N1 of the hydrazone in order to establish robust structure–activity relationships. The results of the biological testing demonstrated that the presence of the hydrazothiazole nucleus bearing at C4 a phenyl ring functionalised at the meta position with a nitro group represents an important pharmacophoric feature to obtain selective and reversible human MAO-B inhibition for the treatment of neurodegenerative disorders. In addition, the most potent and selective MAO-B inhibitors were evaluated in silico as potential cholinesterase (AChE/BuChE) inhibitors and in vitro for antioxidant activities. The results obtained from molecular modelling studies provided insight into the multiple interactions and structural requirements for the reported MAO inhibitory properties.A new series of 4-(3-nitrophenyl)thiazol-2-ylhydrazone derivatives were designed, synthesised, and evaluated to assess their inhibitory effect on the human monoamine oxidase (hMAO) A and B isoforms. Different (un)substituted (hetero)aromatic substituents were linked to  The patients suffering from NDDs share a large plethora of pathogenic mechanisms and symptomatologyHuman MAOs are of great medicinal importance due to their unique role in modulating the function/activity of specific neurotransmitters (i.e. dopamine) in various pathogenic cental nervous system (CNS) conditions ,Selective MAO-B inhibitors could thus restore the dopamine concentration in the basal ganglia and limit ROS-induced neurotoxicity as well-recognised for the clinically used drugs in this class l-deprenyl showed enhanced binding in presymptomatic familial AD patients, and (iii) AD patients are characterised by enhanced astrocytosis. Moreover, MAO-B was reported to be associated with γ-secretase in the regulation of intraneuronal Aβ levels especially in pyramidal neurons as well as glia cells in the frontal cortex and hippocampusPrevious studies have also shown the correlation between MAO-B and Alzheimer’s disease (AD) due to (i) the increase of MAO-B activity in brain and platelets in AD patients, (ii) the MAO-B specific ligand 11C-deuterium-1–37, in order to evaluate their hMAOs inhibition profile, chelating properties, antioxidant activity as well as in silico AChE and BuChE inhibition properties. These activities determine the efficacy of these compounds to work as multi-target drugs.Recently, we designed and synthesised multi-target inhibitors based on thiazol-2-ylhydrazone scaffold with selective MAO-B inhibitory potency for further structural tuning2.1–36) were synthesised in high yields as reported in our previous communications (a). The Hantzsch reaction between the resulting thiosemicarbazone and 2-bromo-3′-nitroacetophenone in the same conditions of solvent and temperature gave the corresponding 4–(3-nitrophenyl)thiazol-2-ylhydrazone derivatives (b). For the synthesis of the thiophene-containing 4-(3-amino)thiazol-2-ylhydrazone derivative (37), reduction of the nitro group was performed using sodium dithionite previously solubilised in a basic aqueous solution and added dropwise to a stirring suspension of compound 3 in tetrahydrofuran at room temperature (c). All the synthesised products were washed with petroleum ether and diethyl ether and purified by chromatography using silica gel as stationary phase and the appropriate mixtures of ethyl acetate and petroleum ether as mobile phase. Characterisation and purity assessment of the synthesised compounds were carried out by melting point determination, spectroscopic methods  and elemental analysis.4–(3-Nitrophenyl)thiazol-2-ylhydrazone derivatives . For a selected compound, the reversibility/irreversibility of MAO inhibition and mechanism of inhibition (e.g. competitive) were evaluated. Furthermore, with the aim to explore the multi-target profile of these derivatives, we performed tests to determine chelating properties, antioxidant activity as well as the AChE and BuChE inhibition profile.Compounds 4.® melting point apparatus SMP1, and are uncorrected. IR spectra were measured with a PerkinElmer Spectrum 100 FT-IR spectrophotometer equipped with universal total reflectance (ATR) accessory with absorption frequencies expressed in reciprocal centimetres. 1H and 13 C NMR spectra were recorded at 400 MHz on a Bruker spectrometer using CDCl3 and DMSO-d6 as the solvents at room temperature. The samples were analyzed at a final concentration of 30 mg/mL. Chemical shifts are expressed as δ units (parts per millions) relative to the solvent signal. Coupling constants J are valued in Hertz (Hz). The processing and analyses of the NMR data were carried out with MestreNova. Elemental analyses for C, H, and N were recorded on a Perkin-Elmer 240 B microanalyzer obtaining analytical results within ± 0.4% of the theoretical values for all compounds. All reactions were monitored by thin layer chromatography (TLC) performed on 0.2 mm thick silica gel-aluminium backed plates . Preparative flash column chromatography was carried out on silica gel . All compounds were recrystallised from ethanol. The yields shown are not optimised. Organic solutions were dried over anhydrous sodium sulphate. Evaporation of the solvent after reaction was carried out on a rotary evaporator .Starting materials and reagents used in the synthetic procedures were obtained from commercial suppliers and were used without further purification. Solvents were freshly distilled before use whenever required. All melting points were measured on a Stuart4.1.1–37 in high yields and purity. With respect to the synthetic approach of compound 36 that is the product of the dimerisation of the parent compound 31, two equivalents of thiosemicarbazide were used in the first step (a) and two equivalents of 2-bromo-3′-nitroacetophenone were used in the second step (b). To obtain the thiophene containing 4-(3-amino)thiazol-2-ylhydrazone derivative (37), sodium dithionite (5.5 eq) was dissolved in a basic solution of water (30 ml) and sodium bicarbonate (5.5 eq.). The resulting solution was added dropwise to a stirring suspension of nitro compound 3 (1.0 eq.) in tetrahydrofuran (50 ml) at room temperature and the reaction was stirred for 2 h (up to completion by TLC). The tetrahydrofuran was evaporated in vacuo, the solid that precipitated from the aqueous phase was collected by filtration, washed with water (20 ml) and petroleum ether (20 ml), and dried to give the desired amino derivative 37. In order to confirm structure and purity of the synthesised test compounds, characterisation was performed using IR, 1H NMR and 13C NMR along with elemental analysis. Melting points were recorded as mp range. The synthesised compounds exist in theory as a mixture of E and Z isomers. However, the chemical-physical data are characteristic for the most predominant and thermodynamically stable E geometric isomer because of the up-shielding observed in the 1H NMR spectra for the NH signal and for the absence of any Z-stabilising functional groups. The Z isomer is also undesirable due to steric interaction between the aryl group and the NH moiety1–37 showed stretching absorption bands at approximately 3310 cm−1 for NH, at 3040 cm−1due to the stretching of Csp2-H, at 1640 cm−1 for the C=N and at 1585 and 1445 cm−1 for C = C.To a stirring solution of the appropriate carbonyl compound (1.0 eq.) in ethanol (50 ml), thiosemicarbazide (1.0 eq.) and acetic acid as the catalyst were added. The reaction was monitored by TLC up to completion, usually reached in 24–72 h. The obtained suspension was filtered, and the solid washed twice with petroleum ether (20 ml) and diethyl ether (20 ml). The thiosemicarbazone (1.0 eq.), thus synthesised, was reacted with 2-bromo-3′-nitroacetophenone previously dissolved in ethanol (50 ml), and the reaction was magnetically stirred at room temperature until completion as monitored by TLC. The resulting 4–(3-nitrophenyl)thiazol-2-ylhydrazone derivative was collected by filtration, washed with petroleum ether (20 ml) and diethyl ether (20 ml), and purified by column chromatography using ethyl acetate:petroleum ether as mobile phase, to give compounds rst step , a and tond step , b. To o4.1.1.1H NMR : δ 6.60–6.61 , 6.82–6.83 , 7.64 , 7.65–7.70 , 7.79–7.81 , 7.93 , 8.14–8.16 , 8.29–8.31 , 8.65 , 12.17 . Anal. Calcd for C14H10N4O3S: C, 53.50; H, 3.21; N, 17.82. Found: C, 53.26; H, 3.01; N, 17.99.Green powder, mp 213–215 °C, 71% yield; 4.1.2.1H NMR : δ 2.28 , 7.38–7.47 , 7.64 , 7.66–7.75 , 7.87–7.89 , 8.15–8.18 , 8.32–8.35 , 8.74 , 11.52 . Anal. Calcd for C14H10N4O2S2: C, 54.87; H, 3.68; N, 17.06. Found: C, 55.06; H, 3.51; N, 17.22.Yellow powder, mp 230–234 °C, 68% yield; 4.1.3.1H NMR : δ 7.09–7.11 , 7.37–7.38 , 7.58–7.60 , 7.64 , 7.68–7.72 , 8.13–8.15 , 8.23 , 8.28–8.30 , 8.66 , 12.19 . 13C NMR : δ 106.9 , 120.4 (Ar), 122.5 (Ar), 129.8 , 130.7 (Ar), 132.0 (Ar), 134.0 , 136.6 (Ar), 137.5 , 139.5 , 145.9 (C=N), 148.2 (Ar-NO2), 148.8 , 168.7 . Anal. Calcd for C14H10N4O2S2: C, 50.90; H, 3.05; N, 16.96. Found: C, 51.13; H, 2.88; N, 17.20.Yellow powder, mp 187–189 °C, 70% yield; 4.1.4.1H NMR : δ 2.34 , 7.05–7.07 , 7.35–7.41 , 7.44–7.50 , 7.65 , 7.69–7.71 , 8.11–8.14 , 8.31–8.35 , 8.70 , 11.41 . 13C NMR : δ 15.0 (CH3), 107.4 , 120.4 (Ar), 122.5 (Ar), 127.1 , 128.1 , 128.4 , 130.7 (Ar), 132.0 (Ar), 136.7 (Ar), 143.7 , 144.6 (C=N), 148.5 (Ar-NO2), 148.8 , 168.7 . Anal. Calcd for C15H12N4O2S2: C, 52.31; H, 3.51; N, 16.27. Found: C, 52.05; H, 3.78; N, 16.43.Yellow powder, mp 270–272 °C, 74% yield; 4.1.5.1H NMR : δ 2.39 , 7.07 , 7.36–7.38 , 7.57 , 7.61–7.65 , 8.16–8.22 , 8.66 , 9.49 . 13C NMR : δ 15.2 (CH3), 107.2 , 120.4 (Ar), 122.4 (Ar), 124.7 , 125.7 , 127.3 , 130.7 (Ar), 132.0 (Ar), 136.7 (Ar), 141.7 , 144.6 (C=N), 148.5 (Ar-NO2), 148.8 , 168.7 . Anal. Calcd for C15H12N4O2S2: C, 52.31; H, 3.51; N, 16.27. Found: C, 52.60; H, 3.27; N, 16.02.Yellow powder, mp 183–185 °C, 85% yield; 4.1.6.1H NMR : δ 2.29 , 7.09 , 7.26–7.29 , 7.67–7.73 , 8.14–8.32 , 8.70 , 11.58 . Anal. Calcd for C15H11ClN4O2S2: C, 47.55; H, 2.93; N, 14.79. Found: C, 47.71; H, 3.17; N, 14.53.Orange powder, mp 227–229 °C, 81% yield; 4.1.7.1H NMR : δ 7.38–7.47 , 7.67–7.74 , 8.06 , 8.15–8.17 , 8.30–8.32 , 8.41–8.43 , 8.69 , 12.31 . Anal. Calcd for C16H12N4O2S: C, 59.25; H, 3.73; N, 17.27. Found: C, 58.99; H, 3.98; N, 17.02.Yellow powder, mp 193–195 °C, 76% yield; 4.1.8.1H NMR : δ 7.63–7.65 , 7.73–7.74 , 7.79–7.82 , 8.04–8.05 , 8.06 , 8.16–8.18 , 8.31–8.33 , 8.45–8.47 , 8.69 , 12.68 . 