N-acetyl/N-thiocarbamoylpyrazoline derivatives were designed and synthesized in high yields to assess their inhibitory activity and selectivity against human monoamine oxidase A and B. The most important chiral compounds were separated into their single enantiomers and tested. The impact of the substituents at N1, C3 and C5 positions as well the influence of the configuration of the C5 on the biological activity were analyzed. Bulky aromatic groups at C5 were not tolerated. p-Prenyloxyaryl moiety at C3 oriented the selectivity toward the B isoform. The results were also corroborated by molecular modelling studies providing new suggestions for the synthesis of privileged structures to serve as lead compounds for the treatment of mood disorders and neurodegenerative diseases.New  Monoamine oxidases, (MAOs) have been widely recognised as important pharmacological targets for the treatment of mood disorders  and neurodegeneration  as the results of their effects on monoamine metabolism and level ,2. Two iP1–P18) based on the 1,3,5-substituted pyrazoline scaffold (P1–P6) or the acetyl (derivatives P7–P18) moiety. These groups possess different steric/electronic properties, and differ in their hydrogen bonds acceptor/donor potential. Position C3 of pyrazoline ring was functionalized with a phenyl ring  was carried out to evaluate how the chiral properties of the single enantiomers influence the inhibitory activity against hMAO enzymes N1-thiocaV), the compounds P1–P5 were selected and mg-quantities of their pure enantiomeric forms were easily isolated by semipreparative enantioselective HPLC on the Chiralpak AD column using pure ethanol elution mode . These outcomes showed that when increasing the length of the alkyl chain, both enzymes are able to recognize one of the two optical isomers as the eutomer and the preferred absolute configuration appears to be (R). Finally, the substitution with the geranyl moiety was considered in order to determine how a branched chain, containing unsaturation and longer length compared to the other derivatives tested, affected the inhibition activity and/or the selectivity. The results showed that compound P6 was ineffective as an inhibitor of hMAO-A, retaining activity in the high micromolar range only against hMAO-B (hMAO-B: IC50P6 = 54.1 µM). Therefore, the excessive increase of the alkyl chain dimensions was detrimental for inhibition potency, although selectivity for hMAO-B was obtained.Further increase of the alkyl chain length led to the compounds moieties . The P4 P7–P18, which are substituted on the N1 position with the acetyl group, have also been tested. Except for compound P7, bearing an allyloxy functional group, all derivatives possessed para-prenyloxy substitution on the phenyl ring bound at the position 3 of pyrazoline ring . Compound P8 was the simplest compound endowed with prenyl group, bearing an unsubstituted phenyl ring on position 5 of pyrazoline ring (the U phenyl). This compound exhibited activity against hMAO-B in the low micromolar range, without activity for hMAO-A .Derivatives U phenyl ring affected both activity and selectivity. Compounds P9–P11 were substituted with methyl groups at respectively the ortho, meta and para positions, while compound P12 contained the 2,4-dimethyl substituted phenyl ring. Derivative P9 showed preference for hMAO-A with an inhibitory activity that was 3-fold higher than that observed for hMAO-B . Shifting of the methyl group from ortho to meta position (P10) resulted in a change of selectivity along with an enhancement of inhibitory activity towards hMAO-B, with an IC50 value in the low micromolar range . For compound P11, bearing the methyl group in the para position, hMAO-A and hMAO-B inhibition was reduced compared to P10 . The 2,4-dimethyl substituted derivative P12 exhibited weak inhibitory activity against both isoforms, with only slight inhibition recorded for hMAO-A . The presence of 2,4-disubstituted phenyl system at the C5 position thus is detrimental for the hMAO inhibition. Similarly, P16, bearing the 2,4-dichloro substituted phenyl ring, did not inhibit either hMAO-A or hMAO-B. It may be concluded that a sterically hindered system on the C5 position of the pyrazoline ring negatively affects the inhibitory activity. This was demonstrated by P17 and P18, which were substituted with bulky naphthyl and biphenyl groups, respectively, and are devoid of inhibition activity against both hMAO-A and hMAO-B.The addition of substituents on the P13–P15, which were substituted with chlorine on the phenyl ring, have been evaluated. Derivative P13, the ortho-chloro substituted derivative, showed similar inhibitory activity against both isoforms . P15, containing a para-chlorophenyl, showed a similar profile, with an inhibitory activity against both hMAO A and hMAO-B, although less potent than P13 . Among the chloro-substituted molecules, the best inhibitory activity against hMAO B was observed when the substituent was placed in the meta position, as previously seen for methyl substituted compounds (P9–P12). In fact, P14 displayed inhibition activity against hMAO-B in the low micromolar range, but it also retained a good activity on hMAO-A .Finally, compounds W ring can be done. We already stated that the excessive size of this substituent was detrimental for the inhibitory activity, as observed for compound P6. However, the increase of the alkyl chain length (compounds P1–P5) also involves the enhancement of lipophilicity leading, at least for the (R)-enantiomers, in the improvement of the affinity against both the isoforms (see P8–P18) could be the perfect compromise with a good balance between lipophilicity and steric hindrance, improving the selectivity against MAO-B, without impairing the inhibitory activity. This is consistent with the bigger hydrophobic pocket observed in the binding site of MAO B, where the lipophilic chains of the ligands could be better accommodated than into MAO-A, as suggested by docking studies. Considering the shared scaffold within compounds P1–P5 and the dimensions of the prenyl group, we can assume that also for derivatives P8–P18, MAO-B isozyme could exhibit chiral recognition for (R)-enantiomers. The use of racemic mixture for the biological evaluation could reduce the real inhibitory activity due to the competition between the two enantiomers for the same active site. Therefore, the results obtained for prenylated derivatives are worthwhile of further investigation.In the light of the above, some considerations regarding the properties (dimensions and lipophilicity) of the alkyl chains bound to the orms see . From thR)-P5, which showed submicromolar activity against hMAO-B and low micromolar potency against hMAO-A, was subjected to the computational protocol in order to evaluate its binding mode into the catalytic site of the two enzymes. As shown in With the aim of providing a rational interpretation of the results obtained from the biological evaluation of the pyrazoline derivatives, molecular modeling studies including robust docking analyses and energy minimizations in explicit water environment were performed . Initially, compound (S)-enantiomer of the inhibitor, which showed a lower activity against both enzyme isoforms. The binding mode predicted for the compound into hMAO-B suggested that, due to the different stereochemistry of the pyrazoline ring, the ligand is not able to form the H-bond network with FAD and Q206 and only the water-bridged interaction with the carbonyl oxygen of the cofactor flavin group and the amino group of K296 is maintained (S)-P5 on hMAO-A showed a more significant reduction, compared to (R)-P5. Accordingly, the ligand was predicted to assume a binding disposition characterized by an inverted orientation of the pyrazoline core, with respect to the binding mode of its (R)-enantiomer. For this reason, none of the H-bonds predicted for the (R)-enantiomer can be formed by compound (S)-P5 and the thioamide group of the ligand can only form an H-bond with Q215 mediated by two water molecules. Moreover, the terminal phenyl ring of the ligand shows only a T-shaped stacking with Y69 in place of the strong π–π stacking with Y398, Y435 and FAD flavin group observed in the predicted binding mode into hMAO-B. Finally, since the position of the ligand is slightly shifted toward the end of the binding cavity, its pentoxyl chain protrudes more deeply into the short channel connected to the solvent and may thus form unfavorable interactions with water.The same computational protocol was then applied to the (intained A. HoweveS)-P5 and (R)-P5 into hMAO-A and hMAO-B, few considerations can be derived. The thioamide moiety of the ligands is important for both the H-bond donor NH2 group and the H-bond acceptor sulfur atom, with this last portion that seems to be a fundamental feature. This is consistent with the observation that derivatives with the thioamide moiety replaced by an acetyl group maintain micromolar activity. The different disposition assumed by the alkoxyl chain of the ligands into hMAO-A and hMAO-B could explain why compounds with longer chains are more selective over hMAO-B than those bearing shorter chains like (R)-P3: a shorter chain would limit not only the favorable hydrophobic interactions with the lipophilic pocket of hMAO-B, but also the possible unfavorable interactions with the solvent when bound to hMAO-A, thus balancing the activity of the inhibitor toward the two enzyme isoforms. Finally, the inactivity of derivatives bearing bulky groups connected to the pyrazoline core such as P16–18 is consistent with the limited space available in proximity of the flavin portion of FAD cofactor.Based on the binding modes predicted for the two compounds . Where mixtures of solvents are specified, the stated ratios are volume:volume. Stuart® melting point apparatus SMP1  has been used to measure the melting points (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 centimeters. 