The synthesis of the first example of azo chromophores containing chromene moieties has also been established using the same methodology. The antimicrobial activity of the new molecules has been tested against seven human pathogens including two Gm+ve, two Gm-ve bacteria, and four fungi, and the results of the inhibition zones with minimum inhibitory concentrations were reported as compared to reference drugs. All the designed compounds showed significant potent antimicrobial activities, among of them, four potent compounds 4b, 4c, 13e, and 13i showed promising MIC from 0.007 to 3.9 µg/mL. In addition, antiproliferative analysis against three target cell lines was examined for the novel compounds. Compounds 4a, 4b, 4c, and 7c possessed significant antiproliferative activity against three cell lines with an IC50 of 0.3 to 2 µg/mL. Apoptotic analysis was performed for the most potent compounds via caspase enzyme activity assays as a potential mechanism for their antiproliferative effects. Finally, the computational 2D QSAR and docking simulations were accomplished for structure-activity relationship analyses. The design of novel materials with significant biological properties is a main target in drug design research. Chromene compounds represent an interesting medicinal scaffold in drug replacement systems. This report illustrates a successful synthesis and characterization of two novel series of  The vast majority of health problems facing the world's population at the present time are cancer and infectious diseases . CancerMeanwhile, bacterial infections remain a serious threat to human lives because of its emerging resistance to existing antibiotics, which is another increasing public health problem. Consequently, there is a vital need for the development of new antimicrobial agents with potent activity against drug resistant microorganisms . The drOxygen atom-containing heterocyclic molecules are one of the main targets in drug design research. In particular, chromene compounds are well known as important components either in biologically active synthetic or natural compounds  . For in4a-i) and 3,5-diamino-1-phenyl-1H-benzo[f]chromene-2-carbonitrile (7a-e). In addition, the present study shows the synthesis of 2-amino-7-hydroxy-4-phenyl-6-(phenyldiazenyl)-4H-chromene-3-carbonitrile (13a-k) compounds as a first example of azo dye incorporating chromene derivatives with the purpose of investigating their possible biological and cytotoxic activities. Structures of the novel molecules were elucidated on the basis of IR, 1H-, 13C- NMR and MS, and their UV-vis behaviors were also examined.In view of the above mentioned benefits and in continuation of our previous work in development synthesis of polyfunctional-substituted heterocyclic compounds with potential biological activity , we repIn continuation to our previous work, lipophilic parameters were found to control the activity of phenyl chromene carbonitrile derivatives . UV-vis measurements were performed on Carry 100. All chemicals were available from Sigma-Aldrich Chemical Co. and were used without purification. Solvents were HPLC grade and were used without further purification, with the exception of ethanol, which was distilled prior to use. 2,7-amino-4-aryl-3-cyano-4H-chromene and 2-amino-4-aryl-7-hydroxy-3-cyano-4H-chromene series were synthesized according to a previous report . The prA mixture of 3-amino-2-naphthol (10 mmol), malononitrile (10 mmol) and aromatic aldehyde (10 mmol) were dissolved in EtOH (15 ml), then piperidine (0.5 ml) was added. The mixture was refluxed for 2-4 hours until complete precipitation occurred. The isolated precipitate was filtered, washed with ethanol and recrystallized from ethanol. Characterization of the new compounds was achieved using NMR, FTIR and mass spectrometry.3,5-diamino-1-(4-fluorophenyl)-1H-benzo[f]chromene-2-carbonitrile (4a)-1: 3320, 3425, 3490 (NH2), 2190 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 5.20 , 5.45  6.85 , 6.91 , 6.97-7.04 , 7.13-7.17 , 7.47 , 7.53 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 37.41(C-4), 57.98 (C-3), 106.85 (C-9), 115.52 (Ar-CH), 115.88 (C-4a), 120.37 (CN), 122.08 (C-5), 122.64 (C-4b), 123.21 (C-6) 124.89 (C-7), 125.66 (C-8), 128.69 (Ar-CH), 132.07 (C-8a), 136.29 (C-10), 138.05 (Ar-C), 141.83 (C-10a), 159.79 (C-2), 161.99 (Ar-C). 19F NMR (DMSO-d6) δ (ppm): -116.11 (Ar-F). MS m\z (%): 331  with base peak at 237.Colorless crystals from ethanol (67 % yield), mp 193 °C; IR (KBr) cm3,5-diamino-1-(4-chlorophenyl)-1H-benzo[f]chromene-2-carbonitrile(4b)-1: 3320, 3425, 3490 (NH2), 2195 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 5.35 , 5.66  7.06-7.13 , 7.23-7.29 , 7.35 , 7.60 , 7.64 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 37.46 (C-4), 57.78 (C-3), 107.11 (C-9), 115.57 (C-4a), 120.43 (CN), 122.22 (C-5), 122.72 (C-6), 123.19 (C-7) 124.98 (C-8), 125.74 (C-4b), 128.71 (Ar-CH), 131.23 (C-8a), 132.13 (Ar-C), 136.30 (C-10), 138.19 (Ar-C), 144.54 (C-10a), 159.89 (C-2). MS m\z (%): 347  with base peak at 236 (100).Colorless crystals from ethanol (73 % yield), mp 187 °C; IR (KBr) cm3,5-diamino-1-(p-tolyl)-1H-benzo[f]chromene-2-carbonitrile(4c)-1: 3320, 3425, 3490 (NH2), 2198 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 2.12 , 5.09 , 5.43  6.79 , 6.90 , 6.98 , 7.13 , 7.45 , 7.52 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 20.58 (CH3), 37.98 (C-4), 58.28 (C-3), 106.74 (C-9), 116.17 (C-4a), 120.54 (CN), 122.02 (C-5), 122.82 (C-6), 123.33 (C-7), 124.83 (Ar-C), 125.64 (C-8), 126.81 (Ar-CH), 129.24 (Ar-CH), 132.08 (C-4b), 135.66 (C-8a), 136.28 (C-10), 138.09 (Ar-C), 142.71 (C-10a), 159.71 (C-2). MS m\z (%): 327  with base peak at 236 (100)Colorless crystals from ethanol (64 % yield), mp 180 °C; IR (KBr) cm3,5-diamino-1-(4-methoxy-phenyl)-1H-benzo[f]chromene-2-carbonitrile(4d)-1: 3320, 3425, 3490 (NH2), 2185 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 3.67 , 5.17 , 5.51 , 6.82 , 6.87 , 6.98 , 7.05-7.12 , 7.22 , 7.54 , 7.63 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 37.50 (C-4), 55.60 (OCH3), 58.34 (C-3), 107.00 (C-9), 116.19 (C-4a), 120.40 (CN), 122.09 (C-5), 122.87 (C-6), 123.34 (C-7) 124.88 (C-8), 125.64 (C-4b), 126.89 (C-8a), 128.74 (Ar-CH), 138.17 (C-10), 146.46 (C-10a), 159.81 (Ar-C), 160.07 (C-2). MS m\z (%): 343  with base peak at 186 (100). Colorless crystals from ethanol (60 % yield), mp 183 °C; IR (KBr) cm3,5-Diamino-1--1H-benzo[f]chromene-2-carbonitrile (4e)-1: 3320, 3425, 3490 (NH2), 2200 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 5.57 , 5.67 , 6.99 , 7.05 , 7.10 , 7.19-7.23 , 7.38 , 7.56 , 7.59 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 35.29 (C-4), 56.63 (C-3), 107.82 (C-9), 115.05 (C-4a), 120.17 (CN), 122.61 (C-5), 122.94 (C-6), 123.06 (C-4b), 125.48 (C-7) 126.34 (C-8), 128.90 (Ar-CH), 129.21 (Ar-CH), 131.57 (Ar-CH), 132.22 (C-8a), 132.47 (Ar-C), 136.64 (C-10), 138.88 (Ar-C), 142.16 (C-10a), 160.37 (C-2). 19F NMR (DMSO-d6) δ (ppm): -111.92, -115.30 (Ar-F). MS m\z (%): 382  with base peak at 237.Colorless crystals from ethanol (69 % yield), mp 278 °C; IR (KBr) cm3,5-Diamino-1--1H-benzo[f]chromene-2-carbonitrile (4f)-1: 3320, 3425, 3490 (NH2), 2202 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 5.49 , 5.54 , 6.95 , 7.03 , 7.10 , 7.18-7.22 , 7.50-7.56 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 32.69 (C-4), 56.57 (C-3), 104.50 (Ar-CH), 107.61 (C-9), 114.66 (C-4a), 120.59 (CN), 122.69 (C-6), 122.79 (C-7), 123.04 (C-8), 125.38 (C-5) 126.29 (Ar-CH), 128.74 (C-4b), 128.88 (C-8a), 131.21 (Ar-C), 132.46 (Ar-CH), 136.67 (C-10), 138.89 (C-10a), 158.55 (Ar-C), 160.32 (Ar-C), 160.64 (C-2). 19F NMR (DMSO-d6) δ (ppm): -111.92, -115.30 (Ar-F). MS m\z (%): 349  with base peak at 237.Colorless crystals from ethanol (69 % yield), mp 282 °C; IR (KBr) cm3,5-Diamino-1-(2-fluoro-3-trifluoromethyl-phenyl)-1H-benzo[f]chromene-2-carbonitrile (4g)-1: 3320, 3425, 3490 (NH2), 2210 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 5.57 , 5.63 , 7.05 , 7.11 , 7.21-7.29 , 7.51-7.57 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 33.31(C-4), 56.06 (C-3), 107.86 (C-9), 114.12 (C-F3), 120.44 (C-4a), 122.61 (CN), 122.98 (C-5), 124.45 (Ar-C), 125.47 (C-6) 125.76 (C-7), 126.37 (Ar-CH & C-8), 132.49 (Ar-CH), 133.97 (C-4b), 134.09 (C-8a), 135.05 (Ar-H), 136.70 (C-10), 138.98 (C-10a), 155.59 (Ar-C), 158.12 (Ar-C), 160.80 (C-2). 19F NMR (DMSO-d6) δ (ppm): -122.89 (Ar-F). MS m\z (%): 399  with base peak at 237.Colorless crystals from ethanol (64 % yield), mp 202 °C; IR (KBr) cm3,5-Diamino-1--1H-benzo[f]chromene-2-carbonitrile (4h)-1: 3320, 3425, 3490 (NH2), 2198 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 3.61 , 3.66 , 5.24 , 5.52 , 6.53 , 6.94 , 7.03 , 7.10 , 7.23 , 7.56 , 7.73 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 39.08 (C-4), 56.30 (OCH3), 58.66 (C-3), 60.41 (OCH3), 104.80 (Ar-CH), 107.52 (C-9), 116.38 (C-4a), 121.06 (CN), 122.65 (C-5), 123.52 (C-6) 123.88 (Ar-C), 125.42 (C-7), 126.14 (C-8), 132.54 (C-4b), 136.70 (C-8a), 138.70 (C-10), 141.85 (C-10a), 153.47 (Ar-C), 160.41 (C-2). MS m\z (%): 403  with base peak at 237.Colorless crystals from ethanol (77 % yield), mp 225 °C; IR (KBr) cm3,5-Diamino-1-thiophen-3-yl-1H-benzo[f]chromene-2-carbonitrile (4i)-1: 3320, 3425, 3490 (NH2), 2196 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 5.38 , 5.50 , 6.84 , 6.95 , 7.01 , 7.12 , 7.23-7.25 , 7.35 , 7.56 , 7.78 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 33.94 (C-4), 57.92 (C-3), 107.26 (C-9), 116.89 (C-4a), 121.05 (CN), 121.09 (Ar-CH), 122.52 (C-5), 123.25 (C-6) 123.54 (C-4b), 125.32 (C-7), 126.05 (Ar-CH), 127.00 (C-8), 127.11 (Ar-CH), 132.43 (C-8a), 136.66 (C-10), 138.29 (Ar-C), 146.33 (C-10a), 160.89 (C-2). MS m\z (%): 319  with base peak at 237.Colorless crystals from ethanol (71 % yield), mp 230 °C; IR (KBr) cmA mixture of 5-amino-1-naphthol (10 mmol), malononitrile (10 mmol) and aromatic aldehyde (10 mmol) were dissolved in EtOH (15 ml), then piperidine (0.5 ml) was added. The mixture was refluxed for 2-4 hours until complete