13C NMR : δ 107.6 , 120.4 (Ar), 122.6 (Ar), 125.2 (Ar), 128.0 (Ar), 128.9 (Ar), 130.3 (Ar), 130.7 (Ar), 132.1 (Ar), 134.0 (Ar), 136.5 (Ar), 137.2 (Ar), 137.6 (C=N), 147.9 (Ar-NO2), 148.8 , 168.6 . Anal. Calcd for C16H11N5O4S: C, 52.03; H, 3.00; N, 18.96. Found: C, 52.27; H, 2.82; N, 19.12.Orange powder, mp 228–230 °C, 79% yield; 4.1.9.1H NMR : δ 7.67–7.73 , 8.15 , 8.17–8.31 , 8.66 , 11.62 . Anal. Calcd for C16H11N5O4S: C, 52.03; H, 3.00; N, 18.96. Found: C, 52.21; H, 3.24; N, 18.74.Yellow powder, mp 222–224 °C, 81% yield; 4.1.10.Characterisation data were in agreement with those reported in the literature4.1.11.1H NMR : δ 2.34 , 7.38–7.45 , 7.67 , 7.69–7.73 , 7.77–7.80 , 8.13–8.16 , 8.31–8.33 , 8.72–8.73 , 11.36 . 13C NMR : δ 14.6 (CH3), 107.3 , 120.5 (Ar), 122.4 (Ar), 126.2 (2 × Ar), 128.9 (2 × Ar), 129.3 (Ar), 130.7 (Ar), 132.0 (Ar), 136.8 (Ar), 138.3 (Ar), 147.4 (C=N), 148.6 (Ar-NO2), 148.8 , 170.6 . Anal. Calcd for C17H14N4O2S: C, 60.34; H, 4.17; N, 16.56. Found: C, 60.12; H, 4.32; N, 16.79.Yellow powder, mp 261–263 °C, 86% yield; 4.1.12.1H NMR : δ 1.08–1.12 , 2.86–2.91 , 7.38–7.46 , 7.69 , 7.70–7.74 , 7.79–7.81 , 8.15–8.17 , 8.32–8.34 , 8.74 , 11.55 . Anal. Calcd for C18H16N4O2S: C, 61.35; H, 4.58; N, 15.90. Found: C, 61.20; H, 4.32; N, 16.13.Yellow powder, mp 215–217 °C, 85% yield; 4.1.13.1H NMR : δ 2.34 , 7.61–7.65 , 7.70–7.78 , 7.91–7.93 , 8.15–8.17 , 8.30–8.31 , 8.71 , 11.51 . Anal. Calcd for C17H13N5O4S: C, 53.26; H, 3.42; N, 18.27. Found: C, 53.11; H, 3.25; N, 18.54.Orange powder, mp 195–200 °C, 87% yield; 4.1.14.1H NMR : δ 2.40 , 7.14 , 7.61–7.63 , 8.15–8.26 , 8.63 , 8.70 , 8.98 . Anal. Calcd for C17H13N5O4S: C, 53.26; H, 3.42; N, 18.27. Found: C, 53.55; H, 3.70; N, 18.09.Orange powder, mp 214–216 °C, 85% yield; 4.1.15.1H NMR : δ 2.41 , 7.71–7.75 , 8.02–8.05 , 8.16–8.18 , 8.28–8.30 , 8.32–8.35 , 8.74 , 11.73 . Anal. Calcd for C17H13N5O4S: C, 53.26; H, 3.42; N,18.27. Found: C, 53.01; H, 3.18; N, 18.43.Orange powder, mp 258–260 °C, 80% yield; 4.1.16.Characterisation data were in agreement with those reported in the literature4.1.17.Characterisation data were in agreement with those reported in the literature4.1.18.Characterisation data were in agreement with those reported in the literature4.1.19.Characterisation data were in agreement with those reported in the literature4.1.20.Characterisation data were in agreement with those reported in the literature4.1.21.Characterisation data were in agreement with those reported in the literature4.1.22.1H NMR : δ 2.41 , 7.71–7.75 , 7.77 , 8.16–8.18 , 8.33–8.35 , 8.59–8.60 , 8.63–8.64 , 8.74–8.75 , 9.22–9.23 , 11.82 . Anal. Calcd for C15H12N6O2S: C, 52.93; H, 3.55; N, 24.69. Found: C, 53.20; H, 3.74; N, 24.84.Yellow powder, mp 243–245 °C, 85% yield; 4.1.23.1H NMR : δ 7.20–7.25 , 7.45–7.47 , 7.62 , 7.70–7.74 , 7.79–7.80 , 8.15–8.17 , 8.23–8.25 , 8.29–8.33 , 8.70 , 11.55 , 11.98 . 13C NMR : δ 106.0 , 112.0 , 112.4 , 120.4 (Ar), 121.0 , 122.1 (Ar), 122.5 , 123.1 , 124.5 , 130.3 , 130.7 (Ar), 132.1 (Ar), 136.6 (Ar), 137.6 , 140.6 (C=N), 148.0 (Ar-NO2), 148.7 , 168.7 . Anal. Calcd for C18H13N5O2S: C, 59.49; H, 3.61; N, 19.27. Found: C, 59.65; H, 3.39; N, 19.49.Yellow powder, mp 253–255 °C, 83% yield; 4.1.24.1H NMR : δ 2.36 , 7.25–7.28 , 7.43–7.47 , 7.63–7.90 , 8.12–8.21 , 8.31–8.50 , 8.78 , 11.08 , 11.48 . Anal. Calcd for C19H15N5O2S: C, 60.46; H, 4.01; N, 18.56. Found: C, 60.70; H, 3.83; N, 18.37.Green powder, mp 209–211 °C, 87% yield; 4.1.25.1H NMR : δ 6.08 , 6.96–6.98 , 7.11–7.13 , 7.23 , 7.63 , 7.68–7.72 , 7.96 , 8.13–8.15 , 8.28–8.30 , 8.66 , 12.17 . 13C NMR : δ 101.9 , 105.1 , 106.7 , 109.0 , 120.4 (Ar), 122.4 (Ar), 122.7 , 129.2 , 130.7 (Ar), 132.0 (Ar), 136.7 (Ar), 142.0 (C=N), 148.4 (Ar-NO2), 148.6 , 148.7 , 149.0 , 169.1 . Anal. Calcd for C17H12N4O4S: C, 55.43; H, 3.28; N, 15.21. Found: C, 55.22; H, 3.04; N, 15.39.Yellow powder, mp 255–256 °C, 84% yield; 4.1.26.1H NMR : δ 2.37 , 7.25–7.29 , 7.31, 7.34–7.37 , 7.61–7.63  8.14–8.16 , 8.32–8.34 , 8.73 , 11.61 . 13C NMR : δ 14.3 (CH3), 106.6 , 107.7 , 111.7 , 120.5 (Ar), 122.0 , 122.5 (Ar), 123.8 , 125.9 , 128.6 , 130.7 (Ar), 132.0 (Ar), 136.7 (Ar), 139.1 (C=N), 148.6 (Ar-NO2), 148.8 , 154.0 , 155.0 , 169.9 . Anal. Calcd for C19H14N4O3S: C, 60.31; H, 3.73; N, 14.81. Found: C, 60.07; H, 3.97; N, 15.00.Pink powder, mp 228–230 °C, 81% yield; 4.1.27.1H NMR : δ 2.28 , 4.28 , 6.89–6.91 , 7.27–7.29 , 7.66 , 7.70–7.74 , 8.15–8.17 , 8.31–8.33 , 8.73 , 11.28 . 13C NMR : δ 14.5 (CH3), 64.5 (CH2), 64.7 (CH2), 107.2 , 114.9 , 117.4 , 119.5 , 120.4 (Ar), 122.4 (Ar), 130.7 (Ar), 131.6 , 132.0 (Ar), 136.7 (Ar), 143.6 (C=N), 144.7 , 147.1 , 148.5 (Ar-NO2), 148.7 , 170.7 . Anal. Calcd for C19H16N4O4S: C, 57.57; H, 4.07; N, 14.13. Found: C, 57.80; H, 3.84; N, 58.02.Orange powder, mp 242–244 °C, 70% yield; 4.1.28.1H NMR : δ 7.59–7.63 , 7.67–7.75 , 7.87–7.89 , 7.99–8.03 , 8.16–8.18 , 8.33–8.35 , 8.70–8.71 , 8.77–8.79 , 12.52 . Anal. Calcd for C20H14N4O2S: C, 64.16; H, 3.77; N, 14.96. Found: C, 63.92; H, 3.90; N, 15.12.Orange powder, mp 212–214 °C, 72% yield; 4.1.29.1H NMR : δ 2.33 , 7.54–7.59 , 7.91–8.00 , 8.26–8.41 , 8.56–8.58 , 8.71–8.75 , 12.14 . Anal. Calcd for C21H16N4O2S: C, 64.93; H, 4.15; N, 14.42. Found: C, 65.10; H, 3.89; N, 14.21.White powder, mp 226–228 °C, 74% yield; 4.1.30.1H NMR : δ 2.36 , 7.53–7.55 , 7.70–7.72 , 7.90–7.94 , 7.96–7.98 , 8.03–8.05 , 8.07–8.09 , 8.22–8.24 , 8.41–8.43 , 8.74–8.75 , 11.54 . Anal. Calcd for C21H16N4O2S: C, 64.93; H, 4.15; N, 14.42. Found: C, 64.74; H, 4.36; N, 14.65.Yellow powder, mp 152–154 °C, 77% yield; 4.1.31.1H NMR : δ 2.44 , 2.66 , 7.10 , 7.64–7.66 , 7.90–7.92 , 8.02–8.04 , 8.15–8.17 , 8.21–8.23 , 8.65 , 10.15 . 13C NMR : δ 14.3 (CH3), 27.2 (CH3), 107.7 , 120.5 (Ar), 122.5 (Ar), 126.2 (2 × Ar), 128.8 (2 × Ar), 130.7 (Ar), 132.0 (Ar), 136.7 (Ar), 136.9 (Ar), 142.4 (C=N), 146.0 (Ar-NO2), 148.8 , 170.3 , 197.9 (C=O). Anal. Calcd for C19H16N4O3S: C, 59.99; H, 4.24; N, 14.73. Found: C, 60.15; H, 4.03; N, 15.00.Yellow powder, mp 205–207 °C, 79% yield; 4.1.32.1H NMR : δ 7.05 , 7.39–7.43 , 7.62–7.67 , 8.14–8.25 , 8.58 , 10.15 . 13C NMR : δ 105.7 , 120.7 (Ar), 122.6 (Ar), 127.4 (2 × Ar), 128.4 (2 × Ar), 128.6 (2 × Ar), 129.7 (Ar), 129.8 (Ar), 130.0 (2 × Ar), 130.2 (Ar), 131.5 (Ar), 131.6 (Ar), 135.3 (C=N), 136.6 (Ar), 147.4 (Ar-NO2), 148.7 , 168.8 . Anal. Calcd for C22H16N4O2S: C, 65.98; H, 4.03; N, 13.99. Found: C, 66.14; H, 4.21; N, 14.17.Yellow powder, mp 166–168 °C, 83% yield; 4.1.33.1H NMR : δ 2.26 , 6.60 , 6.86–6.87 , 7.66–7.79 , 8.15–8.17 , 8.31–8.33 , 8.72 , 11.35 . 13C NMR : δ 14.2 (CH3), 107.3 , 110.6 , 112.3 , 120.4 (Ar), 122.4 (Ar), 130.7 (Ar), 132.0 (Ar), 136.7 (Ar), 139.7 , 144.6 (C=N), 148.6 (Ar-NO2), 148.8 , 152.2 , 170.2 . Anal. Calcd for C20H14N4O4S: C, 59.11; H, 3.47; N, 13.79. Found: C, 58.94; H, 3.24; N, 13.98.Red powder, mp 183–185 °C, 75% yield; 4.1.34.1H NMR : δ 1.24 , 7.25 , 7.37–7.48 , 7.61–7.69 , 8.01–8.03 , 11.40 . Anal. Calcd for C25H18N4O2S: C, 68.48; H, 4.14; N, 12.78. Found: C, 68.30; H, 3.97; N, 12.54.Pink powder, mp 255–257 °C, 72% yield; 4.1.35.1H NMR : δ 2.49 , 4.24 , 4.34 , 4.50 , 4.72 , 6.99 , 7.79 , 8.22–8.32 , 8.58 , 12.35 . Anal. Calcd for C21H18FeN4O2S: C, 56.51; H, 4.07; N, 12.55. Found: C, 56.73; H, 4.29; N, 12.80.Orange powder, mp 156–158 °C, 80% yield; 4.1.36.1H NMR : δ 2.37 , 7.71–7.75 , 7.86 , 7.87–7.88 , 7.89–7.90 , 8.16–8.18 , 8.33–8.35 , 8.75 , 11.45 . Anal. Calcd for C28H22N8O4S2: C, 56.18; H, 3.70; N, 18.72. Found: C, 56.01; H, 3.96; N, 18.55.Orange powder, mp 283–285 °C, 81% yield; 4.1.37.1H NMR : δ 5.15 , 6.50 , 6.99–7.12 , 7.36 , 7.58–7.59 , 8.21 , 12.06 . Anal. Calcd for C14H12N4S2: C, 55.97; H, 4.03; N, 18.65. Found: C, 56.15; H, 3.84; N, 18.37.Red powder, mp 165–167 °C, 71% yield; 4.2.4.2.1.50 values for the inhibition of MAO has been reportedλex = 310; λem = 400 nm)50 values were estimated. All enzyme reactions were carried out in triplicate and IC50 values are given as the mean ± standard deviation (SD).The procedure for the measurement of IC4.2.2.50 values for the inhibition of MAO has been reported50) were incubated for 15 min at 37 °C to a final volume of 0.8 ml in dialysis buffer, potassium phosphate buffer  containing 5% sucrose. Stock solutions of the test compounds were prepared in DMSO and added to the incubations to yield 4% DMSO. The samples were dialyzed for 24 h at 4 °C in 80 ml of dialysis buffer using Slide-A-Lyzer® dialysis cassettes (Thermo Scientific) with a molecular weight cut-off of 10,000 and a sample volume capacity of 0.5–3 ml. The dialysis buffer was replaced at 3 and 7 h after the start of dialysis. Following dialysis, the dialysis samples were diluted twofold with the addition of kynuramine to yield a kynuramine concentration and inhibitor concentration of 50 µM and 2 × IC50, respectively. The reactions (500 µL) were incubated for 20 min at 37 °C and terminated with NaOH  and water (1000 µL). The fluorescence of 4-hydroxyquinoline in these samples was measured as described for the IC50 determination above. As controls, MAO-B was similarly pre-incubated and dialyzed in the absence of inhibitor (negative control) as well as in the presence of the irreversible inhibitor, (R)-(−)-deprenyl The procedure for the measurement of IC4.2.3.50, ½ × IC50, ¾ × IC50, 1 × IC50 and 1¼ × IC50. For each line, kynuramine was used at concentrations of 15–250 μM. The Ki value was estimated from a plot of the slopes of the Lineweaver–Burk plots versus inhibitor concentration .To construct Lineweaver–Burk plots, the enzyme reactions were carried out to a volume of 500 µL and the concentration of MAO-B was 0.015 mg protein/mL. All enzyme reactions and activity measurements were carried out as described above for the dialysis experiments. The first plot was constructed in the absence of inhibitor, while the remaining five plots were constructed in the presence of the following concentrations: ¼ × IC4.3.4.3.1.50, which is the effective concentration at which the absorbance was 0.5, was calculated for each compound and trolox, as a reference drug.The total antioxidant activity of the compounds was evaluated by phosphomolybdenum method according to Zengin et al.4.3.2.The radical scavenging effect of the compounds using the the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was estimated according to Zengin et al.