1H- and 13C-NMR spectra were recorded at 400 and 101 MHz, respectively, on a Bruker spectrometer  using CDCl3 and DMSO-d6 as the solvents at room temperature. Chemical shifts are expressed as δ units (parts per millions) relative to the solvent signal. 1H spectra are reported as follows: δH : chemical shift/ppm , number of protons, assignment). 13C spectra are reported as follows: δC : chemical shift/ppm (assignment). Multiplets are abbreviated as follows: br—broad; s—singlet; d—doublet; t—triplet; q—quartet; m—multiplet. Coupling constants J are valued in Hertz (Hz). Purification on column chromatography was carried out using silica gel . All the operations were monitored by TLC performed on 0.2 mm thick silica gel-aluminum backed plates . Visualization was carried out under ultra-violet irradiation (254 nm). Where given, systematic compound names are those generated by ChemBioDraw Ultra 12.0 following IUPAC conventions . A Perkin-Elmer 240 B microanalyzer  was used to determine elemental analyses for C, H, and N; analytical results were within ±0.4% of the theoretical values for all the tested compounds. Microsomes from insect cells containing recombinant hMAO-A and hMAO-B (5 mg protein/mL) and kynuramine dihydrobromide were obtained from Sigma-Aldrich. Amplex® Red , horseradish peroxidase, (R)-deprenyl hydrochloride, pargyline hydrochloride and H2O2 (3%) were also from Sigma-Aldrich. For fluorescence spectrophotometry, a SpectraMax iD3 multi-mode microplate reader  was employed.Solvents and reagents were used as supplied without further purification (Sigma-Aldrichn-hexane 1:4) gave the title compounds. Characterization data obtained for compounds A3–A5 and A7 were in accordance with [A6 were in accordance with [To a stirring solution of 1-(4-hydroxyphenyl)ethan-1-one (1 eq.) in anhydrous acetone (20 mL) freshly ground potassium carbonate (1.2 eq.) was added. 1-Bromoalkane (1.1 eq.) was added and the reaction stirred at room temperature for 24–48 h, under nitrogen atmosphere. The synthesis was quenched with 50 mL of ice/water and the resulting suspension was extracted with chloroform (3 × 20 mL). The organics were reunited, dried over sodium sulfate and concentrated in vacuo, to give the crude compounds. Purification by column chromatography on silica gel oxy)phenyl)ethan-1-one (A8), yellow-orange oil (72% yield). 1H-NMR : δ 1.59 , 1.66 , 1.73 , 2.07–2.11 , 2.52 , 4.58 , 5.05–5.08 , 5.44–5.47 , 6.91 , 7.90 .n-hexane (2 × 20 mL) giving the title compounds without further purification requirements. For compound C18 the suspension obtained after quenching with 150 mL of ice/water, was extracted with chloroform (3 × 20 mL). The organics reunited were dried over sodium sulphate and evaporated in vacuo. Purification by column chromatography on silica gel (ethyl acetate:n-hexane 1:4) gave the title compounds. Characterization data obtained for compounds C1–C5, C7–C8 were in accordance with [C15 were in accordance with [To a stirring solution of the proper chalcone (1 eq.) in ethanol (10 mL) was added a dispersion of barium hydroxide (1 eq.) in ethanol (40 mL). The appropriate (un)substituted benzaldehyde (1 eq.), dissolved in ethanol (20 mL), was added dropwise and the reaction stirred at reflux for 24–72 h. The synthesis was quenched with 150 mL of ice/water and the resulting suspension filtered. The crude solid was washed with petroleum ether (2 × 20 mL) and nce with ,30, whilnce with .(E)-1-oxy)phenyl)-3-phenylprop-2-en-1-one (C6), white solid (82% yield); mp 66–68 °C. 1H-NMR : δ 1.56 , 1.62 , 1.73 , 2.06 , 4.68 , 5.06 , 5.44 , 7.08 , 7.45 , 7.71 , 7.88 , 7.95 , 8.16 .(E)-1-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(o-tolyl)prop-2-en-1-one (C9), yellow solid (92% yield); mp 72–75 °C. 1H-NMR : δ 1.79 , 1.83 , 2.50 , 4.62 , 5.52 , 7.01 , 7.24-7.33 , 7.49 , 7.71 , 8.06 , 8.12 .(E)-1-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(m-tolyl)prop-2-en-1-one (C10), yellow solid (77% yield); mp 99–101 °C. 1H-NMR : δ 1.74 , 1.76 , 2.36 , 4.65–4.66 , 5.45 , 7.08 , 7.26–7.27 , 7.33–7.35 , 7.65–7.72 , 7.92 , 8.16 .1-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(p-tolyl)prop-2-en-1-one (C11): yellow solid (87% yield); 95–98 °C. 1H-NMR : δ 1.74 , 2.35 , 4.64 , 5.44 , 7.08–8.15 .(E)-3--1-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one (C12), yellow solid (75% yield); mp 102–105 °C. 1H-NMR : δ 1.58–1.59 , 2.37 , 2.47 , 4.62  5.51 , 7.00–7.09 , 7.47 , 7.63 , 8.06 , 8.11 .(E)-3-(2-chlorophenyl)-1-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one (C13), white solid (94% yield); mp 98–100 °C. 1H-NMR : δ 1.79 , 1.84 , 4.62 , 5.52 , 6.99–7.03 , 7.33–7.36 , 7.45–7.48 , 7.53 , 7.76–7.78 , 8.01 , 8.05 , 8.19 .3-(3-chlorophenyl)-1-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one (C14), white solid (94% yield); mp 92–94 °C. δ 1.78 , 1.83 , 4.61 , 5.49 , 7.05–7.08 , 7.30–7.34 , 7.49–7.51 , 7.55 , 7.74–7.72 , 8.00–8.02 , 8.03–8.05 , 8.21 .(E)-3--1-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one (C16), yellow solid (96% yield); mp 127–130 °C. 1H-NMR : δ 1.74 , 1.76 , 4.66 , 5.44–5.47 , 7.09 , 7.54–7.57 , 7.76 , 7.94 , 8.04 , 8.18 , 8.27 .3--1-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one (C17), yellow solid (75% yield); mp 122–124 °C. 1H-NMR : δ 1.74 , 1.76 , 4.66 , 5.46 , 7.09 , 7.39–7.52 , 7.75–7.79 , 7.98–8.03 , 8.19 .(E)-1-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-prop-2-en-1-one (C18), yellow solid (67% yield); mp 82–84 °C. 1H-NMR : δ 1.80 , 1.84 , 4.60–4.64 , 5.51–5.55 , 7.03 , 7.54–7.61 , 7.66 , 7.92–7.94 , 8.11 , 8.28–8.30 , 8.68 .P1–P5 were in accordance with reference [P6 we operated as follows: to a stirring solution of 1-oxy)phenyl)ethan-1-one (1 eq.) and thiosemicarbazide (2.8 eq.) in ethanol (50 mL) was added dropwise a solution of ground potassium hydroxide (2.8 eq.) in hot ethanol (50 mL). The reaction was stirred at reflux for 48 h and then quenched with 200 mL of ice/water. The resulting suspension was extracted with chloroform (3 × 20 mL). The organics reunited were dried over sodium sulfate and evaporated in vacuo. Purification by column chromatography on silica gel (ethyl acetate–n-hexane 1:2) gave the title compound.Synthesis and characterization data for compounds eference . For the3-oxy)phenyl)-5-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (P6), orange oil, 62% yield. 1H-NMR : δ 1.63 , 1.70 , 1.76 , 2.10–2.17 , 3.12 , 3.75 , 4.59 , 5.10–5.13 , 5.48–5.51 , 6.02 , 6.52 , 6.95 , 7.15 , 7.22–7.24 , 7.29–7.33 , 7.66 . 13C-NMR : δ 16.7 (CH3), 17.8 (CH3), 25.7 (CH3), 26.3 (CH2), 39.6 (C(4)H2, pyr), 51.5 (CH2), 64.9 (CH2-O), 95.8 (C(5)H, pyr), 114.7 (2 × Ar), 119.4 (=CH), 123.9 (=CH), 125.3 (2 × Ar), 126.9 (2 × Ar), 128.9 (2 × Ar), 130.4 (Ar), 131.1 (C=), 131.8 (Ar), 136.3 (C=), 141.3 (Ar), 153.1 , 158.7 (Ar), 175.8 (C=S). Calcd. for C26H31N3OS: C, 72.02; H, 7.21; N, 9.69. Found: C, 72.18; H, 7.15; N, 9.61.n-hexane as mobile phase, gave the title compounds. Characterization data for compound P8 were in accordance with [To a stirring solution of the proper chalcone (1 eq.) in acetic acid (15 mL) was added dropwise hydrazine hydrate (4 eq.). The reaction was stirred at reflux for 24–48 h and quenched with 200 mL of ice/water. The resulting suspension was extracted with chloroform (3 × 20 mL). The organics reunited were dried over sodium sulfate and evaporated in vacuo. Purification by column chromatography on silica gel with appropriate mixtures of ethyl acetate and nce with .1-phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (P7), orange solid (71% yield) mp 95–98 °C. 1H-NMR : δ 2.44 , 3.13 , 3.71 , 4.36 , 5.33 , 5.45 , 5.58 , 6.02–6.12 , 6.97 , 7.24–7.26 , 7.32 , 7.70 . 13C-NMR : δ 22.0 (CH3), 42.5 (C(4)H2, pyr), 59.8 (C(5)H2, pyr), 68.9 (CH2-O), 114.9 (2 × Ar), 118.0 (=CH), 124.2 (Ar), 125.6 (2 × Ar), 127.6 (Ar), 128.2 (2 × Ar), 128.9 (2 × Ar), 132.78 (Ar), 142.0 (=CH2), 153.7 , 160.4 (Ar), 168.6 (C=O). Calcd. for C20H20N2O2: C, 74.98; H, 6.29; N, 8.74. Found: C, 74.77; H, 6.19; N, 8.65.1-(3-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-5-(o-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (P9), yellow solid (61% yield); mp 110–112 °C. 1H-NMR : δ 1.78 , 1.83 , 2.47 , 2.48 , 3.00 , 3.74 , 4.57 , 5.52 , 5.74 , 6.96 , 7.01–7.03 , 7.13–7.20 , 7.69 . 