The in vitro antimicrobial activity of the synthesized compounds was investigated against various pathogenic strains; the results obtained were further explained with the help of DFT and molecular orbital calculations. Compound 1b and 1f displayed good antimicrobial activity and synergistic effects when used with kanamycin and amphotericin B. Furthermore, in vitro cytotoxicity of compounds 1b and 1f were studied against HeLa cells  and Hek-293 cells. The results of molecular docking study were used to better rationalize the action and prediction of the binding modes of these compounds.The control of antimicrobial resistance (AMR) seems to have come to a dead end. The major consequences of the use and abuse of antibacterial drugs are the development of resistant strains due to genetic mutability of both pathogenic and nonpathogenic microorganisms. We, herein, report the synthesis, characterization and biological activities of coumarin-thiazole-pyrazole (CTP) molecular hybrids with an effort to explore and overcome the increasing antimicrobial resistance. The compounds were characterized by analyzing their IR, Mass,  Staphylococcus aureus, Staphylococcus enteric, Escherichia coli) and fungal  pathogens are developing resistance against widely prescribed antibacterial and antifungal drugs like amphotericin B (Amp B), fluconazole (FLC), chloroamphenicol and penicillin +; Calcd.: 461.92;Anal. Calcd. for C23H16ClN5O2S: C, 59.80; H, 3.49; N, 15.16. Found: C, 59.85; H, 3.50; N, 15.46.4-(6-Chlorocoumarin-3-yl)-2-diazenyl)pyrazol-1-yl) thiazole (1b). Rf: 0.6; mp 220–222°C;Yield: 88%; IR (cm-1): 1719 (C = O), 1602 (C = N); 1H NMR : 8.30 , 8.08 , 7.65–7.61 , 7.49 , 7.31 , 2.74 , 2.20 ; 13C NMR : 167.47 (C = O), 159.23 (C2-thiazole), 154.49 (C8'-coumarin), 149.56 (C4-thiazole), 147.15 (C4-coumarin), 143.40 (C-Ar), 137.33 (C-Ar), 133.24 (C-Ar), 128.86 (C-Ar), 125.07 (C-Ar), 124.26 (C-Ar), 124.16 (C-Ar), 122.74 (C-Ar), 119.53 (C-Ar), 118.64 (C-Ar), 116.89 (C-Ar), 115.04 (C-Ar), 112.54 (C-Ar), 31.16 (5-CH3 pyrazole), 25.30 (3-CH3 pyrazole); MS (m/z) Found: 542.1, 540.1; Calcd.: 540.82; Anal. Calcd. for C23H15BrClN5O2S: C, 51.08; H, 2.80; N, 12.95. Found: C, 51.00; H, 2.82; N, 13.14.4-(Coumarin-3-yl)-2-diazenylpyrazol-1-yl)thiazole (1c). Rf: 0.6; mp 240–241°C; Yield: 80%; IR (cm-1): 1713 (C = O), 1601 (C = N); 1H NMR : 8.32 , 8.02 , 7.87 , 7.71 , 7.49 , 7.62 , 7.43–7.40 , 2.74 , 2.20 , 2.05 ; 13C NMR : 162.73 (C = O), 157.90 (C2-thiazole), 147.90 (C8'-coumarin), 140.92 (C4-thiazole), 140.62 (C4-coumarin), 139.09 (C-Ar), 134.47 (C-Ar), 133.02 (C-Ar), 132.82 (C-Ar), 130.27 (C-Ar), 128.85 (C-Ar), 128.44 (C-Ar), 128.39 (C-Ar), 128.35 (C-Ar), 126.95 (C-Ar), 126.92 (C-Ar), 125.36 (C-Ar), 123.28 (C-Ar), 118.07 (C-Ar), 117.99 (C-Ar), 113.57 (C-Ar), 35.10 (5-CH3 pyrazole), 29.61 (3-CH3 pyrazole), 14.60 (Ph-CH3); MS (m/z) found: 441.3; Calcd. 441.50; Anal. Calcd. For C24H19N5O2S: C, 65.29; H, 4.34; N, 15.86; Found C, 65.69; H, 4.26; N, 15.85.4-(6-Chlorocoumarin-3-yl)-2-diazenyl)pyrazol-1-yl) thiazole (1d). Rf: 0.6; mp 255–256°C; Yield: 83%; IR (cm-1): 1720 (C = O), 1601 (C = N); 1H NMR : 8.39, 8.00, 7.86 , 7.66 , 7.63 , 7.53 , 7.35 , 2.69 , 2.19 , 1.98 ;13C NMR : 164.37 (C = O), 152.86 (C2-thiazole), 151.56 (C8'-coumarin), 144.30 (C4-thiazole), 143.71 (C4-coumarin), 139.30 (C-Ar), 138.86 (C-Ar), 131.69 (C-Ar), 131.60 (C-Ar), 129.69 (C-Ar), 128.69 (C-Ar), 128.41 (C-Ar), 126.37 (C-Ar), 126.20 (C-Ar), 120.61 (C-Ar), 120.24 (C-Ar), 119.41 (C-Ar), 116.44 (C-Ar), 113.44 (C-Ar), 20.94 (CH3-Ph), 24.37 (5-CH3 pyrazole), 13.86 (3-CH3 pyrazole); MS (m/z) Found: 477.6, 475.8; Calcd. 475.95; Anal. Calcd. for C24H18ClN5O2S: C, 60.56; H, 3.81; N, 14.71; Found C, 60.55; H, 3.71; N, 14.63.4-(Coumarin-3-yl)-2-diazenyl)pyrazol-1-yl)thiazole (1e). Rf: 0.6; mp 210–212°C; Yield: 88%; IR (cm-1): 1717 (C = O), 1600 (C = N); 1H NMR : 8.30 , 8.08 , 7.82 , 7.71 , 7.65–7.61 , 7.47 , 7.28–7.24 , 2.72 , 2.32 ;13C NMR : 164.81 (C = O), 159.22 (C2-thiazole), 154.38 (C8'-coumarin), 149.26 (C4-thiazole), 147.14 (C4-coumarin), 141.59 (C-Ar), 136.64 (C-Ar), 133.14 (C-Ar), 129.23 (C-Ar), 128.82 (C-Ar), 125.07 (C-Ar), 123.35 (C-Ar), 121.87 (C-Ar), 119.40 (C-Ar), 118.67 (C-Ar), 116.81 (C-Ar), 110.77 (C-Ar), 25.08 (5-CH3 pyrazole), 14.71 (3-CH3 pyrazole); MS (m/z) found: 506.1, 504.1 (3:1); Calcd.: 506.37; Anal. Calcd. for C23H16BrN5O2S: C, 54.55; H, 3.18; N, 13.83. Found: C, 54.48; H, 3.12; N, 13.82.4-(6-Chlorocoumarin-3-yl)-2-diazenyl)pyrazol-1-yl) thiazole (1f). Rf: 0.6; mp 235–236°C;Yield: 85%; IR (cm-1): 1719 (C = O), 1605 (C = N); 1H NMR : 8.28 , 8.07 , 7.79 , 7.65–7.61 , 7.28 , 2.84 , 2.21 ; 13C NMR , 159.23 (C2-thiazole), 150.22 (C8'-coumarin), 147.40 (C4-thiazole), 142.56 (C4-coumarin), 139.21 (C-Ar), 133.23 (C-Ar), 128.88 (C-Ar), 125.10 (C-Ar), 124.85 (C-Ar), 124.80 (C-Ar), 122.43 (C-Ar), 119.34 (C-Ar), 118.64 (C-Ar), 116.81 (C-Ar), 113.49 (C-Ar), 110.39 (C-Ar), 25.16 (5-CH3 pyrazole), 15.54 (3-CH3 pyrazole); MS (m/z) found: 497.2, 495.2 (3:1); Calcd.: 496.37; Anal. Calcd. for C23H15Cl2N5O2S: C, 55.65; H, 3.05; N, 14.11; Found: C, 55.64; H, 3.10; N, 14.17.4-(Coumarin-3-yl)-2-pyrazol-1-yl)thiazole (1g). Rf: 0.6; mp 205–206°C; Yield: 82%; IR (cm-1): 1715 (C = O), 1603 (C = N); 1H NMR : 8.34 , 8.08 , 7.82–7.78 , 7.78 , 7.65–7.63 , 7.62–7.57 , 7.33 , 2.79 , 2.24 ; 13C NMR : 167.79 (C = O), 159.35 (C8'-coumarin), 154.35 (C2-thiazole), 148.40 (C4-coumarin), 146.95 (C4-thiazole), 142.99 (C-Ar), 138.95 (C-Ar), 135.97 (C-Ar), 132.95 (C-Ar), 128.78 (C-Ar), 124.99 (C-Ar), 123.43 (C-Ar), 122.59 (C-Ar), 120.05 (C-Ar), 119.79 (C-Ar), 118.76 (C-Ar), 116.75 (C-Ar), 110.52 (C-Ar), 24.83 (5-CH3 pyrazole), 15.22 (3-CH3 pyrazole); MS (m/z) found: 427.3;Calcd.: 427.28; Anal. Calcd.forC23H17N5O2S: C, 64.62; H, 4.01; N, 16.38; Found C, 64.55; H, 4.05; N, 16.45.Escherichia coli MTCC 2961, Staphylococcus aureus MTCC 3160, Enterococcus faecalis MTCC 439, Vibrio cholera MTCC 3906, Streptococcus pyogenes MTCC 442 were cultured in Mullar Hinton broth . The fungal strains Candida albicans NCPF 400034, Candida glabrata MTCC3019, Candida krusei NCPF 44002, Candida parapsilosis NCPF 450002, Candida keyfer, NPCPF 410004 and Candida tropicalis NPCPF420007 were cultured in yeast extract-peptone-dextrose  and RPMI 1640 media . For agar plates, 2.5% (w/v) bacteriological agar  was added to the medium. The strains were stored with 15% glycerol at -80°C as frozen stocks. The cells were freshly revived on respective agar plates from the stock before each experiment .,54.6) waat 0–5°C .1a-1g) exclusively, the condensation of 3,5-dimethyl-4-hydrazoarylpyrazole-1-thiocarboxamides (5) with 6-substituted-3-bromoacetylcoumarins (7a-b) was carried out in refluxing ethanol under alkaline condition  and mass.In order to achieve the synthesis of 4-(coumarin-3-yl)-2-thiazoles(ondition . The str1H NMR spectra of compounds (1a-1g) revealed two singlets in the range δ 8.39–8.29 and 8.00–8.10 ppm corresponds to coumarin 4-H and thiazole 5-H, respectively, thereby confirming the formation of 1. Two Singlets in the range δ 2.19–2.32 and 2.69–2.86 ppm appeared due to 3-CH3 and 5-CH3 group attached at pyrazole ring. It has been well established that 5-CH3 is more deshielded compared to 3-CH3 due to a lone pair of electrons present on the nitrogen atom of the thiazole nucleus and therefore appeared at about δ 2.80ppm [1H NMR along with other spectral data has been given in experimental section.The  2.80ppm ,55. More1a-1g) was evaluated in vitro according to Clinical and Laboratory Standard Institute (CLSI) guidelines using five clinical bacterial strains  and six fungal strains . The antimicrobial activity of synthesized compounds (1a-1g) ranged from 250 μM to 15.67 μM and results have been summarized in Tables 1b and 1f have been found to be the most potent antimicrobial agents among all tested compounds. When used as a standalone antibacterial agent, the MIC of 1b was either comparable (S. aureus) or at least 50% reduced (S. pyogens) compared to KAN. In case of 1f, the MIC is 50% less compared to that of KAN when tested against S. aureus, E. faecalis and S. pyogens.The antimicrobial activity of compounds a-1g was 1b and 1f as standalone found to be more active in comparison to other candidates when Amp B was used as reference drug. The results of antimicrobial activity study revealed that incorporation of halogen atoms (Cl and Br) on coumarinyl and phenyl moieties linked to pyrazolylthiazoles may be responsible for higher potential of 1b and 1f. Generally, substitution of hydrogen by halogen in coumarin derivatives compounds increases the lipophilicity and thus increases the rate of cell penetration, which is an important feature of antimicrobial drug efficiency [1b and 1f significantly increased activity against the bacterial and fungal strains. The results demonstrated that the antimicrobial activity of the investigated compounds was influenced by the physicochemical properties of the type and position of substituent on the aromatic ring.Though MIC was much higher in case of antifungal