+ was produced directly by reacting a 7 mM ABTS solution with 2.45 mM potassium persulphate and allowing the mixture to incubate for 12–16 h in the dark at room temperature. Prior to initiating the assay, the ABTS solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. The sample solution (1 ml) was added to the ABTS+ solution (2 ml) and mixed, and the sample absorbance was recorded at 734 nm after 30 min incubation at room temperature. The corresponding IC50 value, which is the effective concentration at which 50% of DPPH/ABTS radicals are scavenged, was calculated for each compound and trolox, as a reference drug.The scavenging activity of the compounds on the ABTS radical cation  was measured according to the method of Zengin et al.4.3.3.2 , neocuproine , and NH4Ac buffer . Similarly, a blank was prepared by adding the sample solution (0.5 ml) to a premixed reaction mixture (3 ml) without CuCl2. The absorbances of the sample and blank were subsequently recorded at 450 nm after 30 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample.The cupric ion reducing activity (CUPRAC) was determined according to the method of Zengin et al.s-triazine (10 mM) in 40 mM HCl, and ferric chloride (20 mM) in a ratio of 10/1/1 (v/v/v). The absorbance of the sample was subsequently recorded at 593 nm after 30 min incubation at room temperature. Both results were expressed as EC50 values, using trolox as a reference drug.The FRAP (ferric reducing antioxidant power) assay was carried out as described by Zengin et al.4.3.4.2 solution . The reaction was initiated by the addition of 5 mM ferrozine (0.2 ml). Similarly, a blank was prepared by adding the sample solution (2 ml) to FeCl2 solution  and water (0.2 ml) without ferrozine. Then, the sample and blank absorbance were recorded at 562 nm after incubation for 10 min at room temperature. The absorbance of the blank was subtracted from that of the sample. EDTA was used as a positive control and this property was expressed as IC50 value for each compound.Metal chelating activity on ferrous ions was evaluated by the method previously described4.4.3, 4, 13, 37 were built by means the Maestro GUI and their oral absorption and blood–brain barrier permeation were theoretically predicted by the QikPropThe Schrödinger suite 2018-15.5.1.1–37 occurring at the X and Y substituents as shown in Y moiety was effective in order to obtain derivatives that selectively inhibit hMAO-B in the nanomolar range. Among these compounds, the most active towards this isoform were the derivatives 4 (IC50 hMAO-B = 0.0018 μM) and 5 (IC50 hMAO-B = 0.0025 μM), both containing methyl as X group and respectively, thiophen-2-yl and thiophen-3-yl as Y substituent. Comparing the inhibitory activities of compound 4 and compound 3, which contains H as X group, it can be noted that there was only a modest decrease in MAO-B inhibition activity for 3, while a reduction in inhibition activity was more noticeable for hMAO-A (IC50 hMAO-A = 57.2 μM). This increased the SI by approximately 10-fold (from 922 for compound 4 to 8412 for compound 3). A similar result was obtained for compound 6 where substitution on thiophenyl ring led to a reduction in inhibition activity towards hMAO-A compared to derivative 4. Even though hMAO-B inhibition was also slightly reduced, the SI increased significantly (compare 6 vs. 4). Among derivatives possessing furan as Y substituent (1 and 2), only compound 1 exhibited inhibition activity in the nanomolar range for hMAO-B (IC50 hMAO-B = 0.095 μM). The lower activity of 2 suggests that isosteric replacement of the oxygen atom for a sulphur is unfavourable for MAO-B inhibition (compare 2 vs. 4). Within the set of compounds containing the phenyl ring as Y moiety, we observed the best inhibitory activity for derivative 11 (IC50 hMAO-B = 0.0071 μM), which contains methyl as X substituent. The absence of the methyl group  or its elongation to X = ethyl (compound 12) were detrimental for MAO-B inhibition activity. Nitro-substitution on the phenyl ring, as with compounds 8–10 and 13–15, was advantageous when done in the ortho-position , a modification that also improved the selectivity compared with the unsubstituted derivative 11 . On the other hand, substitution with an acetyl moiety in the para-position of phenyl ring reduced activity and selectivity as observed for compound 31 . Compounds substituted with pyridine as Y moiety (16–21) possess nanomolar activity towards hMAO-B (0.014 μM < IC50 hMAO-B < 0.212 μM), while poor potencies were recorded for hMAO-A inhibition, with IC50 in the micromolar range (9.78 μM < IC50 hMAO-A < 156 μM). Most of the compounds possessing a hetero-bicyclic system as Y moiety (compounds 23–27) showed the loss of activity and selectivity, except for compound 33 that contains a coumarin moiety. Coumarin derivatives are well known to inhibit the hMAO enzymes as previously reportedY group was effective only when substituted in the α-position (compare 28 and 29 vs. 30), while the presence of very bulky groups such as phenanthrene (compound 34) reduced activity towards both isoforms . Finally, we evaluated the effect of dimerisation with compound 36 and the reduction of nitro group to obtain amine functionality with compound 37. The IC50 values showed that the dimerisation negatively affected inhibition activity, and this is likely related to the steric hindrance in the MAO active site. Compound 37, on the other hand, showed loss of MAO-B inhibitory activity, which highlights the importance of the nitro group on the meta position for this scaffold (compare 37 vs. 3). Conversely, the MAO-A inhibitory activity was not affected by nitro reduction.The synthesised compounds reported here share a similar structure, with the principal differences among compounds 4 and 37 were selected as test compounds. The reversibility of MAO-B inhibition was evaluated by incubating MAO-B in the presence of the test compounds (at a concentration of 4 × IC50) for 15 min, and subsequently dialyzing the samples for 24 h. The samples were diluted twofold and the residual MAO-B activity was measured. For reversible inhibition, dialysis is expected to reverse inhibition by the test inhibitors and restore enzyme activity to the level of the negative control. The negative control consisted of similar incubation and dialysis in the absence of inhibitor and represented 100% enzyme activity, while as positive control, these studies were carried out in the presence of the irreversible MAO-B inhibitor, (R)-(−)-deprenyl. A final experiment consisted of incubations containing MAO-B and tested inhibitors which were not dialyzed, but maintained for 24 h.Reversibility of MAO inhibition is an important factor to consider when evaluating the inhibition properties of potential inhibitors. Irreversible MAO inhibitors, particularly of the MAO-A isoform, are associated with dangerous adverse effects such as the cheese reaction, which occurs when irreversible MAO-A inhibitors are taken with tyramine-rich food4 and 37, with the residual activity at 61 and 97%, respectively. Compound 37 was thus a fully reversible MAO-B inhibitor, while 4 was partially reversible, which was likely due to tight-binding of this high potency inhibitor to the MAO-B active site. For (R)-(−)-deprenyl, dialysis did not restore enzyme activity with the residual MAO-B activity at 3% of the negative control value. In undialysed mixtures of 4 and 37, inhibition persisted with the residual activity at 33 and 20%, respectively.The results are given in 4 and 37, a set of Lineweaver–Burk plots was constructed for each inhibitor at the following inhibitor concentrations: ¼ × IC50, ½ × IC50, ¾ × IC50, 1 × IC50, and 1¼ × IC50. The substrate, kynuramine, was used at 15–250 µM for each line. The results are given in Ki) was estimated (Ki = −x-axis intercept). For compounds 4 and 37, Ki values of 0.0026 and 1.84 µM are estimated.To provide further support for the reversibility of MAO-B inhibition by compounds 5.2.in vitro by five experimental approaches, using trolox as the reference compound. As it can be seen from the results in It is a well-known that MAO-mediated oxidative metabolism of monoamines leads to the production of hydrogen peroxide as a by-product. Subsequently, this chemical species may be converted to free radicals through the Fenton reaction and thus contributes to the oxidative stress. Uncrontrolled increases in the concentrations of these radicals initiates oxidative damage to several cellular components. Thus, the prevention of ROS generation along with MAO inhibition is an important strategy to prevent or limit neurotoxicity in NDDs. The capability of the most promising MAO inhibitors of this study to act as antioxidant agents has been evaluated The proposed mechanism for the antioxidant activity of this scaffold of compounds is depicted in 5.3.3, 4, 13, and 37, as suggested by the experimental data, was investigated by molecular modelling simulations. In agreement with experimental data, docking results proposed, in general, preferred hMAO-B recognition compared to the A isoform. Indeed, all evaluated compounds presented better theoretical affinity for hMAO-B and thus more productive interactions with this isoform . In fact, for 37 the opposite orientation occurred due to the loss of this productive interaction although contacts with the tyrosine residues and with the internal tryptophan (Trp86) were maintained. In hBuChE, where Ala277 replaces the hAChE Trp286, the compounds better recognised the internal Trp82 (corresponding to Trp86 in hAChE).The top poses obtained for oth ChEs  and 8 di3, 4, and 37 is comparable to known CNS drugs  may be possible which suggests that 4-(3-nitrophenyl)thiazol-2-ylhydrazones are novel multi-target compounds and potential leads for the design of future therapies for NDDs.4-(3-Nitrophenyl)thiazol-2-ylhydrazones were demonstrated to be a very interesting scaffold which can be used to design high potency hMAO-B inhibitors, with IC