13C-NMR : δ 18.3 (CH3), 19.5 (CH3), 21.9 (CH3), 25.9 (CH3), 41.7 (C(4)H2, pyr), 57.0 (C(5)H2, pyr), 65.0 (CH2-O), 114.9 (2 × Ar), 119.2 (=CH), 123.9 (Ar), 124.0 (Ar), 126.6 (Ar), 127.4 (Ar), 128.2 (2 × Ar), 130.7 (Ar), 134.1 (Ar), 138.6 (C=), 139.9 (Ar), 153.8 , 160.7 (Ar), 168.5 (C=O). Calcd. for C23H26N2O2: C, 76.21; H, 7.23; N, 7.73. Found: C, 76.33; H, 7.37; N, 7.66.1-(3-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-5-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (P10), orange solid (67% yield); mp 120–122 °C. 1H-NMR : δ 1.79 , 1.84 , 2.34 , 2.45 , 3.13 , 3.71 , 4.58 , 5.51-5.54 , 5.55 , 6.97 , 7.04–7.08 , 7.14–7.24 , 7.70 . 13C-NMR : δ 18.3 (CH3), 21.5 (CH3), 22.0 (CH3), 25.9 (CH3), 42.5 (C(4)H2, pyr), 59.80 (C(5)H2, pyr), 65.0 (CH2-O), 114.9 (2 × Ar), 119.3 (=CH), 122.6 (Ar), 124.0 (Ar), 126.2 (Ar), 128.2 (2 × Ar), 128.4 (Ar), 128.8 (Ar), 138.5 (Ar), 138.6 (C=), 142.0 (Ar), 153.8 , 160.7 (Ar), 168.6 (C=O). Calcd. for C23H26N2O2: C, 76.21; H, 7.23; N, 7.73. Found: C, 76.33; H, 7.17; N, 7.85.1-(3-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (P11), yellow oil (75% yield). 1H-NMR : δ 1.78 , 1.83 , 2.32 , 2.43 , 3.11–3.15 , 3.67–3.74 , 4.57 , 5.52–5.58 , 6.96 , 7.10–7.14 , 7.68–7.70 . 13C-NMR : δ 18.3 (CH3), 21.1 (CH3), 22.0 (CH3), 25.9 (CH3), 42.5 (C(4)H2, pyr), 59.6 (C(5)H2, pyr), 64.9 (CH2-O), 114.9 (2 × Ar), 119.2 (=CH), 123.9 (Ar), 125.6 (2 × Ar), 128.2 (2 × Ar), 129.5 (2 × Ar), 137.2 (Ar), 138.7 (C=), 139.1 (Ar), 153.8 , 160.6 (Ar), 168.6 (C=O). Calcd. for C23H26N2O2: C, 76.21; H, 7.23; N, 7.73. Found: C, 76.01; H, 7.25; N, 7.88.1--3-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (P12), orange solid (97% yield); mp 117–119 °C. 1H-NMR : δ 1.78 , 1.83 , 2.28 , 2.43 , 2.47 , 3.00 , 3.74 , 4.57 , 5.51 , 5.71 , 6.89–7.01 , 7.68 . 13C-NMR : δ 18.3 (CH3), 19.4 (CH3), 20.9 (CH3), 21.9 (CH3), 25.8 (CH3), 41.9 (C(4)H2, pyr), 56.8 (C(5)H2, pyr), 64.9 (CH2-O), 114.8 (2 × Ar), 119.2 (=CH), 124.0 (Ar), 124.1 (Ar), 127.3 (Ar), 128.2 (2 × Ar), 131.5 (Ar), 133.9 (Ar), 136.9 (Ar), 137.1 (Ar), 138.7 (C=), 153.8 , 160.6 (Ar), 168.6 (C=O). Calcd. for C24H28N2O2: C, 76.56; H, 7.50; N, 7.44. Found: C, 76.41; H, 7.41; N, 7.53.1-(5-(2-chlorophenyl)-3-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (P13), orange solid, (73% yield); mp 131–134 °C. 1H-NMR : δ 1.78 , 1.82 , 2.50 , 3.05 , 3.84 , 4.57 , 5.50 , 5.92 , 6.95 , 7.07–7.09 , 7.21–7.23 , 7.40–7.42 , 7.68 . 13C-NMR : δ 18.3 (CH3), 21.9 (CH3), 25.8 (CH3), 41.5 (C(4)H2, pyr), 57.6 (C(5)H2, pyr), 65.0 (CH2-O), 114.9 (2 × Ar), 119.3 (=CH), 123.7 (Ar), 125.9 (Ar), 127.3 (Ar), 128.2 (2 × Ar), 130.0 (Ar), 131.7 (Ar), 138.6 (Ar), 138.7 (C=), 154.1 , 160.8 (Ar), 168.7 (C=O). Calcd. for C22H23ClN2O2: C, 69.01; H, 6.06; N, 7.32. Found: C, 69.19; H, 6.13; N, 7.17.1-(5-(3-chlorophenyl)-3-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (P14), white solid, (61% yield); mp 85–88 °C. 1H-NMR : δ 1.78 , 1.83 , 2.45 , 3.12 , 3.74 , 4.58 , 5.50 , 5.55 , 6.97 , 7.14 , 7.22–7.29 , 7.69 . 13C-NMR : δ 18.3 (CH3), 21.9 (CH3), 25.8 (CH3), 42.4 (C(4)H2, pyr), 59.4 (C(5)H2, pyr), 65.0 (CH2-O), 114.9 (2 × Ar), 119.2 (=CH), 123.6 (Ar), 123.9 (Ar), 125.8 (Ar), 127.9 (Ar), 128.2 (2 × Ar), 130.2 (Ar), 134.8 (Ar), 138.8 (C=), 143.9 (Ar), 153.8 , 160.8 (Ar), 168.8 (C=O). Calcd. for C22H23ClN2O2: C, 69.01; H, 6.06; N, 7.32. Found: C, 68.84; H, 5.99; N, 7.35.1-(5-(4-chlorophenyl)-3-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (P15), yellow solid (72% yield); 57–60 °C. 1H-NMR : δ 1.78  1.82 , 2.42 , 3.10 , 3.72 , 4.57 , 5.49-5.51 , 5.54 , 6.96 , 7.18 , 7.29 , 7.68 . 13C-NMR : δ 18.3 (CH3), 21.9 (CH3), 25.8 (CH3), 42.3 (C(4)H2, pyr), 59.2 (C(5)H2, pyr), 65.0 (CH2-O), 114.9 (2 × Ar), 119.2 (=CH), 123.7 (Ar), 127.1 (2 × Ar), 128.2 (2 × Ar), 129.0 (2 × Ar), 133.3 (Ar), 138.7 (=C=), 140.5 (Ar), 153.7 , 160.8 (Ar), 168.7 (C=O). Calcd. for C22H23ClN2O2: C, 69.01; H, 6.06; N, 7.32. Found: C, 68.91; H, 6.05; N, 7.41.1--3-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (P16), white solid (80% yield); mp 128–130 °C. 1H-NMR  1.77  1.82 , 2.49 , 2.99–3.04 , 3.79–3.86 , 4.56 , 5.50 , 5.84–5.86 , 6.94 , 7.01 , 7.20 , 7.43 , 7.67 . 13C-NMR : δ 18.3 (CH3), 21.9 (CH3), 25.9 (CH3), 41.4 (C(4)H2, pyr), 57.3 (C(5)H2, pyr), 64.9 (CH2-O), 114.9 (2 × Ar), 119.1 (=CH), 123.5 (Ar), 127.0 (Ar), 127.6 (Ar), 128.2 (2 × Ar), 129.8 (Ar), 132.4 (Ar), 133.8 (Ar), 137.3 (Ar), 138.8 (C=), 154.1 , 160.8 (Ar), 168.8 (C=O). Calcd. for C22H22Cl2N2O2: C, 63.32; H, 5.31; N, 6.71. Found: C, 63.17; H, 5.35; N, 6.76.1--5-methyl-3-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (P17), white solid (63% yield); mp 132–135 °C. 1H-NMR : δ 1.79  1.84 , 2.47 , 3.20 , 3.77 , 4.59 , 5.51–5.54 , 5.65 , 6.98 , 7.32–7.35 , 7.42–7.44 , 7.55–7.57 , 7.72 . 13C-NMR : δ 18.3 (CH3), 22.0 (CH3), 25.9 (CH3), 42.4 (C(4)H2, pyr), 59.6 (C(5)H2, pyr), 65.0 (CH2-O), 114.9 (2 × Ar), 119.2 (=CH), 123.9 (Ar), 126.1 (2 × Ar), 127.1 (2 × Ar), 127.3 (Ar), 127.7 (2 × Ar), 128.2 (2 × Ar), 128.8 (2 × Ar), 138.7 (C=), 140.6 (Ar), 140.8 (Ar), 141.0 (Ar), 153.8 , 160.7 (Ar), 168.7 (C=O). Calcd. for C28H28N2O2: C, 79.22; H, 6.65; N, 6.60. Found: C, 79.41; H, 6.69; N, 6.45.1-(3-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)-5--4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (P18), orange solid (77% yield); mp 78–80 °C. 1H-NMR : δ 1.75 , 1.84 , 2.45 , 3.19 , 3.77 , 4.55 , 5.47–5.52 , 5.63–5.67 , 6.95 , 7.31–7.34 , 7.40–7.45 , 7.68–7.74 , 7.77–7.81 . 13C-NMR : δ 18.3 (CH3), 22.0 (CH3), 25.9 (CH3), 42.5 (C(4)H2, pyr), 60.0 (C(5)H2, pyr), 65.0 (CH2-O), 114.9 (2 × Ar), 119.1 (=CH), 123.5 (Ar), 123.9 (Ar), 124.5 (Ar), 125.9 (Ar), 126.2 (Ar), 127.6 (Ar), 128.0 (Ar), 128.2 (2 × Ar), 129.0 (Ar), 132.9 (Ar), 133.3 (Ar), 138.8 (C=), 139.2 (Ar), 153.9 , 160.7 (Ar), 168.8 (C=O). Calcd. for C26H26N2O2: C, 78.36; H, 6.58; N, 7.03. Found: C, 78.21; H, 6.71; N, 6.99.® Red (200 µM). Stock solutions of the test inhibitors were prepared in DMSO and added to the reactions to yield a final concentration of 4% (v/v) DMSO. The reactions were initiated with the addition of hMAO-A (0.0075 mg protein/mL) or hMAO-B (0.015 mg protein/mL), and incubated at 37 °C for 20 min in a convection oven. At endpoint, the reactions were terminated with the addition of 10 μL pargyline (20 mM) and 10 μL (R)-deprenyl (5 mM) for hMAO-A and hMAO-B, respectively. The concentrations of resorufin in the reactions were determined by fluorescence spectrophotometry  [® Red (200 µM), 4% DMSO as co-solvent, 10 μL pargyline (20 mM) for hMAO-A and 10 μL (R)-deprenyl (5 mM) for hMAO-B. The IC50 values were determined by constructing sigmoidal curves of MAO catalytic activity versus the logarithm of inhibitor concentration. For this purpose, the one site competition model incorporated into the Prism 5 software package  was used. All experiments were carried out in triplicate and the IC50 values are expressed as mean ± standard deviation (SD) [Microsomes from insect cells expressing recombinant hMAO-A or hMAO-B (5 mg protein/mL) served as enzyme sources, and were pre-aliquoted and stored at −70 °C. All enzymatic reactions were carried out to a final volume of 200 µL in black 96-well microtiter plates (Corning). Potassium phosphate buffer  served as reaction solvent. The reactions contained kynuramine (50 µM), various concentrations of the test inhibitor (0.003–100 μM), horseradish peroxidase (1 unit/mL) and Amplex 590 nm) . Resorufion (SD) .P1–P5 were carried out on the commercially available 250 × 10 mm I.D. Chiralpak AD  column using pure ethanol elution mode. The temperature was set at 25 °C. The flow-rate was 2.5 mL min−1. HPLC apparatus consisted of a Perkin-Elmer  200 LC pump equipped with a Rheodyne  injector, a 5 mL sample loop, an HPLC Perkin-Elmer oven and a Perkin-Elmer detector. The signal was acquired and processed by Clarity software . The amounts of racemic samples resolved for single chromatographic runs ranged from 1 to 10 mg.Semipreparative HPLC separations of the enantiomers of 2 to uniquely minimize the positions of the water molecules. In the second stage, the entire system was energy minimized by applying a harmonic potential of 10 kcal/mol·Å2 only to the protein α carbons.The crystal structures of hMAO-A (PDB code 2Z5X) and hMAO-B (PDB code 4A79) were taken from the Protein Data Bank . Moleculp-Prenyloxyaryl moiety at C3 oriented the selectivity toward the B isoform. Moreover, single enantiomers obtained by HPLC enantioseparation were tested. The outcomes highlighted the importance of the C5 stereochemistry, displaying a better hMAO-B recognition of the (R)-enantiomers within this scaffold. In agreement with the experimental results, docking studies suggested that (R)-enantiomers would be able to form better H-bond interactions with respect to (S)-enantiomers and highlighted the limited space availability in the binding site region close to FAD cofactor, which may hamper the accommodation of excessively bulky groups at C5. Moreover, the bigger lipophilic pocket present in hMAO-B compared to that observed in hMAO-A may better welcome longer hydrophobic chains at C3, thus contributing to the selectivity of the corresponding ligands toward the B isoform.This research paper aimed to reinforce the privileged relationship between the pyrazoline scaffold and the hMAO inhibitory activity. These compounds were obtained through a well-established synthetic pathway. The substitution pattern at N1, C3 and C5 positions can finely modulate the inhibitory activity and isoform selectivity of these heterocyclic compounds. Bulky aromatic groups at C5 were not tolerated.