precipitation occurred. The isolated precipitate was filtered, washed with ethanol and recrystallized from ethanol. Characterization of the new compounds was achieved using NMR, FTIR and mass spectrometry.2,7-Diamino-4-(4-tert-butyl-phenyl)-4H-benzo[h]chromene-3-carbonitrile (7a)-1: 3320, 3425, 3490 (NH2), 2190 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 1.23 , 4.80 , 5.76  6.71 , 6.95 , 7.05 , 7.15 , 7.26-7.34 , 7.44 , 7.75 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 31.09 (CH3), 34.10 (C-CH3), 40.48 (C-4), 56.18 (C-3), 107.99 (C-7), 108.34 (C-5), 117.64 (CN), 118.62 (C-4a), 120.77 (C-10), 122.02 (C-8a), 123.43 (C-9), 124.05 (C-6), 125.38, 127.10 (Ar-CH), 127.41 (C-4b), 142.91 (C-8), 144.87 (Ar-C), 149.04 (C-10a), 160.38 (C-2). MS m\z (%): 369  with base peak at 237.Gray crystals from ethanol (75 % yield), mp 210 °C; IR (KBr) cm2,7-Diamino-4-(4-methoxy-phenyl)-4H-benzo[h]chromene-3-carbonitrile (7b)-1: 3320, 3425, 3490 (NH2), 2190 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 3.71 , 4.79 , 5.78 , 6.72 , 6.87 , 6.92 , 7.05 , 7.15 , 7.30 , 7.45 , 7.74 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 40.52 (C-4), 55.44 (OCH3), 56.84 (C-3), 108.43 (C-7), 108.79 (C-5), 114.41 (Ar-CH), 118.15 (C-10), 118.99 (CN), 121.18 (C-4a), 122.41(C-9), 123.89 (C-8a), 124.47 (C-4b), 127.88 (C-6), 129.09 (Ar-CH), 138.45 (Ar-C), 143.18 (C-8), 145.33 (C-10a), 158.53 (Ar-C), 160.60 (C-2). MS m\z (%): 343  with base peak at 237.Gray crystals from ethanol (75 % yield), mp 185 °C; IR (KBr) cm2,7-Diamino-4--4H-benzo[h]chromene-3-carbonitrile (7d)-1: 3320, 3425, 3490 (NH2), 2190 (CN), 3140 (CH); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 5.10 , 7.16 , 7.29 , 7.38 , 7.48 , 7.67-7.72 , 7.93 , 8.24 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 40.91 (C-4), 55.92 (C-3), 114.75 (C-7), 118.02 (Ar-CH), 119.44 (CN), 120.17 (C-6), 120.59 (C-5), 122.78 (C-4a), 123.76 (C-10), 126.05 (C-8a), 127.58 (C-9), 128.18 (C-6), 128.91, 129.88 (Ar-CH), 139.84 (C-4b), 143.33 (C-8), 148.12 (Ar-C), 150.49 (C-10a), 160.62 (C-2), 160.88 (Ar-C); 19F NMR (DMSO-d6) δ (ppm): -116.11 (Ar-F). MS m\z (%): 367  with base peak at 237.Greenish crystals from ethanol (52 % yield), mp 235 °C; IR (KBr) cm2,7-Diamino-4-thiophen-2-yl-4H-benzo[h]chromene-3-carbonitrile (7e)-1: 3320, 3425, 3490 (NH2), 2190 (CN), 3140 (CH), 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 5.21 , 5.80 , 6.74 , 6.95 , 7.07-7.12 , 7.17 , 7.30 , 7.37 , 7.44 , 7.81 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 37.02 (C-4), 57.17 (C-3), 107.99 (C-7), 108.34 (C-5), 117.64 (CN), 118.62 (C-4a), 120.77 (C-10), 122.02 (C-8a), 123.43 (C-9), 124.05 (C-6), 125.38, 127.10 (Ar-CH), 127.41 (C-4b), 142.91 (C-8), 144.87 (Ar-C), 149.04 (C-10a), 160.38 (C-2). MS m\z (%): 319  with base peak at 237.Gray crystals from ethanol (64 % yield), mp 232 °C; IR (KBr) cmAzo dye compounds were prepared using previously reported method -benzene-1,3-diol (12a)-1: 3310 (OH), 1473 (N=N); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 2.42 , 6.38 , 6.54 , 7.29 , 7.65-7.69 , 10.04 , 13.61 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 21.30 (CH3), 103.29, 109.08, 121.28, 129.84, 132.18 (Ar-CH), 134.54, 140.18, 148.00, 156.05, 162.53 (Ar-C). Orange crystals from ethanol (86 % yield), mp 185 °C; IR (KBr) cm4-(4-Methoxy-phenylazo)-benzene-1,3-diol (12b)-1: 3325 (OH), 1500 (N=N); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 3.87 , 6.40 , 6.55 , 6.99 , 7.65 , 7.75, 9.87 , 13.53 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 55.52 (CH3), 103.34, 108.85, 114.41, 123.04, 132.12 (Ar-CH), 134.24, 144.37, 155.56, 161.09, 161.99 (Ar-C).Orange crystals from ethanol (84 % yield), mp 187 °C; IR (KBr) cm4-(4-Nitro-phenylazo)-benzene-1,3-diol (12c)-1: 3340 (OH), 1477 (N=N); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 6.38 , 6.52 , 7.68 , 7.92 , 8.07 , 10.73 , 12.46 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 103.02, 109.75, 121.55, 129.47, 130.49, 132.85 (Ar-CH), 136.98, 153.18, 157.59, 164.07(Ar-C).Reddish orange crystals from ethanol (89 % yield). mp 192 °C; IR (KBr) cm1-[4--phenyl]-ethanone (12d)-1: 3350 (OH), 1450(N=N); 1H NMR (400 MHz): 2.64 , 6.36 , 6.57 , 7.66 , 7.84 , 8.06 , 10.43 , 13.65 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 26.69 (CH3), 103.23, 110.25, 121.10, 129.43, 132.84 (Ar-CH), 134.92, 136.86, 152.65, 157.74, 164.28(Ar-C), 196.79 (C=O).Red crystals from ethanol (84 % yield), mp 220 °C; IR (KBr) cm4--benzoic acid (12e)-1: 3320 (Br OH), 1510 (N=N); 1H NMR (400 MHz): 6.43 , 6.53 , 7.64 , 7.89-8.05 , 11.00 , 12.02 , 12.39 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 103.14, 104.79, 109.88, 121.65, 122.54, 129.43, 130.67 (Ar-CH), 132.09, 153.32, 154.07, 161.35 (Ar-C), 166.79 (C=O).Red crystals from ethanol (84 % yield), mp 300 °C; IR (KBr) cm4-(4-Hydroxy-phenylazo)-benzene-1,3-diol (12i & 12j)-1: 3350 (OH), 1515 (N=N); 1H NMR (400 MHz): 6.32 , 6.47 , 7.62 , 7.73 , 10.15 , 10.36 , 12.49 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 103.56, 107.00, 109.71, 114.74, 118.01, 130.67, 131.99 (Ar-CH), 132.63, 152.17, 156.82, 158.85, 163.47 (Ar-C).Reddish orange crystals from ethanol (84 % yield), mp 210 °C; IR (KBr) cm12a-h (1 mmol), malononitrile (1 mmol), various benzaldehyde in ethanol (10 mL) and piperidine (0.3 mL) was added under rapid stirring. The reaction was refluxed for 5 h and the solid was filtered and recrystallized from ethanol to afford the pure product.To a mixture of aryl azo dyes 2-Amino-4-(4-fluoro-phenyl)-7-hydroxy-6-p-tolylazo-4H-chromene-3-carbonitrile (13a)max= 380 nm; IR (KBr) (cm-1): 3410, 3327 (NH2), 3203 (OH), 2920 (CH3) 2191(CN), 1456 (N=N); 1H NMR (400 MHz) (DMSO) δ: 2.41 , 4.83, 6.77 , 7.04 , 7.11 , 7.23-7.26 , 7.71-7.78 , 7.91 , 11.86 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 21.44 (CH3), 35.80 (C-4), 59.61 (C-3), 108.27 (C-8), 112.03 (CN), 115.08 (Ar-CH), 115.22 (C-5), 120.09 (C-4a), 121.86, 129.04 (Ar-CH), 129.09 (C-6), 129.99, 132.55, 134.11 (Ar-CH), 140.18 (Ar-C), 141.73, 147.71 (Ar-CH), 151.63 (C-7), 151.96 (Ar-C), 159.50 (C-8a), 160.70 (C-2), 162.32 (Ar-C). 19F NMR (DMSO-d6) δ (ppm): -116.24 (Ar-F). MS m\z (%): 400  with base peak at 237.Brown crystals from ethanol (55 % yield), mp 203 °C; UV/Vis (EtOH): λ2-Amino-4-(4-fluoro-phenyl)-7-hydroxy-6-(4-methoxy-phenylazo)-4H-chromene-3-carbonitrile (13b)max= 386 nm; IR (KBr) (cm-1): 3414, 3336 (NH2), 3223 (OH), 2185 (CN), 1464 (N=N); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 3.84 , 4.79 , 6.79  7.03 , 7.07-7.14 , 7.21-7.25 , 7.74 , 7.93 , 12.23 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 35.85(C-4), 55.60 (CH3), 57.08 (C-3), 108.03 (C-8), 112.11 (CN), 114.59 (Ar-CH), 115.01 (C-5), 115.22 (Ar-CH), 120.08 (C-4a), 124.32 (Ar-CH), 125.46 (C-6), 129.01, 134.46 (Ar-CH), 141.23 (Ar-C), 144.80 (Ar-CH), 151.41 (C-7), 151.83 (C-8a), 159.69 (Ar-C), 161.82 (C-2), 162.10 (Ar-C). 19F NMR (DMSO-d6) δ (ppm): -116.26 (Ar-F). MS m\z (%): 416  with base peak at 186.Brown crystals from ethanol (40 % yield), mp 272 °C; UV/Vis (EtOH): λ2-Amino-4-(4-fluoro-phenyl)-7-hydroxy-6-(4-nitro-phenylazo)-4H-chromene-3-carbonitrile (13c)max= 390 nm; IR (KBr) (cm-1): 3387,3313 , 2185 (CN), 1458 (N=N); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 4.79 , 6.80  7.06 , 7.13 , 7.22-7.26 , 7.77 , 8.06 , 12.17 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 35.85 (C-4), 57.17 (C-3), 108.69 (C-8), 112.39 (CN), 115.03 (Ar-CH), 115.24 (C-5), 119.95 (C-4a), 122.49, 124.58, 128.99 (Ar-CH), 129.07 (C-6), 129.35, 135.12, 137.86 (Ar-CH), 141.14 (Ar-C), 152.80 (C-7), 153.13, 153.88 (Ar-C), 159.44 (C-8a), 159.72 (C-2), 162.13 (Ar-C). 19F NMR (DMSO-d6) δ (ppm): -116.19 (Ar-F). MS m\z (%): 431  with base peak at 237.Brown crystals from ethanol (41 % yield), mp 254 °C; UV/Vis (EtOH): λ6-(4-Acetyl-phenylazo)-2-amino-4-(4-fluoro-phenyl)-7-hydroxy-4H-chromene-3-carbonitrile (13d)max= 390 nm; IR (KBr) (cm-1): 3398, 3313 (NH2), 3196 (OH), 2187 (CN), 1450 (N=N); 1H NMR (400 MHz) (DMSO-d6) δ (ppm): 2.62 , 4.79 , 6.81  7.06 , 7.13 , 7.22-7.26 , 7.78 , 8.07 , 12.15 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 26.85 (CH3), 35.85 (C-4), 57.16 (C-3), 108.69 (C-8), 112.41 (CN), 115.04 (Ar-CH), 115.25 (C-5), 119.95 (C-4a), 122.51, 124.44, 128.99 (Ar-CH), 129.07 (C-6), 129.37, 135.15, 137.88, 141.17 (Ar-CH), 152.81 (C-7), 153.17, 153.90 (Ar-C), 159.44 (C-8a), 159.72 (C-2), 162.13 (Ar-C), 197.25 (C=O). 19F NMR (DMSO-d6) δ (ppm): -116.20 (Ar-F). MS m\z (%): 428  with base peak at 109.Brown crystals from ethanol (52 % yield), mp 265 °C; UV/Vis (EtOH): λ4-[2-Amino-3-cyano-4-(2-fluoro-phenyl)-7-hydroxy-4H-chromen-6-ylazo]-benzoic acid (13e)max= 388 nm; IR (KBr) (cm-1): 3420-3385 (NH2), 2230 (CN), 1645 (C=O), 1520 (N=N); 1H NMR (400 MHz) (DMSO) δ: 5.05 , 6.78 , 7.10 , 7.13-7.17 , 7.24-7.27 , 7.78 , 7.95 , 8.03 , 10.92 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 31.35 (C-4), 55.95 (C-3), 108.50 (C-8), 111.69 (CN), 115.85 (Ar-CH), 116.06 (C-5), 120.58 (C-4a), 122.46, 124.85, 129.16, 130.35, 130.58 (Ar-CH), 132.16 (C-6), 135.42 (Ar-CH), 152.61 (C-7), 153.30, 154.32 (Ar-C), 159.16 (C-8a), 160.25 (C-2), 161.69 (Ar-C), 168.61 (C=O).19F NMR (DMSO-d6) δ (ppm): -119.76 (Ar-F). MS m\z (%): 430  with base peak at 237.Brown crystals from ethanol (52 % yield), mp 243 °C; UV/Vis (DMF): λ4-[2-Amino-3-cyano-4-(2-fluoro-phenyl)-7-hydroxy-4H-chromen-6-ylazo]-benzenesulfonic acid (13f)max = 390 nm; IR (KBr) (cm-1): 3450- 3425 (NH2), 2220(CN), 1495 (N=N); 1H NMR (400 MHz) (DMSO) δ: 5.04, 6.80 , 7.08 , 7.11-7.16 , 7.22-7.27 , 7.73 , 7.78 , 7.92 , 11.77 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 31.31 (C-4), 56.01 (C-3), 107.73 (Ar-CH), 108.65 (C-8), 111.55 (CN), 115.86 (Ar-CH), 116.08 (C-5), 120.45 (C-4a), 122.56, 125.01, 127.08, 129.12, 130.34 (Ar-CH), 131.97 (C-6), 135.25 (Ar-CH), 150.69 (Ar-C), 151.04 (C-7), 153.04, 153.65 (Ar-C), 159.15 (C-8a), 160.25 (C-2), 161.59 (Ar-C). 