To explore and compare the content of rehabilitation practices in, respectively, a Danish and a Norwegian region, focusing on how the citizens' rehabilitation needs are met during rehabilitation in the municipalities. Six Danish and five Norwegian cases were followed 12 months after the onset of stroke. Field work and focus group interviews with multidisciplinary teams in the municipalities were conducted. The conceptual frame of the International Classification of Functioning was used to outline general patterns and local variation in the rehabilitation services. Each of the settings faces different challenges and opportunities in the provision of everyday life-supportive rehabilitation services. Rehabilitation after stroke in both settings basically follows the same guidelines, but the organization of rehabilitation programmes is more specialized in Denmark than in Norway. Team organization, multidisciplinarity, and collaboration to assess and target the patients' needs characterized the Danish rehabilitation services. Decentralized coordination and monodisciplinary contributions with scarce or unsystematic collaboration were common in the Norwegian cases. Seamless holistic rehabilitation was challenged in both countries, but more notably in Norway. The municipal services emphasized physical functioning, which could conflict with the patients' needs. Cognitive disturbances to and aspects of activity or participation were systematically addressed by the interdisciplinary teams in Denmark, while practitioners in Norway found that these disturbances were scarcely addressed. The study showed major differences in municipal stroke rehabilitation services in the Northern Norway and Central Denmark Regions—in their ability to conduct everyday life—supportive rehabilitation services. Despite the fact that biopsychosocial conceptions of disease and illness, as recommended in the ICF, have been generally accepted, they seemed scarcely implemented in the political and health managerial arenas, especially in Norway. These national diversities can partly be explained by the size of the municipalities and the available health profiles in delivering patient and family-centred rehabilitation services. The organization of rehabilitation interventions differs within and between European countries . The estHealth care in both Denmark, which has 5.7 million inhabitants, and Norway with 5.2 million comprises two complementary public sectors: the regional specialised hospital services and the primary health care system, each with separate obligations and regulated by distinctive laws and regulations. In Denmark, areas of responsibility and division of labour between regions and councils were reorganised following municipal reform in 2007 and changes in the health legislation in 2006 , which oOnce a patient is discharged from hospital, the responsibility for rehabilitation falls to the municipality. The two countries' respective primary sectors consist of 98 Danish and 428 Norwegian municipalities. Each of these provides primary health care, long-term care services, home-based care, and social care provision, accommodated to the needs of citizens and within the bounds of available economic and professional resources. The legal basis for stroke rehabilitation is regulated by a range of laws. Patients' needs generally require the participation of professionals across several fields, such as health, social care, education, and employment. Furthermore, collaboration and preclarification of the patients' rights are defined by general rules. Thus, the legal framework and sectoral legislation reflect the complexity and diversity of stroke rehabilitation , 10.Denmark provides centralized neurorehabilitation with a large patient volume to achieve specialization. The Danish Health Authority provides general guidelines for the organization and provision of services included in rehabilitation programs following a stroke. The Health Technology Assessment (MTV) report  and the The Norwegian Health Authority provides national guidelines regarding treatment and rehabilitation following a stroke . Unlike The level of access to a contiguous coordinated intervention conducted by a specialized interdisciplinary team that works in collaboration with patients and relatives can have a major influence on rehabilitation outcome , 14. MulThe aim is to describe the citizens' rehabilitation needs present at discharge after stroke and how professionals in the municipalities experience these are met, focusing on regaining a meaningful everyday life. The study is a substudy of a multicenter study “The NORDA-study”, which describes and compares stroke pathways in Norway and Denmark.Qualitative content analysis was suitable to explore and describe the multifaceted phenomena of ongoing relational rehabilitation practices . As therPatients: Eleven individuals, aged 25-65 and suffering from a confirmed diagnosis of stroke with moderate disability, were followed from the time of discharge from hospital until about one year after onset. The inclusion criteria were that they had lived an active, independent life before the injury and that they were discharged to their own home in one of two Danish municipalities. In Norway, we included patients consecutively, comprising their home municipalities, five in all. Professionals included were members of the municipal health services who were involved in service provision to any of the included patients. Exclusion criteria were cognitive and communication changes that made it impossible to share the patients' experiences.Focused field studies of each informant were used to examine interactions between patients and professionals in the municipal health services. This included conversations with professionals about their reflections, with patients about personal aims regarding and experiences of the rehabilitation process , 27. FieOngoing rehabilitation practice from the rehabilitation professionals' perspective was explored by semi-structured focus group interviews, which allowed for exchange and the elaboration of experiences and ideas among colleagues –30. The The study was carried out according to the ethical guidelines for nursing research in the Nordic countries . PermissDenmark: The two Danish municipalities have 61,000 and 48,000 citizens, respectively. In each municipality, following a stroke, all citizens younger than 65 are offered rehabilitation at a health centre. The rehabilitation is organised across employment, social and health administrations as well as professional organisations in an interdisciplinary “Brainteam” affiliated to the health centre.Norway: Patients were included from five different municipalities. Of these, four have fewer than 10,000 and the remaining municipality has 72,000 citizens. In one of the municipalities, the rehabilitation professionals involved were team organized. In the remaining municipalities, professionals worked separately as privately-practising or/and on individual locations.See I started working with him (case 10), it was in March – where he had a referral from a doctor in [the preceding] October, but – the Helfo system [the authority who grants the treatment] rejected speech therapy  ... Only 25 hours, then a new referral from the general practitioner after a long time (case 10)Seamless cross-sectoral rehabilitation services are hallmarks, in both Denmark and Norway. Following discharge from hospital, the municipal health services are required to facilitate coordination between different services. However, we found wide variation. In Denmark, there was a lack of continuity from discharge to municipal rehabilitation in cases 1, 3, 5, and 6. Similar discontinuity was identified in the Norwegian cases 8, 10, and 11 . The munAccording to national recommendations in both countries, professionals in a multidisciplinary neurorehabilitation team are required to have specialist expertise in neurorehabilitation. Team members include physicians, nurses, physiotherapists (PT), occupational therapists (OT), social workers, psychologists, speech therapists, social workers, job consultants, and course coordinators. We found a wide variation in the composition of rehabilitation teams .We do not have such a thing . I'm working on rehabilitation. Everybody is working on everything” (case 7). A privately-practicing PT questioned his own contribution: “… it's not that I can't perform neurological physiotherapy, but I do not really see the value – as the stimulation is rare. It is only provided two or three times a week, and this intensity is not sufficient” (case 10). In the same case, the services became monodisciplinary due to lack of coordination. Both speech therapists and physiotherapists were practicing alone, and they tried independently to support the patient's experienced needs in relation to work. However, their rehabilitation attempts seemed to fail. In describing the patient's return to work, his employer said “… He has been thrown into normal work … supported by the other mechanics … We haven't got any instruction or supervision from professionals. We have only received a telephone call from his hospital case manager” (case10).Sensorimotor changes in the hand and or arm were described in all cases, except for case 2 , but theTo group and abstract the various aspects of the patient's life the professionals described to address during the rehabilitation process, the conceptual framework of the ICF was used to outline rehabilitation work related to body function, activities, and participation as well as environmental and personal factors.The body's functions and anatomy refer to the physiological functions, including mental functions, and the body's anatomical structures. There appeared to be an emphasis on body functions, in that the PTs were involved in first-line work in all of the included cases and, apart from one single case (case 2), it was also the most enduring intervention. The presence of motor function as the backbone of rehabilitation was apparent, despite the absence of disturbances in this area of function. In case 4, no sensorimotor changes were described, but the patient received 38 weeks of continuous physiotherapy from discharge onwards. The initiation and duration might be explained in light of a PT assumption that improved fitness and strength improve the patient's cognitive development. The fitness training was self-organised and could therefore be understood as training in structuring: “[The training] … stimulates him on his circuit and he is stimulated with physical activity and [the aim is to do] something to support the cognitive development” (case 2), well-being “… to exercise and doing some activity that gives her some energy” (case 5), and participation “… if we could keep him continuing the fitness training … he will be in a team 2-3 times a week” (case 6), concurrently with the prevention of a new stroke.Physiotherapy was conducted as individual self-training and as group exercises for patients with similar challenges, and the PTs and OTs emphasized the importance of time for the social aspect to facilitate recognition and participation .A physical focus appears in both countries, despite the prevalence of severe cognitive disturbances. Physiotherapy was offered as first-line rehabilitation, and it was also the most enduring service.The ICF defines activity as the execution of a task or action by an individual …”. However, interventions addressing cognitive changes were random. A Norwegian speech therapist related: “We have worked completely systematically, where we started with clothes, colors, days, months, family members, work colleagues, and this is what we are still working on. He has a much reduced working memory and major word-finding difficulties” (case 10). The patient is assigned 54 hours of speech therapy, but the therapist mainly focused on his severely