The historical developments of endovascular neurosurgery in the Netherlands are described. TM stents were inserted. Flow characteristics were evaluated with dye. In the second part of the study 10 experimental aneurysms of the cervical carotid artery in beagles were treated with stents. Interestingly, the positioning of the stents was evaluated with angioscopy, as the stents were barely visible under fluoroscopy. All 10 aneurysms occluded and all but one carotid artery remained patent.Endovascular Neurosurgery in The Netherlands started in 1992 when André Grotenhuis at Radboudumc, Nijmegen studied the effect of balloon-expandable stents in the treatment of experimental carotid aneurysms  in neurovascular arteriovenous shunts during his year in Toronto and continued these efforts in Leiden after his return . The eluBesides this clinical research, the group in Nijmegen has begun to focus on the complex mechanism of aneurysm wall inflammation and break down, causing rupture. This is studied in various animal models .Since The Netherlands is a relatively small country (around 17 million inhabitants in an area less than a third of that of the state of New York), nationwide research initiatives are also feasible and the MRCLEAN trial, looking into the endovascular treatment of acute ischemic stroke, is a very successful recent example of this . EndovasDue to the growing awareness that neurovascular diseases were being treated by different specialties, whose efforts would benefit from nationwide collaboration, 2015 saw the birth of the Dutch Neurovascular Society (“Netherlands Neurovasculair Genootschap”). This society brought together neurovascular neurosurgeons, vascular neurologists, and neurointerventional radiologists. Within the context of this society, the abovementioned pioneering led to the gradual acceptance of non-radiologists in the field of neurointerventional procedures in The Netherlands. The Dutch Neurovascular Society developed and approved training standards for neuroendovascular training and, thus, further opened up the neurointerventional field to non-radiologists such as neurologists and neurosurgeons. As a result, 5 Dutch neurologists/neurosurgeons have now followed a neuroendovascular training program at 5 different Dutch neurointerventional sites. In addition, young neurosurgeons from abroad  have also done a hybrid neurovascular fellowship at the Radboudumc in Nijmegen.In order to monitor, and eventually improve, quality of care for neurosurgical patients in The Netherlands, the Dutch Society of Neurosurgery initiated a nationwide registry, , in 2010. This was spearheaded by Jeroen Boogaarts and one of the four patient subgroups in this registry is the group of patients who suffered a subarachnoid hemorrhage. The results of endovascular neurosurgery are among the many outcome parameters being evaluated with this database. Such initiatives demonstrate the ability of medical specialists, from different specialties and from different medical centers, within a country to combine forces in an effort to improve the quality of their health care program.Especially with the increasing need for (emergency) endovascular treatments, such collaboration also becomes increasingly important within one center or one region. In many centers in The Netherlands, neurointerventionalists have combined forces with body interventionalists to allow a 24/7 coverage for stroke treatment. Consequently, one can come across interventional neurologists, interventional neurosurgeons, interventional neuroradiologists, and/or interventional body-radiologists working side-by-side, in the angiosuite. In some parts of the world, this would not be surprising, but in Europe this is still quite a unique situation.Despite these developments, it remains difficult to envision what lies ahead. Due to lengthy training programs and persisting political barriers for cross-training, it is still not self-evident for a neurosurgeon to enter the neuroendovascular field. And once a neurosurgeon has been trained successfully, he/she requires a position where both open and endovascular neurosurgery may be performed. This requires cooperation from two departments in the appointment of such an individual as in The Netherlands any X-ray-guided procedure falls under the responsibility of the Department of Radiology. In The Netherlands, there has not yet been a precedent where such an individual decided to dedicate him-/herself fully to endovascular treatment. Therefore, it is unclear what the implications of such a decision would be for his/her license to practice.According to the authors more neurosurgeons should be dual-trained and the political barriers need to be removed for the following reasons.A young Dutch neurosurgeon interested in the neurovascular field can be trained in open neurovascular techniques without any political barrier. As the case-load in most, if not all Dutch Neurosurgical Centers, however, has become so low  it is questionable whether enough experience can be obtained in open vascular neurosurgery. Beside that the efficiency of an open neurosurgeon being on call just for clipping is very low considering the case-load. It seems that this discourages young neurosurgeons for choosing the neurovascular subspecialty. This can be solved either by further centralization of neurovascular neurosurgery or by dual-training or by doing both. It is the experience of the authors and of others that dual-training clearly leads to cross-pollination and thus improvement of the skills in both endovascular and open neurovascular techniques .Dual-trained neurosurgeons can offer a more efficient and less expensive 24/7 emergency service for neurovascular diseases as compared to a service where this is performed by interventional radiologists and open only neurosurgeons.As there are no results yet from the above-mentioned Dutch database we cannot shed light whether a dual-trained individual is better or an open-only neurosurgeon.As of 2017 there are 3 dual trained interventional neurosurgeons in The Netherlands among a total of 26 practicing neurointerventionalists.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacteroides, Escherichia-Shigella, Phascolarctobacterium, Incertae-Sedis  and Incertae-Sedis (Erysipelotrichaceae Family) could be the mechanism by which bran-fried AR enhanced the therapeutic effect. Correlation analysis revealed that the modulation on intestinal microbiota was closely related to the secretion and expression of cytokines and gastrointestinal hormones. These findings can help us to understand the role and significance of bran-fried AR against SDS.Atractylodis Rhizoma (AR), a kind of well-known traditional Chinese medicine (TCM), has a long history of being used to treat spleen-deficiency syndrome (SDS). Stir frying with bran is a common method of processing AR, as recorded in the Chinese Pharmacopoeia, and is thought to enhance the therapeutic effect in TCM. Our previous studies have confirmed that bran-fried AR is superior to raw AR in terms of the improvement of gastrointestinal tract function. However, the biological mechanism of action is not yet clear. Here, we report the difference between raw and bran-fried AR in terms of the modulatory effect of intestinal microbiota. We found that the composition of intestinal microbiota of SDS rats changed significantly compared with healthy rats and tended to recover to normal levels after treatment with raw and bran-fried AR. Nine bacteria closely related to SDS were identified at the genus level. Among them, the modulatory effect between the raw and bran-fried AR was different. The improved modulation on  Qi and blood [Spleen-deficiency syndrome (SDS) is a common clinical syndrome of traditional Chinese medicine (TCM). According to the TCM theory, the spleen governs the transportation and transformation in the organism, which is the foundation of acquired constitution and the biochemical source of nd blood . The maind blood . Splenicnd blood ,4. Many nd blood ,6.Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz. It has the functions of drying dampness, invigorating the spleen, dispelling wind-cold infections and improving eyesight [Atractylodis Rhizoma (AR), called CangZhu in China, is the dried rhizome of eyesight . AR, witeyesight  and streeyesight  to a greIn our previous research, we compared the difference between raw and bran-fried AR in terms of chemical composition, pharmacological action, absorption, distribution, metabolism, and excretion ,11,12. YWe speculated that AR could improve the intestinal microbiota imbalance caused by SDS and that the modulatory effect of raw and bran-fried AR was different. To prove this conjecture, we duplicated the model of SDS by a compound factor method, which was composed of poor diet, excessive fatigue and severe diarrhea caused by bitter and cold Chinese medicine. 16S ribosomal DNA (rDNA) gene sequencing technology was used to study the composition of intestinal microbiota in SDS rats before and after treatment with raw and bran-fried AR. From this, the processing principle of bran-frying could be explained by comparing the difference in the modulatory effect on intestinal microbiota between raw and bran-fried AR.Atractylodes lancea (Thunb.) DC. by Professor Liang Xu from the School of Pharmacy, Liaoning University of TCM . Bran-fried AR was made in our laboratory using the same batch of raw AR in accordance with the method outlined in the Chinese Pharmacopoeia . Specifically, 30 g of bran was put into a heated pot, which was hot enough to make the bran smoke immediately. As soon as possible, 200 g of AR was added to the pot and stir-fried quickly until the surface was dark yellow. The bran-fried AR was removed and cooled down, while the bran was sifted out. Voucher specimens  were deposited in the Herbarium of Liaoning University of TCM . Both the raw and bran-fried AR were crushed into a fine powder and sieved through a 160 mesh stainless steel sieve. Raw and bran-fried AR powder suspensions (375 mg·mL−1) were then prepared with normal saline for reserve. Finally, senna leaf (Batch number: Z13020471), purchased from the Outpatient Department of TCM of the Affiliated Hospital of Liaoning University of TCM , was boiled for 30 min, filtered by gauze and concentrated into a decoction (0.5 g crude drug per milliliter) for reserve.AR, collected from the Herb Planting Base of Luotian , was identified as the dried rhizome of n = 10 per group): control group (CG), model group (MG), raw AR group (RA) and bran-fried AR group (BFA).Specific pathogen Free (SPF) male Sprague Dawley (SD) rats  were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (license number: SCXK(Liao)2010-0001, Liaoning, China) and housed at a certified animal experimental laboratory with a 12 h light/dark cycle, a constant temperature of 25 ± 1 °C, and 55–75% relative humidity. Animals were allowed free access to food and water. After acclimatization for one week, the rats were randomly assigned to four groups (−1) on odd-numbered days. On even-numbered days over this period, the rats were forced to swim exhaustedly and were only fed cabbage (freely). For days 15–21 of the experiment, the rats were intragastrically administered the senna leaf decoction (10 mL·kg−1). This model spanned 3 weeks, during which the sawdust bedding was wetted. From the beginning of the 22nd day of the experiment, the RA and BFA groups were respectively intragastrically administered raw and bran-fried AR powder suspension (3750 mg·kg−1) for seven consecutive days, during which the control and model groups were gavaged with saline daily. All of the studies on animals were approved by the Animal Ethics Committee of the Affiliated Hospital of Liaoning University of TCM .For all of the groups except the control group, the rat model of SDS was established via a compound factor method, which involved an irregular diet, excessive fatigue and severe diarrhea caused by bitter and cold Chinese medicine. For days 1–14 of the experiment, the rats were intragastrically administered lard  after overnight fasting. Abdominal aorta blood samples were collected and centrifuged under the condition of 4000 r·min−1 for 10 min at 4 °C. Serum was separated and stored at −80 °C until analysis. Enzyme-linked immunosorbent assay (ELISA) was used for quantitative detection of serum tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), immunoglobulin G (IgG), Na+/K+-ATPase, motilin (MTL), gastrin (GAS), trypsin (TRY), amylase (AMS), and somatostatin (SS). Each operation was carried out in strict accordance with the kit’s instructions. All kits were purchased from Shanghai Kexing Trading Co., Ltd .When the administration was over, all animals were anesthetized by intraperitoneal injection of chloral hydrate . A UV spectrophotometer and agarose gel electrophoresis were used for concentration and purity testing. DNA sample quality was available when the total amount of DNA was greater than 500 ng. The V3 and V4 region of the 16S rDNA gene was amplified with specific barcoded primers. The primer sequences were as follows: 341F: 5′-CCTACGGGNGGCWGCAG-3′; 785R: 5′-GACTACHVGGGTATCTAATCC-3′ . The PCRhttp://www.arb-silva.de/). The classification was based on Bergey’s taxonomy and the default threshold value was set at 80%. In a single sample, the microbial abundance at each taxonomic level was calculated by the percentage of all sequencing items to the total sequencing items at that level. Alpha diversity indices, including the Chao index and Shannon index, were calculated by Mothur software. OTU sequences were aligned to the SILVA database by PyNAST software. The weighted UniFrac distance matrix between communities was generated by QIIME software. Principal Co-ordinates Analysis (PCoA) of the weighted UniFrac matrix was