screening, the compounds ficiency . SubstitS. aureus and candida sp. are responsible for major opportunistic infections in clinical settings. There are growing concerns about the ability of S. aureus to acquire resistance, even towards the newest antibacterial available in the market [C. keyfer infections mostly occur in patients with hematologic malignancies or carcinomas who are already undergoing highly cytotoxic chemotherapy [S. aureus [Furthermore, it is noteworthy here that e market . Similarotherapy . KAN has. aureus . Likewis. aureus ,63. Emer. aureus ,65. Ther. aureus .1b or 1f can act as an adjunct to potentiate a reduction in MIC of KAN and Amp B against clinical strains S. aureus and C. Keyfer, respectively. A checkerboard dilution assay has been used to determine the fraction inhibitory concentration index (FICI) for the compounds 1b and 1f, when used in combination with KAN and Amp B against S. aureus and C. Keyfer, respectively. As evident from S. aureus with compounds 1b, and also with 1f. Likewise, in case of Amp B, there is a 5-fold reduction in MIC with compound 1b and ~8 fold reduction with compound 1f against fungal pathogen C. keyfer.As discussed above, we further evaluated whether the compounds 1b and 1f have comparable cytotoxicity  at MIC concentrations  and Hek-293 cells  indicated that the compounds trations  and negl1b and 1f are active as an adjuvant at much lower concentrations , we believe that these compounds can act as an ideal adjuvant to increase the efficacy of KAN, Amp B or possibly another drugs too. Our experiments with another antifungal agent fluconazole showed that usage of 1b and 1f as adjunct reduced the MIC ~5-fold with 1b and 8-fold with 1f against C. keyfer substantiating the broad spectrum effects of these derivatives as adjunct. Compound 1f consistently demonstrated the most potent activity as a standalone as well as in combination to other drugs as an adjunct. So we evaluated this compound for further analysis including a time kill kinetic assay.Since compounds S. aureus and C. keyfer were rapidly reduced after treatment with the 1f (MIC conc. 15.67 μM) and 1f (MIC conc. 62.50 μM), respectively. Maximum killing of S. aureus and C. keyfer cells with 1f were observed after 24 h in comparison to untreated control. Samples withdrawn at the indicated time points were evaluated for colony forming units (CFUs) at MHA (Muller Hinton Agar) and YPEDA (Yeast Extract Peptone Dextrose Agar) medium. At synergy concentrations of treatment with 1f (0.5 μM of KAN and 07.81 μM of 1f) against S. aureus and (0.097 μM of Amp B and 31.25 μM of 1f) against C. keyfer showed killing after 12 h (S. aureus) and 8 h (C. keyfer), respectively . A change in both S. aureus and C. Keyfer cell morphology was observed in treated with compound 1f at MIC conc. (at 15.67 μM for S.aureus and 62.50 μM for C. keyfer). Increased roughness and cell wall damage was observed within 12 h of incubation . The MIC value of Amp B against C. keyfer is 12.50 μM. More promisingly, when used as an adjunct to standard antimicrobial drugs, compounds 1b and 1f significantly enhanced the efficacy of these drugs KAN, Amp B and FLC by significantly reducing the MIC values of these drugs.As a result, compounds (1a-1g), using Khon-Sham’s DFT method that undergo the gradient-corrected hybrid density functional B3LYP method and the 6–31+g bases set [The electron donating and receiving ability of a molecule can be determined by considering HOMO and LUMO energy of that molecule. Higher the energy of HOMO higher will be its electron donating ability. A full geometry optimization was performed for all compounds ases set . It has ases set , HOMO-LU1b and 1f showed illustrious LUMO energy of -2.647 eV and -2.648 eV respectively and also exhibited higher antibacterial & antifungal activity than the other compounds. It was observed that compounds with more stabilized LUMO orbitals have magnificent biological activity /2. This descriptor is used as a measures of resistance to change in the electron distribution or charge in a molecule [HOMO+ ELUMO) /2, which is defined as the power of an atom in a molecule to attract electrons towards it [HOMO+ELUMO)/2. Electrophilicity index (ω), is a measures of the propensity or capacity of a species to accept electrons was introduced by Parr and is calculated using the electronic chemical potential and chemical hardness and given as ω = μ2/2η[1a-1f are summarized in 1a and 1f are comparatively soft molecule with high polarizability than other compounds of this series. The high electronegativity (4.41 eV) and electrophilicity (5.51 eV) of 1a and 1f indicates its high power to withdrawn electrons and hence to act as an electrophile.The chemical reactivity indices like chemical hardness (η), electronegativity (χ), electronic chemical potential (μ), and electrophilicity Index (ω), were also calculated for all the synthesized molecules. The stability and reactivity of a chemical system is represented by chemical hardness which is given by η =  with a root-mean-square deviation (RMSD) of <1 results .1a-1g were performed in the binding pocket of DNA gyrase B (PDB ID:5L3J). Four basic parameters-Glide score, Glide energy, H-bonds and non-bonded interactions  were used to define the binding affinity of ligands 1a-1g for DNA gyrase B. Glide score with more negative value represents minimum energy for the formation of complex between ligand and receptor (Glide energy). The docked poses for compounds 1b, 1g and KAN in the active site of DNA gyrase B are shown in Figs -1 to -84.78 kcalmol-1) and the energy ranges are more than that of reference compounds KAN and CPF  with DNA gyrase B are shown in In the present manuscript, an attempt has been made to study the interactions of novel coumarin-thiazole-pyrazole (CTP) hybrids with DNA gyrase B. For this purpose molecular docking studies of all the synthesized compounds  and CPF . It has (1a-1g) may prove good lead as antibacterial drugs by targeting DNA gyrase B. For drug formulation pharmacokinetic properties are likewise a significant prospect and should be brought into thoughtfulness. The pharmacokinetic parameters of all synthesised hybrids have been studied along with standard drug KAN and CPF using QikProp [(1a-1g) were seems to be acceptable and were incorporated in 1a-1g was highly satisfactory with a high percentage (>80%) of Human Oral Absorption as compared to the reference, indicating their possibilities in oral drug formulation for the treatment of S. aureus infection.Pharmacodynamics results indicated that compounds  QikProp . The majThe bowel-blood barrier was predicted using Caco-2 cell permeability (QPPCaco) as model along with Lipinski’s rule of 5  all the 1b and 1f displayed significant antimicrobial activity without expressing any significant mammalian cell cytotoxic effect. Compound 1b showed the similar level of antibacterial activity with MIC 31.25 μM and 1f found best agent with MIC 15.67 μM particularly against S. aureus when compared with standard drug, kanamycin. Interestingly, as an adjuvant to standard antimicrobial drugs, compounds 1b and 1f significantly enhanced the efficacy of the standard drugs KAN, Amp B and FLC by significantly reducing the MIC values of these drugs.In conclusion, some new coumarinyl linked pyrazolylthiazoles have been synthesized and explored for their potential as standalone or adjuvant antimicrobial agents against pathogenic strains. Compounds 1b and 1f may act as lead compounds for the development of potent antibacterial and antifungal agents and as adjuvant they have potential to unlock the microbial resistance to antibiotics.The results demonstrated that the antimicrobial activity of the investigated compounds was influenced by the physicochemical properties of the type and position of substituent on the aromatic ring. The results based on electron microscopic investigations of cell morphology and cellular structural integrity suggest that the antimicrobial activity of these compounds may in part be attributed to initial cell wall damage, leading to action on the intracellular components of cells, ultimately leading to cell death. The result of DFT studiy of compounds were correlated with the observed antimicrobial activities which indicate that coumarin moiety plays an important role in antimicrobial activities. Moreover, the molecular docking studies further helped in supporting the experimental results and the ADME properties predicted further strengthen the path towards oral drug formulation. Therefore, it is concluded that the compounds S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig(TIF)Click here for additional data file.S13 Fig(TIF)Click here for additional data file.S14 Fig(TIF)Click here for additional data file.S15 Fig(TIF)Click here for additional data file.S16 Fig(TIF)Click here for additional data file.S17 Fig(TIF)Click here for additional data file.S18 Fig(TIF)Click here for additional data file.S19 Fig(TIF)Click here for additional data file.