19F NMR (DMSO-d6) δ (ppm): -119.77 (Ar-F). MS m\z (%): 466  with base peak at 237.Brown crystals from ethanol (59 % yield), mp 290 °C; UV/Vis (DMF): λ4-[2-Amino-4-(4-tert-butyl-phenyl)-3-cyano-7-hydroxy-4H-chromen-6-ylazo]-benzenesulfonic acid (13g)max = 386 nm; IR (KBr) (cm-1): 3430-3400 (NH2), 2225 (CN), 1510 (N=N); 1H NMR (400 MHz) (DMSO) δ: 1.23 , 4.73 , 6.81 , 7.05 , 7.11, 7.32 , 7.73-7.79 , 7.93 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 31.53 (CH3), 34.53 (C-CH3), 36.37 (C-4), 57.61 (C-3), 108.74 (C-8), 113.13 (CN & C-5), 120.73 (C-4a), 122.46 (Ar-CH), 124.95 (C-6), 125.67, 127.02, 127.15, 135.23, 142.42 (Ar-CH), 149.30, 150.71 (Ar-C), 150.92 (C-7), 152.76 (Ar-C), 153.32 (C-8a), 160.08 (C-2). MS m\z (%): 504  with base peak at 237.Brown crystals from ethanol (67 % yield), mp 330 °C; UV/Vis (DMF): λ4-[2-Amino-4-(4-butyl-phenyl)-3-cyano-7-hydroxy-4H-chromen-6-ylazo]-benzenesulfonic acid (13h)max = 385 nm; IR (KBr) (cm-1): 3450, 3425 (NH2), 2210 (CN), 1510 (N=N); 1H NMR (400 MHz) (DMSO) δ: 0.88 , 1.26-1.31 , 1.47-1.55 , 2.49 , 4.73 , 6.81 , 7.05 , 7.07-7.13 , 7.73-7.80 , 7.93 , 11.93 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 14.14 (CH3), 22.23 (CH2), 33.52 (CH2), 34.85 (CH2), 36.51 (C-4), 57.65 (C-3), 108.73 (C-8), 113.12 (CN & C-5), 120.67 (C-4a), 122.43 (Ar-CH), 125.10 (C-6), 127.01, 127.40, 128.74, 135.19, 141.08 (Ar-CH), 142.66 (Ar-C), 150.86 (C-7), 152.72 (Ar-C), 153.31 (C-8a), 160.03 (C-2). MS m\z (%): 504  with base peak at 237.Brown crystals from ethanol (63 % yield), mp 275 °C; UV/Vis (DMF): λ2-Amino-4-(4-tert-butyl-phenyl)-7-hydroxy-6-(4-hydroxy-phenylazo)-4H-chromene-3-carbonitrile (13i)max = 387 nm; IR (KBr) (cm-1): 3350- 3300 (NH2), 2215 (CN), 1520 (N=N); 1H NMR (400 MHz) (DMSO) δ: 1.23 , 4.72 , 6.75 , 6.87 , 7.01 , 7.11 , 7.31 , 7.70 , 7.80 . 31.52 (CH3), 34.50 (C-CH3), 36.40 (C-4), 57.63 (C-3), 107.81 (C-8), 113.03 (CN), 116.58 (Ar-CH & C-5), 120.90 (C-4a), 124.96 (Ar-CH), 125.61 (Ar-CH & C-6), 127.12, 135.03, 142.61 (Ar-CH), 143.74 (Ar-C), 149.19 (C-7), 151.61 (Ar-C), 152.63 (C-8a), 160.28 (C-2), 162.13 (Ar-C). MS m\z (%): 440  with base peak at 237.Brown crystals from ethanol (79 % yield), mp 255 °C; UV/Vis (DMF): λ2-Amino-4-(2-fluoro-phenyl)-7-hydroxy-6-(4-hydroxy-phenylazo)-4H-chromene-3-carbonitrile (13j)max= 387 nm; IR (KBr) (cm-1): 3330-3310 (NH2), 2215 (CN), 1520 (N=N); 1H NMR (400 MHz) (DMSO) δ: 5.04 , 6.76 , 6.88 , 7.05 , 7.11-7.17 , 7.22-7.27 , 7.71 , 7.81 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 31.23 (C-4), 56.00 (C-3), 108.03 (C-8), 111.28 (CN), 115.79 (Ar-CH), 116.00 (C-5), 116.48 (Ar-CH), 120.51 (C-4a), 124.96, 125.04, 126.05 (Ar-CH), 129.06 (Ar-C), 130.29 (Ar-CH), 132.07 (C-6), 134.79, 144.00 (Ar-CH), 151.88 (C-7), 152.35 (Ar-C), 159.08 (C-8a), 160.35 (C-2), 161.61 (Ar-C). 19F NMR (DMSO-d6) δ (ppm): -119.70 (Ar-F). MS m\z (%): 402  with base peak at 237.Red crystals from ethanol (62 % yield), mp 260 °C; UV/Vis (DMF): λ2-Amino-4-(2-fluoro-phenyl)-7-hydroxy-6-(3-hydroxy-phenylazo)-4H-chromene-3-carbonitrile (13k)max= 388 nm IR (KBr) (cm-1): 3475 (NH2), 2220 (CN), 1515 (N=N); 1H NMR (400 MHz) (DMSO) δ: 5.04, 6.81, 6.93, 7.10 , 7.13-7.17 , 7.25-7.27 , 7.31-7.35 , 7.41 , 7.78 , 9.81 , 12.41 . 13C NMR (125 MHz) (DMSO-d6) δ (ppm): 31.23 (C-4), 55.94 (C-3), 107.73 (Ar-CH), 108.60 (C-8), 111.42 (CN), 115.39, 115.80 (Ar-CH), 116.02 (C-5), 118.78 (Ar-CH), 120.35 (C-4a), 124.97, 126.97 (Ar-CH), 129.04 (Ar-C), 130.50 (Ar-CH), 131.85 (C-6), 134.80 (Ar-CH), 152.16 (C-7), 152.97, 158.63 (Ar-C), 159.14 (C-8a), 160.18 (C-2), 161.51 (Ar-C). 19F NMR (DMSO-d6) δ (ppm): -119.74 (Ar-F). MS m\z (%): 402  with base peak at 237.Reddish brown crystals from ethanol (68 % yield), mp 280 °C; UV/Vis (DMF): λThe microorganism inoculums were uniformly spread using sterile cotton swabs on a sterile Petri dish malt extract agar (for fungi) and nutrient agar (for bacteria). One hundred cubic millimeters of each sample was added to each well . The systems were incubated for 24-48 h at 37 °C (for bacteria) and at 28 °C (for fungi). After incubation, the microorganism growth was observed. Inhibition zones of the bacterial and fungal growth were measured in millimeters. Tests were performed in triplicate  and simultaneously the cells were treated with 100 µL from different dilutions of tested sample in fresh maintenance medium and incubated at 37 °C. A control of untreated cells was made in the absence of tested sample. Positive controls containing doxorubicin were also tested as a reference drug for comparison. Six wells were used for each concentration of the test sample. Every 24 hours an observation under the inverted microscope was made. The number of the surviving cells was determined by staining the cells with crystal violet , the concentration required to cause toxic effects in 50 % of intact cells, was estimated from graphic plots.Human colon carcinoma (HCT-116), human hepatocellular carcinoma (HEPG-2), adenocarcinomic human alveolar basal epithelial cell (A-549), and human breast adenocarcinoma (MCF-7) cell lines were obtained from the American Type Culture Collection . The cells were grown on RPMI-1640 medium supplemented with 10 % inactivated fetal calf serum and 50 µg/ml gentamycin. The cells were maintained at 37 °C in a humidified atmosphere with 5 % COary (2007) (Klancnary (2007). The ceann, 1983; Gangadeann, 1983) followe4a, 4b, and 4c) and a reference (Doxorubicin) drug at IC50 concentration was added. After 48 h a Caspase-Glo 3/7 Assay (Promega) was performed according to the manufacturer's instructions. After adding 100 μL of Caspase 3/7 Glo Reagent, cells were incubated for 2.5 h at room temperature. The luminescence was measured using a multi-plate reader  with an integration time of 1 second per well. The values are the means ± SD of three sets of experiments.To measure caspase-3 and -7 activities, a luminescent Caspase-Glo 3/7 assay was performed. HCT116 and MCF-7 cells were seeded onto black 96-well plates at a density of 3,500 cells/well. After 24 h the compounds that were tested  3 mM Tris, pH 7.5, 24 mM KCl, 2 mM MgCl2, 6.5 % (w/v) glycerol, 0.01 mg/ml bovine serum albumin (BSA), 2 mM DTT (dithiothreitol), 9 mM ATP, and 5 % DMSO solution containing the compounds). For E. coli topoisomerase IV ATPase assay, the final assay composition was 10 nM topoisomerase IV (a complex consisting of two ParC and two ParE subunits), 40 mM HEPES-KOH, pH 7.6, 100 mM Potassium Glutamate, 25 mM Magnesium Acetate, 10 g/ml single-stranded DNA, 0.2 mg/ml BSA, 10 mM DTT, 0.5 mM ATP, and 5 % DMSO solution containing the compounds. The reactions were started by the addition of the ATP, and the reaction mixtures were allowed to incubate at 30 °C for 60 mins. Reactions were stopped by adding Malachite green solution . Color was allowed to develop for 5 min, and the absorbance at 600 nm was measured spectrophotometrically. The half-maximum (50 %) inhibitory concentration (IC50) values were determined from the absorbance readings using no-compound and no-enzyme controls. The values reported are the averages of at least four independent experiments.Gyrase B and ParE monomers have only modest ATPase activity which is enhanced in the case of the holo enzymes and stimulated further by DNA. The phosphate released following conversion of ATP into ADP can be detected by the addition of malachite green solution and measured by monitoring the increase in absorbance at 600 nm. Topoisomerases were purchased from Inspiral is Ltd . For the In vitro experiments were carried out at the University of Prince Edward Island and approved by the Animal Care and Biosafety Committees, which adheres to the guidelines of the Canadian Council on Animal Care (protocol #14-041 and 15-028). Mixed neocortical cultures, containing both neurons and glia, were prepared from fetal rats at 17-18 days gestation. Briefly, embryonic tissue was extracted from untimed pregnant Sprague-Dawley rats by caesarean section and transferred to ice-cold Hanks Balanced Salt Solution (Gibco). Cortical brain tissue was carefully isolated by fine dissection using aseptic techniques. Cortices were minced in cold HBSS using a sterile razor blade prior to digestion with 0.0125 % trypsin. Dissociated cells were plated in poly-L-lysine  coated 96 well plates at a seeding density of 50000 cells/well in warm (37 °C) Dulbecco's Modified Eagle Medium (Gibco) containing 10 % iron-supplemented bovine calf serum (Hyclone) and 1 % antibiotic/antimycotic (Gibco). Cultures were allowed to adhere overnight in a humidified 37 °C incubator having 5 % CO2 and atmospheric oxygen. The following day, the media was replaced with warmed Neurobasal A Medium (Gibco) containing 0.5 mM L-glutamine (Gibco) and supplemented with 1 % B27, N2 and antibiotic/antimycotic (Gibco). Half-volume media changes were made every 4 days thereafter with experiments being carried out on 14-16 day old cultures. After 14 days in culture, the medium was replaced with deoxygenated unsupplemented glucose-free Neurobasal A containing either 10 or 100 µM of the novel compounds T3, T4, T5, T8, T9, T11, or T12. Control wells contained glucose-free medium plus vehicle - either 0.1 % ethanol or 0.1 % DMSO. Cultures were transferred to a humidified 37 °C incubator having 5 % CO2, 1.1 % O2 and the balance N2 for a period of 24 hours. Following OGD, an equal volume of normal supplemented Neurobasal A containing identical drug concentrations was added and cultures were transferred to normoxic conditions (5 % CO2 and atmospheric oxygen) for an additional 24 h. Cell viability was assessed by measuring LDH release from damaged cells. Control cultures were prepared in separate plates having supplemented Neurobasal A and being maintained in 5 % CO2 and atmospheric oxygen for the duration of the experiment. Toxicity was assessed in wells treated with 10 µM of each of the test compounds listed above. After 24 h an equal volume of culture media as added to each well to account for handling effects on experimental outcomes. Toxicity was assessed after an additional 24 h in culture by measuring LDH release from treated cells. At the end of each experiment, cell injury was assessed by measuring lactate dehydrogenase (LDH) released from damaged cells into the culture medium . LDH measurements represent the average of 7 replicate treatments/dose. The effect of OGD and drug treatments on LDH was expressed as a percent of LDH release compared to OGD and vehicle groups. Treatments were compared using a one-way ANOVA followed by Bonferroni post-hoc analysis. Differences were considered significant if p < 0.05. 