carried out by R software. LEfSe software was used to analyze the difference in relative species abundance between the control group and model group. Bacteria with linear discriminant analysis (LDA) scores greater than 3 and p < 0.05 were selected as the biomarkers of SDS, for which the modulatory effect of raw and bran-fried AR were compared.Off-line paired-end sequences were joined by FLASH software, after which unmatched, mismatched and low abundance sequences were removed by Mothur software. UCHIME software was used to remove chimeric sequences to obtain clean sequences. The sequences were clustered into operational taxonomic units (OTUs) according to their sequence similarity by UCHIME software. The threshold value of similarity was set at 97%; that is, the sequences with similarity higher than 97% were clustered into an OTU. Sparse sequences were discarded in the process of OTU clustering. The OTU sequences were systematically classified by an RDP classifier referring to the SILVA database   , IL-6, IgG, and SS (p < 0.01) as well as increased serum levels of Na+/K+-ATPase, MTL, GAS, TRY, and AMS (p < 0.01) in the SDS rats. Among the AR treated groups, bran-fried AR was significantly superior to raw AR in regulating the serum levels of SS, MTL, GAS, TRY, and AMS (p < 0.01). Although bran-fried AR was also superior to raw AR in regulating the serum levels of TNF-α, IL-6, IgG, and Na+/K+-ATPase, there were no significant differences.Compared with the control group, rats in the SDS model group had significantly higher expression in the serum levels of TNF-α, IL-6, IgG, and SS ( < 0.01) A–C,I, an < 0.01) D–H. Compp < 0.01). After the treatment with raw and bran-fried AR, the Chao and Shannon index values of these two groups were significantly higher than that of the model group. Furthermore, the Chao and Shannon index values of the bran-fried AR group were significantly higher than that of the raw AR group (p < 0.05).In the chart of the Shannon rarefaction curve, the curve of each sample tended to be smooth, suggesting the sequencing depth was sufficient to capture the biodiversity present in the samples A. Alpha The weighted UniFrac took into account phylogenetic distances between the OTUs, so that it captured differences not only at the level of individual phylotypes, but also at levels of higher taxonomic category. The weighted UniFrac PCoA A,B reveaBacteroidetes, Firmicutes, Tenericutes, Proteobacteria, Actinobacteria, Spirochaetae, and Cyanobacteria , while the abundance of Spirochaetae was significantly decreased . After administration of raw and bran-fried AR, the abundance of Proteobacteria and Spirochaetae tended to recover to the normal level. Specifically, the modulatory effect of bran-fried AR was found to be significantly superior to that of raw AR . Compared with the control group, the abundance of Tenericutes , Actinobacteria , and Cyanobacteria  changed significantly in the model group, although there were no favorable modulatory effects following administration of raw or bran-fried AR . Additionally, as the dominant bacteria at the phylum level the abundance of Bacteroidetes and Firmicutes did not significantly change in the model group compared with the control group. However, the ratio of Bacteroidetes to Firmicutes significantly decreased after administration of raw and bran-fried AR (p < 0.05) .At the phylum level, we found that the majority of the intestinal microbiota consisted of species from bacteria C. There Ruminococcus, Bacteroides, Parasutterella, Escherichia-Shigella, Phascolarctobacterium, Enterobacter, Acinetobacter, Incertae-Sedis , and Incertae-Sedis (Erysipelotrichaceae Family), were finally identified and considered to be closely associated with SDS. A cladogram was obtained from the LEfSe analysis of 16S sequences from each fecal sample. This cladogram, shown in Linear discriminant analysis (LDA) effect size (LEfSe) is a software that can perform linear discriminant analysis among different groups to find communities or species that exist at statistically significant differences. Typically, the horizontal axis is the LDA score value, while the vertical axis consists of taxa with significant differences among groups, ranked according to the size of their LDA score value. The longer the length of the bar graph, the more significant the difference is. Different colors of bar graph may be used to indicate that a specific taxon corresponds to a higher abundant sample group. On the basis of the result of LEfSe analysis between the control group and the model group A, nine bParasutterella (p < 0.05). Conversely, bran-fried AR was significantly superior to raw AR in the regulation of Bacteroides, Escherichia-Shigella, Phascolarctobacterium, Incertae-Sedis  and Incertae-Sedis (Erysipelotrichaceae Family) (p < 0.05). There was no significant difference between raw and bran-fried AR in the regulation of Ruminococcus, Enterobacter or Acinetobacter.The abundance of all bacteria and biomarkers at the genus level among the four groups is shown in Ruminococcus, Escherichia-Shigella and Phascolarctobacterium were significantly and positively correlated with TNF-α, IL-6, IgG and SS, and significantly and negatively correlated with MTL, GAS, TRY and AMS. In addition, Bacteroides and Escherichia-Shigella were significantly and negatively correlated with Na+/K+-ATPase —such as butyrates—in the intestine. SCFAs play an important role in maintaining intestinal homeostasis. Butyrates are able to bind the G-protein coupled receptors GPR43, which are mainly expressed in the colonic epithelium, and thus can aid in maintaining normal intestinal permeability while suppressing mucosal inflammation [Parasutterella occupies a specific intestinal niche and may be involved in the metabolism of bile acid and cholesterol [Escherichia-Shigella and Enterobacter are common conditional pathogens. When the intestinal microbiota is balanced, they coexist harmoniously with beneficial bacteria. When the normal microecological balance is broken, they can cause endogenous infection [Bacteroides, Escherichia-Shigella, Phascolarctobacterium, Incertae-Sedis  and Incertae-Sedis (Erysipelotrichaceae Family). This may therefore be one of the mechanisms by which bran-fried AR enhanced the therapeutic effect.Based on the changes in composition of intestinal microbiota at the phylum level, we have a preliminarily understanding of the possible mechanism by which bran-fried AR enhanced the therapeutic effect, but it is not in-depth. As such, we further identified nine intestinal bacteria, such as ammation . Parasutlesterol . Escherinfection . The chaIn this study, we improved the understanding of the mechanisms through which AR treats SDS. Furthermore, we revealed possible mechanisms by which bran-fried AR enhances its therapeutic effect by comparing the modulatory effect on intestinal microbiota of raw and bran-fried AR in a rat model of SDS. However, the material basis of this modulatory effect remains unclear. In recent years, a large number of literatures have reported that polysaccharides in TCM can improve intestinal microbiota ,40. Liu Bacteroides, Escherichia-Shigella, Phascolarctobacterium, Incertae-Sedis  and Incertae-Sedis (Erysipelotrichaceae Family). These findings can help our understanding of the role and significance of bran-fried AR. In addition, the research method of this experiment can provide new ideas for the study of mechanism of action and processing principle of TCM.Our findings suggest that the biological mechanism by which AR treats SDS may be related to the modulation of intestinal microbiota. The mechanism by which bran-fried AR enhanced the therapeutic effect could be associated with improved modulation on


Astragalus membranaceus (Fisch.) Bge. Var. mongohlicus, (Bge.) Hsiao, and Astragalus membranaceus (Fisch.) Bge. (Fam. Leguminosae), whose extracts of dried roots are processed via homogenization-assisted negative pressure cavitation extraction. Geographic factors and extraction methods have impacts on the pharmaceutical and chemical profiles of AR. Therefore, the levels of the major bioactive constituents of AR, including polysaccharides, saponins, and flavonoids, may not be consistent in different batches of extract, and the pharmaceutical efficacy of these bioactive ingredients may vary depending on the source. Therefore, the present review mainly focuses on the consistency of the available sources of AR and extracts and on the investigation of the biological functions and mechanisms of action of AR and of its major bioactive constituents. Furthermore, it will also include a discussion of the most popular AR composite formulae to further elucidate their chemical and biological profiles and understand the pharmaceutical value of AR.Astragali Radix (AR), a Chinese materia medica (CMM) known as Huangqi, is an important medicine prescribed in herbal composite formulae (Fufang) by Traditional Chinese medicine (TCM) practitioners for thousands of years. According to the literature, AR is suggested for patients suffering from “Qi”- and “Blood”-deficiencies, and its clinical effects are reported to be related to anti-cancer cell proliferation, anti-oxidation, relief of complications in cardiovascular diseases, etc. The underlying cell signaling pathways involved in the regulation of these various diseases are presented here to support the mechanisms of action of AR. There are two botanical sources recorded in China Pharmacopoeia :  Huangqi  is one of the most popular herbal medicines in traditional Chinese medicine (TCM), firstly recorded in “Shennong Bencao Jing” . AR tastes sweet and tepid and is extensively used to tonify spleen and lung functions according to the TCM treatment principles. TCM considers that AR can enrich “Qi” , treat stagnant blood flow due to qi deficiency, and improve “Yin” deficiency by promoting diuresis to remove oedema due to inadequate transformation of dampness and “Qi”. In the modern pharmaceutical principle, AR enhances the immune system and the blood circulation. Details of these properties and their mechanisms of action will be reviewed and summarized in the following paragraphs.Astragalus L. consists of 278 species, 2 subspecies, 35 varieties, and 2 forma found predominantly within China . I. IAstragrphology . Furtheromal DNA ,5. Indeegholicus . FurtherA. membranaceus has pharmaceutical properties. Kim and co-workers compared the chemical and genetic composition of aerial parts, including flower and stems, and root parts of A. membranaceus. A total of 10 mevalonate pathways involved in astragaloside biosynthesis were found and identified by Illumina/Solexa HiSeq2000 }-glucoside, and calycosin 7-O-β-d-(6″-acetyl)-glucoside and (−)-methylinissolin 3-O-β-d-glucoside, showed inhibitory effects on NO production in Raw 264.7 cells after LPS-induced chronic inflammation [50 values ranging from 2.7 μM to 6.1 μM [50 value of 20.1 μM. Furthermore, the AR flavonoids calycosin and calycosin-7-O-β-d-glucoside accelerated glomerular endothelial cell apoptosis rate [The effects of flavonoids derived from AR on the immune functions were determined in Raw 264.7 cells . Four neammation . The inhammation . These to 6.1 μM . Isoliqusis rate . Immunomsis rate . AR flavsis rate ,61,62,63The AR herbal extract dramatically decreased total cholesterol and LDL cholesterol and aortic fatty streak area levels; on the other hand, it exhibited the potential ability of increasing HDL cholesterol levels in atherosclerotic rabbits . BesidesAR showed promising effects in improving biochemical and histological changes of heart failure . The theIntraperitoneal injection of 50% CCl4 twice a week for two months and intraperitoneal injection of dimethylnitrosamine (DMN) are the typical methods for inducing liver fibrosis in Sprague–Dawley rats ,69. The The angiogenic function of the polysaccharide-enriched AR fraction was revealed in zebrafish. A cocktail containing 300 nM VEGFR tyrosine kinase inhibitor II was applied to Tg(fli-1a: EGFP)y1 and Tg(fli-1a:nEGFP)y7 embryos for 3 h, to induce the loss of blood vessels. After challenging with a polysaccharide-enriched AR fraction, a rescue effect was shown by a statistically significant increase of blood vessels in a dose-dependent manner .O-β-d-glucoside) by monitoring the expression of erythropoietin (EPO) and its upstream regulator, hypoxia-induced factor (HIF-1α) in cultured HEK293T cells. These four flavonoids could upregulate EPO and HIF-1α at both the transcriptional and the translational levels [Furthermore, our group has investigated the erythropoietic functions of the four major flavonoid constituents of AR , the Adjuvant (Chen CMM), the Assistant (Zuo CMM), and the Guide (Shi CMM), according to the different roles they play in the prescription. At times, some CMM may have two or more roles depending on their diverging properties. The four CMMs, supplementing one another, exert a curative role together. However, not every prescription is composed of four kinds of CMMs; there may be less or more than four, with the Emperor CMM being the dominant one, depending on the diseases, the characteristics of the CMMs, and the therapeutic needs. AR is often used as the Emperor CMM with little or no toxicity in most TCM formulae. The following three examples illustrate the works our team has researched in detail.In the clinical practice of TCM, AR is often combined with other CMMs in composite herbal formulae (Fufang) for the treatment of various diseases, according to patients’ individual constitution and symptoms. Indeed, its roles in the different prescription formula are not the same. The principles for composing a TCM prescription, as first described in the Huang Di Nei Jing (The Inner Canon of the Yellow Emperor), stipulate that the prescription may include four different