reduced memory function without involving other health professional groups. The employer experienced a gap between the patient's needs and the rehabilitation services: “It is very limited what he can do … with us. We try to keep him a little physically active for as long as possible. In one way, the language is not a problem for him, but it may be more to remember and such things which are more critical” (case 10). None of the professionals mentioned the possible cognitive disturbances described at admission. The impression of inconsistent assessment and lack of provision targeted to cognitive changes were supported by the experiences of a therapist in the reablement team: “We received specific feedback from the rehabilitation service  that they can't conduct activity training … especially the occupational therapists are supposed to work with cognition. But they don't” (case 8).Professionals articulated the interaction with equals as a subsidiary goal in group-based training, to facilitate the patient's cognition and realization their condition. Therefore, both PTs and OTs were present at the training sessions to promote social interaction and to support the patients in cases 2 and 3. An OT explained that “The rehabilitation services addressing activity were integrated in daily life, work, or leisure activities in the majority of both Danish and Norwegian cases. In both settings, the sensorimotor challenges appeared to be afforded a higher priority than cognitive challenges. Limited coordination between professionals resulted in fragmented and nonevidence-based rehabilitation services. This was due to monodisciplinary interventions and contributions from unsupervised employer and colleagues, which characterised the Norwegian cases. Interdisciplinary team organisation was prevalent in the Danish cases and the individualized interventions related to cognition were mainly compensatory. Group interventions and relations with equals were arranged to improve cognition and realize the changes.The ICF category of participation refers to a person's involvement in everyday life situations and represents the societal perspective of functioning. Within this framework there are different roles related to the family, interpersonal interactions and relationships, employment, social life, and recreation and leisure activities. In the Danish cases, spouses and cohabitants were routinely asked to participate in planning and follow-up meetings. In addition, they might be offered training evenings about changes following a brain injury  and invited to participate in a group of relatives to exchange experiences (cases 2 and 4). Relatives' participation in rehabilitation is considered to be valuable: “We made some clear agreements about further training, and he was so fortunate that his wife wanted to join” (case 2). Addressing social interaction with a broader group was considered but appeared to be less prominent: “… talked a little (with him) about the social … we thought about the circle of interaction. He had a family, but he seemed to be a little bit lonely” (case 8). In case 4, the children's wellbeing was addressed; the pedagogue supported the patient to inform the children's school classes about her condition. To facilitate the patients' interaction during physical training, they were grouped in relation to the similarity of challenges and ability to benefit from each other: “We had a really good group of gentlemen who fit really well together. Yes, they could really benefit from each other” (case 2). “We talked a lot about what other people think - … talked to her about informing people, in relation to her experiences of people rejecting her and that they are not reaching out” (case 4). However, the Norwegian professionals assumed there were unmet needs: “And in this case, relatives have got a new cohabitant/man in the family. It's a whole new person. I doubt that they take care of anything” (case 10). In case 9, soon after discharge, the patient was offered reablement, but declined. The professionals concluded that “She still has some trouble with anxiety. Particularly related to leaving the house … she needs as much everyday coping on the mental side” (case 9).Reintegration into the social network was challenged by uncertainty and stigmatization. This was addressed in case 4, where the pedagogue described the following:Family integration was present in the Danish cases; the partners were routinely invited to planning and evaluation meetings and some of them also to training sessions. We were unable to identify any similar strategies in the Norwegian cases, but the professionals discussed the possibilities for broader social reintegration with some patients. Group training with peers, exchange of experience groups for patients and relatives, and individual support were described in the Danish settings, in order to facilitate social interaction. This aspect was not described by the Norwegian professionals. “The prerequisite for the goals is the motivation behind training” (case 10). Correspondingly, they acknowledge and praise when the patients are active, persistent, and responsible: “She has a positive energy and spreads it … [she has] been able to transfer the guidelines we have given her home … [and integrate them in her activities] … she really understands how to put training into her everyday life” (case 1). Another highlighted a patient's approach, this time in combination with will and effort: “[He was] … very optimistic … he was also very clear in the conversation with me: ‘my goal, I will return to work as a professional driver … so I exercise … You have some guts in yourself'” (case 7).According to the ICF, the personal factors of pre- and poststroke personality consist of an individual's traits that are essential to the person's behavior and ability to cope . SurprisHome visits to adapt the environment to the patients' needs were common in Denmark, but confined to one case in Norway, despite similar challenges in both groups. In all Danish cases and the Norwegian case 8, a PT and an OT visited the patients' home in order to assess needs and suggested environmental adaptation of furniture, fixtures and fittings as well as the need for aids. The need for assistive aids was also assessed by an OT in Norwegian cases 7, 8, and the 9, but there was no follow-up or involvement of rehabilitation provision. Personal aspects were acknowledged by professionals in both countries, but systematic strategies to identify and improve these were absent.This qualitative study explores and compares the rehabilitation efforts after stroke in two Danish and five Norwegian municipalities. Homogeneity in the two compared groups is not confirmed as the included participants suffered from a broad variety of changed functions. Both aspects impede the need for great caution in relation to generalize our findings to the entire national populations.Rehabilitation outcome depends on access to a contiguous, coordinated intervention conducted by a specialized interdisciplinary team that collaborates with patients and relatives , 14. SurIn the early weeks after a stroke, patients have varied and complex needs which require expertise from different groups of health care professionals. Both Danish municipalities have a coordinator and a specialised brain injury rehabilitation team to address various aspects of functional changes. In Norway, one patient was enrolled in reablement, while the other cases were provided generalist services in stroke rehabilitation. This entails varying competencies and resources, comparable to national findings in 2012 . The varNational diversity was found in present health profiles, time resources and integration of rehabilitation services in the patients' everyday lives and home environment. In the Danish municipalities, the integration of rehabilitation provision in valued daily life activities was dominant, while work life movements, counting, and writing in authentic environments were more obvious in the Norwegian cases. A key emphasis on body function appears in the included cases in both settings. This indicates a discrepancy in relation to the ICF which relies on a biopsychosocial (BPS) concept of disease and illness, as formulated by Engel . The maiIn relation to the ICF framework of activity and participation, the patients' personal experiences and preferences related to their changed life condition should be taken into consideration . UnderstRehabilitation after stroke basically follows the same guidelines in both settings, but the organization of rehabilitation courses is more team organized in the Danish than in the Norwegian settings. Volume and centralization seem to be pivotal in conducting rehabilitation that addresses the ICF aspects of human life influenced by a stroke. Team organization, multidisciplinarity, and collaboration to assess and target the patients' needs characterised the rehabilitation services in the two Danish municipalities. Decentralized coordination and monodisciplinary contributions with scarce or unsystematic collaboration were common in the five Norwegian municipalities. Seamless cross-sectoral services are key contributors to holistic rehabilitation. This was challenged in both countries, but most notably in Norway due to unsystematic coordination and waiting lists for privately-practising therapists. The municipal provision emphasized physical functioning in duration and intensity, which might conflict with the patients' needs, as described at discharge from hospital. Cognitive disturbances and aspects of activity or participation were systematically addressed by the interdisciplinary team in the Danish cases, while practitioners experienced a lack of multidisciplinary collaboration in the Norwegian municipalities, where these disturbances seemed to be scarcely addressed. The patterns of diversity between rehabilitation efforts in Danish and Norwegian municipalities may partly be explained by the variation in population density, geographical extent, available health profiles, time resources, and utilisation of the usual environment.Greater consideration should be given to the aspects of activity and participation in the context of community settings. This is in order to determine how patients with stroke and those in their closest networks can be supported in becoming less dependent on public services and, as far as possible, able to understand and manage their own everyday lives. This requires an accordance between the ideology of the ICF and clinical rehabilitation practice in the political and the managerial arenas of the health care system.