4c as a representative example in 5 ml of methanol and 0.5 ml of diluted HCl. The solution was then mixed with equivalent amount of ferric chloride reagent with stirring for 1 h. The absorbance for reaction mixture and single compound was measured using UV-Visible Spectrophotometer Evolution 201 (Thermoscientific).Spectroscopic analysis of chelation property of novel compounds were identified using chemical reaction with ferric chloride. It was done by dissolving of 0.5 gm of compound All molecular descriptors supplied by the program MOE were computed for QSAR analysis. QSAR-Contingency (Molecular Operating Environment (MOE), Chemical Computing Group, 2012; Hogg et4a-c, and 7c with the selective pharmacologically important drug targets that overexpressed in breast and colon cancers using docking module implemented in MOE software (Molecular Operating Environment (MOE), Chemical Computing Group, 2012chromene-3-carbonitrile 7a-e.The new compounds have been synthesized using Knoevenagel condensation followed by Michael addition adducts . Two ne4a-i and 7a-e were characterized using FT-IR and NMR spectroscopic techniques. For instance, the FT-IR spectroscopy showed a characteristic absorption band between 2185 and 2210 cm-1 for the CN group, while the NH2 stretches were in the range of 3320-3490 cm-1. The 1H NMR spectra of chromene compounds 4a-i in DMSO-d6 showed signals at 5.09-5.67 ppm for 4-H pyran, while the singlets at 5.43-5.66 ppm are corresponding to NH2-10. The NH2-2 has a signal overlapped with the aromatic protons in the range of 6.84-7.78 ppm for all chromene derivatives except for compounds 4a and 4g, which had the protons of NH2-2 as a singlet at δ 6.85 and 7.05 ppm, respectively. The 13C NMR spectra showed a signal at δ 32.96-39.08 ppm corresponding to C-4, while C-3 appeared in the range of 56.06-58.66 ppm. The C-2 resonance showed signal at 159.71-160.89 ppm and the CN resonance was in the range of 120.17-122.61 ppm. It is also important to mention that chromene molecule 7c has been reported previously in literature  moiety. The 1H NMR spectra showed the presence of signals for H-4, appearing as a singlet in the range of δ 4.72-5.05 ppm, while the NH2-2 protons appeared in the range of 7.01-7.10 ppm. The rest of the aromatic protons of the chromene moieties and the arene rings appeared downfield in the range of 7.07-8.07 ppm. The 13C NMR spectra showed the resonance of C-4 at 31.23-36.51 ppm, while the CN resonance at 111.28-113.13 ppm and C-2 resonance at 159.72-161.82 ppm. And finally, the 19F NMR spectra showed the presence of the florin signals between -116.19 and -119.77 ppm. Compounds 12a-k and their azo chromene counterparts' 13a-k was performed in DMF in order to explore their λmax values. The obtained data showed that the formation of the chromene azo derivatives did not alter the λmax values of their azo precursors due to the non-conjugated system of the chromene moieties. All azo compounds and their azo chromene analogues displayed λmax in the range of 390-404 nm. A UV-vis study of azo dyes in vitro studies. The PASS is an in silico tool used for predicting biological activity spectra for natural and synthetic substances, which is based on the structure-activity relationships knowledgebase for more than 260,000 compounds with known biological activities including drugs, drug candidates, pharmaceutical leads and toxic compounds  and inactivity (Pi). The higher the Pa value, the lower the predicted probability of obtaining false positives in biological testing. The PASS analysis revealed that target compounds have high activity scores as apoptotic agonists with Pa values in the 0.851-0.721 range. This prediction agrees with previous published work  has been utilized in order to predict the spectrum of activity . In thial., 2015). The PAal., 2010; Tharejaal., 2010). The obStreptococcus pneumoniae (RCMB 010010) and Bacillus subtilis (RCMB 010067), two Gram-negative bacteria: Pseudomonas aeruginosa (RCMB 010043) and Escherichia coli (RCMB 010052), and four fungi: Aspergillus fumigatus (RCMB 02568), Syncephalastrum racemosum (RCMB 05922), Geotricum candidum (RCMB 05097), and Candida albicans (RCMB 05036). Ampicillin, ciprofloxacin, gentamicin and amphotericin B were used as positive control drugs . While compounds 7b and 7d were also found to be more active against Gm+ve, Gm-ve bacteria, and fungi with an IZ ranging from 18-23 mm. In the case of antibacterial activity against Gm+ve bacteria, most of the compounds were found to be comparable to the positive controls, having an MIC ranging from 3.9 to 0.49 µg/ml. All the newly synthesized compounds described in this report were screened for both antibacterial and antifungal activity via the reported agar diffusion well method . The insen, 2001). The ob13e, 13j, and 13j with MIC values of 0.007, 0.12, 0.49, and 3.9 µg/ml. Generally, the investigation of antimicrobial activity of the novel derivatives showed, in part, that some of them were more potent compared to the positive controls used against Gm+ve bacteria and certain strains of fungi.The chromene azo derivatives showed moderate inhibitory activity against all tested microorganisms especially compound in vitro cytotoxic activity was performed by MTT assay  activity. Thus, most of synthesized compounds showed promising antiproliferative activity compared to previously reported work  againstal., 2007; Zheng eal., 2007; El-AgroH-chromene series were identified as apoptosis inducers via caspase activation . Furthe4a, 7c, and 13b were evaluated against DNA gyrase from Escherichia coli in the DNA supercoiling assay for studying their mechanism. The results are presented in Table 5Based upon the antimicrobial screening data, target compounds in vitro culture, the cytotoxic effect of the chromene-based compounds  was assessed by lactate dehydrogenase (LDH) release from cells pretreated with different concentrations after 24 and 48 h of incubation  release in the medium is an enzymatic indicator that illustrates the loss of membrane integrity, apoptosis, or necrosis of a cell. Using a primary mixed neuronal Figure 9A. By incrFigure 9B. IncreasFigure 9B. These r4c as a representative of one of our target compounds. The data are presented in Figure 10max equal 361 nm which is shifted bathochromically due to the complexation. The absorption spectrum was observed to change from 281 and 342 nm for the drug alone, to 361 nm for the drug complex. This behavior occurs due to the possibility of monocomplexation, dicomplexation or tricomplexation reactions and indicates that these chemical compounds have the ability to chelate with metals as part of their biological cytotoxic mechanism. The tendency of the target drugs to work through a chelation mechanism with metals like iron and calcium, which would be similar to the behavior of doxorubicin as reference drug, was performed experimentally. The experiment was done by reaction of ferric chloride as metal source and compound 2), F value, and standard deviation. Three 2D QSAR equations were built using a set of 28 synthesized compounds with regard to each showing anticancer bioactivity in terms of molecular descriptors. These descriptor panels include: total polar surface area (TPSA), van der Waals volume (vdw_vol), partition coefficient (logP(o/w), number of hydrogen bonding acceptor (a_acc), total hydrophobicity surface (Q_VSA_HYD), molar refractivity (SMR), and van der Waals surface area (vdw_area). The QSAR models were generated from up to 6 descriptors describing the biological activity of anticancer agents and agree with the rule of thumb; 5:1 ratio of compounds to descriptors. The statistical characteristics of all models are grouped in Table 650 for all antiproliferative screening. The observed and predicted data of the training set of compounds , with exception of 4e, 4i, 7c, 13b, and 13f as testing sets, are presented in Table 7In this study, the best multi-linear regression analysis was utilized a stepwise search tool for finding out n-parameter regression models (n is the number of descriptors) for relating of structures to activity based upon different parameters coefficients values like . The bemax values. The new compounds have been evaluated for their antimicrobial and antiproliferative activities. Several of the target compounds showed potent antibacterial and antifungal effects towards some strains. In addition, they displayed more potency as anticancer agents with IC50 values in low micromolar concentrations. Certain studies have been employed to clarify their mechanisms and proved that these series could play their rule through the apoptotic effect by activation of caspases. As well, SAR analyses were discussed and explained by application of different computational methods.New series of chromene compounds have been prepared via multi-components reaction of 3-amino-2-naphthol with malononitrile and aromatic aldehydes in the presence of piperidine. The structures of the obtained compounds were confirmed using FT-IR, NMR and mass spectroscopy. Using the same methodology, a novel series of chromene containing azo chromophores was successfully synthesized via two steps' reaction. First, preparing the azo dye derivatives followed by the established methodology of the multi-components chromene approach. UV-vis of the new materials shows that the incorporation of the chromene moieties didn't alter their λThe authors would like to express their appreciation to the Deanship of Scientific Research at Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia for financial project support 6305/1435.The authors declare no conflicts of interest.