CMMs. They are the Yang Qi” of spleen and lungs and tonifying “Wei Qi” of the stomach [Yu Ping Feng San (YPFS), an ancient TCM prescription, consists of AR, Atractyldis Macrocephalae Rhizoma, and Saposhnikoviae Radix; here, AR plays the major functions in YUPS by enriching “ stomach ,85. Our  stomach . The co-Emperor function. The administration of BYHWD in chronic denervation models significantly increased the axonal regenerative abilities and neurite outgrowth [Buyang Huanwu prescription (BYHWD) is another popular ancient formula consisting of seven CMMs  which is designed to treat complications after cerebrovascular accident, paralysis, stroke, and their related complications. In this formula, AR also plays the utgrowth . This foutgrowth ,88,89.Emperor role. Previous studies have confirmed that DBT is able to improve the cardiovascular function by stimulating NO production in endothelia cells [Danggui Buxue Tang (DBT), consisting of two CMMs (AR and ASR), is a widely prescribed classical and simple formula. The pharmaceutical value of this prescription has been reported widely . AR actsia cells . Immune ia cells . On the ia cells . Furtheria cells . In vivoThe research on composite formulae is only possible with the advances in biomedical, chemical, and computational technology, by employing multidisciplinary approaches for investigating evidence-based aspects of TCM practice. Research linking a specified starting CMM, the relatively new systems biology, and experience-based TCM principles is vital to elucidate the complexity of TCM prescriptions . More reA. membranaceus and A. membranaceus var. mongholicus were compared. Homogenization-assisted negative pressure cavitation extraction is considered the optimal extraction method for polysaccharide-enriched AR to obtain consistent chemical ingredients for biological investigations. The pharmaceutical values of AR were summarized. The key values include immunomodulation, anti-oxidation activation, cardiovascular protection, inhibition of liver fibrosis, stimulation of blood regeneration, anti-obesity action, and pain-relieving properties. We believe that AR is one the most important natural medicines of TCM herbal composite formulae, and its promising biological functions should be further explored and tested for clinical application in conditions where conventional medicines may not be efficacious.The chemical compositions of various plant parts of


LSAT is a web-based microsatellite SSR marker designer tool specific for the Liliaceae family. It is developed using HTML, CSS, PHP,Perl and Java scripts. It works without extra add-ons on standard browsers. LSAT provides SSR primer designing service using theweb interface. It helps in SSR mining and primer design. LSAT is user friendly with customizable search parameters producing visualoutput having download options. The current version of LSAT is backed by two data sets, namely, lily EST (Expressed Sequence Tag)from NCBI and lily nr (non redundant) with 4,099 and 216,768 unigenes, respectively. LSAT will be updated regularly uponavailability of additional data (either EST and/or transcriptome) on Liliaceae.http://210.110.86.160/Lsat/Lsat.htmlLSAT is available for free at  Microsatellite markers are extensively used in plant breeding dueto its co-dominant nature, genomic abundance, and high degreeof polymorphism. With the advancement of sequencingtechnology, large number of sequences from wide range of plantspecies is made available in the public domains. This is a valuableresource for designing SSR marker. It could be a potential tool forQTL mapping and marker-assisted breeding of Lilium sp. Highpolymorphic SSRs were developed and validated for geneticdiversity and population structure in lily accessions study .DevelopLSAT input interface was developed using HTML and JAVAscript. LSAT is backed by two main data sets as shown in The customized input information is received by a PHP script togenerate the SSR search parameters file and the sequences datafile together to subsequently invoke MISA for running SSRsearch. The modified version of the MISA script generates two output files. One with statistical summary and another with SSRinformation like SSR characteristics and 200 bp flanking regionsin either side of each SSR locus. The PHP script will generate aHTML page with visualized SSR results for download. Theinterface further prompts users for primer design. Thus, a PHPscript runs primer3 (http://primer3.sourceforge.net/) with thehelp of several PERL scripts loaded on to the system to designSSR primer. Thus, the designed SSR primers are visualized as anoutput.LSAT is freely available for use. It is a user friendly, fast and arobust web-server of SSR marker specific for Liliaceae family. Itprovides a Graphical User Interface (GUI) with customizedparameters of SSR design with known statistical information.This is the unique feature of LSAT compared to other existingSSR tools. The information on SSR will help fast screening of SSRprimers prior to help in wet-lab validation. We plan to improveLSAT with increased the number of datasets on SSR at thebackend. We also plan to add user requested datasets to theinterface. The interface will be upgraded frequently withadvanced scripts by displaying results in graphicalrepresentation having advanced features for SSR marker.


All TNBC samples were stained with the same anti-AR antibody using the same immunohistochemistry protocol, and samples with ≥1% of AR-positive nuclei were deemed AR-positive TNBCs. Results: AR status shows population-specific patterns of association with patients’ overall survival after controlling for age, grade, population, and chemotherapy. We found AR-positive status to be a marker of good prognosis in US and Nigerian cohorts, a marker of poor prognosis in Norway, Ireland and Indian cohorts, and neutral in UK cohort. Conclusion: AR status, on its own, is not a reliable prognostic marker. More research to investigate molecular subtype composition among the different cohorts is warranted.Background: The androgen receptor (AR) has emerged as a potential therapeutic target for AR-positive triple-negative breast cancer (TNBC). However, conflicting reports regarding AR’s prognostic role in TNBC are putting its usefulness in question. Some studies conclude that AR positivity indicates a good prognosis in TNBC, whereas others suggest the opposite, and some show that AR status has no significant bearing on the patients’ prognosis. Methods: We evaluated the prognostic value of AR in resected primary tumors from TNBC patients from six international cohorts {US ( Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by the lack of estrogen (ER), progesterone (PR), and Her2 receptors. This designation masks the heterogeneity of this patient population and the challenge of stratifying them for optimal treatment selection ,2. Due tStudies have explored the prognostic role of AR in TNBC to better understand androgen action in TNBC, identify actionable factors that drive outcomes, and determine if testing for AR status should become part of routine clinical practice for TNBCs. However, there are conflicting reports about AR’s prognostic value in TNBC. While some studies report that AR-positivity is associated with better prognosis ,14, othep = 0.02; AR expression varied widely in our cohorts . In the US cohort, 25% of all cases were AR-positive: Twenty percent among African Americans (AA) and thirty percent among those of European decent  and Nigerian (p = 0.01) cohorts among all patients (p = 0.08), Norway (p = 0.08), and India (p = 0.02) There was no statistically significant difference in OS between AR-positive TNBCs and AR-negative TNBCs in the UK (p = 0.79) cohort among all patients, or among adjuvant chemotherapy-treated patients  present in the cohorts, and perhaps other covert modifiers of AR biology. Candidate modifiers in different populations might include patients’ biogeographic ancestry, AR splice variants, epigenetic factors, tumor microenvironment, and repeat length polymorphisms (RLPs) in the AR gene. For example, among the alternatively spliced transcripts known to be generated from the AR gene, the constitutively active variant AR-V7 is often expressed in TNBC cells along with the full-length mRNA, but it regulates a transcriptional program distinct from full-length AR, and may affect outcomes. At present, little is known about the expression of AR-V7 and other splice variants in TNBC tumors in different populations. Few studies have examined the RLPs or tumor microenvironment in AR positive/negative TNBC tumors and correlated these features with the tumors’ molecular subtypes. Moreover, amplification of and mutations in the AR gene have been shown to drive castration- resistant prostate cancer; thus, further analysis of the mutational status of the AR gene and its downstream signaling components would shed some light on the biology of AR in different TNBCs arising in different populations. The aforementioned under-studied potential modifiers of AR biology could affect not only patients’ prognosis following chemotherapy, but also responses to AR-targeting agents, and are, therefore, very clinically relevant.Another potential confounder of our results may be the molecular apocrine (MA) nature of the TNBCs in our cohorts. Studies have shown that AR binds ER-binding cis-regulatory elements in MA tumors and drives expression of genes normally regulated by ER. More importantly, a study of 58 transcriptionally defined MA tumors found that a significant proportion of these tumors showed expression of AR mRNA via qRT-PCR even though they were AR-negative via IHC . These in = 420), UK (n = 239), Norway (n = 104), Ireland (n = 222), Nigeria (n = 180), and India (n = 242). Resection samples from primary tumors were formalin-fixed and paraffin-embedded (FFPE). Samples from Nigeria, US, and Norway were processed at GSU, and the samples from the UK, India and Ireland were processed at their respective institutions.All aspects of this study were approved by Institutional Review Boards of the institutions involved (IRB number: H19306). Materials and data were shared in accordance with the stipulations of Material Transfer Agreements and Data User Agreements between Georgia State University (GSU) and the other participating institutions as listed in Clinicopathological data . Heat-induced retrieval of antigen epitopes was performed in citrate buffer (pH 6.0) using pressure cooker at 15 psi for 30 min. Next, the samples were quenched using hydrogen peroxide for 20 min, followed by blocking with the Ultra-Vision protein block  for 10 min. Samples were then incubated for 60 min at room temperature with anti-AR primary antibody  at 1:50 dilution. Next, the samples were incubated with MACH2 HRP-conjugated secondary antibody  for 30 min. The AR antigen was visualized using the Betazoid 3, 3-diaminobenzidine Chromogen Kit . The tissue sections were counter stained with Mayer’s hematoxylin for 1 min. Slides were then dehydrated in alcohol, cleared in xylene, and mounted with mounting medium. Appropriate negative and positive controls were used during staining.Nuclear AR staining is indicative of active receptors, because AR can translocate to the nucleus upon ligand binding. Therefore, the stained slides were scored for the percentage of tumor-cell nuclei that showed AR-positivity. TNBC samples were considered AR-negative if they had <1% of the tumor nuclei positive for AR; samples that had AR in ≥1% of the nuclei were considered AR-positive. Samples from the Nigerian, US, and Norwegian cohorts were stained and scored centrally (at GSU) and the samples in the UK, Indian, and Irish, cohorts were stained and scored at the respective institutions by two independent pathologists without prior knowledge of the patients’ pathologic or outcome data. For cross validation of the IHC scores, stained slides of grade- and stage-matched cases (representing 20–30% of each cohort) were selected from each cohort . The slides were reviewed and re-scored at Nottingham University Hospital by two pathologists who were blinded to the clinical annotation and previous IHC scores. The scores from the second round of scoring showed >99% concordance with the original IHC scores.p < 0.05 considered statistically significant. Statistical analyses were carried out with SAS 9.4® software . Differences between clinicopathological proportions were determined using the χ2 test. Differences between baseline IHC biomarker expressions or continuous clinicopathological variables were evaluated via a 2-tailed t-test. Univariate Kaplan-Meier curves were used to investigate the effect of AR status (positive vs. negative) on OS, and the log-rank test was used to assess the statistical significance of between-group survival differences. Multivariable Cox Proportional Hazard models were used to adjust for grade, chemotherapy, population, and age with significance being determined with the Wald chi-square test. For all clinical survival analysis, we used the OS, which was calculated as the time interval from surgery until death, and death was used as an event.Bar graphs were plotted using Excel and SPSS. Experimental groups were compared using Student’s t-test, and p-values were calculated with Our findings, therefore, provide a strong impetus to pursue future studies that integrate transcriptomic and genomic information to better parse and refine the classification of TNBCs. Deeper dissection of the pathways upstream/downstream of AR, as well as transcriptomic, genomic, and epigenetic profiling of samples from different patient populations, could move us from the currently used coarse categories towards finer signatures. Signatures that may reclassify AR positive/negative TNBCs into more clinically actionable subgroups.