Gait analysis can provide valuable information on a person’s condition and rehabilitation progress. Gait is typically captured using external equipment and/or wearable sensors. These tests are largely constrained to specific controlled environments. In addition, gait analysis often requires experts for calibration, operation and/or to place sensors on volunteers. Alternatively, mobility support devices like rollators can be equipped with onboard sensors to monitor gait parameters, while users perform their Activities of Daily Living. Gait analysis in rollators may use odometry and force sensors in the handlebars. However, force based estimation of gait parameters is less accurate than traditional methods, especially when rollators are not properly used. This paper presents an evaluation of force based gait analysis using a smart rollator on different groups of users to determine when this methodology is applicable. In a second stage, the rollator is used in combination with two lab-based gait analysis systems to assess the rollator estimation error. Our results show that: (i) there is an inverse relation between the variance in the force difference between handlebars and support on the handlebars—related to the user condition—and the estimation error; and (ii) this error is lower than 10% when the variation in the force difference is above 7 N. This lower limit was exceeded by the 95.83% of our challenged volunteers. In conclusion, rollators are useful for gait characterization as long as users really need the device for ambulation. Currently, a high percent of population presents some form of disability. For example, in Europe, 18% of the population over 16 years old report moderate disabilities , early dGait analysis is usually performed in a controlled, instrumented area. Data capture can rely on sensors in the walking surface, such as force plates in a treadmill  or pressGait analysis has also been performed using wearable sensors. Users may carry a number of sensors on various part of their bodies, such as pressure sensors under the feet , inertiaTo improve usability and to perform gait analysis in daily life, sensors could be attached to a device that people use on a daily basis. One solution is to use inertial sensors in a smartphone , but assInstrumented or smart rollators have already been used for gait analysis using optical information, where the users’ feet were tracked by Microsoft Kinect sensors   and TimeRollator based gait analysis approaches typically present two major weaknesses. First, solutions are usually tested using healthy volunteers for simplicity. Unfortunately, healthy people do not usually present the same behavior as challenged users when they handle assistive devices. Much work has focused on comparing healthy gait and the specific gait of challenged volunteers due to e.g., Parkinson’s , stroke Nevertheless, force based gait estimation is quite appealing because: (i) it is not constrained to any specific environment; (ii) it does not require expert intervention; and (iii) users do not need to wear sensors themselves. In addition, data can be processed on the fly to provide continuous information on gait, anytime, anywhere. We already proposed a method for gait analysis using a rollator in . The preThis study consisted of two stages. In stage 1, volunteers were allowed to move freely using a smart rollator for support a. These In stage 2, a group of volunteers was asked to walk with the smart rollator over an instrumented treadmill using a smart walker, wearing optical markers, to obtain the estimation error of the force based gait analysis method. These tests were more challenging and uncomfortable and had to be conducted at a specific facility, so the number of volunteers with disabilities measured in this stage was limited.The full test group for stages 1 and 2 included 43 volunteers from three different institutions. Most challenged volunteers  were either inpatients from Fondazione Santa Lucia (FSL) in Italy or rehabilitation patients from the Hospital Regional Universitario of Malaga (HRU) in Spain. These 29 volunteers were on average All 43 volunteers were evaluated using the Tinetti scale  to separUniversitat Politecnica de Catalunya [®  rollator frame. It includes a steel force transducer in the vertical stem of each handle to measure exerted forces on all three axes. Strain gauges connected in an additive full bridge are bonded to the transducer where stress is higher. The signal goes through a programmable gain instrumentation amplifier and is digitalised using a microcontroller. The system provides an accuracy of: 0.1 N (pushing); 0.2 N  and 0.05 N (resting force). In addition, each wheel has an encoder to estimate odometry. It includes an embedded system for data filtering and preprocessing that supports a wireless connection. All volunteers in both stages of our tests used the i-Walker rollator  is a system based on optical active-markers, which are recorded by external cameras. The markers coordinates in space were determined by the Optotrak software  with an accuracy below one millimeter and were sampled at 100 Hz. Volunteers from VUA walked on a treadmill with markers attached to their heels a. FigureForce plates in the treadmill: Volunteers in stage 2 walked with the rollator on a treadmill equipped with force plates a, which In this study, three different systems were used for gait analysis:All three motion capture systems can return sets of spatiotemporal gait parameters depending on the employed methodology. The i-Walker is the most limited system because it relies on forces and odometry only. Hence, the i-Walker cannot return complex parameters like those related to body posture. All three capture systems, however, may detect the heel strike. Hence, we used heel strike as the basis to obtain the same spatiotemporal gait parameter set simultaneously from all three systems and, in doing so, assess our rollator based methodology.i-Walker: Force based heel strike detection in the i-Walker is based on the fact that when a person’s heel strikes, the handlebar force at the corresponding side grows, whereas the handlebar force in the opposite side decreases a. Hence,Optotrak: Optotrak data can be used to estimate heel strike in a straightforward method. Volunteers had a marker on each heel a. When h strikes c.Treadmill: When a user heel strikes on the treadmill, the CoP X (left–right) coordinate moves in the direction of the heel strike. Hence, heel strike can be detected as an inflection point in X coordinate time series c.Heel strike was determined differently with each capture system.Step time (Stride time (Number of Step (Time required (Cadence (Step length (Stride length (d): sum of all step lengths.Distance were split into three groups depending on their condition, as detailed in a further subsection: healthy, mild and severe disabilities. Then, we evaluated the average, variance, maximum and minimum of um fdiff d were nop-value for the normality of the data per groups and for each comparison of p-value of We checked the normality of the data using the Lilliefors test. After that, we performed the F-test for equality of variances for each pair of groups  to test the null hypothesis that the two samples had the same variance. We found inequality of variance in some comparisons. Hence, we also performed a non-parametric two-sided Wilcoxon rank sum test for each pair of groups to test observed differences in their averages. Volunteers in the severe group clearly presented differences with respect to healthy volunteers both in averages and variation. The hypothesis of equal variance was also rejected for these variables. On the other hand, the hypothesis that mildly challenged volunteers have a similar median compared to healthy volunteers was rejected only for the maximum. The same occurred for the variance hypothesis. Finally, the hypothesis that severely challenged volunteers have a similar median compared to the mildly challenged volunteers was rejected in all cases. In addition, the hypothesis of equality of variances was rejected for the variation and the maximum of These results show that there are substantial differences in In stage 2, volunteers were asked to walk on a treadmill equipped with force plates using the i-Walker for support while they were tracked by the Optotrak system. These tests allowed us to further elaborate on the differences between healthy and challenged user groups.ρ was characterized by its average All three systems were synchronized to extract the gait parameters previously described simultaneously. Results from the reference systems were used to evaluate the error in the rollator based estimation. The error function for each spatiotemporal parameter was equal to the difference between the value obtained from the rollator and the one obtained from one or the other reference system at each sample. The error function was determined for each volunteer and each spatiotemporal gait parameter separately. Subsequently, the error for the whole set of volunteers for a given spatiotemporal gait parameter The Optotrak system measured at 100 Hz with a spatial error under one millimeter. To take the Optotrak error into account, The treadmill had a higher sampling rate than the Optotrak: it measured the CoP, and, consequently, heel strike, at 200 Hz. However, in our experiments, we found that the CoP returned by the treadmill was not always accurate. Cadence: Stride time: Stride length: Step time: Step length: Walking Velocities: As described previously, our force based gait analysis algorithm searches for maximum differences between handlebar forces to estimate spatiotemporal heel strikes. When volunteers bear more weight on the rollator, the variation of these forces is higher and heel strike detection is more accurate. bove 7 N . The maxbove 7 N  when fdiAfter relative error in gait parameter estimation is analyzed using all three different capture systems, one can immediately find major differences between healthy users and challenged subjects using these systems. In most cases, challenged users bear more weight on the rollator, even when healthy users are purposefully trying to do so. In addition, challenged users bear weight asymmetrically on the handlebars, i.e., Results from stage 2 suggest that, as long as the difference between the weight that people bear on the rollator’s handlebars is large enough, all gait parameters can be obtained using the methodology proposed in  using onIt is interesting to note that when healthy people are feigning a challenged behavior, they might bear more weight on the rollator than challenged volunteers. However, the variance of the force difference on the handlebars is reduced, meaning that they are using the rollator like a trolley rather than as a support device. Actually, In general, tests with healthy volunteers to validate assistive devices are not recommended because systematic differences in sensor readings may cause bias. However, if it is impossible to work with challenged volunteers, at the very least, it is necessary to compare the system input instance for healthy volunteers with reported inputs for mildly to severely challenged volunteers. If instances are similar, it can be concluded that healthy volunteers are feigning challenged behavior acceptably for the test.There are alternative methods to obtain gait parameters in rollators using using onboard RGB-D cameras instead of force sensors . HoweverIn this study, we analyzed the validity of a force based methodology for gait analysis using a smart rollator. The aims of this study were to determine whether this methodology can be applied to any types of users regardless of their condition and to determine the estimation error. In the first stage, we studied the differences in weight bearing on rollator handlebars between 12 healthy volunteers and 31 challenged volunteers presenting a variety of musculoskeletal and/or neurological disabilities. Despite obvious gait differences, healthy volunteers are often used in studies on assistive devices. In some studies, they are asked to emulate a challenged user’s behavior by bearing more weight on the devices and walking slower. However, our study shows that, even in this case, there are major differences in terms of how people bear weight on a rollator depending on their condition.In a second stage, we focused on determining how users need to bear weight on the walker for accurate gait analysis and which estimation errors appear in these cases. To achieve this goal, we extracted the same spatiotemporal gait parameters from heel strikes detected simultaneously using: (i) the i-Walker smart rollator; (ii) an  Optotrak system; and (iii) a treadmill equipped with force plates. These tests were done on a subset of 12 healthy volunteers and two challenged volunteers. These volunteers walked on the treadmill wearing Optotrak markers on their heels and using the i-Walker.First, we found out that heel strike on the treadmill could not be reliably detected for challenged users because they do not bear weight symmetrically on both handlebars. Hence, estimation errors were obtained by comparing gait parameters extracted from the rollator with those extracted from the Optotrak system, which reportedly presents a spatial error lower than 1 mm.Our conclusions support the idea that force based gait analysis on a rollator is accurate (less than 10% error) as long as peak force differences between the handlebars are over 7 N. This condition was satisfied by 95.83% of our volunteers presenting mild to severe disabilities (Tinetti scores under 24). Only 37.5% of challenged volunteers with a Tinetti score above 24, mostly including people at the end of their rehabilitation period, satisfied this restriction. Thus, force based gait analysis using a rollator would not be advisable for this group. Finally, 66.67% of our healthy volunteers met this restriction. In general, tests with healthy volunteers to validate assistive devices are not recommended because systematic differences in the sensor readings may cause bias.