When a small molecule binds to the androgen receptor (AR), a conformational change can occur which impacts subsequent binding of co-regulator proteins and DNA. In order to accurately study this mechanism, the scientific community needs a crystal structure of the Wild type AR (WT-AR) ligand binding domain, bound with antagonist. To address this open need, we leveraged molecular docking and molecular dynamics (MD) simulations to construct a structure of the WT-AR ligand binding domain bound with antagonist bicalutamide. The structure of mutant AR (Mut-AR) bound with this same antagonist informed this study. After molecular docking analysis pinpointed the suitable binding orientation of a ligand in AR, the model was further optimized through 1 μs of MD simulations. Using this approach, three molecular systems were studied: (1) WT-AR bound with agonist R1881, (2) WT-AR bound with antagonist bicalutamide, and (3) Mut-AR bound with bicalutamide. Our structures were very similar to the experimentally determined structures of both WT-AR with R1881 and Mut-AR with bicalutamide, demonstrating the trustworthiness of this approach. In our model, when WT-AR is bound with bicalutamide, Val716/Lys720/Gln733, or Met734/Gln738/Glu897 move and thus disturb the positive and negative charge clumps of the AF2 site. This disruption of the AF2 site is key for understanding the impact of antagonist binding on subsequent co-regulator binding. In conclusion, the antagonist induced structural changes in WT-AR detailed in this study will enable further AR research and will facilitate AR targeting drug discovery. AR is expressed in various tissues of different species and regulates many physiological functions including bone density, cognition, muscle hypertrophy, prostate growth and differentiation . AR and activity . AR is aactivity . Both stFigure 1). Among these 12 helices, H12 plays a major role in AR activation and undergoes a considerable conformational change due to the binding of agonist or antagonist in the ligand binding pocket. During agonist or antagonist binding, H12 functions like a “lid” which closes or moves away from the ligand binding pocket, respectively   in the Protein Data Bank (PDB1) . Wild ty1) . To fillDetermining the conformation change of a protein induced by a ligand using crystallography is at best time consuming but often infeasible. Several researchers employed molecular dynamics (MD) simulations to characterize H12 structural changes due to antagonists or agonist binding in the AR ligand binding pocket. Three AR complex structures were studied to understand the antagonist binding induced structural changes of the AF2 site. R1881 and bicalutamide are, respectively, well-known as an agonist and an antagonist for AR. Structures of AR bound with R1881 and bicalutamide were downloaded from PDB: WT-AR-R1881  and Mut-AR-bicalutamide . The third AR complex structure, WT-AR-bicalutamide, was absent from PDB and thus was generated using the induced fit molecular docking (IFD) method . The IFD method explores both possible binding poses of a ligand in a receptor active site as well as the associated conformational changes of the side chains near the active site. MD simulations are an important tool to study receptor–ligand interactions at an atomic level for a given time frame. MD simulations optimize three-dimensional complex protein structure bound with a ligand obtained from X-ray crystallography or molecular docking. Here, we leveraged the advantages of IFD and MD simulations together to understand the subtle structural changes in WT-AR due to anti-androgen binding and also to elucidate key co-activator binding residues in the WT-AR AF2 site. Each AR complex structure was subjected to 1 μs of MD simulations to resolve important AF2 site residue reformation during the binding of small molecules in WT-AR. Our results will enable design of improved prostate cancer treatments and facilitate endocrine disruption chemical risk assessment through AR-mediated responses.Rigid docking (only giving flexibility to ligands) might fail to produce a precise ligand pose due to rigidness of the protein. In contrast, IFD gives flexibility to adjust not only the active site but also the side chain orientations of the protein to fit the pose and conformation of the bound ligand . Hence, 2 from the The IFD ,b moduleThe following steps were involved in the IFD employed here :(i) The protein was refined using the Protein Preparation module.(ii) Each ligand was docked (Glide module) in a defined region using a softened potential to produce 20 different poses (default setting).(iii) A sidechain prediction (Prime module) within a given distance of the ligand was conducted for each complex.(iv) The defined region of the protein-ligand complexes was minimized.(v) The refined protein-ligand complexes were re-docked using Glide by specifying the lowest energy structure.(vi) The IFD score (binding energy) was calculated for each complex.3 was used to add hydrogen atoms and to build the missing side chains, residues, and loops. The OPLS-2001 force field   were retce field  was usedce field  and dockce field  generatece field  by selecce field .Proteins are dynamic in nature. Thus, understanding atomic level motion is required to capture their profound dynamic mechanisms . MD simu- ions. The Particle Mesh Ewald (PME) , WT-AR-R1881, (PDBID: 1E3G) , and theld (PME)  and SHAKld (PME)  algorithld (PME)  and Visuld (PME) . The AmbTable 1). Among these 5 complex structures, Model-1, Model-3, and Model-4 showed a π–cation interaction with Trp741 and Phe764. Trp741 had van der Waals interactions favorable for agonist binding in the ligand binding pocket of WT-AR . To address this open question, we conducted IFD. Flexibility was given to the active site residues and the ligand during Glide docking. The whole WT-AR-bicalutamide system was refined using the Prime module to predict the suitable binding orientation of bicalutamide in the ligand binding pocket of WT-AR. Among the 20 models generated for WT-AR-bicalutamide, the top 5 models were selected based on their IFD/Glide scores and checked for residue interactions  to analyze the structural changes in WT-AR due to bicalutamide binding in the ligand binding pocket using MD simulations. All trajectory files obtained from the MD simulations were examined for stability and fluctuation of the systems. Metrics of root mean square deviation (RMSD) and root mean square fluctuation (RMSF) were calculated for all systems to measure their energetic stability and the spatial fluctuation of residues, respectively. Figure 2A plots the RMSD values of the three systems during the 1 μs simulations. The RMSD values converged in the last 100 ns, indicating that the systems had reached a stable state. The WT-AR-R1881 and Mut-AR-bicalutamide systems were stabilized with an RMSD value of around 2.0 Å, while the WT-AR-bicalutamide system had a higher RMSD value of about 2.5 Å. An average structure was calculated from the last 100 ns for each of the three systems. The structure with the lowest RMSD value compared with the average structure in last 100 ns was selected as a representative structure for each of the systems to elucidate the structural changes of WT-AR induced by bicalutamide.We used the three molecular systems listed in Figure 2B revealed that WT-AR-bicalutamide had a larger RMSF value compared with WT-AR-R1881 and Mut-AR-bicalutamide near the C-terminal of LBD (mostly near H12). The average RMSF value for WT-AR-bicalutamide, Mut-AR-bicalutamide, and WT-AR-R1881 was 1.29, 1.25, and 1.11 Å, respectively. Five residues  in AR had an RMSF of >2.0 Å  and were considered to be flexible residues. These five residues were present in the loop region of AR . The RMSF values of the active site residues were small, demonstrating the stability of the AR active site.Root mean square fluctuation plots were used to analyze flexibility of the residues in AR in the 1 μs MD simulations. Examination of the RMSF plots in Figure 3A). Comparison of WT-AR-bicalutamide with WT-AR-R1881 showed a distortion at the end of H10 due to bicalutamide binding. Several residues in H10 were changed into a loop, which enabled more flexible movement. The structural conversion of H11 into a loop moved H12 away from the AR ligand binding pocket. Moreover, structural changes were observed when comparing WT-AR and Mut-AR bound with bicalutamide . During bicalutamide binding, H11 was retained in the Mut-AR structure but was changed into a loop in the WT-AR structure . As expected, Mut-AR-bicalutamide had a similar 3D structure to WT-AR-R1881.The AR ligand binding pocket accommodates both agonists and antagonists. Most antagonists bind in this site and alter the function of AR. The representative structures of WT-AR-R1881 and WT-AR-bicalutamide obtained from the MD simulations were superimposed to examine the difference between the two systems. Several major structural changes were identified in WT-AR due to the bicalutamide binding compared with agonist binding (R1881)  and 1.02 Å for Mut-AR-bicalutamide . This indicates that the selected representative structures do not deviate much from the X-ray crystal structures. Furthermore, the orientations of R1881 and bicalutamide were also similar to the crystal structures. The overlay of bicalutamide from the Mut-AR X-ray crystal structure and the representative WT-AR structure from MD simulations had an RMSD value of 5.2 Å . This comparative analysis confirmed that the representative structures of WT-AR-bicalutamide obtained from the MD simulations are reliable and were not obtained by chance. Therefore, the representative structure of WT-AR-bicalutamide could be reliably used to elucidate the structural changes in WT-AR due to antagonist binding.Superimposition of the X-ray crystal structures and the representative structures from our MD simulations had an RMSD value of 1.10 Å for WT-AR-R1881 . Among these residues, few had a considerable deviation in their side chain. The side chain distances of Glu897 (CD), Gln738 (CD), Met734 (SD), Val716 (O), Lys720 (CG) were 3.8, 4.2, 2.2, 2.0, and 2.2 Å, respectively, between the WT-AR-R1881 and WT-AR-bicalutamide. These residues also had different conformations between WT-AR-bicalutamide and Mut-AR-bicalutamide as depicted in Figure 5B, with respective side chain distances of Glu897 (CD), Gln738 (CD), Met734 (SD), Val716 (O), Lys720 (CG) as 3.2, 0.5, 1.8, 1.1, and 3.0 Å. Val716, Lys720, and Gln733 were previously experimentally proven to form a charge clump in the AF2 site, which interacts with co-activator proteins  from the MD simulations shed light on critical residue displacements which prevent co-activator binding. Val713, Val716, Lys717, Lys720, Phe725, Met734, Met894, Glu897, and Ile898 were considerably different between WT-AR-bicalutamide and WT-AR-R1881 , indicating the mutant residues turned the antagonist into an agonist. However, WT-AR-bicalutamide had a very different electrostatic potential surface  compared with the other two structures due to structural changes in the AF2 site caused by the antagonist binding. Five residues  played an important role in bicalutamide binding induced WT-AR AF2 site structural changes. The binding of R1881 in the active site of WT-AR formed a positive (blue) and negative (red) binding region in the AF2 site . Proximal residue contact closed the positive  and negative  binding sites of the AF2 site in WT-AR-bicalutamide . The critical residues in the Mut-AR-bicalutamide AF2 site  showed a similar type of change compared with Mut-AR-R1881. Previously, it was experimentally proven that the charge clump was formed by residues Lys720 and Glu897  were optimized by MD simulations using Amber 14. Our results clearly pinpointed residues Val716, Lys720, Gln733 and Met734, Gln738, and Glu897 as playing a pivotal role in the formation of the AF2 site in AR. Structural changes or movement of these residues due to bicalutamide binding changed the structure of the AF2 site, making it unsuitable for co-activator protein binding. The electrostatic potential map clearly revealed that the movement of these residues due to bicalutamide binding disturbed the positive and negative charge clump in the AF2 site of WT-AR. The positive clump in the AF2 site was distorted due to the movement of residues Lys720, Val716, and Gln733. Experimental validation is needed to confirm the mechanism by which bicalutamide binding induced WT-AR AF2 structural changes impact recruitment of co-factors.SS and HH conceived the experiment(s). SS, BP, and WGe conducted the experiments. SS, BP, and WGo analyzed the results. SS, WT, HH, and RK wrote the manuscript. All authors reviewed and approved the manuscript.The content is solely the responsibility of the authors and does not necessarily represent the official views of the Food and Drug Administration. The findings and conclusions in this article have not been formally disseminated by the US Food and Drug Administration (FDA) and should not be construed to represent the FDA determination or policy.The 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.