Pairwise comparisons indicated a significantly greater CoP ellipse area for both the AR (p = 0.002) and VR (p = 0.005) conditions when compared to the normal laboratory condition. Furthermore, there was a significant difference in stride length (p<0.001) and cadence (p<0.001) between conditions. No statistically significant difference was found in the hip, knee and ankle joint kinematics between the three conditions (p>0.082), except for maximum ankle plantarflexion (p = 0.001). These differences in CoP ellipse area indicate that users of head-mounted VR/AR devices had difficulty maintaining a stable position on the treadmill. Also, differences in the gait parameters suggest that users walked with an unusual gait pattern which could potentially affect the effectiveness of gait rehabilitation treatments. Based on these results, position guidance in the form of feedback and the use of specialized treadmills should be considered when using head-mounted VR/AR devices.What was once a science fiction fantasy, virtual reality (VR) technology has evolved and come a long way. Together with augmented reality (AR) technology, these simulations of an alternative environment have been incorporated into rehabilitation treatments. The introduction of head-mounted displays has made VR/AR devices more intuitive and compact, and no longer limited to upper-limb rehabilitation. However, there is still limited evidence supporting the use of VR and AR technology during locomotion, especially regarding the safety and efficacy relating to walking biomechanics. Therefore, the objective of this study is to explore the limitations of such technology through gait analysis. In this study, thirteen participants walked on a treadmill in normal, virtual and augmented versions of the laboratory environment. A series of spatiotemporal parameters and lower-limb joint angles were compared between conditions. The center of pressure (CoP) ellipse area (95% confidence ellipse) was significantly different between conditions ( Over the past two decades, the application of virtual reality (VR) technology in a healthcare setting has become increasingly popular. It has been incorporated into clinical practices such as in the rehabilitation of stroke survivors, as well as patients with cerebral palsy and multiple sclerosis –3. ThereIn current clinical practice, gait retraining often includes treadmill training under the supervision of practitioners or through provision of real-time biofeedback. It is a widely adopted technique that aims to permanently correct faulty gait patterns and has been found to be effective in both walking and running gait modifications –12. For Walking is normally an automatic process. It has been suggested that conscious modification to walking patterns could affect gait retraining adaptations . A previAlthough multiple studies have reported positive results of gait retraining using VR among various patient groups within the lab ,5,22,23,Another concern for gait rehabilitation would be the regularity and quality of gait. Through studying spatiotemporal gait parameters, some studies have reported that walking in a projected VR environment can induce gait instability even in healthy participants ,25. NowaAn alternative to VR is Augmented Reality (AR), which does not fully immerse the user in a simulated environment but includes virtual elements that are superimposed on a real-world view . For exaThis study was designed to assess whether the use of commercially available HMD in VR and AR settings were suitable for clinical gait retraining. Specifically, the aim was to quantify the limitations of current VR and AR technology based on two practical concerns for clinical applications: 1) safety: the ability of the user to maintain a relatively stable position within the treadmill and 2) natural gait patterns: deviation of walking biomechanics from that of normal-treadmill walking. We hypothesized that there would be variations in the control of body position relative to the treadmill between both VR and AR conditions when compared with normal-treadmill walking. Also, based on altered gait biomechanics reported with the use of HMD in an over-ground setting , we hypoA total of 13 participants  were recruited for this study through convenient sampling, which is a comparable sample size to previous studies –32. PartParticipants were asked to walk at a self-selected pace for four minutes to allow for treadmill adaptation prior to data collection . AnthropControl: Treadmill walking without the HMD;Virtual reality (VR): Immersive 360° panoramic image of the laboratory captured by the Samsung Gear 360 Cam , set up instructions and image file used are provided in the supporting information .Augmented reality (AR): Real-time display through the rear camera of the HMD, set up instructions are provided in the supporting information .For the AR and VR conditions, participants wore a head-mounted VR device . The immersive VR/AR environment within this study refers to the panoramic display in a first-person perspective with complete visual obstruction to the real-world environment. The HMD used in this study weighs a total of 470 g, which is comparable to typical commercial HMD models (HTC VIVE Pro: 555 g  and Oculwww.randomizer.org). To ensure safety, participants were supported by an overhead safety harness providing 0% bodyweight support. The experimental setup is indicated in The test sequence was randomized using a web-based software (Ground reaction force and coordinates of the center of pressure (CoP) were sampled through the instrumented treadmill at 1,000 Hz. Marker trajectories were sampled at 200 Hz using an 8-camera motion capture system . The instrumented treadmill and motion capture system were synchronized and were set for data collection for three minutes after the treadmill reached the testing speed.A threshold of 10 N was used to define initial contact and toe-off based on the vertical component of the ground reaction force data . Stride The raw marker trajectories were low-pass filtered at 12 Hz with a fourth order Butterworth filter . Lower-ld was calculated to evaluate the effect size. Statistical tests were performed using SPSS Version 22 .One-way repeated measures ANOVAs were used to compare CoP ellipse area, mediolateral and anteroposterior boundaries of the CoP ellipse, stride length, cadence, and lower-limb joint angles between the three conditions. Pairwise comparisons with Bonferroni correction were conducted when necessary. Cohen’s p = 0.002). Pairwise comparisons indicated a significantly greater CoP ellipse area for both the AR  and VR  conditions when compared to Control. Moreover, both the mediolateral  and anteroposterior  boundaries of the CoP ellipse were found to be significantly different between conditions. Pairwise comparisons indicated that the mediolateral boundary  and AR condition . Similarly, the anteroposterior boundary  and AR conditions . Apart from that, the anteroposterior boundary in the AR condition was also significantly greater than Control .Individual CoP ellipses were plotted for each condition . The meaboundary  was signboundary  was alsop<0.001) and cadence  were significantly different between conditions. Pairwise comparisons indicated that the stride length in the AR  and VR  conditions were significantly shorter when compared to Control, while cadence was higher in the AR  and VR  conditions when compared to Control.Individual mean values for spatiotemporal and kinematic parameters under each condition are presented in p>0.082). The maximum ankle plantarflexion angle was significantly different between conditions . Pairwise comparisons indicated a significantly smaller plantarflexion angle for both the AR  and VR  conditions when compared to Control.The purpose of this study was to assess the use of an HMD in VR and AR settings based on the ability of the user to maintain a relatively stable position on the treadmill and the effect on walking biomechanics compared to that of normal-treadmill walking. The CoP ellipse area was significantly greater in both the AR and VR conditions compared to Control and stride length and cadence in the AR and VR conditions were also found to be significantly different from Control. Regarding lower-limb joint kinematics, no significant differences were found between conditions in the hip and knee joint angles. However, the maximum ankle plantarflexion angle was found to be smaller than Control in both the AR and VR conditions.In this study, the CoP 95% confidence ellipse was computed to indicate the position of the participant within the treadmill during the data collection period. The CoP ellipse area could reflect the ability of the participant to maintain a stable direction during locomotion and also their sense of orientation under different conditions. The high variability observed in the VR and AR conditions reflects a discrepancy among individuals responding to immersive VR and AR using an HMD . OverallThe mediolateral boundary of the CoP ellipse was significantly larger in the VR condition when compared to Control. Interestingly, such a difference was not observed between the AR and Control and participants were better able to control their mediolateral sway under the AR than under the VR condition. Visual information helps in the control of locomotion  and humaThe anteroposterior boundary of the CoP ellipse was significantly greater in both the AR and VR conditions compared to Control, despite the shorter stride lengths found in both treatment conditions. A reasonable explanation for such changes would be the anteroposterior shifting in the participant’s body position relative to the treadmill. The increase in the anteroposterior boundary might also be explained by the lack of depth perception in both treatment conditions. In reality, an object is projected onto the left and right eye at a slightly different angle, which provides information for the brain to perceive depth. The real-time visual display in AR setting and the panoramic image in the VR setting were both monoscopic, meaning that the same image was being displayed to both eyes. The monoscopic display was perceived as a flat surface, like a painting, and therefore depth information was not available. Past research has found that distances tend to be under-perceived in virtual environments ,46 and tRegarding walking biomechanics, differences were observed between the VR and AR conditions when compared with Control. Reduced stride length and increased cadence are examples of gait modifications commonly utilized to increase stability. In general, these gait adjustments are adopted to reduce perturbations to the body and therefore reduce fall risk . SpecifiPast studies have demonstrated that the ankle plantorflexors, such as the gastrocnemius and soleus, contribute to bodyweight support and propulsion ,50. The There are VR devices and software specially designed for gait rehabilitation, which may also require the concurrent use of motion tracking devices or specially designed treadmills. However, such devices may not be readily available inside a rehabilitation clinic. The HMD used in this study was a lightweight, commercially available model and the setup was simple and did not require specific technical modifications. However, the conditions within this study cannot represent all HMD applications in gait rehabilitation and the aim of this study was to identify areas of improvement for VR and AR treatment using HMD. The large CoP area and boundaries suggest that the use of an HMD on a conventional treadmill may be rather dangerous if guidance on position is unavailable, and therefore, hardware and software enhancement might be necessary. Solutions like using IMU-based position and orientation tracking  or an omvs. AR, motorized vs. non-motorized treadmill, HMD vs. projector-based VR), the naturalness of gait should be measured for each specific application and the length of the adaptation period likely depends on the setting as well. For example, some previous studies using HMD in a VR setting did not report the length of an adaptation period [Moreover, our findings on altered walking biomechanics provides an example of the limitations for VR and AR applications. A natural gait pattern is the underpinning for the effectiveness of gait retraining, and while the disruption of gait patterns may be inconsistent across various HMD devices and under different settings Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 FigIndividual center of pressure ellipses under the (a) control condition, (b) augmented reality condition and (c) virtual reality condition. Each color represents a different participant. The same color is used across conditions.(TIFF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 FileAR: augmented reality; VR: virtual reality.(XLSX)Click here for additional data file.