We postulated that the computationally derived and optimized complementarity-determining region 3 (CDR3) of Nb is sufficient for its interaction with receptor. Sequence-related Nb-families preferring the agonist-bound active conformation of β2-AR were analysed using the informational spectrum method (ISM) and β2-AR:NDP complexes studied using protein-peptide docking and molecular dynamics (MD) simulations in conjunction with metadynamics calculations of free energy binding. The selected NDP of Nb71, designated P3, was 17 amino acids long and included CDR3. Metadynamics calculations yielded a binding free energy for the β2-AR:P3 complex of ΔG = (−7.23 ± 0.04) kcal/mol, or a Kd of (7.9 ± 0.5) μM, for T = 310 K. In vitro circular dichroism (CD) spectropolarimetry and microscale thermophoresis (MST) data provided additional evidence for P3 interaction with agonist-activated β2-AR, which displayed ~10-fold higher affinity for P3 than the unstimulated receptor (MST-derived EC50 of 3.57 µM vs. 58.22 µM), while its ability to inhibit the agonist-induced interaction of β2-AR with β-arrestin 2 was less evident. In summary, theoretical and experimental evidence indicated that P3 preferentially binds agonist-activated β2-AR.This study aimed to design and functionally characterize peptide mimetics of the nanobody (Nb) related to the β GPCRs comprise the largest family of cell-surface receptors, as well as the most intensively studied drug target family, with implications in almost every major disease category2. In particular, GPCR crystallography has experienced impressive progress in recent years. Since the structures of rhodopsin and β2-adrenergic receptor (β2-AR) were resolved in 20003 and 20075, respectively, over 200 structures of more than 50 GPCRs have been solved (reviewed in6); yet data about the conformational changes associated with their activation is still sparse. As the biological activity induced by the binding of a ligand to orthosteric or allosteric sites on a GPCR is mediated by the stabilization of specific receptor conformations7, the emerging theme in GPCR activation/signalling is the role of the structural conformation of the receptor in G-protein/effector protein selection. The approaches used to obtain detailed descriptions of GPCR activation dynamics include molecular simulations and the use of Nbs that bind conformational epitopes, which occur only in native proteins9. Nbs were instrumental in solving (i) the first structure of activated β2-AR in complex with the Nb designated Nb80 and slowly dissociating agonist BI16710710, (ii) agonist-occupied β2-AR:Gs heterotrimer complex10 and (iii) β2-AR in complex with its low-affinity agonist adrenaline and the nanobody Nb6B9, which exhibited improved affinity and slower dissociation11. The same approach also facilitated the crystallization of agonist-bound active-state structures of other GPCRs (for reviews see13). Additionally, β2-AR-specific Nbs transiently expressed as “intrabodies” in HEK-293 cells retain their conformational specificity and have been used as a tool to study GPCR signalling via G-protein and β-arrestin recruitment14. Nbs that stabilize the active conformation, such as Nb80, bind to the intracellular domain of a GPCR that is otherwise occupied by Gα subunit or β-arrestin15, primarily through the third complementarity-determining region (CDR3), whereas CDR1 should stabilize only this interaction16. Therefore, we hypothesized that approximately only 25% of the length of original Nbs that is computationally derived and optimized is sufficient for its interaction with the receptor. The main objective of our study was, therefore, the computational design and functional characterization of peptide mimetics of the Nb CDR3 related to β2-AR, i.e., Nb-derived peptides (NDPs). We used the following computational approaches to test our hypothesis: (i) the informational spectrum method (ISM), a virtual spectroscopy method for investigations of protein-protein interactions and the structure/function relationship of proteins18 to design NDPs and to arrange a sequence of amino acids of NDPs that are functionally related to Nbs from the camelid family that are related to β2-AR, with reference to the informational properties of Nbs; (ii) explicit membrane molecular dynamics (MD) of derived NDPs docked into the intracellular space of the β2-AR active conformation to compute the protein-peptide binding free energy; and (iii) a novel computational approach in molecular dynamics stimulation that was introduced in 2002, known as metadynamics20 and implemented in the NAMD program21 with a CHARMM27 force field23, to examine the whole molecular conformational space and calculate the free energy during MD simulation. One selected computationally characterized NDP was then experimentally tested (i) by assessing its ability to bind β2-AR using techniques for studying protein-protein interactions, i.e., circular dichroism (CD) spectroscopy to detect changes in the conformation of interacting proteins24 and microscale thermophoresis (MST), a powerful analytical technique for characterizing biomolecular interactions based on the movement of molecules in microscopic temperature gradients (reviewed in25); and (ii) by interfering with its function using the previously developed bioluminescence resonance energy transfer (BRET)-based β-arrestin 2 recruitment assay26. Based on our results, molecular simulation-based theoretical calculations of binding free energy values are highly consistent with the experimental data, particularly the MST-derived half-maximal effective concentration (EC50) of the derived P3 for the agonist-activated β2-AR.Nanobodies (Nbs) are recombinant, antigen-specific, single-domain, variable fragments of camelid heavy-chain-only antibodies with a broad range of diagnostic, therapeutic and research applications2-AR and information about the characteristics of the interaction between β2-AR and the Nbs, as well as to identify the key domain of Nb involved in receptor targeting ]. Nbs that showed a clear preference for agonist (BI-167107)-occupied β2-AR belonged to family B14. Here, we analysed the extensively characterized Nb80 from family B that binds agonist-activated β2-AR10 and Nb84 and Nb71 from Nb families C and MISC, respectively, the latter two of which display a preference for binding active β2-AR conformations14. Common biological characteristics of the group of proteins that share common information are represented by peaks in their consensus informational spectrum (CIS)18. A cross-spectral analysis of Nbs stabilizing an active β2-AR conformation showed that Nbs shared common information corresponding to the informational spectrum (IS) frequency F(0.216) , indicating their interaction  Fig. . An ISM b71 Fig. , revealeion Fig. . This fi2-AR that exhibited the greatest contributions to the amplitude and signal-to-noise ratio at the characteristic frequency and therefore were responsible for the interaction(s) described by the particular spectral characteristic. By performing computational peptide scanning of Nb71 at F(0.216), we identified the region encompassing amino acid residues (aar) 88–104 16 are presented in Table CABS-dock docking results were the output of the 1,000 top conformations of β2-AR:P3 was subjected to MD simulations to determine its stability. Its structure is shown in Fig. 2-AR:P3 complex remained stable and intermolecular interactions persisted, with eventual breaking and re-arranging on peptide terminals due to its flexibility. The interactions observed in the β2-AR:P3 complex are presented in Fig. 30, which has also been successfully applied in bimolecular simulations dedicated to the study of protein-protein interactions (reviewed in20).A prepared complex of β2-AR:P3 complex after the MD production phase to estimate the binding free energy between β2-AR and the peptide. For the centroid of β2-AR aar, we chose the backbone carbon atoms Arg63, Asn69, Arg131, Ile135, Tyr141, Thr274, Ser329 and Pro330. We chose all residues of the peptide. The initial distance between the two centroids in the docked conformation was 4,890 Å, and we altered this distance by up to 60 Å. The choice of protocol was not trivial, and the main reason was the flexibility of the peptide ligand. The peptide itself possessed high flexibility, giving rise to slow equilibration and a noisy potential of mean force (PMF). After numerous preliminary metadynamics simulation experiments, we concluded that extraction of the peptide to an intracellular water layer along the z-axis of the system, parallel to the axis of the receptor and perpendicular to the cell membrane was an appropriate protocol. A good starting peptide conformation was identified by assessing its conformation after 100 ns of production where the P3 preserved its orientation and a similar binding pattern that between β2-AR and Nb80.  and Ala271 (TM6) during production and the metadynamics phase .We performed well-tempered metadynamics simulations of the β80. Fig.  and 3. Ri.e., its projection on the z-axis between receptor and peptide, is presented in Fig. i.e., we estimated the binding free energy of the β2-AR:P3 complex as ΔG = −7.23 kcal/mol, or Ki = 7.9 μM at 310 K. The standard error of the calculation was 0.04 kcal/mol, so the final result was written as ΔG = (−7.23 ± 0.04) kcal/mol, or Kd = (7.9 ± 0.5) μM.The distance evolution, The peptide conformational energy minima were meant to be achieved during the 100 ns simulation time. However, due to high conformational freedom of the peptide producing a noisy PMF profile, we had no choice but to select an area within approximately 20 to 30 Å to average the PMF, as it represents the unbound peptide form. The complete metadynamics simulation movie is in Supplementary Material 2-AR and P3.In subsequent steps, we attempted to provide experimental evidence for the computationally characterized interaction between the β2-AR and P3 is presented in Fig. 2-AR/Rluc8 were titrated with P3 in the presence or absence of the β2-AR agonist isoproterenol. Comparison of the graphs in Fig. 2-AR/Rluc8-transfected HEK-293 cells that were preincubated with the β2-AR agonist isoproterenol  than in untransfected HEK-293 cells, in which low endogenous expression of the β2-AR was indicated by the very sensitive cAMP ALPHAscreenTM assay31 and the low expression of the β2-AR mRNA32.Based on the CD spectra for P3 recorded in the far-UV range, this peptide does not have a distinctive secondary structure and is rather unstructured , without the need for a prior protein purification/labelling step. MST-derived dose-response curves for the interactions of isoproterenol and P3 with β2-AR are shown in Fig. 50 of the well-validated β-adrenoceptor agonist isoproterenol for β2-AR was in the submicromolar range, i.e., 0.275 ± 0.065 µM, which corresponded well to the relatively low affinity of β2-AR agonists10. On the other hand, P3 displayed EC50 values of 58.22 ± 13.59 µM and 3.57 ± 0.81 µM for non-activated and agonist-activated β2-AR, respectively. Isoproterenol-activated β2-AR thus displayed an approximately 10-fold higher affinity for P3 than unstimulated β2-AR  was reduced by approximately 50% when the experiment was performed with homogenized or permeabilized cells relative to the maximal signal when the experiment was performed with whole cells (BRETmax of ≈40 vs. 80 mBU), whereas the potency of isoproterenol and pindolol remained comparable among the various conditions  were also highly consistent with previously reported data26. Figure 2 antagonist dose-response curves generated in the presence of increasing concentrations of the adrenergic receptor antagonist pindolol. The potency of pindolol  at inhibiting the isoproterenol-induced BRET2 signal was consistent with a previously reported range26. The hydrophilic NDP P3 showed no effect on the isoproterenol-induced BRET2 signal in whole cells, but when either homogenized or permeabilized cells were used, the peptide caused a small dose-dependent reduction (up to 10%) in the isoproterenol-induced BRET2 signal . Researchers have also postulated that Nb71 inhibits agonist-mediated β-arrestin recruitment to the β2-AR by blocking receptor phosphorylation14. Therefore, we also hypothesized that the computationally derived NDP of the Nb71 CDR3 is not able to block receptor phosphorylation. The β-arrestin 2 Arg393Glu, Arg395Glu mutant used in our study should bind to the receptor regardless of its phosphorylation status due to the reversed charge of an amino acid in the polar core (Arg393Glu)34, and this mutation prolongs the lifetime of the receptor:β-arr2 complex due to disruption of β-arrestin 2:AP-2 binding (Arg395Glu34). Similarly, peptidomimetics of the Nb80 CDR3 loop were recently shown to only moderately inhibit isoproterenol-induced cAMP production29, suggesting that additional residues outside the CDR3 loop are important for effective interference with the Gα/β-arrestin