Rickettsia asembonensis in fleas from domestic animals. We characterized 5 complete genomic regions  and conducted multilocus sequence typing and phylogenetic analyses. The molecular isolate from Peru is distinct from the original R. asembonensis strain from Kenya.While studying rickettsial infections in Peru, we detected  Rickettsia asembonensis belongs to a group of R. felis–like organisms (RFLOs) that are similar, yet distinct, from their closest known relative, R. felis  collected in the Peruvian Amazon (gltA) and variable  genes.C. felis flea as described  showed fewer substitutions than variable genes . The 17kDa gene exhibited no mutations along its 480-nt open reading frame (ORF), whereas the gltA gene exhibited 3 mutations along its 1,314-nt ORF. Two mutations in gltA encoded silent changes, whereas the third encoded a conservative lysine-to-glutamic acid change at position 290. In the variable group, ompB exhibited no mutations along its 4,947-nt ORF; ompA and sca4 exhibited 5 each. ompA had 2 conservative changes  and 2 nonconservative changes (tyrosine-to-aspartic acid at position 162 and arginine-to-glycine at position 1280). sca4 had 1 conservative change (glutamine-to-histidine at position 608) and 3 nonconservative changes . On the basis of these data, we conclude that the Peru molecular isolate is distinct from the original Kenya strain.Comparison of the consensus sequences we generated (GenBank accession nos. KY650696–KY650700) with those of strain NMRCii (GenBank accession no. JWSW01000078.1) . As expegltA gene. Although reference sequences are available for multiple Rickettsiaceae, R. asembonensis sequences are limited in number and length . As expected, the Peru isolate groups with RFLOs, including other R. asembonensis isolates and R. senegalensis  and 3 variable  genes of an R. asembonensis molecular isolate from Peru. Although characterization of 1 isolate is not sufficient to evaluate strain diversity within Peru, much less among American strains, these sequences represent a major contribution toward the expansion of availability of much needed genomic information. Our multilocus sequence typing and phylogenetic analyses indicate that the Peru isolate is closer to American strains than to the original strain from Kenya. Characterization of additional isolates, derived from a variety of ectoparasites in which R. asembonensis has been detected, is needed to further validate our findings and to conduct in-depth diversity studies. In turn, these results should help decrease the chronic underdiagnosis of rickettsial diseases throughout the Americas.Rickettsia asembonensis molecular isolate.Reference sequences used for phylogenetic analysis of Peru


To understand private consumer and clinician preferences towards different rehabilitation modes following knee or hip arthroplasty, and identify factors which influence the chosen rehabilitation pathway.Mixed methods cross-sectional study involving 95 semi-structured interviews of consumers (patients and carers) and clinicians  in Sydney, Australia, during 2014–2015. Participants were asked about the acceptability of different modes of rehabilitation provision, and factors influencing their chosen rehabilitation pathway. Interviews were in person or via the telephone. Qualitative analysis software was used to electronically manage qualitative data. An analytical approach guided data analysis.Pre-operative preferences strongly influenced the type of rehabilitation chosen by consumers. Key factors that influenced this were both intrinsic and extrinsic, including; the previous experience of self or known others, the perceived benefits of the chosen mode, a sense of entitlement, the role of orthopaedic surgeons and influence of patient preference, a patient’s clinical status post-surgery, the private hospital business model and insurance provider involvement. The acceptability of rehabilitation modes varied between clinician groups.No one rehabilitation mode provided following arthroplasty is singularly preferred by stakeholders. Factors other than the belief that a particular mode was more effective than another appear to dominate the pathway followed by private arthroplasty consumers, indicating evidence-based policies around rehabilitation provision may have limited appeal in the private sector.The online version of this article (doi:10.1186/s12913-017-2379-9) contains supplementary material, which is available to authorized users. Over the past 15 years there has been a steady increase in the rates of primary total knee arthroplasty (TKA) and total hip arthroplasty (THA) procedures undertaken annually in Australia –5. TheseIn the context of TKA and THA no high-level evidence supporting or contesting the benefits of inpatient rehabilitation over outpatient or home programmes was available at the time this study was instigated. One Canadian randomised controlled trial (RCT) that combined hip and knee patients compared 18 days of inpatient rehabilitation to eight domiciliary sessions (physiotherapy in the home). No significant differences were shown in the outcomes measured, which included the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Short Form-36 and patient satisfaction .In Australia, inpatient rehabilitation following arthroplasty surgery is most commonly utilised by private consumers i.e. those who are privately insured or elect to cover some or all of the costs of their healthcare, in contrast to public patients who have their healthcare needs met within the public health system, without payment. Recent estimates indicate that a median 40% of privately insured patients per surgeon were transferred to inpatient rehabilitation in 2014 following TKA, though this figure ranges from 0% to 100% . This coGiven that there are serious questions about the long-term sustainability and cost effectiveness of broad and untargeted inpatient rehabilitation provision following arthroplasty surgery, a greater understanding of factors shaping consumer and clinician preferences, as well as the manner in which decisions relating to rehabilitation following surgery are made, will be invaluable when reviewing current services or designing new healthcare delivery systems . UtilisiAll participants were volunteers and provided written, informed consent. Data collection occurred in two phases. Phase 1 involved consumers and was nested within a larger multicentre, observational study . Consecutive eligible private patients about to undergo arthroplasty surgery and their carers were invited to participate while attending a pre-operative admission clinic at one of two private arthroplasty hospital providers in New South Wales, Australia. Patient eligibility included having a principal diagnosis of osteoarthritis and was about to undergo either a unilateral or bilateral TKA or THA. Carer eligibility included being identified as the primary carer for one of these individuals. Sites with different business models were chosen, to investigate whether these may have an impact on a patient’s treatment after surgery.Phase 2 involved key clinicians from three disciplines involved in the care of knee or hip arthroplasty recipients. A separate computer-generated randomisation list was created for each of the individual groups: orthopaedic surgeons, rehabilitation specialists and physiotherapists. For orthopaedic surgeons and physiotherapists, the hospitals listed in the Australian Orthopaedic Association National Joint Replacement Registry were used to generate a random sample of sites and a surgeon and physiotherapist at each identified site were invited to participate. For rehabilitation specialists and physiotherapists working in the sub-acute rehabilitation services, a list provided by the Australasian Rehabilitation Outcomes Centre was used to generate a random sample of sites, with the same method of recruitment and random sample generation utilised. All clinician participants had been responsible for the care of knee or hip arthroplasty recipients in the private healthcare sector in New South Wales within the last 12 months.The determination of an appropriate sample size in qualitative research is a key component of the legitimacy of analysis and conclusions drawn . HeterogData for both consumers and clinicians were collected from one-to-one semi-structured interviews conducted between January 2014 and February 2015. For consumers, the semi-structured interviews were developed by clinicians familiar with the pre-admission and post-surgery settings, while for clinicians they were developed in consultation with an expert panel comprising an OS, RS and PT. Both were piloted before use. For patients, eight open-ended questions were posed which covered the following areas: the rehabilitation they had received, the process of decision-making they had undertaken, who had been involved in this decision, influencing factors, and options they had available . The elucidation of these codes was assisted by a process of listening to and reading transcriptions of the audiotapes, as well as consulting colleagues and perusing relevant literature. QSR’s NVivo qualitative analysis software  was utilDescriptive statistics were generated for the demographic, clinical and quantitative components of the study.Respondent characteristics for consumer participants are listed in Table Two overarching factors and their components Table  were ideThese factors related to the patient participants’ own perceptions of their situation, options and needs.‘I had the medical service there if I needed it, I had pain relief on hand if I needed it, and it was a simple matter of walking down to the gym and hydro pool… twice a day and getting the regular exercise.’ (Patient 32)‘…just having the medical staff and the monitoring going on… so that she’s able to get fully mobile and gain the confidence to be able to get around, shower, and that sort of stuff’. (Carer 5)Whether their own, or as reported by family and friends, a positive personal experience with inpatient rehabilitation was a common factor for patients who preferred this mode of rehabilitation delivery. Patients and carers frequently described the convenience of having everything at hand, from medical support to medication and meals:‘Being with the other patients and chatting and comparing notes was excellent too, ‘cause you felt that you weren’t alone.’ (Patient 20)Patients also appreciated the opportunity to compare progress, and interact with others in the same situation as themselves:‘I think [outpatient rehabilitation] works very well for most [of] my public patients… they go straight home.’ (OS 7)However, in the same way that a personal positive experience with inpatient rehabilitation was a common factor for patients who preferred that mode of rehabilitation delivery, a personal positive experience with a mode other than inpatient rehabilitation often led patients and carers towards a preference for these modes. This highlighted the finding that patients and carers tended towards a preference for the same mode of therapy received if they were to have surgery again. This theme was also a factor for clinicians who rated modes other than inpatient rehabilitation highly, whether from the private or public health sector. As one OS stated:‘[I recommend inpatient rehabilitation] if they’re old, if they’ve got significant comorbidities, if they lack family support, if they have more than 12 steps, if they’re slow while they’re in hospital.’ (OS 18)‘if they live alone, they are older, more deconditioned, they are slower, not motivated, or not able to comprehend what we are trying to get across to them as easily as some.’ (PT 2)There was a strong sense from many of the consumers and clinicians that inpatient rehabilitation was an essential component of treatment. Several of the reasons for this perception revolved around clinical or social issues, and in some instances personal factors such as ‘laziness’ or a need for motivation. Factors frequently mentioned to have influenced a choice of inpatient rehabilitation included weakness, advanced age, the presence of significant comorbidities, the home environment, and a lack of support at home:‘[Inpatient rehabilitation] can really improve the joint range of motion, pain management, confidence in managing mobility and functioning and preparing them better to be discharged home after rehabilitation.’ (RS 4)The RS interviewed also expounded the benefits of inpatient rehabilitation‘Inpatient rehab is a huge advantage because lots of our patients come from two, three hours away… and so by the time they drive [to the PT] and drive back, they’ve lost the benefit of the actual physio treatment session.’ (OS 13)The option of inpatient rehabilitation was also identified as an excellent option for people living in a rural environment.However, convenience was often a factor which led patients and carers towards a mode other than inpatient rehabilitation. The reasons for this perceived convenience were varied, from finding it easier to attend a private PT nearby (Patient 27) or having transport provided for outpatient therapy (Carer 19) to having a preference for the PT to come to one’s own home.Competing priorities also played a part, particularly when patients had a role as a carer. For one woman (Patient 6) it was the needs of her daughter with a bi-polar disorder that led her to a preference for an early discharge with outpatient one-to-one therapy follow up, while for others it was the needs of a husband and household pets (Patient 5), or the prospect of a loss of income with a prolonged admission (Patient 38).‘Staying away for a longer period of time is really hard for [my kids] and, yes, it would have been easier to have [the PT] actually coming to the house.’ (Patient 31)While outpatient services were well utilised, domiciliary physiotherapy was arranged in a number of cases, allowing patients to be treated in their own home. Others to whom it was not offered would have preferred it as an option if available:Almost every patient who expressed a preference for a mode other than inpatient rehabilitation expressed that their preference was linked in some way to getting back to the familiar environment of their own home. For some it was simply being able to sleep in one’s own bed, others again spoke of the support of family, friends and neighbours. And while many patients and carers found the hospital environment of inpatient rehabilitation conducive to their recovery, that setting led others towards a preference for other modes.‘…the fund was paying for [inpatient rehabilitation], so I was prepared to get the benefit of the whole thing’. (Patient 10)Consumers identified inpatient rehabilitation as a tangible way of getting value for the money they had put towards their private health insurance:‘We do get a cohort of people who want their entitlement; they've paid for it…’ (OS 19)to PT:‘…they’ve paid their private health insurance premiums for however many years, they think, “well, why shouldn’t I get to go [to inpatient rehabilitation], other people get to go. Yeah my knee might be good or my hip might be good but I want to go as well”.’ (PT 10)On some occasions this sense of entitlement ran alongside the perception that inpatient rehabilitation would also be advantageous for their recovery from surgery, but in others it seemed to be the primary motivating force for their preference. Clinicians verified this sense of entitlement, from OS:‘I mean, to a large extent, they’re paying for private health insurance, so they’re entitled to go to [inpatient] rehab.’ (OS 12)In some instances, clinicians themselves shared these sentiments:Consumers who expressed a preference for modes other than inpatient rehabilitation often spoke of it as unnecessary, given their individual circumstances. Some saw it as an option for ‘old’ people, while others referred to the support they had available at home as making an early discharge to that environment more advantageous to their recovery. For some, it was simply fondness for their own home environment, or dislike of the hospital environment, that led them in this direction.‘because [the patient] had transport provided so that he could stay here [at home] and just go and have his physio.’ (Carer 19)When compared to those with a preference for inpatient rehabilitation, this group of consumers tended to describe a more supportive home environment, with services such as transport, cleaning and meal preparation more frequently available. Many patients interviewed indicated that they would have utilised transport, cleaning and meal services if these had been available. There was evidence of these being offered or available in some cases, and this having had an influence on decisions made. For example, when asked why inpatient rehabilitation was considered unnecessary, one carer responded:A number of patients also indicated that they had utilised local community transport services for appointments.There were varied experiences reported by patients following surgery, but most shared a number of common characteristics. Although the information provided at different sites varied, most patients reported attending a pre-admission visit to their nominated hospital, meeting with their surgeon prior to and after surgery, and having some physiotherapy intervention after discharge. However, there were a number of extrinsic factors both before and after surgery which led patients towards different modes of rehabilitation following arthroplasty. These factors were explored in the clinician interviews.99% of my patients go to inpatient rehab… I tell them they’re going to go.’ (OS 14)‘As a group, OS reported differences of opinion regarding the perceived clinical value of inpatient rehabilitation following knee arthroplasty, and different approaches to recommendations made to their patients about post-operative care. For some surgeons, it was a case of one-size fits all:‘My words vary with every patient I see. My default position is that you go home and access outpatient facilities. However, if [they] are old, live alone, [or] have medical issues, then I understand that inpatient physio might be more appropriate’ (OS 3)Others tailored the mode to the individual, with the default set as an alternative mode and inpatient rehabilitation only suggested if deemed clinically or socially advantageous:‘I think [inpatient rehabilitation] is a big part of what sells my joints to patients. I know surgeons who sent patients home one or two days after surgery [and] I get a lot of their patients.’ (OS 17)One surgeon, who expressed a conviction that inpatient rehabilitation had a positive impact on the clinical outcomes for his patients, also implied that this approach had the effect of attracting patients to his services:‘If the patient is really keen on it, I don’t say no. I don’t feel strongly about it, that they should or shouldn’t go.’ (OS 7);‘…a patient will come and say “I want rehab post-op” and you think “Oh, you’re too fit and healthy, you don’t need that”, but if they want it, they get it.’ (OS 10)However, while these variations in approach existed within the group, the impact of a consumer preference for inpatient rehabilitation on the decision made was consistently reported by all OS interviewed. All indicated that they would go along with a stated preference for inpatient rehabilitation, even if they didn’t see a clinical need for it:In many occasions I’d prefer the patient to go home, but you’re caught between a rock and a hard place a lot of the time.’ (OS 12)‘This was even the case when it directly contravened the clinicians own personal preferences:The patient’s clinical status was reviewed in the acute phase of their recovery with OS and PT playing a part, and occasionally also nursing staff. Depending on their condition and rate of recovery at this point, any decision regarding path to rehabilitation type that had been made prior to surgery was either enacted, clarified or changed to take into consideration an unforeseen occurrence. The latter included post-operative complications, as well as better-than-expected patterns of recovery. RS reported playing virtually no part in this decision, which was confirmed by patients, OS and PT.‘I think it was just sort of a done deal that you went to rehab after.’ (Patient 9)The journey towards or away from inpatient rehabilitation may have been influenced by the model of service delivery in place at the hospital in question. At one site, a number of patients interviewed went through surgery and outpatient therapy afterwards without even knowing that inpatient rehabilitation was an available option. Alternatively, at the second site, all patients interviewed were aware of inpatient rehabilitation as an option, and it was often presented as ‘part of the package’ at preadmission sessions. Although many patients indicated they had already made a decision in regards to their preferred mode of rehabilitation before attending these sessions, others were clearly influenced by this approach:‘…up here, the inpatient therapy, they can do their own thing… There is certainly a bit of encouragement … from the hospitals, because there is a financial benefit for them as well… The strike rate for people going to [inpatient] rehab [here] is almost one hundred percent, and I guess the reality is maybe 60% may not need it. Maybe they’ll do just as well at home. It’s just the reality.’ (OS 12)‘In our facility most of the patients are going straight into rehab. It’s like a normal routine.’ (PT 2)Clinicians confirmed this approach at other sites, and indicated that patients admitted for therapy to particular rehabilitation inpatient units may have had no significant clinical indicators for such therapy.‘There are people that come to rehab that don’t really need to come to rehab. It’s not really that anything bad happens because of it, just that they don’t really need to be here.’ (PT 5)A number of clinicians indicated that patients admitted for therapy to particular rehabilitation inpatient units may have had no significant clinical indicators for such therapy.‘It can get pretty expensive by the time they come in and pay the gap all the time.’ (PT 6)There were references to health funds through both consumer and clinician interviews, but in only one case was there evidence of direct involvement on the choice of mode of rehabilitation delivered. In that instance Patient 33 described how his health fund had arranged domiciliary therapy provision for him following surgery, as part of his coverage. The other references to health funds generally alluded to the fact that the funds would cover inpatient rehabilitation as a treatment option after surgery and, as detailed earlier, knowledge of this cover did lead to a sense of entitlement in some. However, other patients elected a mode other than inpatient rehabilitation even when aware they were covered for this option. When questioned, some patients were unclear of their entitlements in regards to health cover after surgery. One of the PTs commented on the accumulation of out-of-pocket expenses for patients who attended private outpatient therapy.Considering the concerns around the long-term sustainability and cost effectiveness of broad and untargeted inpatient rehabilitation provision following arthroplasty surgery, the findings of this study are timely, and provide a clear description of factors shaping consumer and clinician preferences, as well as how decisions relating to rehabilitation following surgery are decided upon.”This study clearly exposes the many factors which influence private consumer and clinician preferences for particular modes of rehabilitation and associated decision-making following arthroplasty. These factors generally relate to clinical and social matters, but also include a sense of entitlement and extrinsic influences. The pattern of consumer preference tending towards the mode of rehabilitation they received mirrors those outlined by Naylor et al.  in a preA recent randomised, controlled trial, published after this study concluded, indicated that inpatient rehabilitation was not more effective than a monitored home-based program when measured across a range of outcomes up to 52 weeks post-surgery among adults undergoing uncomplicated total knee arthroplasty . These fWhen exploring possible reasons for this situation, a number of potential dilemmas may be responsible. One reason may be the additional time it would take within an orthopaedic consultation to provide information and talk through other options available; a review of 38 studies looking at the most frequently reported barrier to implementing shared decision making in clinical practice was time constraints . AnotherA separate factor influencing the treatment pathway after arthroplasty may be the business model of the site in question. The site at which inpatient rehabilitation was generally presented as ‘part of the package’ was run by a healthcare organisation which owned both the acute and rehabilitation facilities. The second, where patients went through surgery and associated treatment without knowledge of inpatient rehabilitation as an option, was owned by an organisation which did not have a business interest in any local rehabilitation facilities. Further study in this area may establish whether this pattern exists on a wider scale.The clear preference for inpatient rehabilitation by RS is largely unsurprising and revolved primarily around the perceived clinical benefits of this mode. The fact that these same RS benefit personally from the provision of this service was acknowledged during the interviews. Their preferences in this regard, however, seemed to have limited influence on the decision to send patients to inpatient rehabilitation, as they were generally not a part of the decision pathway leading to that setting.This study has provided a unique opportunity to articulate stakeholder attitudes towards various rehabilitation types, and the factors which guide consumer and clinician decisions relating to the utilisation of different rehabilitation modes following arthroplasty. An understanding of consumer and clinician preferences for rehabilitation, particularly inpatient rehabilitation, should help to inform ongoing and future models of care delivery, hand-in-hand with new evidence of effectiveness as it emerges. This will be particularly crucial to consider if alternative, less costly models of care are to be developed for, and acceptable to, the private sector.This study indicates that no one mode of rehabilitation provided following knee or hip arthroplasty is singularly preferred by stakeholders. Consumers may have a preference for the more expensive rehabilitation approaches, but if such modes are shown not to be more effective, then there is a need to explore less costly modes of rehabilitation following arthroplasty. If change is to be enacted, clinicians involved in the decision-making process will need to consider the comparative effectiveness of rehabilitation types, as well as the clinical and social needs of their patients, and play a more active role in promoting appropriate options to their patients


Typha latifolia) and hybrid cattail (Typha x glauca). The objective of this batch equilibrium study was to compare glyphosate absorption by the shoots and rhizomes of native versus hybrid cattails. Although it has been previously reported for some pesticides that passive diffusion is greater for rhizome than shoot components, this is the first study to demonstrate that the absorption capacity of rhizomes is species dependent, with the glyphosate absorption being significantly greater for rhizomes than shoots in case of native cattails, but with no significant differences in glyphosate absorption between rhizomes and shoots in case of hybrid cattails. Most importantly, glyphosate absorption by native rhizomes far exceeded that of the absorption occurring for hybrid rhizomes, native shoots and hybrid shoots. Glyphosate has long been used to manage invasive hybrid cattails in wetlands in North America, but hybrid cattail expansions continue to occur. Since our results showed limited glyphosate absorption by hybrid shoots and rhizomes, this lack of sorption may partially explain the poorer ability of glyphosate to control hybrid cattails in wetlands.Wetlands in the Prairie Pothole Region of North America are integrated with farmland and contain mixtures of herbicide contaminants. Passive nonfacilitated diffusion is how most herbicides can move across plant membranes, making this perhaps an important process by which herbicide contaminants are absorbed by wetland vegetation. Prairie wetlands are dominated by native cattail ( N (phosphonomethyl) glycine] and MCPA (2-methyl-4-chlorophenoxyacetic acid) are among the most widely applied herbicides in North American agriculture, and are frequently detected in surface waters, particularly in wetlands that are integrated with farmland in the Prairie Pothole Region of North America  species, which is notable, considering that these plants often dominate wetlands. Prairie wetlands in North America are dominated by a mix of native cattail (Typha latifolia), the European introduced cattail (Typha x angustifolia) and hybrid cattail (Typha x glauca) from the two parent species. The native (Typha latifolia) and the hybrid species (Typha x glauca) were chosen to be tested in this study.Conventional physical and chemical technologies for removing contaminants from surface water aim at removal before utilization, for example, flocculation, membrane filtration Shkinev . Some teSince the primary pathway for organic contaminants to enter plants is via passive absorption  from American Radiolabeled Chemicals Inc., USA, and analytical grade glyphosate (99.9% purity) and MCPA (99% purity) from Sigma-Aldrich Co., St. Louis, MO, USA. Glyphosate and MCPA properties are given in Table Herbicides purchased were [phosphonomethyl-14C] glyphosate  and [2-methyl-4-chlorophenoxyacetic acid 4 Bq/L 14C-labelled glyphosate. MCPA stock solutions consisted of 10 μg/L or 1 mg/L analytical-grade MCPA and 1.67 × 104 Bq/L 14C-labelled MCPA. We included MCPA in this study so that we could compare the sorption of glyphosate against that of another herbicide that is frequently detected in Prairie wetlands. Solutions (15 mL) were added to 0.2 g plant samples in 50 mL Teflon tube in triplicates, and a previous batch equilibrium study  of supernatant (duplicates) were added to scintillation vials (7 mL) containing 5 mL 30% Scintisafe scintillation cocktail . Vials were lightly shaken before radioactivity was measured. The radioactivity in the initial solution and centrifuged supernatant were measured by Liquid Scintillation Counting (LSC) with automated quench correction (#H method)  and a maximum counting time of 10 min.pl (L/kg) of glyphosate or MCPA was calculated by:i is the concentration of radioactivity in the initial solution; Ce is the concentration of radioactivity in the centrifuged supernatant; V is the volume of solution; Wpl is the oven-dry weight of plant sample.The plant-solution partition coefficients Kpl values. Two-way ANOVA in PROC GLIMMIX was used to quantify the effect of concentrations  and plant treatments  on Kpl values at 48 h.Statistical analysis was conducted by using SAS software version 9.3 for Windows (SAS Institute Inc. 2002–2010). Repeated measure analysis and multiple means comparison (Tukey’s) tests were utilized in PROC GLIMMIX to determine the effect of shaking time and plant treatment on Kpl values than hybrid rhizomes, and than native or hybrid shoots , MCPA Kpl values remained at a relatively low level (<8 L/kg) for all cattail treatments. However, similar to the glyphosate results, regardless of whether the initial solution contained 10 μg/L or 1 mg/L MCPA, native rhizomes demonstrated significantly greater MCPA Kpl values than hybrid rhizomes. Thus, it is possible that in addition to glyphosate, other herbicides are less likely to be absorbed by hybrid than native rhizomes.Sorption data for glyphosate and MCPA obtained at 48 h were further analyzed to help understand the different behaviors of the four cattail treatments at the two chosen concentrations Fig. . Statistpl values for native rhizomes are of the same magnitude as K values reported for other organic materials and soils.Our results highlight the potential of native rhizomes as a sorbent for glyphosate, giving optimal mixing conditions. Table