In previous research, Hugan Qingzhi, a traditional Chinese medicine, was shown to have protective effects against hepatic steatosis. However, its activity against non-alcoholic fatty liver disease (NAFLD) and the mechanisms by which it exerts its effects remain unknown. In the present study, the effects of Hugan Qingzhi on free fatty acid (FFA)-induced L02 cells were examined. The techniques of iTRAQ labeling, together with strong cation exchange-non-liquid chromatography–tandem mass spectrometry (SCX-non-LC-MS/MS) analysis and serum pharmacology, were used to evaluate the effects of Hugan Qingzhi-medicated serum on FFA-induced L02 hepatocyte injury. Results identified 355 differentially expressed proteins following FFA treatment, compared with a control group; 359 altered proteins in the Hugan Qingzhi high dose + FFA treatment group, compared with the FFA treatment group; and 365 altered proteins in the Hugan Qingzhi high dose + FFA treatment group, compared with the control group. Based on the Kyoto Encyclopedia of Gene and Genomes pathway enrichment analysis, it is concluded that several pathways including those of microbial metabolism in diverse environments, fatty acid metabolism, peroxisome proliferator activated receptor signaling, and mitogen-activated protein kinase signaling are closely associated with the effects of Hugan Qingzhi-medicated serum in FFA-induced L02 hepatocyte injury. Furthermore, several differentially expressed proteins, including heat shock protein 27 (HSP27), acetyl-CoA acetyltransferase 1, calnexin, and integrin-linked kinase, were validated by western blotting. A target-specific HSP27 siRNA was used to investigate further the function of HSP27, and it was found that HSP27 might have a key role in the observable effects of Hugan Qingzhi-medicated serum in FFA-induced L02 hepatocyte injury. The results not only confirmed that Hugan Qingzhi exhibits a significant protective effect in FFA-induced L02 hepatocyte injury, but also suggest insights into the mechanism of such protective effects. Non-alcoholic fatty liver disease (NAFLD) is defined as the presence of hepatic steatosis in the absence of any secondary causes of hepatic accumulation, such as significant alcohol consumption, steatogenic drugs, or hereditary disorders. Clinically, metabolic syndrome and its many components such as obesity, diabetes mellitus, and dyslipidemia  are closWithin recent years, proteomics has been widely used in the life sciences to detect differentially expressed proteins and novel targets, and discover the associated mechanisms of action . Isobariin vitro system does not reflect the actual efficacy of the drug  is a traditional Chinese medicine used for treatment of NAFLD. Its therapeutic effects on NAFLD have been confirmed in previous studies . It consthe drug . To miniironment . Thus, sPreparation of HQT has been described in previous studies . The metTable 1) , and fenofibrate-medicated serum (FF) were produced according to methods described in a previous study . Thirty Table 1) . One houTable 1) . All ani2. The cells were divided into six groups as follows: the control group; FFA  group; FFA + 10% low dose HQT-medicated serum group (HL); FFA + 10% moderate dose HQT-medicated serum group (HM); FFA + 10% high dose HQT-medicated serum group (HH); and FFA + 10% fenofibrate-medicated serum (FF). To verify whether the differences observed with the HQT-medicated serum were attributable to HQT rather than the serum, 10% vehicle serum control was added to the control and FFA groups. The final concentration of FFA in each group was 1 mM.Human hepatocyte cell line L02 was obtained from China Cell Culture Center  and cultured in RPMI-1640 Medium , containing 10% fetal bovine serum  at 37°C, under 5% COAccording to a previously described method , the cyt5 per well and allowed to grow to the desired confluence. The cells were then treated for 24 h with Gibco RPMI-1640 containing various concentrations of HQT-medicated serum  or fenofibrate-medicated serum (FF), before stimulation with 1 mM FFA for 24 h.To estimate the protective effects of HQT in FFA-induced L02 cell injury, several biochemical parameters, such as triglycerides (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), super-oxidase dismutase (SOD), and glutathione (GSH) were measured using a colorimetric assay kit from Nanjing Jiancheng Bioengineering Institute . L02 cells were cultured in a 6-well plate at a density of 1 × 105/well. The cells were treated for 24 h with Gibco RPMI-1640 containing HQT-medicated serum , or fenofibrate-medicated serum (FF), before stimulation with 1 mM FFA for 2 h, and incubation for 30 min with DCFH-DA (10 μM) at 37°C. Levels of intracellular ROS were determined by measuring the fluorescence density of DCF using a SpectraMax M5 multifunctional microplate reader .Levels of intracellular reactive oxygen species (ROS) in L02 cells were measured quantitatively and qualitatively using 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), a non-polar dye that can diffuse into cells and then hydrolyze to non-fluorescent 2′,7′-dichlorofluorescein (DCFH). ROS can oxidize cellular DCFH to highly fluorescent dichlorofluorescein (DCF), thus levels of intracellular ROS can be measured by the intensity of fluorescence of DCF. In the present study, cells were seeded in 6-well plates at a density of 1 × 10Oil Red O staining was performed to facilitate lipid visualization and quantification. L02 cells were fixed with 4% paraformaldehyde and stained with 0.7% Oil Red O solution for 30 min at 25°C. Cells were then washed and counterstained with hematoxylin for 5 min. Images were acquired using the Olympus BX51 image system .Expression of IL-6 and TNF-α were analyzed using ELISA kits  according to the manufacturer’s instructions. Absorbance was read at 450 nm using a SpectraMax M5 multifunctional microplate reader .Human HSP27-specific siRNA and siRNA-control were designed and synthesized by RiboBio Inc.  for transfection. The sense and antisense strands of HSP27-specific siRNA were 5′-AUGAGACUGCCGCCAAGUAdTdT-3′ and 5′-UACUUGGCGGCAGUCUCAUdTdT-3′, respectively. Cells were plated in 6-well plates at 30–50% confluence, then transfected with 50 nM siRNA-targeting HSP27 or 50 nM siRNA-control using Lipofectamine 2000 . After 6 h, 1 mL of Gibco RPMI-1640 containing 10% fetal bovine serum was added to each well. Cells were transfected with siRNA for 24 h and then treated with, or without FFA and HQT-medicated serum.g for 10 min at 4°C, cell pellets were collected and lysed in buffer containing 8 M urea, 2 M thiourea, 4% CHAPS, 5% DNA enzyme, and 5% RNA enzyme. The lysates were then intermittently sonicated for 30 s. The resulting lysate was centrifuged at 12,000 × g for 1 h at 4°C, and the supernatant was collected. Total protein concentration was measured using the Bradford method. All samples were stored at -80°C until further analysis.L02 cells were washed twice with ice-cold PBS and harvested by scraping in cold PBS after the various treatments for each group . After centrifugation at 1000 × g for 20 min at 4°C, followed by air-drying and dissolution with 100 μL TEAB dissolution buffer. The Bradford method was used to quantify each protein sample. From each sample, 100 μg of protein was reduced, alkylated, and then digested by Trypsin  at 37°C for 16 h, with the ratio of protein : trypsin = 20:1, and labeled using the iTRAQ Reagent-8 plex Multiplex Kit  according to the manufacturer’s protocol. Two biological replicates were carried out for the control group, and three biological replicates for the FFA and HH groups. The control group samples were labeled with 113 and 114; FFA group samples were labeled with 115, 116, and 117; HH group samples were labeled with 118, 119, and 121.Proteins were precipitated following the addition of a fourfold volume of cold acetone containing 10 mM DTT for about 2 h at -20°C, and centrifugation at 12,000 × 4HCO3 in 3% acetonitrile, pH 10), and were then loaded onto the HPLC device at a flow rate of 1 mL/min with buffer B  at gradients of 0–8% for 5 min; 8–45% for 25 min; 45–80% for 15 min; and 80–100% for 5 min. The eluted fractions were collected at 1-min intervals and pooled into 12 fractions, then desalted with a Strata X C18 column  and vacuum-dried.The iTRAQ labeled peptides were mixed in equal amounts and fractioned by an HPLC system  using the Durashell C18 column . The dried peptides were dissolved in 100 μL buffer A (20 mM NHLiquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis was performed on the AB Sciex non-LC-MS/MS (Triple TOF 5600 plus) system. Peptides were loaded onto a 20 μm PicoFrit emitter (New Objective) packed to 12 cm with Magic C18 AQ beads  for the stationary phase. Chromatography was performed with a 90-min gradient from 2 to 30% . MS1 spectra were collected in the range 350–1500 m/z for 250 ms. The 20 most intense precursors with charge states 2–5 were selected for fragmentation. MS2 spectra were collected in the range 50–2000 m/z for 100 ms. Precursor ions were excluded from reselection for 15 s.t-test (two-tailed or unpaired), and the minimum values among replicates were treated as the final P-values. Data were expressed as mean ± SD. For protein abundance ratios measured with iTRAQ, we took fold changes ≥1.2 (or ≤0.83) and P-values < 0.05 were taken as the thresholds to identify significant changes.The original MS/MS file data were submitted to the ProteinPilot Software v 4.5 for analysis. The Paragon algorithm  was inte1. GO functional classification was performed with the Blast2GO software (v4.5 pipeline5). The KEGG databases2 were used to access current knowledge on biochemical pathways and other types of molecular interactions. Both GO and KEGG pathway enrichment analyses were used to uncover the enriched pathways of significantly altered proteins and to identify their functions.For bioinformatic analyses, differentially expressed proteins were classified based on annotations from the