood using standard protocol from AXYGEN .5′-TGCGCGAAGTGATCCAGAACC-3′ and AR-R 5′-CTCATCCAGGACCAGGTAGCC-3′ were chosen to amplify a region containing the repeat polymorphism of about 250 base pairs 2, 0.2 µl Platinum® Taq DNA polymerase , 10 µl of Gene Amp Fast PCR Mix from ABI , and distilled water to bring to final volume to 25 µl. PCR was performed according to a standard protocol  was used for positive control for AR. PBS was used to replace the primary antibody and served as the negative control.Breast cancer tissue microarrays were prepared and immunohistochemically stained for AR expression. A rabbit polyclonal to AR antibody  was used to evaluate AR expression. Sections from FFPE specimens were cut at 4 µm thick, were transferred onto adhesive glass slides, and incubated for 30 min at 62°C. Sections were dewaxed and rehydrated according to routine procedure, then pretreated for antigen retrieval in Ethylene Diamine Tetraacetic Acid (EDTA) buffer (pH 8.0) in a microwave oven for 20 min. Endogenous peroxidase activity was blocked using 3% HThe criteria for AR positivity were based on the intensity and percentage of tumor cells showing expression. The intensity was graded as negative, weak, moderate or strong. Tumors that had more than 10% of cells exhibiting weak, moderate or strong intensity of expression were considered positive.P<0.05 was considered to be significant.The relationship among clinicopathological features, sex hormone receptor status, and CAG repeat lengths was analyzed with a chi-square test. Overall survival (OS) was calculated from the date of surgery until death or the date patients were last known to be alive. The disease-free survival (DFS) was calculated from the date of surgery until relapse or the date patients were last known to be alive. Univariate survival analyses were based on Kaplan-Meier method. The relative importance of multiple prognostic factors on survival was estimated using the Cox proportional hazard regression model. Statistical analysis was performed using SPSS 16.0 statistical software . We detected CAG repeat length of bloods and tumor tissues in 31 MBC patients. The test results of two paired samples were the same in each patient .P = 0.001). It was thus clear that controls were more likely to exhibit short CAG repeat sequence than MBC patients .The lengths of the PCR products obtained varied between 206 bp and 278 bp . The median number of CAG repeats for 81 MBC patients was 20.5. This study used allele lengths of <21 and ≥21 as “short” and “long” CAG repeat sequence cut-points. AR protein was expressed, mainly in cell nuclei with weak, moderate and strong intensity. The AR staining results were as follows: 14 cases showed negative and 67 cases were positive inmunostaining . The posP = 0.004).P = 0.033). AR expression was related to ER (P = 0.023) and PR expression . The 5-year DFS for the entire group was 45.7%. The 5y-DFS, similarly to 5y-OS, was found to differ with respect to CAG repeat length and was significantly decreased in long CAG repeat sequence patients . The 5y-OS and 5y-DFS were significantly decreased for MBC patients with tumors staining positively for AR . Furthermore, the 5y-OS and 5y-DSF rates were also significantly decreased for MBC patients in lymph node metastases. The 5y-OS was 80.6% for lymph node negative group and 44% for lymph node metastases (P = 0.001). The 5y-DSF was 71.0% for lymph node negative and 30% for lymph node metastases (P = 0.001). Shorter 5y-DFS was correlated with increasing Ki-67 (P = 0.048 for 5y-DFS). There was no difference in 5y-OS rate with respect to Ki-67 status (P = 0.98).Multivariate survival analysis was performed by testing adverse factors identified in univariate analysis in the Cox model. Only CAG repeat lengths  and lymph node status  retained prognostic significance for 5y-OS. AR expression  and lymph node status  had prognostic significance for 5y-DFS.MBC remained an extremely rare disease. Although less frequent, it tended to be more aggressive than its female counterpart The role of AR expression as a prognostic factor was controversial. In MBC, Pich The involvement of AR in MBC development had been also investigated at the DNA level We initiated to test CAG repeat length of blood and tumor tissue in the same MBC patient. Interestingly, the test results of two paired samples were the same in each patient. As the incidence of MBC was low, the size of our study cohort was small. Although there were only 31 MBC patients who had paired samples, these consistent results supported two paired samples information substituted for one another. We had observed statistically significant difference between the CAG repeat length of MBC patients and controls. In addition, no males in the control group had alleles containing more than 28 CAG repeats. Only one of the MBC patients had an allele containing 