AR genomic structural rearrangements (AR-GSRs) as a class of molecular alterations occurring in one third of CRPC-stage tumours. AR-GSRs occur in the context of copy-neutral and amplified AR and display heterogeneity in breakpoint location, rearrangement class and sub-clonal enrichment in tumours within and between patients. Despite this heterogeneity, one common outcome in tumours with high sub-clonal enrichment of AR-GSRs is outlier expression of diverse AR variant species lacking the ligand-binding domain and possessing ligand-independent transcriptional activity. Collectively, these findings reveal AR-GSRs as important drivers of persistent AR signalling in CRPC.Molecularly targeted therapies for advanced prostate cancer include castration modalities that suppress ligand-dependent transcriptional activity of the androgen receptor (AR). However, persistent AR signalling undermines therapeutic efficacy and promotes progression to lethal castration-resistant prostate cancer (CRPC), even when patients are treated with potent second-generation AR-targeted therapies abiraterone and enzalutamide. Here we define diverse  Castration-resistant prostate cancer frequently presents with persistent androgen receptor signalling. Here, the authors find that the androgen receptor is subject to genetic rearrangements, resulting in variants with ligand-independent activity. Genomic interrogation of castration-resistant prostate cancer (CRPC) tumours has provided a catalogue of mutations and copy number alterations that are associated with disease spread and evolution during androgen receptor (AR)-targeted androgen deprivation therapy (ADT)1234679AR gene. There are efforts underway to develop approaches for monitoring AR amplification or mutation, which could inform more precise use of first- and second-generation AR-targeted therapies, indicate the need for alternative therapies such as taxanes, or assist in prioritization of patients for clinical trials. For example, detection of AR amplification or mutations in cell-free DNA isolated from blood of CRPC patients is associated with resistance to abiraterone and enzalutamideThe most common documented alterations in the AR pathway in CRPC, occurring in ∼60% of tumours, are amplification or mutation of the Expression of AR messenger RNA (mRNA) splicing variants (AR-Vs) lacking the AR ligand-binding domain has emerged as an additional mechanism of resistance to first- and second-generation AR-targeted therapies. In particular, detection of AR-V7 mRNA in circulating tumour cells from CRPC patients treated with abiraterone or enzalutamide is associated with primary resistance and reduced overall survival131516171819202119324AR gene rearrangement events2627AR gene rearrangements are well-characterized in these models, the relevance of AR gene rearrangements to clinical CRPC has been unclear. To address this, in this study we conduct a deep sequencing analysis of AR in tissues derived from metastatic CRPC, localized CRPC and therapy-naive prostate cancer. Our results demonstrate that AR gene rearrangements are frequent and diverse in clinical disease. By integrating these findings with AR expression data, we demonstrate that AR gene rearrangements with high allelic enrichment drive outlier expression of unique AR-V species with constitutive transcriptional activity and protein structures resembling AR-V7. In conclusion, AR gene rearrangements represent an important mechanism of AR-V expression in clinical CRPC.In CRPC models where AR-Vs have been shown to promote resistance, expression levels of AR-Vs relative to full-length AR are high and have been linked to specific AR gene locus is located on the X chromosome and contains multiple repetitive DNA stretches including long- and short-interspersed nuclear elements. We developed a liquid-phase AR bait panel with enhanced coverage features that we hypothesized would provide greater sensitivity for detection of structural aberrations in this high repeat-content locus , defined as rearrangement events having at least one breakpoint mapping within the AR gene body  displaying at least one AR-GSR event  in CRPC metastases that displayed AR amplification . Although AR-GSRs did not occur in the six CRPC metastases with AR missense mutations, tests for mutual exclusivity between AR-GSRs and AR missense mutations did not reach statistical significance . Notably, AR-GSR-positive metastases C-6A, C-6B and C-9A did not harbour AR amplification or mutation events. This showed that AR-GSR positivity could re-classify a subset of CRPC metastases that would otherwise have been deemed to harbour a normal AR gene.Next, we analysed AR DNA-seq data with a structural variant detection pipeline to develop a list of candidate ene body . This apSR event . AR-GSRsAR gene alteration, we performed AR DNA-seq with six additional CRPC specimens obtained from transurethral resection of the prostate (TURP) procedures and 21 hormone-naive prostate cancer specimens obtained from prostatectomies .To understand the context of this new class of ectomies . Similarpecimens . Conversnegative . CollectAR-GSR break fusion junctions were variable across the AR gene body and associated with diverse deletion, duplication, inversion and translocation events  3, the 3′-terminal exon spliced in AR-V7 mRNA. Conversely, levels of AR-V7 and full-length AR were significantly higher in tumours that displayed an increase in AR gene copy number compared with tumours that displayed a single AR gene copy . To test this scenario further, we next focused on subject C-12, where two tumour sites harboured a series of shared and exclusive AR deletion and duplication events  of tumours C-12A and C-12B, and mapped RNA-seq reads against a C-12A-specific genome assembly harbouring this 379 kb on event . We creaese bins . The diam region . SplicinAR exon 3 and these AR upstream exons  and AR DNA-binding domain (DBD) core  core . These ABD) core . In addiBD) core . These rAR exon 3  bait library using the SureSelect QXT reagent kit (Agilent) as per the manufacturer's recommendations. The SureSelect bait library was created using the SureSelect DNA Standard Design Wizard (Agilent) with 5 × tiling density, moderately stringent masking and MaximizePerformance parameters selected (Agilent). Post-capture sequencing libraries were pooled and diluted to 10 pM for flow cell clustering and sequenced in 2 lanes of an Illumina HiSeq 2500 with 2 × 150 bp settings for CRPC metastases, or 1 lane of an Illumina HiSeq 2500 with 2 × 125 bp settings for localized prostate cancer.http://picard.sourceforge.net). Discordantly mapped read pairs and split reads were extracted as recommended by LUMPY instructions (https://github.com/arq5x/lumpy-sv). LUMPY (v0.2.8)AR-GSRs were defined as structural variants supported by both LUMPY and DELLY with at least one of the breakpoints mapping within the AR gene body . Candidate AR-GSRs with at least ten supporting paired-end reads and ten supporting split reads were selected for further validation. Tumour P-4A displayed three candidate AR-GSRs that met these criteria, but inspection of these and other structural variant calls from P-4A revealed that all were deletions with breakpoints at known splice donor/acceptor junctions in wild-type AR mRNA, indicating this sample was contaminated with AR complementary DNA tary DNA . This saAR-GSR call  were inspected manually in integrative genomics viewerAR-GSR was due to deletion, duplication, inversion or translocation. PCR primers are listed in Data supporting each AR gene  copy number was determined for each tumour specimen by calculating the ratio of normalized mean AR read coverage of a tumour sample versus normalized mean AR read coverage for an AR copy number alteration negative tumour sample (C-10A). Mean AR read coverage was normalized based on five control regions included on the SureSelect capture panel: C1 (chr9:98258995-98264381), C2 (chrX:16153017-16159789), C3 (chr14:105249980-105255049), C4 (chr15:40514992-40520036) and C5 (chr15:67390102-67395049). The average mean coverage of each region was calculated by BedToolsAR sequence variants were called using FreeBayes v0.9.14-14-gb00b735 , while incorporating Phase Lock Gel Heavy tubes (5 Prime) into the protocol, to better facilitate phase separation. RNA yield and purity was obtained on a NanoDrop 2000 (Thermo Fisher Scientific). RNA Integrity was evaluated on a 2100 Bioanalyzer (Agilent Technologies). Total RNA was subjected to RT–PCR using TaqMan primers and probes designed to specifically amplify and interrogate the exon 4/5 splice junction , the exon 3/CE3 splice junction AR-V7;  and the 6 reads per sample.For RNA-seq experiments, RNA was subjected to Illumina-based RNA-seq using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) as per the manufacturer's recommendations. RNA-seq libraries were sequenced with an Illumina HiSeq 2500 with 2 × 50 bp settings, resulting in 70–100 × 10AR architecture design based on the exact break fusion junction signature(s) arising from a single AR-GSR event. To prevent errors from reads mapping to loci represented in both the derivative chromosome and hg19 chromosomes, the endogenous AR locus (chrX) and other appropriate endogenous sequences were masked. RNA-seq reads from selected tumours were mapped to these ‘tumour-specific' reference genome assemblies using HISAT2 (ref. AR-GSR. ComradTumour-specific reference genome assemblies were developed by adding a derivative chromosome to the hg19 reference genome. This derivative chromosome represented a rearranged AT2 ref.  or TopHaAT2 ref. . Bins weImmunohistochemistry experiments were performed as describedPlasmid constructs harbouring full-length AR-V7 (AR 1/2/3/CE3), ARv567es (AR 1/2/3/4/8), -5746 PSA-LUC and 4XARE-E4-LUC have been described−1 streptomycin (pen/strep) in a 5% CO2 incubator at 37 °C. VCaP cells were cultured in DMEM medium (Invitrogen) with 10% FBS and pen/strep in a 5% CO2 incubator at 37 °C. Aliquots of cell culture supernatants from cells in active culture were evaluated regularly for mycoplasma contamination using a PCR-based method as describedLNCaP , VCaP  and DU145  cells were obtained from American Type Culture Collection (ATCC) and cultured according to ATCC protocol. ATCC ensures authenticity of these human cell lines using short tandem repeat analysis. LNCaP and DU145 cells were maintained in RMPI 1640 (Invitrogen) with 10% fetal bovine serum (FBS), 100 units per ml penicillin and 100 μg mlRenilla and 0.42 μg AR-V expression plasmid) were mixed with 4 × 106 LNCaP cells in a 4 mm gap-width electroporation cuvette and subjected to a 305 V, 10 ms pulse using a Square Wave Electroporator (BTX/Harvard Apparatus) before plating in RPMI medium supplemented with 10% charcoal-stripped, steroid-free FBS (CSS). VCaP and DU145 cells were transfected using Superfect (Qiagen) as per the manufacturer's recommendations exactly as described5 cells the day prior. For all reporter-based experiments, 24 h post transfection, cells were re-fed with serum-free medium containing 1 nM dihydrotestosterone (Sigma) or 0.1% ethanol as vehicle control in combination with 30 μM enzalutamide  or 0.1% DMSO as vehicle control for 24 h. Cells were harvested in 1 × passive lysis buffer provided in a Dual Luciferase Assay Kit (Promega). Activities of the firefly and Renilla luciferase reporters were assayed using a Dual Luciferase Assay Kit as per the manufacturer's recommendations. Transfection efficiency was normalized by dividing firefly luciferase activity by Renilla luciferase activity. Data presented represent the mean±s.e.m. from three independent experiments, each performed in duplicate.LNCaP cells were transfected by electroporation exactly as described5 cells per dish in RPMI with 10% FBS and pen/strep. Cells were transduced the next day by addition of 0.5, 2, 8 or 32 μl of lentivirus directly to tissue culture medium. Cells were re-seeded 120 h post transduction on 96-well plates in RPMI+10% CSS+pen/strep, and subjected to cell proliferation assays using a BrdU ELISA kit (Roche) according to the manufacturer's recommendations.Lentivirus encoding GFP, AR 1/2/3/4-ups/5a-ups, AR 1/2/3/4-ups/5b-ups, AR 1/2/3/4-ups/5c-ups and AR 1/2/3/4-chr11 was prepared using a standard third-generation packaging system in 293T cells. Briefly, 293T cells were co-transfected with lentivirus vectors and packaging vectors pCMVΔR8.91 and pMD.G (ref. 31) at a ratio of 4:3:1 using Lipofectamine 2000 (ThermoFisher Scientific). Medium containing lentivirus was collected from 48 to 96 h post transfection, pooled and concentrated using Lenti-X Concentrator (Clontech). LNCaP cells were seeded in 6 cm dishes at 4 × 10Transfected cells were harvested in 1 × Laemmli buffer and subjected to western blot analysis as describedAR-GSRs or copy number increase were performed using one-sided Mann–Whitney U-tests. Mann–Whitney U-tests were selected based on sample distributions being non-normal in Shapiro–Wilks goodness-of-fit tests. Sample sizes were not chosen to ensure adequate power, to determine a pre-determined effect size. Statistical calculations were performed using GraphPad Prism 5 software.Tests for higher expression of AR or AR-V7 mRNA in tissues positive for The RNA-seq and AR DNA-seq data that support this study are available through the NCBI database of Genotypes and Phenotypes (dbGaP) under accession phs001223.v1.p1. All other relevant data are available within the article file or How to cite this article: Henzler, C. et al. Truncation and constitutive activation of the androgen receptor by diverse genomic rearrangements in prostate cancer. Nat. Commun.7, 13668 doi: 10.1038/ncomms13668 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-14 and Supplementary Tables 1-9.Illumina HiSeq 2500 Sequencing Metrics Per Sample.Tier 1 AR-GSR Calls Identified by LUMPY and DELLY.Fusion Transcripts Identified by Comrad in Subjects C-9, C-12, and C-14.