UniProt KnowledgebaseTM Total Protein Extraction Kit , according to the manufacturer’s instructions. Total protein concentrations were determined by the Bradford method, and equal amounts of proteins from different groups were separated via 12 or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) following standard protocols. Primary antibodies including anti-human polyclonal antibodies that recognize ACAT1, CANX, ILK, and GAPDH purchased from Proteinteach Group ; rabbit anti-human phospho-HSP27 (Ser15) antibody and HSP27 antibody purchased from Affinity Bioscience (USA); and secondary antibodies, goat anti-rabbit horseradish peroxidase (HRP)-IgG purchased from Zhongshan Golden Bridge Biotechnology Co, Ltd.  were used in the present study.Cell culture medium was removed from each well, rinsed twice with PBS, and lysed with EnoGenet-test (paired or unpaired) or one-way analysis of variance (ANOVA) with the GraphPad Prism software . Data are presented as mean ± standard deviation (SD). Differences were considered to be statistically significant at p < 0.05.Statistical significance was analyzed using the Student’s p > 0.05), compared with the vehicle control group (Table 2), indicating that each medicated serum treatment has no cytotoxic effects on L02 cells.First, LDH release in supernatant was measured to evaluate the cytotoxic effects of each medicated serum on L02 cells. The results showed that 10% vehicle serum, 10% HQT-medicated serum , and 10% fenofibrate-medicated serum treatments have no significant difference on LDH release . In contrast, SOD activity and GSH levels were reduced . In comparison with the FFA group, the HQT-medicated serum group exhibited not only a down-regulation of MDA production and intracellular ROS, but also an up-regulation of SOD activity and GSH levels in a dose-dependent manner. HQT-medicated serum also showed an ability to inhibit FFA-induced intracellular triglyceride (TG) accumulation  in L02 hepatocytes. In the Oil Red O staining assay, treatment of L02 cells with FFA resulted in a considerable increase in TG accumulation, whereas treatment with HQT-medicated serum significantly attenuated TG accumulation , a finding that is consistent with those presented in Figure 1E. In comparison with the FFA group, AST and ALT levels in the culture supernatant of the HQT-medicated serum treatment group were significantly reduced . In the assay to detect TNF-α and IL-6, both showed a significant increase in FFA-treated cells, compared with the control group, whereas cells treated with HQT-medicated serum showed a marked inhibitory effect . These results all suggest that HQT-medicated serum can regulate oxidative stress and inflammatory cytokine production, as well as ameliorate intracellular TG accumulation to achieve an anti-NAFLD effect.Production of MDA in the culture supernatant, activity of SOD, and intracellular levels of GSH and ROS were all measured in the present study to evaluate whether HQT-medicated serum protects L02 cells injured by FFA. The results showed that production of MDA in the culture supernatant and intracellular ROS stimulated by FFA for 24 h were significantly increased compared with the control group  contained at least two unique peptides. Moreover, 4268, 4269, and 4269 proteins were quantified in FFA:C, HH:C, and HH:FFA, respectively. Further analysis identified that there were 355 , 365 , and 359  differentially expressed proteins in FFA:C, HH:C, and HH:FFA, respectively , metabolic process (9.91%), biological regulation (7.89%), regulation of BP (7.41%), CC organization or biogenesis (6.73%), localization (4.97%), and developmental process (5.52%), among others. The eight main CC subcategories were: cell (16.69%), cell part (16.69%), organelle (16.13%), organelle part (13.27%), membrane-enclosed lumen (8.82%), macromolecular complex (9.03%), extracellular region part (8.77%), and extracellular region (8.85%). The four major MF subcategories were: binding (50.64%), catalytic activity (25.11%), enzyme regulator activity (4.61%), and structural molecular activity (7.00%).GO analysis was performed to functionally classify the differentially expressed proteins (DEPs), with the proteins categorized according to biological process (BP), cellular components (CC), and molecular functions (MF) using the Blast2GO software (v4.5 pipeline5). As shown in The DEPs were further characterized in FFA:C, HH:C, and HH:FFA, respectively, using GO enrichment analysis , HH:C , and HH:FFA , respectively . The results detected by immunoblotting analysis were consistent with those of iTRAQ-identification, indicating that the protective effects of HQT-medicated serum in L02 hepatocyte damage induced by FFA are associated with expression and function of the respective proteins.Several iTRAQ-identified proteins were selected for validation by western blotting, including acetyl-CoA acetyltransferase 1 (ACAT1), integrin linked kinase (ILK), heat shock protein 27 , and calnexin (CANX). These proteins were annotated into four signaling pathways: fatty acid metabolism (ACAT1), PPAR signaling pathway (ILK), MAPK signaling pathway (HSP27), and protein processing in the endoplasmic reticulum . Results showed that in comparison with the control group, ACAT1 and CANX were up-regulated by FFA treatment alone, whereas ILK and phosphorylated HSP27 were down-regulated. In comparison with the FFA group, expression of these proteins was reversed in the HQT-medicated serum group , IL-6 (p = 0.03), and TNF-α (p = 0.28) production induced by FFA were weakened, compared with the FFA group. When combined, these data suggest that activation of HSP27 inhibits FFA-induced ROS, IL-6, and TNF-α production, and that the protective effects of HQT-medicated serum in FFA-induced L02 cell damage are associated with its regulation of HSP27.To determine the involvement of HSP27 in FFA-stimulated L02 cells and the effects of HQT-medicated serum, specific siRNA against HSP27 was applied to L02 cells. As shown in A previous study demonstrated that the anti-hepatic steatosis effect of HQT in L02 hepatocytes inhibits SREBP-1 expression and increases CPT-1 and ACOX-1 expression via activation of the AMPK and PPAR-α pathways . Althougde novo synthesis and uptake from food. Output entails fatty acid oxidation and esterification to produce TG. TG are either incorporated into lipoproteins or stored as lipid droplets (Accumulation of intrahepatic TG (hepatic steatosis) is one of the most remarkable and characteristic changes associated with NAFLD, and usually occurs when the rate of fatty acid input is greater than that of the output. There are two aspects to fatty acid input: droplets . Therefodroplets . On the droplets . Over prdroplets . Therefoin vitro model of NAFLD established in the present study is very similar to that of clinical NASH with ballooning degeneration (Accumulation of TG within hepatocytes caused by abnormalities in lipid metabolism and disposition is the “first hit” in development of NAFLD . An addineration . FurtherA growing body of evidence has shown that hepatocyte death plays a pivotal role in transition of NAFLD to NASH. Abnormal accumulation of fatty acids in the liver stimulates ROS generation, which can directly induce DNA/protein damage and promote hepatic failure via activation of stress-related signaling pathways, including the MAPK and PPAR pathways . In the 2+ storage for synthesis, folding, and maturation of most secreted and trans-membrane proteins. Disturbed hepatic cholesterol homeostasis and free cholesterol accumulation in the liver results in increased oxidative stress, leading to ER stress that is closely associated with the development of NAFLD and entails fat accumulation, insulin resistance, inflammation, and apoptosis among other conditions (The ER is the cellular site of Canditions . Moreovenditions . In the In the proteomics data, KEGG enrichment analysis revealed that among all DEPs, a total of 31, 21, and 35 proteins from FFA:C, HH:FFA, and HH:C, respectively, were enriched in microbial metabolism in diverse environments. This indicates that development of NAFLD is closely related to microbial metabolism in the host. However, no follow-up evaluations, such as determination of the diversity of gut microbiota, were performed in the present study to confirm these results. A growing body of evidence suggests that gut microbiota act as regulators of energy homeostasis and fat deposition, thereby implicating them in the development of obesity and metabolic disease . Moreovein vivo study.Using the combined strategy of iTRAQ with LC-MS/MS and subsequent investigation, this study confirmed that HQT has excellent protective effects in FFA-induced L02 hepatocyte injury. These effects were associated with the reduction of lipid accumulation, oxidative stress, and inflammation, and inhibition of ER stress and hepatocyte death. Subsequent validation experiments proved that ACAT1, HSP27, ILK, and CANX might be associated with the protective effect of HQT in the FFA-induced L02 hepatocyte injury model. Moreover, bioinformatic analysis of the DEPs results showed that microbial metabolism in diverse environments was strongly associated with the protective effect of HQT in FFA-induced L02 hepatocytes, suggesting a key role for the gut microbiota in progression of NAFLD. Consequently, the present study not only expands our understanding of the pathogenesis of NAFLD, and provides new insights for its treatment, but further elucidates the underlying mechanism of the effects of HQT in NAFLD treatment. Further studies are needed to clarify the role of DEPs in the effects of HQT in NAFLD treatment, in particular in primary cells or even FX and XY are responsible for the experiment operation. WT, CX, and MY are responsible for assisting the experimental design and experiment operation. BZ guided the design of the experiment and is responsible for modification of the paper.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.