Prostate cancer is the most commonly diagnosed cancer affecting men in the United States. The prostate is a hormone-dependent gland in which androgen hormones testosterone and dihydrotestosterone bind to and activate the androgen receptor, initiating nuclear translocation of androgen receptor and a subsequent signaling cascade. Due to the androgen dependency of the prostate, androgen deprivation therapies have emerged as first line treatment for aggressive prostate cancer. Such therapies are effective until the point at which prostate cancer, through a variety of mechanisms including but not limited to generation of ligand-independent androgen receptor splice variants, or intratumoral androgen production, overcome hormone deprivation. These cancers are androgen ablation resistant, clinically termed castration resistant prostate cancer (CRPC) and remain incurable. First-generation antiandrogens established androgen receptor blockade as a therapeutic strategy, but these therapies do not completely block androgen receptor activity. Efficacy and potency have been improved by the development of second-generation antiandrogen therapies, which remain the standard of care for patients with CRPC. Four second-generation anti-androgens are currently approved by the Food and Drug Administration (FDA); abiraterone acetate, enzalutamide, and recently approved apalutamide and darolutamide. This review is intended to provide a thorough overview of FDA approved second-generation antiandrogen discovery, treatment application, strategies for combination therapy to overcome resistance, and an insight for the potential future approaches for therapeutic inhibition of androgen receptor. Localized PC is primarily treated by either radical prostatectomy and/or radiation therapy and is often well-managed on these regiments. Metastatic or recurrent PC is usually treated with hormonal therapy, or androgen deprivation therapy (ADT), also termed therapeutic castration. These therapeutic interventions work by inhibiting the testosterone production of the testes and prostate tumors blocking AR, which is largely impactful as over 80% of PC is androgen dependent (Prostate cancer (PC) has long been the most commonly diagnosed non-cutaneous cancer in men in the United States, and currently has the second highest cancer-associated deaths after lung cancer . Throughependent . ADT incependent .CRPC is often metastatic and is responsible for the majority of PC-associated deaths. Treatment options for CRPC are antiandrogen therapies, taxane-based chemotherapies, sipuleucel-T (provenge) vaccine, or radium-223. Antiandrogens differ from LHRH antagonists by blocking specific aspects of androgen signaling. The first-generation antiandrogens bicalutamide, nilutamide, or flutamide exclusively target AR translocation to the nucleus and prevent downstream signaling, while second-generation antiandrogens enzalutamide, apalutamide and darolutamide improve upon this mechanism, and abiraterone acetate prevents androgen biosynthesis.The original discovery of a link between androgen ablation and prostatic disease was made in 1786 by John Hunter who demonstrated effects of surgical castration on animals and secondary sex organ sizes , 5. HoweThe first therapeutic attempts at AR inhibition were the development of first-generation antiandrogens; that is, steroidal analogs meant to block AR ligand activation. However, developed resistance to these inhibitors became quickly apparent. It is believed that the primary drug resistance mechanisms arise from AR amplification, point mutations, expression of AR splice variants which are ligand independent, intratumoral androgen production or downstream signaling mechanisms . It was Prior to development of second-generation antiandrogens, the most effective treatment strategy for CRPC included taxane chemotherapeutic agent docetaxel, and subsequently cabazitaxel , 17. AnoThe development and FDA approval of second-generation, or next-generation antiandrogen treatments has since significantly altered the armamentarium of PC, specifically in treatment of CRPC. Second-generation antiandrogens have increased specificity to the androgen receptor over other steroidal receptors, act at a higher affinity than the previous generation, are exclusively antagonistic to the AR, and in turn elicit no androgen withdrawal syndrome. These compounds have greatly increased patient lifespan, extended metastasis free overall survival, decreased circulating and intratumoral androgens and serum PSA. Even with these advances CRPC has not been eradicated. It is important to fully understand the successes and downfalls in implementation of antiandrogen therapy to patients to look to the future of CRPC treatment.In contrast to androgen receptor blockade, another method of androgen signaling inhibition is the upstream blockade of androgen production. Androgens are produced in the testes, the adrenal glands as well as intratumorally. They are processed by the cytochrome p450 enzyme 17R-hydroxylase-17,20-lyase (CYP17A1). Androgens are then released and circulate through the body. Targeting the biosynthesis mechanism of testosterone through inhibition of CYP17A1, also produced in the testes and adrenal glands, would inhibit production of DHT and decrease endogenous androgen levels. The FDA approved abiraterone acetate will be described in this section, but it is not the only member of this class of inhibitors. Ketoconazole, an FDA approved antifungal compound is also an androgen biosynthesis inhibitor. However, ketoconazole has toxicity due to non-specificity to CYP17A1, though it has also been tested as treatment in PC (Other compounds that have been tested as biosynthesis inhibitors for PC include orteronel (TAK-700), a non-steroidal inhibitor of 17,20-lyase that was tested in clinical trials for metastatic CRPC, as well as galeteronel/TOK-001/VN-124. Galeteronel is a unique dual androgen antagonist and biosynthesis inhibitor that was well-tolerated in Phase 1 trials , 22. HowOther names: Zytiga, Yonsa.The discovery of abiraterone, predecessor to abiraterone acetate, was fueled by the potential of CYP17 inhibition as a treatment for PC. Previously the antifungal ketoconazole which also inhibits CYP17 was determined to inhibit PC growth, with the limitations of low potency, causing adrenal insufficiency, and a general short half-life in the body , 26. AbiAbiraterone acetate was the first of the second-generation antiandrogens to be approved by the FDA in April 2011 . In contPhase 1 and 2 studies were performed on a small cohort of 30 chemotherapy-naïve patients, some of which had previously undergone ketoconazole treatment (NCT00473746) . No doseFor metastatic PC, abiraterone acetate was tested in chemotherapy refractive PC  and chemotherapy-naïve patients . COU-AA-301 enrolled 1,195 patients over 147 sites in 13 countries. Patients with metastatic CRPC were included with progression following chemotherapy on docetaxel, and observed an overall increase in median overall survival from 10.4 months in placebo controlled group to 15.4 months , 35. Pro4-abiraterone (D4A)  .A). Enzalutamide discussed herein was the first of the class to be characterized and has the highest risk of seizures at low doses. The recently approved apalutamide and darolutamide provide reductions in brain penetrance and reduced association with clinical risk of seizure, demonstrating promising advances in the field of antiandrogen therapy.The most utilized mechanism of antiandrogens is blockade of AR signaling by sequestration of AR itself, thus preventing nuclear translocation and subsequent signaling to AR target genes . While fOther names: MDV3100, XTANDI.Enzalutamide was the first characterized second-generation antiandrogen. Developed in the laboratories of Drs. Charles Sawyers and Michael Jung, enzalutamide was isolated from a mutagenic screen of non-steroidal agonist RU59063 . CompounIn 2012, enzalutamide was approved by the FDA for the treatment of men with metastatic CRPC . EnzalutA), which was an important object of investigation in development of later therapies such as apalutamide -guided chemistry . This wapression . Furtherpression . Ultimatpression . FurtherThe advantages of apalutamide over enzalutamide included greater efficacy and higher tumor to plasma ratio and four-fold lower concentrations in the central nervous system, suggesting its decreased ability to permeate the blood brain barrier (BBB), potentially indicating a lower risk of seizure activity .Phase I testing was performed on a small cohort of 30 patients with metastatic CRPC and was well-tolerated at all doses . UltimatThe safety of apalutamide for metastatic CRPC was evaluated in patients who had either undergone treatment with abiraterone acetate plus prednisone (AAP), or AAP-naïve patients . Apalutamide was again safe, well-tolerated, and demonstrated therapeutic benefit in in patients with metastatic CRPC. Apalutamide treatment was incredibly effective in patients naïve for AAP treatment with an 80% reduction in PSA decline compared to a 22% decline in post-AAP therapy patients . The TITOther names: ODM-201, BAY1841788, NUBEQA.in vivo growth of VCaP CRPC xenografts, and exhibited low penetrance of the BBB in mice and rats . The first instance of clinical combination therapy was in HIV as an answer to the rapid rate of mutation of the disease-causing retrovirus. Termed highly active antiretroviral therapy (HAART), it combined multiple antiretroviral drugs preventing the growth of the virus, and is still used to date . Early tSince the treatment with first-generation antiandrogens, resistance associated with androgen withdrawal has been commonly characterized. Greater than 95% of testosterone production in the male body is generated in the testes, yet adrenal, prostatic, and intratumoral androgens play a great role in resistance as minute overexpression of AR is enough to counteract androgen withdrawal, sensitizing tumors to small amounts of androgen to activate AR signaling [reviewed –70]. FurMutation is one common mechanism by which androgen withdrawal experiences resistance. It was first reported in 2003 by Hara et al. who discovered a mutation of AR which could cause resistance to bicalutamide by allowing an AR antagonist to agonist switch . SeveralWhile second-generation antiandrogens have taken a huge stride in treatment of advanced PC, 20–40% of patients still do not respond to these therapies, as measured by PSA , 42, 77,Mechanisms which have been proposed to prevent therapeutic resistance include drug cycling, intermittent hormonal therapy as opposed to continuous, and combination therapy.Combination therapies have become more common in recent years in many medical fields, especially oncology. Due to resistance mechanisms, including those just described, antiandrogens are often not prescribed as a single therapy. Antiandrogens were originally given in combination with ADT in a combined androgen blockade (CAB). Prior to the development of second-generation antiandrogens, first-generation antiandrogens were already being combined with medical androgen suppression. A large meta-analysis performed on data from PC patients who received combined androgen receptor blockade consisting of androgen suppression with a first-generation antiandrogen noted a mild improvement in 5-year survival . AnotherIn contrast to other more rapidly dividing cancers, chemotherapy for PC treatments is usually performed later in the disease progression. However, in late stage PC, taxane chemotherapy is a common standard of care. Chemotherapy has long since been given in conjunction with ADT such as in the CHAARTED trial specifically treating docetaxel with ADT for patients with high volume disease (NCT00309985) . Lookingin vitro sensitizes cells to radiation  . In this trial sipuleucel-T was administered safely either concurrently or sequentially with AAP, and patients had similar immunologic prime-boost effects, commonly associated with effectiveness of the vaccine . While iiewed in ].in vitro  , 102. Abin vitro . Niclosain vitro –110. Chrin vitro . TMPRSS2in vitro . An inhiin vitro . Monoamiin vitro , 114. MAin vitro . MAOAI ireatment –119.The future of healthcare is moving toward personalized precision medicine rather than blanketed therapy to determine best practice treatment strategies for individual patients. Genomic sequencing of PC patient samples led to the discovery of mutations involving genes such as SPOP, BRCA1, or BRCA2, FOXA1, chromosomal translocations of TMPRSS2:ERG/ETS, and point mutations of AR itself , 121. CuAnother shift in treatment paradigm for aggressive PC is the increased incidence of neuroendocrine PC (NEPC). It is believed the increasing frequency of NEPC in patients (currently up to 25% of CRPC patients) , commonlNew treatments targeting different aspects of the androgen receptor signaling cascade have more exploratory potential. The ideal antiandrogen compound would be a directed inhibitor toward the N-terminal AR domain inhibiting all AR isoforms described to date including full length AR as well as ligand-independent splice variants. 5α-reductase inhibitors finasteride and dutasteride are in development, and part of standard of care treatment for benign prostatic hyperplasia (BPH). However, in PC, the use of these compounds which prevent the conversion of testosterone to DHT, have been minimally implemented and could be explored further [reviewed in ]. SimilaPotential for antiandrogens as standalone therapeutic agents seems to have plateaued for use in advanced PC. Until such time as antiandrogens are able to inhibit all splice variants of the androgen receptor, it is far more likely that the next wave of therapeutic investigation will be focused on the combination of antiandrogen therapy with other treatments such as chemotherapy.MR and TS conceptualized the manuscript. MR researched the literature. MR, SM, and TS wrote and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.