interactions. Alternatively, the relative inefficiency of P3 in the BRET recruitment assay might have been due to the lower affinity of P3 (micromolar range) than purified β-arrestin for phosphorylated β2-AR, which is in the nanomolar range (a reported Kd of 1.8 nM)35.Because the proteinlls Fig.  instead nal Fig. , with anin vitro experimental data used to design and characterize NDPs. It is a small step towards obtaining a better understanding of GPCR dynamics at the molecular level in the context of GPCR interactions with their protein partners. The combined use of experimental and computational techniques represents a powerful framework for achieving progress in this direction and could lead to further modification and optimization of NDPs as efficient modulators of GPCRs and other applications, including drug discovery and therapy.In summary, this study presents evidence obtained from a combination of computer-based methodological approaches supported by Molecular biology and cell culture reagents for were from Sigma-Aldrich  and Gibco Invitrogen Corporation . Pindolol and isoproterenol were from Sigma-Aldrich. Coelenterazine 400a from Biotrend Chemikalien GmbH . Selected NDPs (P1-P4) were custom-synthesized at Biomatik Corporation, Cambridge, Ontario, Canada.36 and has been successfully applied to the structure-function analysis of different proteins18, the prediction of new protein interactors37 and the identification of protein domains responsible for long-range interactions38.The principle of the ISM has been thoroughly explainedComputational peptide scanning was utilized to define linear protein regions responsible for the interaction(s) described by the particular spectral characteristic. The sequence of Nb71 was scanned by the ISM algorithm with overlapping windows of different lengths to identify regions with the highest amplitudes at the predefined Fourier frequency.2-AR used for the bioinformatics analysis was retrieved from the UniProt database with accession number P07550. The sequences of Nbs are presented in US patent US20130137856 and the PDB entry 3P0G FASTA sequence.The sequence of human β2-AR was obtained from the RSCB protein databank (PDB entry code 3P0G). All lipids, water molecules, and ions and Nb80 were removed. Only the P0G ligand was retained.The active state crystal structure of the β39. Using the given protein receptor 3D structure, binding site and peptide sequence, a docking search for the binding site is performed that allows for full flexibility of the peptide and small fluctuations in the receptor backbone. The output of the simulation is the three-dimensional coordinates of the protein in complex with the ligand accompanied by full docking process trajectories and CABS force field docking scores, including energies of the receptor, ligand and their interaction. The binding site is indirectly defined by unlikely-to-bind regions of β2-AR, which exclude all aar regions except intracellular loops. The number of simulation cycles was set to 50. The best solutions, including the peptide in the intracellular space and lowest CABS-dock energy, were selected for further MD simulations.Peptide-protein docking was conducted using the online CABS-dock server40 and van der Waals parameters, whereas force constants were obtained from the Hessian equation calculated after geometric optimization of the HF/6–31 G(d) level of theory in Gaussian 03 W41. All parameters were generated in the VMD Parameterize extension42.Ligand was assigned CGenFF force field atomic charges (ParamChem)2-AR-agonist-NDP complex, with peptide coordinates obtained from the docking output, was inserted into a 70 × 70 Å 2-oleoyl-1-palmitoyl-sn-glyecro-3-phosphocholine (POPC) lipid bilayer. A 10 Å water layer was added from the positive side of the z-axis and 60 Å from the negative side. Bad-contact water molecules were removed from the lipid membrane bilayer using the appropriate tcl script. Additionally, the system was neutralized with 0.15 M NaCl, resulting in a 61,183  atom ensemble. The system was subject to a 10,000 step energy minimization, 250 NVE ps equilibration, and 100 ns NPT MD production. Pressure and temperature were set to 1 bar and 310 K, respectively, using a Berendsen thermostat, and the applied integration step was 1 fs. In all simulations, periodic boundary conditions with particle-mesh Ewald calculations were implemented. The cut-off was set to 12 Å. A CHARMM2223 force field was used for protein and lipids, and CGenFF44 was used for ligands.The β19 and later improved to well-tempered metadynamics20. Before metadynamics simulation, one must determine the collective variables that will be varied during simulation and in regard to which the PMF will be calculated. We chose one variable, the distance between centroids of protein amino acid (aa) and peptide residues. Their atoms belonged to backbone C of the binding aar in the intracellular loops of the receptor and all residues in the peptide. The lower boundary  was set to the initial distance between centroids of receptor-ligand atom groups, which was obtained from the coordinates of the docked structure optimized by MD production. The upper boundary  was set to the distance at which the peptide was located sufficiently far away from the receptor in the water layer close to the side of the PBC cell. In this way, we ensured that no intermolecular interactions between the protein and peptide were present along all three coordinate axes. During our metadynamics simulation, the peptide pushed towards the intracellular water layer, parallel to the z-axis of the cell and perpendicular to the cell membrane. The resulting change in free energy between initial and equilibrated states of the peptide in the water layer was designated the binding free energy of the complex. Collective variable trajectory frequency (frequency of generating free energy files) was set to 10,000 ps. The lower wall constant ) was set to 120.0, the upper wall constant  was set to 180.0, and width (the force resolution) was set to 0.1. For the main atoms, we selected backbone carbon atoms from the receptor, and for the reference, we selected backbone carbon atoms from the peptide. The hill weight  was set to 0.1 kcal/mol, the hill width was set to 1.0 Å, and the new hill frequency was set to 100 ps. Bias temperature was set to 1550 K. All free energy files generated during simulation were collected. The total metadynamics simulation time was 40 ns, and the integration step was 1 fs. Descriptive statistical analysis of PMF and the corresponding Figures was performed using Origin 8 .Metadynamics is a powerful method for calculating free energy. It was initially developed by Laio and Parrinello2-AR (HAβ2-AR) cDNA and human β-arrestin 2 N-terminally tagged with the green fluorescent protein variant 2 GFP2 (GFP2/β-arr2) were from cDNA Resource Center (www.cdna.org) and from BioSignal Packard Inc., Montreal, Canada, respectively. C-terminal Renilla luciferase 8 (RLuc8) tagged HAβ2-AR (β2-AR/RLuc8), C-terminal GFP2-tagged β2-AR (β2-AR/GFP2) and mutant GFP2/β-arr2  were described in previous studies46.Human HA-tagged β47. Transient transfections were performed when cells reached ~90% confluence using the Lipofectamine®-Plus™ Reagent. The expression levels of RLuc8- and GFP2-tagged constructs were monitored by recording total luminescence and fluorescence as previously described48.HEK-293 cells  were routinely maintained and passaged as described previously280 = 1.5)  were dialysed against PBS buffer, pH 7.2, containing 1% glucose and 0.1% DMSO. P3 was solubilized in 0.1% DMSO in DPBS supplemented with Ca2+/Mg2+, 1 g/L glucose, and 36 mg/L sodium pyruvate buffer. The concentration of the P3 stock solution was 1 mM. CD spectra of a 10-fold dilution of the initial cell lysate in the presence of P3 at final concentrations of 0, 5, 10, 20, and 30 µM were recorded. The density of HEK-293 and β2-AR/Rluc8-transfected HEK-293 cells were equal, as determined spectrophotometrically. In the case of agonist addition (10 µM), the cell lysate was incubated with agonist for 5 minutes at 25 °C before the CD spectra were recorded. Each CD spectrum was subtracted from the CD spectrum of the buffer.All CD spectra were recorded on a Jasco J-1500 spectropolarimeter at 25 °C using a 1 mm quartz cuvette in the UV range of 250–200 nm. The CD spectra were measured every 0.5 nm with a scanning rate of 10 nm/min. Lysed cells (A2 flask were transiently co-transfected with constructs encoding β2-AR/GFP2 (5.0 μg), and cell lysates were prepared after 48 h as described below. The 75 cm2 flasks were transferred to ice, and cells were washed once with ice-cold DPBS. Then, 750 µL of NP40 lysis buffer was added to each 75 cm2 flask and incubated on ice for 5 minutes. Cells were scraped from the 75 cm2 tissue culture flasks using cell scrapers , and detached cells were incubated on ice for an additional 20 minutes. Cell lysates were transferred to cold 1.5 mL tubes and clarified by centrifugation . Next, 700 µL of cleared lysate was transferred to a new cold 1.5 mL tube, and the level of β2-AR/GFP2 was verified by measuring the total fluorescence. Ten microlitres of cell lysate were used as the target, while the non-fluorescent ligands isoproterenol and P3 were titrated in a series of 1:1 dilutions in MST buffer containing 0.05% Tween 20. Ten microlitres of the serial dilution of the ligand were mixed with 10 μL of the cell lysate. After a short incubation at room temperature, samples were centrifuged and loaded into Monolith™ NT.115 MST Premium Capillaries  and measured using a Monolith NT.115pico and MO. The control software was set to room temperature (25 °C) . Data were analysed using MO.Affinity Analysis software  at the standard MST-on time of 5 s and presented as a bound fraction. The change in the normalized fluorescence (ΔFnorm) value of each point was divided by the amplitude of the fitted curve to calculate the bound fraction, resulting in values ranging from 0 to 1. This approach is independent of both the initial Fnorm value and the amplitude of the binding curve and thus enabled us to compare the EC50 values of interactions with different amplitudes.HEK-293 cells plated in a 75 cm48, with some modifications. Because P3 binds to intracellular receptor domains, BRET assays were performed using either homogenized cells or permeabilized cells. HEK-293 cells cultured in a 75 cm2 flask were transiently co-transfected with constructs encoding β2-AR/RLuc8 (0.1 μg) alone or together with the double GFP2/β-arr2 Arg393Glu, Arg395Glu mutant (4.9 μg). Homogenized/permeabilized cells were prepared 48 h after transfection. Homogenized cells were washed twice with DPBS, scraped from 75 cm2 tissue culture flasks using cell scrapers  and pelleted by centrifugation at 1,000 rpm for 5 minutes. The cell pellets were resuspended in 1 mL of DPBS supplemented with Ca2+/Mg2+, 1 g/L glucose, and 36 mg/L sodium pyruvate, incubated on ice for 10 minutes, and homogenized using a glass homogenizer (BDH).We used a previously described BRET-based βarr2 recruitment assay2 signal) was calculated. The results were reported in milliBRET units (mBU); BRET2 ratio × 1000. The expression levels of RLuc8- and GFP2-tagged constructs in each experiment were assessed by measuring the total luminescence and fluorescence as described above. Determinations were performed in triplicate. The obtained data were transferred to GraphPad Prism 7.0  and BRET EC50/IC50 values (nM ± SEM) generated using a sigmoidal dose-response curve fit.Cells were permeabilized by adding 0.01% Triton™ X-100 in DPBS and incubating for 15 minutes at 37 °C. Homogenized/permeabilized cells were then diluted in supplemented DPBS such that the luminescence signal in 180 μL of DPBS containing homogenized/permeabilized cells distributed in 96-well microplates  was approximately 30,000 arbitrary units. After the coelenterazine 400a was injected , the luminescence signals were recorded . Signals at 410 nm and 515 nm were recorded sequentially, and the 515/410 ratio (BRETSupplementary material