This report has been prepared by the European Academy of Allergy and Clinical Immunology Task Force on Allergic Rhinitis (AR) comorbidities. The aim of this multidisciplinary European consensus document is to highlight the role of multimorbidities in the definition, classification, mechanisms, recommendations for diagnosis and treatment of AR, and to define the needs in this neglected area by a literature review. AR is a systemic allergic disease and is generally associated with numerous multi-morbid disorders, including asthma, eczema, food allergies, eosinophilic oesophagitis (EoE), conjunctivitis, chronic middle ear effusions, rhinosinusitis, adenoid hypertrophy, olfaction disorders, obstructive sleep apnea, disordered sleep and consequent behavioural and educational effects. This report provides up-to-date usable information to: (1) improve the knowledge and skills of allergists, so as to ultimately improve the overall quality of patient care; (2) to increase interest in this area; and (3) to present a unique contribution to the field of upper inflammatory disease. This report was prepared by the European Academy of Allergy and Clinical Immunology (EAACI) Task Force on “Allergic Rhinitis (AR) comorbidities”. This was initiated, based on the rationale that AR is rarely found in isolation and needs to be considered in the context of systemic allergic disease associated with numerous comorbid disorders including asthma, chronic middle ear effusions, sinusitis, lymphoid hypertrophy with obstructive sleep apnea, disordered sleep, and consequent behavioural and educational effects.2 response to allergen exposure, resulting in symptoms, often beginning with atopic dermatitis (AD) and progressing to asthma and rhinitis  [AR, which has increased in prevalence over several decades, now affects 10–30% of the population, with the greatest frequency found in children and adolescents [AR is associated with numerous multi-morbid disorders. Those occurring in children have already been discussed in the EAACI Task Force on Paediatric Rhinitis . This paco-occurring with a primary disease or disorder; or the effect of such additional disorders or diseases [Multimorbidity is the presence of one or more additional disorders (or diseases) diseases . When thAR is an organ-specific manifestation of allergic disease. As such, it coexists with other organ-specific disorders that have a common allergic basis. It is therefore rarely found in isolation but frequently has associated multi-morbid disorders .Disorders which are part of the spectrum of allergic diseases, e.g. asthma, AD, food allergy, anaphylaxis;Disorders anatomically related to the nose: conjunctivitis, sinusitis, middle ear problems, throat and laryngeal effects;Sleep problems and secondary effects on concentration and behaviour; andTurbinate hypertrophy.These can be subdivided into:Although more common in paediatric practice, the occurrence of multi-morbidities in adults is significant and has important implications for quality of life, and work attendance and performance. It is likely that those with severe chronic upper airways disease (SCUAD) suffer more severe co-morbid effects.A European study of over 20,000 children showed that the co-existence of eczema, asthma and rhinitis in the same child is more frequent than expected if they were independent entities. Those children with one of these diseases at age 4 were 4–7 times more likely to have two or three of them at age 8. Children with two or three allergic diseases at 4 years are 30–60 times more likely to have two or three of these diseases at age 8 [The association between asthma and rhinitis in adults has been recognized for some decades, since the pioneering study of Brydon , who shoIn fact the majority of inflammatory asthma sufferers have some form of upper airway disease (ARIA 2001) , either Several possible relations exist between AR and asthma: (a) AR may be statistically associated with asthma; (b) AR may exacerbate coexisting asthma; and (c) AR may have a causal role in the pathogenesis of asthma.Lack of nasal function, i.e. purifying, warming and humidifying inspired air;Nasobronchial reflex ;Rhinovirus adhesion theory -1 expression) ; and“Migration” of T cell responses to other tissues after initial sensitization. Braunstahl  has showThe idea of postnasal drip (carriage of inflammatory cytokines/mediators from nasopharynx to lower airways) has been largely abandoned, since the ‘drip’ travels to the gut, by virtue of the larynx, not to the lower airway, unless the subject is deeply unconscious.Several possible causal mechanisms have been postulated to explain a link between AR and asthma:Certainly the presence of rhinitis, both allergic and non-allergic, is a risk factor for subsequent asthma development .The co-existence of rhinitis is associated with poor asthma control in adults, adolescents and children . Recent In children, there are clear data concerning the co-occurrence of AD and AR, largely from birth cohorts .In one Taiwanese study , AR was In a Croatian study , the ageTaken together these observations suggest that sensitization in later onset allergy is not via the skin, but by inhalant allergens acting via the respiratory tract mucosa .While some data suggest that the greater extent of allergic disease promotes more severe reactions , 24, othIn patients with asthma, AD and AR, the risks of systemic glucocorticoid bioavailability are highest, since treatment is likely to be directed to three sites: skin, bronchi and nose. Absorption occurs (from least to greatest rate) in the nasal mucosa, followed by the bronchi, then the skin. Alternative means of treatment  may need to be considered.AR can be associated with primary food allergy; however, it is more frequently associated with secondary food allergy, also known as pollen food syndrome (PFS). Some medical professionals refer to pollen food syndrome as oral allergy syndrome (OAS), although strictly speaking the two are not the same. When the term OAS was first used in 1987 it had no connection with pollen allergy but referred to any allergic symptoms in the mouth that often preceded more serious symptoms. The term pollen food syndrome is preferred when referring to those allergy symptoms to food that are linked to pollen allergy, are limited to the mouth and throat, and are usually mild. . The mosThose sensitised to both birch and grass pollens are more likely to develop pollen food syndrome , 29. TheIn one Italian study of pollen sensitive AR subjects aged 4–18 years old, a longer AR duration was significantly associated with moderate-to-severe AR symptoms (p = 0.004), and with co-morbidities such as asthma p 0.030), PFS was present in 24% (p < 0.001) , PFS was.Interestingly, birth order effects differ for different allergic disorders in a large Japanese study of 14,669 schoolchildren aged 7–15 years . There wPFS is usually a mild type of food allergy that occurs upon contact of the mouth and throat with raw fruits or vegetables containing epitopes also present in a pollen to which the subject is sensitized .PFS is a problem in patients sensitized to various pollen allergens. There was clear association between PFS and polyvalent airborne allergy (69%). Cross-reactivity patterns were typical . Subcutaneous SIT significantly alleviated PFS symptoms associated with ingestion of the responsible fruit and vegetables in patients .The treatment is avoidance of the food(s) causing reaction(s). Adrenalin is only indicated if severe reactions are described in the medical history, or if there are reactions to processed food. For primary food allergy an experimental study of SIT using peanuts was suspended because of adverse reactions .SIT for food allergy is reaching the point where it may soon be used routinely in clinical practice. Sublingual immunotherapy is effective for desensitization with a very favorable adverse event profile. Epicutaneous immunotherapy is also effective, most notably in younger children, with a high rate of local reactions. Oral immunotherapy demonstrates high efficacy, but with a higher risk of gastrointestinal and systemic adverse events. The need for long-term application to sustain desensitization is currently unclear. Immunomodulatory adjuvants may be added to enhance or diminish the immunogenicity of proteins, whereas genetic modifications of food allergens are designed to limit the risk of adverse reactions and address the issues of standardization and supply .The most common pollen-fruit cross-reaction is the birch-apple syndrome . Mauro eEoE is currently defined as a “chronic, immune/antigen-mediated esophageal disease characterized clinically by symptoms related to esophageal dysfunction and histologically by eosinophil-predominant inflammation” .Symptoms related to esophageal dysfunction;Eosinophil-predominant inflammation on esophageal biopsy, which is required for diagnosis, characteristically consisting of a peak value of ≥15 eos per high power field (eos/hpf) ;Response to treatment  supports, but it is not required, for diagnosis  .EoE is a clinicopathologic disorder diagnosed by clinicians taking into consideration both clinical and pathologic information:EoE is commonly associated with other atopic diatheses  . In adulAcid suppression Approximately one-third of patients with suspected eosinophilic esophagitis have a good clinical and histologic response to proton pump inhibitors  [ce high) .Oral prednisone (a synthetic corticosteroid drug) may be useful to treat EoE if topical steroids are not effective or in patients who require rapid improvement in symptoms  .Esophageal dilation Dilation of esophageal strictures is effective for relieving dysphagia, but has no effect on underlying inflammation [ammation , 47.Other experimental treatments Prostaglandin D2 receptor antagonist [(Azathioprine or 6-mercaptopurine) [tagonist , leukotrtagonist , Mepoliztagonist , purine opurine) , 51.Patients without symptomatic and histologic improvement after topical steroids might benefit from a longer course of topical steroids, higher doses of topical steroids, systemic steroids, an elimination diet or esophageal dilatation  .EsophagAllergic conjunctivitis is the typical conjunctival reaction in AR. It occurs following exposure to allergens. Ocular symptoms occur in 50–70% of patients with rhinitis, being more common with outdoor than with indoor allergens . Pitt etEye symptoms include itching, watering, redness and difficulty with vision due to these. Ocular symptoms are reduced by nasal air filters , suggestSome INS reduce eye symptoms as well as nasal ones, the recent molecules appear more consistently effective . The comThe extent of co-occurrence of rhinosinusitis is disputed and is likely to be different in acute rhinosinusitis (ARS), chronic rhinosinusitis (CRS) and CRS with nasal polyposis.When considering the role of allergy in sinus disease, it can be speculated that nasal inflammation induced by IgE-mediated mechanisms favours the development of acute and/or chronic sinus disease , 59.Several mechanisms could explain the link between allergic inflammation and sinus disease. Allergic inflammation of the nasal mucosa may give rise to mucosal congestion leading to impaired mucus drainage at the ostiomeatal complex in predisposed patients. Ostiomeatal pathology is considered to be essential to the generation of sinus-related symptoms .Certainly, AR sufferers experience common colds (which are a form of acute rhinosinusitis) more severely and for longer than those without underlying minimal persistent inflammation . The conSymptoms of IgE-mediated allergic inflammation should be asked for during history taking in patients with CRS and specific allergy evaluation should be performed in case of clinical suspicion. With regard to treatment, it is recommended that anti-allergic therapy is added to the treatment of patients with chronic sinus disease and concomitant allergy .Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with high concentrations of IgE in nasal polyp (NP) tissue. CRSwNP often coexists with asthma and this group is particularly characterized with tissue eosinophilia and high local IgE levels . TherefoStaphylococcus aureus enterotoxins (SAE) [In mucosal tissues, mRNA for the ε-chain of IgE was associated with a significant proportion of B cells , 66. Recns (SAE) , 65–67. ns (SAE)  demonstrCRSwNP is an IgE mediated disease; however, the role of atopy is less clear.In one hospital population of children with chronic OME, over 80% had rhinitis . A populOME is much rarer in adults and is usually found in association with more severe rhinosinusitis  rather than with rhinitis .The Eustachian tube exerts a major function in middle ear homeostasis via its role in the ventilation and protection of the middle ear and mucociliary clearance. The Eustachian tube contains an allergic inflammatory infiltrate in AR patients . It is tConcomitant occurrence of allergic diseases and primary immune deficiencies was reported by Klemola . KlemolaBecause of the pathophysiological associations of AR with OME, treatments focusing on allergic inflammation may be helpful in the management of OME . A meta-Atopic status and nasal disease should be evaluated in recurrent or chronic OME patients who have had no response to antibiotic therapy, and INS could be used as an adjunct to treatment of some OME patients. Further studies are needed to elucidate whether atopic status or rhinitis itself may influence the development of OME and the extent of ear involvement in various forms of rhinosinusitis.The adenoid tissue is a peripheral lymphoid organ located in the nasopharynx, forming part of Waldeyer’s ring. It contributes to the development of immunity against inhaled micro-organisms in early life. The volume of the adenoid increases with age and is maximal at 5–6 years, followed by a gradual decrease in volume by the age of 8–9 years . SymptomChildren with AR appear to have a greater susceptibility to AH than non-allergic children, with IgE-mediated inflammation of the nasal mucosa likely playing a role in both conditions .Adenoid hypertrophy in adults is rare and may indicate underlying malignancy or infection, such as human immune deficiency virus (HIV), rather than allergy.Treatment of AR with intranasal corticosteroids has been shown to improve various parameters associated with adenoid hypertrophy .In a recent study of 51 subjects with AR, half had hyposmia . Half ofReduced olfaction is more common in chronic rhinosinusitis, especially when NPs are present .AR may have a negative impact on olfactory function and is considered to be a sinonasal-related origin of chemosensory dysfunction . This isOlfactory function is a global perception of orthonasal and retronasal stimuli and of a stimulation of intranasal trigeminal nerve endings. The trigeminal function is enhanced in patients with AR and a link exists between chemosensory trigeminal function and neuroinflammation . AntidroSystemic corticosteroid is the first-line-treatment for olfactory dysfunction of sinonasal origin but use must be short term because of side-effects. Topical corticosteroid does not reach the same efficacy but has a good safety profile . Nasal cAntihistamines also have a positive effect on the olfactory function even if prescribed for a short period of time . OlfactoThe passage of mediators, cytokines and secretions backwards from the nose should not reach the larynx, unless epiglottic function is disturbed. However, many patients with AR do experience throat symptoms, including irritation, the sensation of difficult to shift mucus and cough . In a reEdema of the laryngeal mucosa, laryngeal erythema and candidiasis may all be found in a minority of patients treated with inhaled glucocorticosteroids , but areGastro esophageal reflux (GER) may masquerade as CRS . AssociaThe effect of AR on sleep can impair quality of life . PatientOne study  has demoPatients with AR frequently complain of disordered sleep, daytime somnolence and inability to concentrate. Recent studies document daytime somnolence in children with AR. Craig et al.  reportedBaraniuk et al. , in a stAR can reduce driving performance . It can In a study involving major examinations, sedating antihistamines increased the likelihood of dropping an examination grade . The medIn AR patients, nasal obstruction is a bothersome symptom which is most commonly due to inferior turbinate hypertrophy. The inferior turbinate is the initial deposit point for allergens and undergoes dynamic changes through the allergic cascade, which results in nasal obstruction. Targeting the inferior turbinate to augment the nasal airway is the mainstay of surgical treatment in AR .The turbinates are tiny shelf-like bony structures that project into the nasal passageways. They help warm, humidify, and clean the air that passes over them. If turbinate hypertrophy develops, it causes persistent nasal congestion and, sometimes, pressure and headache in the middle of the face and forehead. This condition may require surgery .Turbinate hypertrophy which is multimorbidity of the AR is primarily treated by allergen avoidance and medical treatment, but when these measures fail to control symptoms then surgery to the inferior turbinates of nose can be performed . OutfracDiagnosis of multi-morbidities of AR are shown in Table A physical examination of all organ systems potentially affected by allergies, with emphasis on the upper respiratory tract, should be performed in patients with a history of rhinitis.During the treatment of AR, multi-morbidities (co-morbidities) of AR should be considered. Treatment of AR according to guidelines may cause decreasing the nasal symptoms; and may cause improvement of the co-morbid problems; see Fig. This EAACI Task Force Report on AR Multi-morbidities has defined and classified the multi-morbidities associated with AR, together with providing recommendations for diagnosis and treatment. The information provided here should help improve allergists’ knowledge and skills, ultimately improving the overall quality of patient care, as well as helping to raise the profile of this important area of work.