14 repeats, compared to 6 of the controls. The length of this CAG repeat had been investigated in several studies of MBC patients and controls previously This study revealed the following important findings: (a) there was a significant difference in the CAG repeat lengths between MBC patients and controls; (b) long CAG repeat lengths were more common in patients than controls and there was a strong trend toward short CAG repeat lengths being more prevalent in controls than in cases; (c) long CAG repeat sequence presented at the T2-4 stage compared with short CAG repeat sequence in MBC patients but did not differ with respect to other clinicopathological features; (d) there was a significant difference by ER and PR status regarding AR expression in the MBC patients; (e) the presence of long CAG repeat sequence and AR-positive expression were associated with shorter survival of MBC patients.To conclude, the findings presented in this study indicate that the CAG repeat length within the AR gene might be one useful molecular biomarker to identify males at increased risk of breast cancer development. The CAG repeat length and AR expression were two independent prognostic indicators in MBC patients. Larger studies were required to define the importance of AR (CAG)n repeats status and AR expression in MBC further and we had agreed to contribute our data to this.Figure S1We detected CAG repeat length of bloods and tumor tissues for 31 MBC patients. The test results of two paired samples were the same in each patient.(DOC)Click here for additional data file.


The obstetrical literature is dominated by Randomised Controlled Trials (RCTs), with the vast majority being analysed using an intention-to-treat (ITT) approach. Whilst this approach may reflect well the consequence of assignment to therapy and hence the ‘trialists’perspective’, it may fail to address the consequence of actually receiving therapy (the patient’s perspective).This review questions the ubiquitous adherence to the ITT approach, and gives examples of where this may have misled the maternity care professions. It gives an overview of techniques to overcome potential deficiencies in result presentation, using method effectiveness models such as ‘Per Protocol’ (PP) or ‘As-Treated’ (AT) that may give more accurate clinical meaning to the presentation of obstetrical results. It then proceeds to cover the added benefits, considerations and potential pitfalls of the use of Instrumental Variable (IV) models in order to better reflect the clinical context.While ITT may achieve statistical purity, it frequently fails to address the true clinical or patient’s perspective. Though more complex and potentially beset by problems of their own, alternative methods of result presentation may better serve the latter aim. Each of the other methods may rely on untestable assumptions and therefore it is wisest that study results are presented in multiple formats to allow for informed reader evaluation. In the hierarchy of statistical trials, the RCT has been on the throne for over half a century . BradforRandomisation and adherence/compliance are critical for ITT to produce a good estimate of efficacy. Randomisation “provides a guarantee against biased estimates of treatment effects due to differences in the distribution of known and unknown confounding factors” . ITT proITT can successfully provide valid estimates for the effect on outcome of assignment to therapy, though these reflect the effect of randomisation to treatment rather than the true effect of taking treatment . Though In clinical practice there are frequent deviations from ‘ideal’ trial conditions, provoking compromises in RCT design to try and overcome this disconnect. Pragmatic trials compare two treatment strategies and tend to reflect the situation in current clinical practice, using less selected participants and being conducted under more realistic conditions with lower adherence to the assigned treatment . ExplanaA further alternative, when the issue is that of missing data resulting in incomplete adherence to ITT, is the use of (multiple) imputation analysis (MIA), a technique used to generate plausible values for missing data, using a set of rules for combining individual estimates and standard errors . Guideliclinical efficacy . For clractice” , given tractice” .ITT does not reflect the whole complexity of patient care and clinical events and may not appear satisfactory unless it yields a positive result. The comparison using ITT is regarded by most as conservative, with most commonly a bias towards the null hypothesis, or dilution due to contamination of the treatment groups ,13. The . Also, literature regarding use of epidurals for analgesia in labour has been clouded by a blind reliance on ITT in RCTs, even in the face of significant crossover. In a major RCT comparing epidural with non-epidural analgesia, 31% of women counselled agreed to participate, with 42% of those agreeing in the antenatal clinic being randomised, then 33% not receiving their intended epidural and 28% from the non-epidural group receiving an epidural [In the trade-off between bias and precision, different schools of thought exist and to date ITT has been almost ubiquitous in the obstetrical literature, despite its apparent shortfalls. One of the clearest and most clinically detrimental examples of this is the Term Breech Trial, where ITT contributed to conclusions that do not appear to be clinically justified, were unsubstantiated at 2 year maternal and neonatal follow up and have had worldwide ramifications in maternity care -22. The epidural . The priepidural . When muepidural  has bothepidural . ‘Clinicepidural .human beings and the real world”, and allow a number of behavioural evaluations [Per Protocol (PP) or As-Treated (AT) approaches. Both make the assumption that the probability of taking the treatment is random with respect to all predictors of outcome [When evidence-based medicine needs to be applied to real clinical cases and to the patient’s perspective, ME models have been proposed to be more relevant to clinical decision making than efficacy studies . They maluations . Common  outcome . Clinica outcome , it takeA PP analysis includes all subjects who were, in retrospect, eligible for enrolment in the study without major protocol violations, who received an acceptable amount of test treatment, and who had some minimal amount of follow up . SelectiThe ultimate goal of outcome research is to tell which treatment or intervention is associated with the best outcome, whether maximum benefit or minimum harm to patients. As this may involve establishing if there is a causal association between that treatment or intervention and outcome, researchers concerned with the effects of non-compliance have often used the AT approach , which aUnless alternative methods are considered through the RCT process, blind adherence to ITT risks becoming a self-fulfilling prophecy . The datThere are a number of different causal inference approaches that adjust for unmeasured confounders (i.e. hidden bias) in order to improve upon the shortcomings of the method-effectiveness models. These include inverse probability (IP) weighting, g-estimation and instrumental variable (IV) methods . Inverseinstrumental variable (IV) model, a particular form of g-estimation that does not require measurement of any confounders is a commonly used causal approach to estimate the true trial effect [The l effect , and mayl effect . It is ol effect . The IV l effect . It doesl effect . The IVsl effect . It mustl effect . An IV al effect .Whist technically more complex, a growing body of literature supports and explains the use of IV methods to determine effectiveness in a number of trial situations ,36, corrAs well as the IV approach, researchers have proposed the use of four mutually exclusive groups using a new estimator referred to as a stratified method of moments estimator ,9. TheseWhilst RCTs provide the most scientifically and statistically accurate method for evaluating the influence of treatment or interventions, obstetrical studies are dominated by the use of ITT analysis to present their data. In fact, though ITT may correlate well with the scientific or statistical aims of a study or of society, it frequently does not answer the needs of the patient. Deviations from pure adherence to the intervention under evaluation undermine the value of ITT, and mean that patients may benefit from alternative methods of data presentation.In publishing RCT results, supplementation of ITT evaluation with ‘As Treated’ or ‘Per Protocol’ starts to provide more relevant information for patients , especiaThese concepts have progressed well in many disciplines but are yet to be reflected in obstetrical medicine and maternity care, where non-compliance and treatment contamination are so common. A number of well publicised trials have misled clinical practice because of over-reliance on ITT in the face of non-adherence after randomisation. It therefore would be more appropriate to present results of randomised controlled trials in multiple formats to allow clinicians to discuss both the statistical and clinical relevance of findings. It is clear that there is no single perfect method for presentation of data from RCTs, and each should be approached with clear knowledge of their limitations.RCT: Randomised controlled trial; ITT: Intention to treat; ME: Method-effectiveness; PP: Per protocol; AT: As-treated; IV: Instrumental variable; IP: Inverse probability; MIA: Multiple imputation analysis.The author declares that they have no competing interests.AW devised and wrote the paper.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/13/15/prepub