Background: Androgen receptor (AR) has a role in regulating malignancies and gender disparities in hepatocellular carcinoma (HCC). Recently, TLR4 activation is demonstrated to be required for HCC progression; however, whether and how TLR4 interacts with AR is largely unknown.Methods: The tumorigenesis was detected in female and male mice induced by DEN/CCL4, then TLR4 and AR signals were detected in liver tissues by qPCR and FACS. The proliferation, colony formation and migration of HCC cell treated with TLR4 agonist LPS, or/and androgen DHT were evaluated in vitro. Furthermore, the expression of TLR4 and AR was detected by IHC in tissue microarray of HCC, and correlation of AR and TLR4 was defined.Results: Male mice are more susceptible to develop HCC than female mice. Meanwhile, we found baseline TLR4 levels were higher in male mice than in female mice. AR expression in male mice was increased by treatment with DEN/CCL4. And, AR was constitutively expressed in human HCC cell lines. Dihydrotestosterone (DHT) stimulated TLR4 expression in both HepG2 and HepG2 2.15 cells, which could be blocked by silencing AR. On the other hand, treatment with LPS stimulated AR expression, but it was blocked by treatment with TLR4 antagonist and in cells deficient for TLR4. DHT treatment exacerbated TLR4-induced cellular proliferation, colony formation, migration, and invasion of HepG2 cells. The positive relationship between AR and TLR4 was confirmed in human HCC samples.Conclusions: DHT-AR-TLR4 signaling enhances the development of HCC cells and facilitates their migration and invasion, demonstrating a mechanism underlying gender disparity in HCC. Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death, and is notably more prevalent in men than in women, with a male-to-female ratio ranging from 2:1 to 8:1 The androgen receptor (AR) is a member of the nuclear steroid receptor superfamily The role of the gut-liver axis in the development of liver diseases, including non-alcoholic steatohepatitis and HCC, has recently attracted a great deal of attention . TLR4 showed a positive relationship with AR expression in human HCC tissues. Further investigation demonstrated that DHT-AR is critical for TLR4 signal-mediated promotion of HCC, suggesting that TLR4 is essential for gender disparities observed in HCC. These findings provide new insight for improving the efficacy of HCC treatment in the clinic.Based on the importance of TLR4 and AR in HCC development, we wanted to know whether and how TLR4 crosstalk occurs with sex hormone receptors, especially AR, during the development of HCC. Here, we report that the baseline expression of TLR4 was higher in male mice than in female mice. This gender disparity was further increased by treatment with DEN/CCL4(i.p.) injections of 20 mg/kg DEN for three weeks , followed by weekly i.p. injections of 100 μl CCL4 dissolved in olive oil for six weeks. The procedure was divided into three stages; early (7-21 weeks), middle (22-42 weeks), and late , as previously described A mouse model of DEN-induced HCC was generated (described in Supplementary Methods). 6-week-old C57BL/6 mice were obtained from HFK Bioscience Co, Ltd . Beginning at fourteen days of age, female and male mice (5 mice per group) received weekly intraperitoneal 2. LPS isolated from Escherichia coli (0111:B4), natural AR ligand dihydrotestosterone , and estrogen (E2) (E2758) were purchased from Sigma-Aldrich . The TLR4 signaling inhibitor, TAK-242, was obtained from Invivogen .The HCC cell lines HepG2, H7402, Hepa1-6, and HepG2.2.15 were cultured and maintained in our laboratory. All cell lines were grown in DMEM , containing 1% penicillin-streptomycin and supplemented with 10% fetal bovine serum (FBS). Cell cultures were incubated at 37°C in 5% CO5 HepG2 or Hep1-6 cells/well were plated into 12-well plates, and were treated with DHT or LPS. Total RNA from cells and liver tissues was extracted using the Trizol reagent , and was used to generated cDNA using Moloney Murine Leukemia Virus Reverse Transcriptase  according to the manufacturer's protocol. cDNA amplification was performed using real-time PCR with FastStart Universal SYBR Green Master  on an iCycleriQ real-time PCR system . GAPDH and β-actin genes were used to normalize gene expression. The primers used in this study are described in Table 1.5×10TM 549-conjugated goat anti-rabbit secondary antibody . Quantification was performed using Image J and Photoshop software, analyzing at least 10 images per mouse for AR staining.Immunofluorescence was performed using primary antibodies to AR , and DyLight3 cells/well were plated into 96-well plates. After sample preparation, 20 µL of MTT reagent was added, and cells were incubated at 37 °C for another 3h. Absorbance at 570 nm was recorded by a scanning multi-well spectrophotometer .The MTT cell proliferation reagent  was used to evaluate cell growth, according to the manufacturer's instructions. Briefly, 5×10Cells or liver tissue was homogenized using lysis buffer with containing a protease inhibitor cocktail . Total protein (30 µg) from each sample was separated on a 10% SDS-polyacrylamide gel, and then transferred onto PVDF membranes. Membranes were incubated for 1 h in blocking buffer containing 5% milk, then incubated with anti-β-actin , anti-AR , or rabbit anti-TLR4 , diluted 1:2000 overnight at 4 °C. After overnight incubation, membranes were incubated with peroxidase conjugated goat anti-rabbit IgG  for 1 h at room temperature. Proteins were visualized using Immobilon Western Chemiluminescent HRP Substrate  and detected with Alpha Ease FC software  A human hepatoma tissue microarray (OD-CT-DgLiv02 and HLiv-HCC060CD-01) was obtained from Outdo Biotech . Paraffin-embedded and formalin-fixed mouse liver samples were cut into 5 μm sections, which were then processed for immunohistochemistry as described 5 cells/well were plated into 12-well plates. Cells were treated with DHT or LPS. Twenty-four hours later, cells were washed, harvested, and re-centrifuged in 200 μl 1× annexin-binding buffer. Cells were incubated with 5 μl Alexa Fluor® 488 Annexin V and 2.5 μl of propidium iodide (PI)  at room temperature for 15 min, then analyzed by flow cytometry .1.5×10The migration assay was performed using 24-well transwell inserts with a pore size of 8 μm . Initially, HepG2 cells treated with DHT or LPS were seeded into the upper chamber in serum-free medium. DMEM with 10% FBS was added to the lower chamber, as a chemoattractant. After 6 h incubation at 37 °C, the cells that traversed the membrane were fixed in methanol and stained with 0.1% crystal violet. For quantification, the number of migrated cells was calculated by counting at least five random separate fields.For the clonogenic colony formation assay, 500 cells/well were seeded in 6-well plates. After 14 days, visible colonies were observed using the naked eye, fixed with 4% formaldehyde, and stained with 0.1% crystal violet. Colonies with a diameter greater than 1 mm were counted.t-test. Statistical significance was determined as ** P < 0.01 and * P < 0.05 compared with control.Statistical analysis was performed using paired Student's 4 . The tumors induced by this treatment demonstrated typical features of HCC in male mice , and the spleens of male and female mice did not show any significant differences (data not shown). Moreover, compared to female mice, there was a profound increase in tumor number and size in male mice 44 weeks after the initial DEN/CCl4 treatment (n=5) . qPCR analysis revealed that expression of Ki67, proliferating cell nuclear antigen (PCNA), Acta2, and alpha-fetoprotein (AFP) was markedly increased in male mice treated with DEN/CCl4, and displayed expression profiles characteristic of HCC (n=5) . By IHC analysis, Ki67 protein expression was higher in the liver tissue from male mice treated with DEN/CCl4 than in female mice . These data confirm that there is a gender disparity associated with the development of HCC.The incidence and mortality of liver cancer incidence is significantly higher in male mice than in female mice Figure ). Following DEN/CCL4 injection, we observed TLR4 positive hepatocytes were increased in liver tissue of male mice (n=5) ; this was accompanied by an elevation of AR mRNA . Furthermore, NF-κB activation in liver tissue from male mice were confirmed by western blot assays . These findings indicate there may be a relationship between TLR4 and AR in HCC development.Previous reports demonstrate that AR promotes gender disparities in cancer Figure S2). Immunofluorescence staining confirmed the expression of AR in HepG2 and HepG2.2.15 cells . HepG2 and HepG2.2.15 cells were treated with androgen DHT or estrogen (E2) for 48 h. Treatment with DHT elevated TLR4 the expression of TLR4, while E2 treatment down-regulated TLR4 in HepG2 cells . A similar trend in TLR4 expression changes was observed in HepG2.2.15 cells . Meanwhile, FACS assay revealed the percentage of TLR4 positive HepG2 cells was increased by DHT treatment . However, silencing of AR by siRNA abolished the DHT-induced up-regulation of TLR4 in HepG2 cells . We stimulated HepG2 cells with the TLR4 agonist, LPS, for 24 h; the expression of AR at both the mRNA and protein levels was increased ; this increase in AR was abrogated by the TLR4 inhibitor TAK242 . In addition, the level of Ki67 in WT male mice treated with DEN/CCL4 was higher than that in WT female mice; this was profoundly decreased in TLR4-/- mice . These results suggest that the positive regulation between AR and TLR4 signaling is critical for HCC initiation and development.To clarify the relationship between AR and TLR4 signaling in HCC, we studied the human HCC cell lines HepG2 and HepG2.2.15, which constitutively express AR at a relatively high level . Compared to control, DHT- and LPS-treated cells, the proliferation of HCC cells was augmented by treatment with a combination of LPS and DHT (LPS+DHT) ; simultaneously, the proportion of HepG2 cells in S-phase was increased , but the apoptosis of HepG2 cells was not affected by DHT+LPS . Furthermore, colony formation assays revealed that both DHT and LPS stimulated colony growth  and enhanced the migratory ability  of HepG2 cells, which was further enhanced by treatment with a combination of LPS and DHT. However, these phenomena were not observed in Hepa1-6 cells, which exhibited low levels of AR . Therefore, these data suggested that AR signaling exacerbates TLR4-induced proliferation, colony formation, and migration of HCC cells.To determine if AR-associated signaling facilitates TLR4-induced HCC development, HepG2 cells were treated with 100 nM DHT or 100 nM E2. After stimulation for 6 h, we observed that DHT promoted the wound healing capacity of HCC cells, while E2 did not show significant effects , and we found that the expression of TLR4 was positively correlated with AR in HCC tumors . Furthermore, we found that the expression of AR in male is higher than that in female HCC. And the similar trend was observed for detection of TLR4 .These results support the positive relationship between TLR4 and AR in HCC development.To extend our findings in mice to human HCC, we analyzed TLR4 and AR expression using tissue microarray, containing matched pairs of tumor and peritumoral liver tissue from 22 male and 8 female patients with primary HCC. We found that TLR4 and AR were expressed more highly in HCC tumor tissue than in adjacent liver tissue . In addition, in line with our findings, estrogen restricts TLR4 and TNF-α, but not TLR3 and NLRP3 responses, contributing to a proinflammatory hepatic microenvironment in males, which imparts increased risk to men with chronic viral hepatitis to progress to cirrhosis or HCC TLR4 promotes HCC via several mechanisms, including increasing expression of NANOG, Caspase-1, Ephrin-A1, NO, and BCL6 and increasing the number of T regulatory lymphocytes Figure 3), the exact molecular mechanism has not been enucleated, we still need further study to explore it.Previous studies found that AR activation could convene more than 200 kinds of regulatory molecules, including transcriptional factors, kinases, molecular chaperones, cytoskeleton proteins, and molecules related to histone modification. Importantly, PGE and STAT3 are among them, and we preciously found that TLR4 signaling could promote the proliferation and induce multidrug resistance of HCC cells to chemotherapeutics, in which COX-2/PGE2/STAT3 positive feedback loop Figure 4).Our study demonstrates that AR can promote HCC tumorigenicity; this is supported by evidence that mice lacking hepatic AR had less hepatocarcinogenesis after exposure to a higher dose of DEN Similar to the gender disparities seen in human HCC, gender differences are also observed in mouse models of HCC, including both genetically or chemically induced HCC. Sex hormones and inflammatory responses have been implicated in the gender disparity of HCC; such studies have often used a mouse model that develops HCC upon exposure to DEN. This model incorporates chronic injury, inflammation, fibrogenesis, and CCl4-mediated increases in endotoxin levels Figure 6), the relationship of these molecules with survival time needs further clarification. Targeting AR with ASC-J9® or other small molecules may have potential to be developed to suppress HCC progression more effectively in near future In addition, various immune cells in the tumor microenvironment influence the progression of HCC Our data suggest that DHT-AR-TLR4 signaling enhances the development of HCC cells and facilitates their migration and invasion. These findings suggest that AR and TLR4 are potential therapeutic targets in the treatment of HCC, although further mechanistic studies are warranted.Supplementary figures.Click here for additional data file.