Despite rapid discoveries in molecular biology of renal cell carcinoma (RCC) and advances in systemic targeted therapies, development of new diagnostic and therapeutic strategies is urgently needed. The androgen receptor (AR) has been shown to hold prognostic and predicitve value in several malignancies. Here, we studied a possible association between AR expression and prognosis in patients with RCCs.Low AR expression levels were associated with occurrence of distant metastasis and higher tumor stage in papillary and clear-cell RCCs. Importantly, multivariate Cox regression analyses revealed that AR is an independent prognostic factor for cancer-specific survival.The expression of AR was measured by immunohistochemistry and assessed by digital image analysis using a tissue microarray containing tumor tissue of a large and well-documented series of RCC patients with long-term follow-up information. Chi-squared tests, Kaplan-Meier curves and Cox regression models were used to investigate the possible relationship between AR expression and clinico-pathological characteristics and patient survival.Patients affected by AR-positive tumors exhibit a favorable prognosis by multiple Cox regression, while loss of AR expression is related to aggressive disease. Therefore, assessing AR expression offers valuable prognostic information that could improve treatment selection for metastatic disease. Moreover, our findings highlight a potential therapeutic use of AR pharmaceuticals in patients with RCCs. Recent insights into the biology of renal cell carcinoma (RCC) have led to the introduction of novel targeted therapies for kidney cancer. Despite marked clinical effects some patients are inherently resistant and most patients eventually acquire resistance. Hence, the American Cancer Society estimates 14,240 deaths related to kidney and renal pelvic cancer in the United States for 2016 [The steroid and nuclear receptor superfamily (NR) functions as DNA-binding transcription factors that regulate gene expression. Androgen receptor (AR) as member of the NR is bound to heat shock proteins which act as inhibitors, and is released and activated upon androgen binding. Ligand binding leads to AR translocation from the cytosol to the nucleus and transcriptional regulation of target genes . HoweverMoreover AR expression has been recognized in kidneys and kidney cancer , 15. Ha In this study, we systematically examined the expression of androgen receptor (AR) in RCCs and took advantage of a large hospital-based series of renal cell carcinomas with long-term follow-up information.Immunostains were performed on tissue microarrays containing tumor and corresponding normal renal tissue from 932 patients with renal cell carcinomas. In total 546 cases could be successfully scored for AR expression by immunohistochemistry. The remaining cases were excluded from further analyses either because of insufficient tumor tissue, poor tissue preservation or missing patient information. Figure The median time of follow-up was 7.67 years . By the end of follow up 182 patients had died from RCC, median time of follow-up among these patients was 7.81 years . The clinical and pathological features of the study population are summarized in Table n = 69) and clear-cell (n = 477) RCCs.An average of 1595 cells could be evaluated per core. Figure The mean of positive ccRCC tumor cells was 9% (median: 3%). Maximum percentage of positive cells was 72%, minimum 0%. The majority of ccRCCs  showed no AR expression or only in a small subpopulation of tumor cells (0 to 5%). In contrast, only 10 tumors (2%) showed more than 50% AR-positive tumor cells  in papRCCs  and presence of distant metastasis (P = 0.013). For example, percentage of AR-positive tumors was 46% in pT1 compared to 28% in pT3 carcinomas. No consistent association of AR expression with differentiation, lymph node metastasis, and ECOG performance status was observed  for AR by immunohistochemistry decreased with higher tumor stage  in metastatic disease (11%) compared to localized disease (55%). Moreover, percentage of papRCCs positive for AR decreased with higher tumor stage and dedifferentiation. For instance, AR-positive tumors constitute only 33% of high-grade carcinomas compared to 61% of low-grade carcinomas  in patients affected by tumors positive for AR compared to tumors with low/no AR expression. Subgroup analyses of non-metastatic disease also revealed a significantly higher survival rate in patients affected by AR-positive RCCs Figure .To further validate the findings, a multivariate analysis was performed using the Cox proportional hazards model. Differences in cancer-specific survival remained statistically significant after adjustment for established prognostic factors  in the multiple regression analysis for ccRCC patients, and confirmed AR expression in over 5% of the tumor cells  as an independent favorable prognostic marker. This also applies for the subset of cases with non-metastasized ccRCCs  for cancer-specific survival. Kaplan-Meier plots are depicted in Figure After adjustment for prognostic factors, AR expression also remained statistically significant for patients affected with papRCCs . Apart from AR expression only metastatic disease was confirmed as a significant prognostic factor, whereas grade of malignancy, tumor extent, regional lymph node metastasis, the ECOG Performance Status, gender, and age were not correlated with the clinical outcome (Table n = 15) died whereas 147 out of 375 patient (39.2%) without increase of AR mRNA levels deceased (P < 0.05), Kaplan-Meier curves are depicted in To further elucidate the relationship between AR expression and patient prognosis we took advantage of the Cancer Genome Atlas (TCGA) data on clear-cell renal cell carcinoma. Cohort used: KIRC-TCGA from 2016-01-28 ,20. WhenKaplan-Meier analyses revealed, that ccRCC patients with elevated AR expression of over 5% showed a significantly longer time to progression than low/no AR expressing tumors, in papRCC-patients a trend can be observed .in vitro experiments using different RCC cell lines. Using AR immunohistochemistry of cytoblocks we were able to identify Caki2 cells as AR espressing cell lines with over 80% of the tumor cells expressing the receptor. ACHN cells were found to be mostly AR negative  or interleukin-2 (IL-2). These new targeted agents show impressive initial responses, but resistance is eventually observed and cure rarely occurs . AlthougAndrogen receptor is a well known therapy target in prostate cancer treatment. In fact, already in 1941 Huggins and Hodges demonstrated that hormonal manipulation can result in antitumor activity in prostate cancer  and mediImportantly, the present study shows that patients affected by tumors with increased AR expression have a favorable clinical course with a 5-year cancer-specific survival rate of 80% in ccRCC patients (90% papRCC) compared to 67% affected by ccRCCs with low AR expression (69% papRCC). These findings are in line with previous reports based on smaller collectives, showing that AR-positive tumors are associated with a significantly better outcome , 18 and Despite mounting evidence that AR participates in the tumorigenesis and/or progression of diverse tumors, the mechanisms of how AR signaling contributes to carcinogenesis are poorly understood . Most pr® platform as a state-of-the-art, computer-based, automated digital image analysis system in order to minimize potential errors associated with manual quantification.Together these studies and our data highlight differences in AR biology depending on the (tissue) context. Furthermore, variations in the methods of detection of AR expression have to be considered. We used HALOin vitro data demonstrate that AR signaling increased cell viability and survival in AR postive RCC cells. Interestingly, a recent study revealed that AR activation increased the proliferation rate of RCC cell lines and promoted cell migration and invasion in vitro and RCC progression and invasion in RCC xenografted mouse models via AR-HIFα-VEGF signaling. Importantly, ASC-J9, a newly developed AR degradation enhancer suppressed RCC progression in vitro and in two RCC mouse models without obvious side effects [in vivo and in vitro studies have to provide more detailed insights into the mechanisms of AR signaling in RCCs. Interestingly, a Phase II disease-oriented drug trial including 28 patients using flutamide, a synthetic, non-steroidal antiandrogen, showed partial remission in one patient and stabilization of disease in two patients [Our  effects . Howeverpatients .In conclusion, our data demonstrate that AR is expressed in a substantial fraction of clear-cell and papillary RCCs. Correlation with clinical and pathologic findings indicate functional relevance and highlights AR expression as an independent prognostic biomarker. Further studies are needed to ascertain if AR expression is of predictive value and if new AR therapeutics are a treatment option in RCC patients.Tissue samples from 932 patients with primary renal cell carcinomas treated at the Department of Urology at the University of Heidelberg between 1987 and 2005 were collected. The human tissue samples were provided by the Tissue Bank of the National Centre for Tumour Diseases Heidelberg after approval by the Ethics Committee of the University of Heidelberg. Further details have been described previously .A tissue microarray containing 932 primary tumor and corresponding normal tissue samples of 932 patients was created. The tumors were graded according to the three-tiered nuclear grading system  and pathAfter heat-induced antigen retrieval using the target retrieval solution ULTRA Cell Conditioning  tissue microarray slides were stained with a ready to use anti-Androgen Receptor (SP107) rabbit monoclonal primary antibody . Staining was performed using an automated staining system BenchMark ULTRA  in accordance with the manufacturer’s instructions, the following solutions were used: OptiView DAB IHC Detection Kit (760–700), Hematoxylin I (790–2208), Bluing Reagent (760–2037).® platform from Indica Labs  including the TMA module and the CytoNuclear v1.4 module. In short, 5–10 representative cores were used to define staining parameters such as minimum nuclear optical density (OD), minimum staining OD, nuclear and cellular size and roundness, etc. As erythrocytes showed false positivity, a tissue classifier was trained to distinguish between tumor tissue and blood filled vessels and extravasal erythrocytes. The latter were excluded from automated image analysis. The TMA module was used to automatically exclude missing or erroneous cores, further more cores with advanced tumor necrosis, scarring or non-tumor tissue were excluded by SMG manually. With the parameters fixed, the cores were analyzed and the percentage of AR-positive tumor cells was calculated. Results from automated tissue analysis were manually controlled on a set of randomly selected cores. Positive cells were defined having a minimal OD of the staining of greater than 0.090. Intensity and quantity of immunoreactive tumor cells was further calculated based on the following system: the intensity ranged from 0, negative (Min OD: 0–0.090), 1, low (Min OD: 0.090–0.190), 2, medium (Min OD 0.190–0.273), to 3, high (Min OD: > 0.273). For further analysis tumor cells were grouped in a two-tiered sytem (positive 1-3) and negative (0), to account for inter- and intraindividual variations of AR expression and to simplify the analysis. The quantity of positive tumor cells was measured continuously and was presented as positive tumor cells in relation to all cells. Further details on the analysis parameters are given in Prior to image analysis, TMA slides were digitalized using the NanoZoomer-Series Digital slide scanner . Digital image analysis was performed using the HALOCell culture of RCC cell lines (Caki2 and ACHN) were established according to standard experimental protocols. In short, Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with fetal calf serum (FCS) and antibiotics was used and cells were kept at a 40–60% confluence rate. 5 µM of Cl-4AS-1, a steroidal androgen receptor agonist  were used to treat the RCC cell lines for 48 h with subsequent cell viability measurements. Cells with DMSO (solvant) containing medium served as controls. MTT and Cresylviolett (KRV) assays were performed according to the manufacturer’s protocols and absorbance was measured using a ELISA plate reader .http://www.rproject.org). For count data, Fisher’s exact test (two-sided) was used. Probability values < 0.05 were considered to indicate a statistically significant result.Survival was calculated from the date of nephrectomy to two different events: cancer-specific survival  and time to progression .Association between survival times and AR expression was first assessed by log-rank tests and represented by Kaplan-Meier plots. In order to account for the influence of established prognostic factors, hazard ratios (HRs) and 95% confidence intervals (CIs) were adjusted for patient gender and age, tumor extent, lymph node metastasis, distant metastasis, grade of malignancy, and ECOG Performance Status in a multiple Cox proportional hazard regression. Data were analysed using the R software package  was used for normalization and Z-score was calculated with formula: /SD normal.


The search is limited to English-language articles published between January 1980 and September 2016. Results show that existing robot-assisted ankle rehabilitation techniques can be classified into wearable exoskeleton and platform-based devices. Platform-based devices are mostly developed for the treatment of a variety of ankle musculoskeletal and neurological injuries, while wearable ones focus more on ankle-related gait training. In terms of robot design, comparative analysis indicates that an ideal ankle rehabilitation robot should have aligned rotation center as the ankle joint, appropriate workspace, and actuation torque, no matter how many degrees of freedom (DOFs) it has. Single-DOF ankle robots are mostly developed for specific applications, while multi-DOF devices are more suitable for comprehensive ankle rehabilitation exercises. Other factors including posture adjustability and sensing functions should also be considered to promote related clinical applications. An ankle rehabilitation robot with reconfigurability to maximize its functions will be a new research point towards optimal design, especially on parallel mechanisms.This review aims to compare existing robot-assisted ankle rehabilitation techniques in terms of robot design. Included studies mainly consist of selected papers in two published reviews involving a variety of robot-assisted ankle rehabilitation techniques. A free search was also made in Google Scholar and Scopus by using keywords “ankle Conventional rehabilitation programs for musculoskeletal and neurological disabilities require cooperative and intensive efforts from both therapists and patients , 2. ThisSaglia et al.  have sumA systematic review by Zhang et al.  has demoZhang et al.  also con∗,” “robot∗,” “rehabilitat∗,” or “treat∗.” The search is limited to English-language articles published between January 1980 and September 2016. Valuable references listed in relevant publications were further screened. Since this review focuses on comparative analysis of existing ankle rehabilitation devices in terms of robot design, only one of the studies with the same robot-assisted ankle rehabilitation technique will be included and discussed. The inclusion criterion is that if the study clearly presented the robot design, and the most recent one will be selected when there are multiple studies meeting the inclusion criterion. Included prototypes are either intelligently controlled or have multiple DOFs and have also been experimentally validated. Ankle devices specially designed for assessment [Included studies mainly come from the selected papers in a systematic review  that focsessment , 11 willsessment –14. It sWearable ankle rehabilitation robotic systems usually have one to three DOFs , 6, 15. A variety of platform-based robotic systems have been proposed and developed based on the consideration of ankle anatomy. The simplest representation of ankle motion is a hinge joint in the sagittal plane. A platform following this model is the ankle stretching device developed by Zhang et al. . This isSaglia et al.  have devFor an ankle rehabilitation robotic system with three rotational DOFs, the parallel mechanism is more often employed than serial mechanisms due to some features (safe workspace and large torque generation capacity). The multi-DOF ankle robot with AaEE has been considered to be suitable for comprehensive ankle therapy. Two typical examples of such robot platforms were developed by the rehabilitation robotics group at the University of Auckland , 16. TheWhile the robot developed by Jamwal et al.  and ZhanTo ensure comprehensive ankle exercises in a three-dimensional space, the requirement of three rotational DOFs can be a prerequisite of an optimal ankle rehabilitation robot. However, an ankle device with two DOFs for ankle (dorsiflexion/plantarflexion and inversion/eversion) is also acceptable since ankle adduction/abduction is limited and primarily controlled by rotation of the leg . TherefoWith regard to the torque capacity of different ankle robots, many factors affect their performance, including the device structure and the selection of actuators. We focus on comparing and discussing these ankle devices with consistent rotation centers in terms of actuation capacity. The torque capacity of the motor-driven devices directly depends on the driving ability of the motor and the friction of the robot joint. A typical motor-driven ankle training device is the one developed by Zhang et al. . Its outIn terms of robot functionality, the installation of a variety of sensing components also affects the robot design. Wearable devices are more difficult to be integrated with a sensing system due to the operation in a gait pattern . If goinIn addition to the selection of robot structure, optimization techniques should be also applied to the robot to have a suitable workspace and improve its torque generation capacity. Dimensional synthesis is one of the most difficult issues in the field of parallel robots with or without actuation redundancy. To deal with the optimal design of a redundantly actuated parallel robot used for ankle rehabilitation, Wang et al.  presenteRobot-assisted ankle rehabilitation exercises can be delivered in a variety of ways to reduce motor impairment and enhance functional motor outcomes. While existing robot-assisted ankle rehabilitation techniques have shown great potential for treating ankle disabilities , few canBy comparing a variety of ankle rehabilitation robots, it was found that an optimal robotic design for comprehensive ankle rehabilitation should be characterized with appropriate workspace and actuation torque, aligned rotation center between the robot and the ankle joint, and adjustable robot posture. However, this will definitely increase the cost due to complex robotic design. The authors also believe that no “one-size-fits-all” design exists for optimal robot-assisted ankle rehabilitation techniques, and therapy should be essentially tailored to each patient's needs and disabilities. Some examples of optimal ankle rehabilitation robots for certain applications are summarized in Additionally, Siegler et al.  have demHowever, two more design issues should be considered towards optimal ankle rehabilitation robots. One is the fixation issue of the human foot and shank, and a commonly used method is using straps attaching the foot on a footplate and the shank on a fixed leg holder. Again, since the anatomical ankle joint is not an ideal hinge joint , the rigAn attempt was made to ensure that a variety of robotic designs proposed for ankle rehabilitation were reviewed. In this review, we assumed that the same robotic prototype reported in different studies and dates by the same group had the same design, although some minor improvements  could have been made. However, other research may exist in which the ankle was not identified as a key term within the article; instead, some studies described the ankle complex as lower extremity or lower limb. Only articles after 1980 were included in this study as robot-assisted ankle rehabilitation techniques were quite limited before then. We included published journal and conference papers with a clear description of the robot design but did not include those written in languages other than English. Some studies may therefore have been excluded on this basis, leading to a potentially incomplete search.An optimal ankle rehabilitation robot design must be characterized with aligned rotation center as the ankle joint, appropriate workspace, and actuation torque.The number of robot DOFs depends on specific applications. The single-DOF robot is mostly developed for a special application such as ankle stretching along dorsiflexion and plantarflexion, while multi-DOF devices are more suitable for comprehensive ankle rehabilitation exercises.Other factors, including the adjustability of robot posture, sensing functions, the fixation of the human foot and shank on the robot, and mechanical stops, also affect their clinical applications. Adjustable robot posture can enable its use on a large population of patients with varying ankle disabilities. Robots with all required sensing functions allow for the implementation of advanced interactive training. The leg holder should be designed with some flexibilities to accommodate the difference between the robot structure and the human anatomy. Mechanical stops must be set for training safety especially on robotic systems whose workspace is larger than the actual ankle motions.Ankle rehabilitation robots with reconfigurability will be a new research area towards optimal design with appropriate workspace and actuation torque, especially on parallel mechanisms. Multiobjective optimization techniques can be involved to make robots in optimal kinematic and dynamic performance.This review focuses on the design analysis of existing ankle rehabilitation robots. Although most robot-assisted ankle rehabilitation techniques have been demonstrated to be effective for ankle physical therapy, they have drawbacks in the design that have impeded their applications in a wide range. Comparative analysis indicates the following: