Neurological diseases causing motor/cognitive impairments are among the most common causes of adult-onset disability. More than one billion of people are affected worldwide, and this number is expected to increase in upcoming years, because of the rapidly aging population. The frequent lack of complete recovery makes it desirable to develop novel neurorehabilitative treatments, suited to the patients, and better targeting the specific disability. To date, rehabilitation therapy can be aided by the technological support of robotic-based therapy, non-invasive brain stimulation, and neural interfaces. In this perspective, we will review the above methods by referring to the most recent advances in each field. Then, we propose and discuss current and future approaches based on the combination of the above. As pointed out in the recent literature, by combining traditional rehabilitation techniques with neuromodulation, biofeedback recordings and/or novel robotic and wearable assistive devices, several studies have proven it is possible to sensibly improve the amount of recovery with respect to traditional treatments. We will then discuss the possible applied research directions to maximize the outcome of a neurorehabilitation therapy, which should include the personalization of the therapy based on patient and clinician needs and preferences. According to the World Health Organization (WHO), neurological disorders and injuries account for the 6.3% of the global burden of disease (GBD) , 2. WithStandard physical rehabilitation favors the functional recovery after stroke, as compared to no treatment . HoweverThe great progress made in interdisciplinary fields, such as neural engineering , 10, hasRobots for neurorehabilitation are designed to support the administration of physical exercises to the upper or lower extremities, with the purpose of promoting neuro-motor recovery. This technology has a relatively long history, dating back to the early 1990s . Robot dEndpoint robots for the upper extremity, include Inmotion2  , KINARM Typical lower limb exoskeletons range from large systems, equipped with treadmill and weight support, and intended for hospital use, like the Lokomat  and the LOPES system , to moreOne common feature of rehabilitation robots, is that they are equipped with movement and/or force sensors, so that they integrate functionalities both for the assessment  and theIn spite of the increasing volume of published studies, the number of high-quality clinical trials on robot-assisted therapy is still relatively low. A large multi-center RCT comparing robot therapy, intensive physical therapy, and usual care  confirmeA major limitation of endpoint robotic approach is that the improvement is limited to the body regions involved in training. In a clinical setting, robotic rehabilitation may be cost and time-consuming, and for this reason, it is difficult to imagine the combination of different endpoint robotic devices in the patient who have an impairment that affects multiple body areas, i.e., post-stroke hemiplegia. Moreover, early robots for neuroehabilitation were specifically aimed at substituting labor-intensive physical rehabilitation with minimal human intervention, producing an automatic and repetitive treatment. This initial trend, however, minimizes the importance of both therapist knowledge and patient–physician relationship. However, the ability to precisely quantify sensorimotor performance during exercise in terms of movement kinematics and exchanged forces is leading to a new revolution in rehabilitation, toward evidence-based and knowledge-driven approaches. Modern rehabilitation devices automatically adapt task difficulty and assistance modalities to individual performance . In the Another stimulating challenge is the development of lightweight robots suitable for the use outside of the hospitals, in domestic or community environments and in conjunction with ADL, e.g., over ground walking in unstructured environments. This implies a modular structure, which facilitates donning and transportability.Non-invasive brain stimulation techniques are a promising adjuvant strategy for enhancing post-injury recovery. In recent years, more than 1,400 studies were performed in humans, with at least one-fifth of these focusing on stroke rehabilitation. NIBS techniques involve modulation of the central nervous system by electrically activating neurons in the brain  and can Related to rehabilitation, one of the major challenges is to design interventions that are efficient, promote motor learning, consolidate skills, and augment retention. For example, NIBS approach to stroke rehabilitation has focused on excitation of the unaffected hemisphere, of the affected hemisphere, or inhibition of unaffected hemisphere, also combining neuromodulation of both hemispheres . To dateSome ethical and technical considerations deserve discussion. First, use of NIBS, calls for greater caution on pediatric population, given the higher stakes and uncertain future effects for brains still undergoing rapid and formative development . Second,Careful monitoring is particularly important when considering that, despite researchers’ discussion of and explicit warnings against unsupervised use of NIBS , brain sIn recent years, it is possible to include also neural interfaces among the strategies for neurorehabilitation and indeed the use of these systems in clinical applications is increasing , 58.A neural interface is essentially a system mediating the communication between the brain and an external device , 60. SevOver the past decade also invasive brain-machine interfaces and neural prostheses in general have been the subject of extensive research with promising findings for the treatment of neuro-related impairments . The devNeural prosthesis can be combined with functional electrical stimulation , 89. In in vitro preparations  and patients’ biosignals, such as EEG or EMG (electromyography) Figure A.An example of such a multimodal approach was shown to the general public during the world cup in 2014, when the kick off was given by a paraplegic man using a lower limbs exoskeleton controlled by brain activity. Two years later, it was demonstrated that the combined use of gait rehabilitation with a BMI was able to induce partial neurological recovery in paraplegic patients . This reExperiences where assisted locomotion has been used in conjunction with neuromodulation are already present in the literature. Spinal tDCS was applied in patients with SCI undergoing assisted locomotion using driven gait orthosis  . ResultsAlthough the combined effect of neuromodulation with robotic therapy still needs to be clinically validated , it cleapersonalized treatment. This is not just intended to focus on a particular disease and address the symptoms shared by different populations of patients, but is truly envisioned as a personalized method for a single individual. The idea of a patient-tailored approach is not new: standardized algorithms have been proposed for stroke, based on the clinical history of the patient, time elapsed after insult, topography of the lesion, type, and severity of functional impairment . Personalization calls for flexible solutions, such as combining the main technologies, in order to adapt to the different patient’s features and preferences. And this is exactly the direction which should be undertaken in neurorehabilitation.MS, LDM, and MC conceived the manuscript. MC and LDM coordinated the activities. MS and MC prepared the figures. MS, MC, RDI, and LA revised the manuscript. All the authors wrote and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with one of the authors RI.


All the compounds have been evaluated for their in vitro antioxidant activities using DPPH free radical scavenging assay as well as FRAP assay. The result showed promising antioxidant activities having IC50 values in the range of 4.74 ± 0.08 to 92.20 ± 1.54 μg/mL taking ascorbic acid (IC50 = 4.57 μg/mL) as standard reference. In this study, compounds 20b and 20t, the most active compound of the series, showed IC50 values of 6.89 ± 0.07 μg/mL and 4.74 ± 0.08 μg/mL, respectively in comparison with ascorbic acid. In addition, the detailed SAR study shows that electron-withdrawing group increases antioxidant activity and vice versa. Furthermore, in the FRAP assay, eight compounds  were found more potent than standard reference BHT (C0.5FRAP = 546.0 ± 13.6 μM). The preliminary cytotoxic study reveals the non-toxic nature of active compounds 20b and 20t in non-cancerous 3T3 fibroblast cell lines in MTT assay up to 250 μg/mL concentration. The results were validated via carrying out in silico molecular docking studies of promising compounds 20a, 20b, and 20t in comparison with standard reference. To the best of our knowledge, this is the first detailed study of C-3 tethered 2-oxo-benzooxazines as potential antioxidant agents.The discovery of C-3 tethered 2-oxo-benzooxazines as potent antioxidants is disclosed. All the analogs  Electrospray ionization mass spectrometry (ESI-MS) and HRMS were recorded on Xevo G2-S Q Tof  Spectrometer. Column chromatography was performed over Merck silica gel (particle size: 60-120 Mesh) procured from Qualigens? (India), flash silica gel (particle size: 230-400 Mesh). All chemicals and reagents were obtained from Sigma Aldrich (USA), Merck (India) or Spectrochem (India) and were used without further purification.All glass apparatus were oven dried prior to use. Melting points were taken in open capillaries on complab melting point apparatus and are presented uncorrected. Ultrasonic irradiation was performed in a Elmasonic S 30 (H) ultrasonic water bath cleaner and the reaction vessel was positioned in the maximum energy area in the cleaner and the removal or addition of water was used to control the temperature of the water bath. Infrared spectra were recorded on a Perkin-Elmer FT-IR Spectrum 2 spectrophotometer 16a-h  were taken in toluene (50 ml) and NaH  was added carefully. After stirring this reaction mixture at 0°C for 30 min dimethyl oxalate  were added and reflux for 6 h. The progresses of the reaction were monitored by TLC using 9:1 Hexane/ethyl acetate as an eluent. After completion of reaction, the reaction mixture was quenched with distilled water and extracted with ethyl acetate (3 × 50 ml); then with distilled water (2 × 10 mL) followed by brine solution (2 × 20 mL). The organic layer was combined and dried over anhydrous Na2SO4 and the organic solvent was removed under reduced pressure to give the crude product. The crude products were purified by recrystalization using EtOAc/Hexane (v/v = 20:80), which afforded the pure desired diketo-ester 17a-h in 78-92% yields. Compounds 17a-h was used for next step without any further purification.Substituted acetophenone 17a-h  in MeOH:THF: H2O , added LiOH.H2O  into the reaction mixture and stirred it for 4 h at room temperature. The progress of the reaction was monitored by TLC. After completion of the reaction, it was quenched with 3N HCl solution and extracted with ethyl acetate (3 × 30 mL); then with distilled water (2 × 10 mL) followed by brine solution (2 × 20 mL). The combined organic layer was dried over anhydrous Na2SO4 and evaporated under vacuum to afford the corresponding crude product. These crude products were further purified by recrystalization with EtOAc/Hexane, which afforded diketo-acids 18a-h in excellent yields (up to 97%). Compounds 18a-h were used for next step without any further purification.To a solution of 18a-h  in water (2.0 mL) was added compound 19a-f  and the reaction mixture was irradiated under ultrasonic sonicator at 80°C temperature for about 75-90 min (depending upon the substrate employed). The progress of the reaction was checked by TLC using 9:1 Hexane/ethyl acetate as an eluent. After completion of reaction, the reaction mixture was extracted with ethyl acetate (3 × 50 ml); then with distilled water (2 × 10 mL) followed by brine solution (2 × 20 mL). The organic layers were combined and dried over anhydrous Na2SO4 and the organic solvent was removed under reduced pressure to give the crude product. The crude products were purified either by recrystalization using Hexane/EtOAc (v/v = 90:10) or by flash column chromatography method over silica gel using 7.5:2.5 to 9:1 Hexane/ethyl acetate as an eluent which afforded the pure desired (Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b]-oxazin-2-one 20a-ab having good yields (80–98%).To a solution of the compound f  = 0.85; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 185–186°C; FT-IR  3434, 1754, 1614, 1594, 1270, 1113; 1H NMR  δ 8.00 , 7.55–7.46 , 7.21–7.05 ; 13C NMR  δ 191.6 (C=O), 156.3 (O=C-O), 141.3 (C=CH), 139.1 (Ar-C), 138.3 (Ar-C), 132.8 (NH-C), 128.8 (Ar-C-NH), 127.7 (Ar-C), 126.0 (Ar-C), 124.0 (Ar-C), 123.8 (Ar-C), 117.2 (Ar-C), 116.0 (Ar-C), 94.7 (C=CH); HRMS (ESI) calcd. for C16H11NO3 [M+H]+: 266.0739; found 266.0734.Yellow solid; yield: 51.97 mg (98%), Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8:2) as an eluent; m.p. 200–203°C; FT-IR  3435, 1756, 1602, 1112; 1H NMR  δ 7.99 , 7.17 , 7.09–7.05 , 7.02–6.95 , 3.87 ; 13C NMR  δ 190.5 (C=O), 163.5 (Ar-C-OCH3), 156.6 (O=C-O), 141.2 (Ar-C-N), 138.6 (Ar-C-O), 131.2 (C=CH), 130.0 (Ar-C), 125.9 (Ar-C), 124.0 (Ar-C), 123.7 (Ar-C), 117.2 (Ar-C), 115.8 (Ar-C), 114.0 (Ar-C), 94.7 (C=CH), 55.6 (O-CH3); HRMS (ESI) calcd. for C17H13NO4 [M+H]+: 296.0845; found 296.0849.Yellowish solid; yield: 51.2 mg (88%), Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 185–187°C; FT-IR  3434, 1761, 1555, 1622, 1174; 1H NMR  δ 8.00–7.98 , 7.58–7.55 , 7.50-7.47 , 7.13–7.03 ; 13C NMR  δ 191.8 (C=O), 155.8 (O=C-O), 139.8 (Ar-C-N), 138.4 (Ar-C-O), 138.1 (C=CH), 133.0 (Ar-C), 131.1 (Ar-C-Cl), 128.9 (Ar-C), 127.8 (Ar-C), 124.8 (Ar-C), 123.8 (Ar-C), 118.3 (Ar-C), 115.8 (Ar-C), 95.7 (C=CH); HRMS (ESI) calcd. for C16H10ClNO3 [M+2]+: 301.7085; found 301.7089.Yellowish solid; yield: 56.93 mg (95%), Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 155–157°C; FT-IR  3434,1754,1634,1601,1495, 1226,1160; 1H NMR  δ 8.06-8.02 , 7.20-7.13 , 7.08-7.04 ;13C NMR  δ 190.4 (C=O), 167.2 (Ar-C-F), 155.8 (O=C-O), 139.8 (Ar-C-N), 138.6 (Ar-C-O), 131.3 (Ar-C-Cl), 130.5 (C=CH), 130.4 (Ar-C), 124.8 (Ar-C), 123.9 (Ar-C), 118.4 (Ar-C), 116.2 (Ar-C), 115.9 (Ar-C), 115.8 (Ar-C), 95.4 (C=CH); HRMS (ESI) calcd. for C16H9ClFNO3 [M+H]+: 318.0255; found 318.0259.Yellowish solid; yield: 59.08 mg (93%); Rf  = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 182–185°C; FT-IR  3434, 1761, 1631, 1586, 1088; 1H NMR  δ 7.93 , 7.45 , 7.13–7.00 ; 13C NMR  δ 190.4 (C=O), 155.7 (O=C-O), 139.8 (Ar-C-Cl), 139.4 (Ar-C-N), 138.7 (Ar-C-O), 136.3 (C=CH), 131.2 (Ar-C), 129.2 (Ar-C), 129.1 (Ar-C), 124.6 (Ar-C), 124.0 (Ar-C), 118.3 (Ar-C), 115.9 (Ar-C), 95.3 (C=CH); HRMS (ESI) calcd. for C16H9Cl2NO3 [M+2]+: 334.9959; found 334.9956.Yellowish solid; yield: 63.9 mg (96%); Rf  = 0.85; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 135–137°C; FT-IR  3432, 3075, 2923, 1626, 1583, 1105; 1H NMR  δ 7.54 , 7.47 , 7.34 , 7.17-7.09 , 6.82 ; 13C NMR  δ 192.1 (C=O), 155.4 (O=C-O), 139.9 (Ar-C-Cl), 138.4 (Ar-C-Cl), 137.7 (Ar-C-N), 137.2 (Ar-C-O), 132.7 (C=CH), 131.3 (Ar-C), 130.8 (Ar-C), 130.7 (Ar-C), 127.6 (Ar-C), 124.4 (Ar-C), 118.4 (Ar-C), 116.1 (Ar-C), 99.3 (C=CH); HRMS (ESI) calcd. for C16H8Cl3NO3 [M+2]+: 368.9570; found 368.9577.Yellowish solid; yield: 65.9 mg (89%); Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 175–177°C; FT-IR  3436, 2924, 1755, 1632, 1583, 1007; 1H NMR  δ 7.85 , 7.62 , 7.13 – 7.00 ; 13C NMR  δ 190.5 (C=O), 155.6 (O=C-O), 139.8 (Ar-C-N), 138.8 (Ar-C-O), 136.8 (C=CH), 132.1 (Ar-C-Cl), 131.2 (Ar-C), 129.3 (Ar-C-Br), 128.1 (Ar-C), 124.6 (Ar-C), 124.0 (Ar-C), 118.3 (Ar-C), 115.9 (Ar-C), 95.2 (C=CH); HRMS (ESI) calcd. for C16H9BrClNO3 [M+2]+: 377.9454; found 377.9458.Yellowish solid; yield: 70.7 mg (93%); Rf  = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9.5:0.5) as an eluent; m.p. 160–162°C; FT-IR  3434, 2925, 1624, 1766, 1494, 1178; 1H NMR  δ 7.94–7.92 , 7.30 , 7.14-7.04 , 2.44 ;13C NMR  δ 191.6 (C=O), 156.0 (O=C-O), 144.0 (Ar-C-CH3), 139.8 (Ar-C-N), 138.2 (Ar-C-O), 135.6 (C=CH), 131.2 (Ar-C), 129.7 (Ar-C), 128.0 (Ar-C), 125.0 (Ar-C), 123.7 (Ar-C), 118.3 (Ar-C), 115.8 (Ar-C), 95.9 (C=CH), 21.8 (-CH3); HRMS (ESI) calcd. for C17H12ClNO3 [M+H]+: 314.0506; found 314.0509.Yellowish solid; yield: 56.5 mg (90%); Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 178-180°C; FT-IR  3434, 1764, 1628, 1594, 1018; 1H NMR  δ 7.99-7.97 , 7.10-7.06 , 7.02–6.99 , 6.97-6.94 , 3.87 ; 13C NMR  δ 190.5 (C=O), 163.7 (Ar-C-OCH3), 156.1 (O=C-O), 139.6 (Ar-C-N), 137.8 (Ar-C-O), 131.1 (C=CH), 130.9 (Ar-C), 130.1 (Ar-C), 125.0 (Ar-C), 123.4 (Ar-C), 118.2 (Ar-C), 115.6 (Ar-C), 114.1 (Ar-C), 95.7 (C=CH), 55.6 (-OCH3); HRMS (ESI) calcd. for C17H12ClNO4 [M+2]+: 331.7345; found 331.7349.Yellowish solid; yield: 56.6 mg (86%); Rf  = 0.85; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9.5:0.5) as an eluent; m.p. 145–147°C; FT-IR  3433, 2930, 1770, 1624, 1596, 1128; 1H NMR  δ 8.02 , 7.17–7.07 , 6.98–6.90 , 2.36 ; 13C NMR  δ 190.0 (C=O), 166.9 (Ar-C-F), 156.4 (O=C-O), 139.3 (Ar-C-CH3), 136.1 (Ar-C-N), 134.7 (Ar-C-O), 130.3 (C=CH), 130.2 (Ar-C), 124.9 (Ar-C), 123.3 (Ar-C), 116.9 (Ar-C), 116.2 (Ar-C), 115.9 (Ar-C), 115.8 (Ar-C), 94.1 (C=CH), 21.1 (CH3); HRMS (ESI) calcd. for C17H12FNO3 [M+H]+: 298.0801; found 298.0807.Yellowish solid; yield: 53.7 mg (90%); Rf  = 0.80; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 142–145°C; FT-IR  3436, 2913, 1755, 1618, 1570, 1083; 1H NMR  δ 7.53 , 7.45 , 7.33–7.30 , 7.10–7.08 , 6.94–6.92 , 6.73 , 2.36 ; 13C NMR  δ 191.6 (C=O), 155.9 (O=C-O), 139.5 (Ar-C-CH3), 139.2 (Ar-C-Cl), 137.5 (Ar-C-Cl), 137.2 (Ar-C-N), 136.2 (Ar-C-O), 132.5 (C=CH), 130.7 (Ar-C), 130.6 (Ar-C), 127.4 (Ar-C), 125.4 (Ar-C), 123.0 (Ar-C), 117.0 (Ar-C), 116.4 (Ar-C), 98.0 (C=CH), 21.1 (CH3); HRMS (ESI) calcd. for C17H11Cl2NO3 [M+2]+: 349.0116; found 349.0112.Yellowish solid; yield: 65.4 mg (94%), Rf  = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8:2) as an eluent; m.p. 180–182°C; FT-IR  3452, 1755, 1625, 1581; 1H NMR  δ 7.99 , 7.06 , 7.00 , 6.96 , 6.88 – 6.86 , 3.88 , 2.34 ; 13C NMR  δ 190.4 (C=O), 163.4 (Ar-C-OCH3), 156.8 (O=C-O), 139.3 (Ar-C-CH3), 138.7 (Ar-C-N), 136.0 (Ar-C-O), 131.3 (C=CH), 129.9 (Ar-C), 124.4 (Ar-C), 123.6 (Ar-C), 116.8 (Ar-C), 116.0 (Ar-C), 114.0 (Ar-C), 94.5 (C=CH), 55.6 (OCH3), 21.1 (CH3); HRMS (ESI) calcd. for C18H15NO4 [M+H]+: 310.1001; found 310.1009.Yellowish solid; yield: 57.7 mg (93%), R−1) 3417, 1767, 1626, 1583, 1282, 1177; 1H NMR  δ 8.00 , 7.58-7.47 , 7.13 , 7.07 , 6.85 , 2.34 ; 13C NMR  δ 191.7 (C=O), 155.7 (O=C-O), 138.6 (Ar-C-N), 138.1 (Ar-C-O), 136.9 (C=CH), 136.6 (Ar-C-CH3), 132.9 (Ar-C), 128.9 (Ar-C), 128.2 (Ar-C), 127.8 (Ar-C), 124.7 (Ar-C), 115.5 (Ar-C), 110.2 (Ar-C-Br), 95.2 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H12BrNO3 [M+2]+: 359.0001; found 359.0008.Yellowish solid; Yield: 65.7 mg (92%); Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 190–192°C; FT-IR  = 0.80; Purification of crude product was done by recrystalization using EtOAc/Hexane; m.p. 230–232°C; FT-IR  3417, 1771, 1631, 1285, 1159; 1H NMR  δ 8.03-7.99 , 7.17-7.13 , 6.99 , 6.84 , 2.34 ; 13C NMR  δ 190.1 (C=O), 167.0 (Ar-C-F), 164.5 (Ar-C-F), 155.6 (O=C-O), 138.7 (Ar-C-N), 136.9 (Ar-C-O), 136.6 (C=CH), 134.5 (Ar-C-CH3), 134.4 (Ar-C), 130.4 (Ar-C), 130.3 (Ar-C), 128.3 (Ar-C), 124.6 (Ar-C-Br), 116.1 (Ar-C), 115.9 (Ar-C), 115.5 (Ar-C), 110.2 (Ar-C), 94.8 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H11BrFNO3 [M+2]+: 376.9906; found 376.9909.Yellowish solid; Yield: 67.6 mg (90 %); Rf  = 0.85; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 224–226°C; FT-IR  3417, 1763, 1626, 1561, 1294, 1127; 1H NMR  δ 7.54 , 7.47 , 7.33 , 7.18 , 6.88 , 6.79 , 2.35 ; 13C NMR  δ 191.9 (C=O), 155.2 (O=C-O), 138.7 (Ar-C-Cl), 137.5 (Ar-C-N), 137.3(Ar-C-O), 137.0 (C=CH), 136.8 (Ar-C-CH3), 132.7 (Ar-C-Cl), 130.8 (Ar-C), 130.7 (Ar-C), 128.8 (Ar-C), 127.5 (Ar-C), 124.3 (Ar-C), 115.7 (Ar-C), 110.3 (Ar-C), 98.8 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H10BrCl2NO3 [M+2]+: 426.9221; found 426.9227.Yellowish solid; Yield: 79.7 mg (94%); Rf  = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9.5:0.5) as an eluent; m.p. 259–260°C; FT-IR  3417, 1768, 1629, 1562, 1283, 1138; 1H NMR  δ 7.86 , 7.62 , 7.16 , 7.00 , 6.86 , 2.35 ; 13C NMR  δ 190.4 (C=O), 155.6 (O=C-O), 139.0 (Ar-C-N), 137.0 (Ar-C-O), 136.9 (C=CH), 136.7 (Ar-C-CH3), 132.2 (Ar-C), 129.3 (Ar-C), 128.5 (Ar-C), 128.1 (Ar-C), 124.5 (Ar-C), 115.6 (Ar-C), 110.3 (Ar-C), 94.8 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H11Br2NO3 [M+2]+: 436.9106; found 436.9100.Yellowish solid; Yield: 77.6 mg (89%); Rf  = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 219–220°C; FT-IR  3417, 1764, 1630, 1602, 1281, 1182; 1H NMR  δ 7.86 , 7.26–7.24 , 7.08 , 7.00 , 6.80 , 2.39 , 2.30 ; 13C NMR  δ 191.4 (C=O), 155.8 (O=C-O), 143.9 (Ar-C-CH3), 138.3 (Ar-C-N), 136.8 (Ar-C-O), 136.5 (C=CH), 135.6 (Ar-C-CH3), 129.6 (Ar-C), 128.0 (Ar-C), 127.9 (Ar-C), 124.8 (Ar-C), 115.4 (Ar-C), 110.1 (Ar-C), 95.3 (C=CH), 21.8 (CH3), 20.9 (CH3); HRMS (ESI) calcd. for C18H14BrNO3 [M+2]+: 373.0157; found 373.0152.Yellowish solid; Yield: 70.7 mg (95%); Rf  = 0.75; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 211–213°C; FT-IR  3446, 3072, 1758, 1621, 1515, 1183; 1H NMR  δ 8.32 , 8.13 , 7.12 , 7.01–6.96 , 2.37 ; 13C NMR  δ 188.8 (C=O), 155.9 (O=C-O), 149.9 (Ar-C-NO2), 143.3 (C=CH), 140.5 (Ar-C-N), 139.7 (Ar-C-O), 136.4 (Ar-C), 128.6 (Ar-C), 125.8 (Ar-C), 124.0 (Ar-C), 123.9 (Ar-C), 117.2 (Ar-C), 116.7 (Ar-C), 94.1 (C=CH), 21.1 (CH3); HRMS (ESI) calcd. for C17H12N2O5 [M+H]+: 325.0746; found 325.0748.Yellowish solid; yield: 57.2 mg (82%), Rf  = 0.75; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 207–209°C; FT-IR  3448, 3069, 1758, 1621, 1515; 1453; 1H NMR  δ 8.32 , 8.15 , 7.26 – 7.17 , 7.04 ; 13C NMR  δ 188.9 (C=O), 155.8 (O=C-O), 150.1 (Ar-C-NO2), 143.3 (Ar-C-N), 141.7 (C=CH), 140.5 (Ar-C-O), 128.7 (Ar-C), 126.2 (Ar-C), 125.1 (Ar-C), 124.1 (Ar-C), 123.4 (Ar-C), 117.5 (Ar-C), 116.6 (Ar-C), 94.3 (C=CH); HRMS (ESI) calcd. for C16H10N2O5 [M+H]+: 311.0590; found 311.0596.Yellowish solid; yield: 52.2 mg (84%); Rf  = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 198–200°C; FT-IR  3436, 2928, 1762, 1625, 1581, 1142; 1H NMR  δ 8.03-7.96 , 7.61-7.49 , 7.31 , 7.15 ; 13C NMR  δ 192.1 (C=O), 155.1 (O=C-O), 145.2 (Ar-C-NO2), 144.9 (C=CH), 137.7 (Ar-C-N), 137.6 (Ar-C-O), 133.4 (Ar-C), 128.9 (Ar-C), 127.9 (Ar-C), 124.7 (Ar-C), 118.9 (Ar-C), 118.0 (Ar-C), 111.5 (Ar-C), 96.9 (C=CH); HRMS (ESI) calcd. for C16H10N2O5 [M+H]+: 311.0590; found 311.0593.Yellowish solid; yield: 55.21 mg (89%); Rf  = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8:2) as an eluent; m.p. > 250°C; FT-IR  3435, 3107, 1759,1622, 1594, 1156; 1H NMR  δ 8.73 , 8.11 , 7.92 , 7.44 – 7.36 , 6.92 ; 13C NMR (100 MHz) δ 188.7 (C=O), 166.6, (Ar-C-F), 156.0 (O=C-O), 145.9 (Ar-C-NO2), 144.6 (C=CH), 139.5 (Ar-C-N), 135.1 (Ar-C-O), 130.9 (Ar-C), 125.8 (Ar-C), 118.9 (Ar-C), 117.7 (Ar-C), 116.6 (Ar-C), 116.4 (Ar-C), 113.0 (Ar-C), 94.5 (C=CH); HRMS (ESI) calcd. for C16H9FN2O5 [M+H]+: 329.0495; found 329.0490.Yellowish solid; yield: 61.71 mg (94%); Rf  = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7:3) as an eluent; m.p. 195–197°C; FT-IR  3435, 2926, 1599, 1758, 1633, 1594; 1H NMR  δ 8.02–7.93 , 7.29 , 7.10 , 6.98 , 3.89 ; 13C NMR  δ 190.7 (C=O), 164.0 (Ar-C-OCH3), 155.3 (O=C-O), 145.2 (Ar-C-NO2), 144.9 (Ar-C-N), 137.0 (Ar-C-O), 130.7 (C=CH), 130.3 (Ar-C), 124.9 (Ar-C), 118.6 (Ar-C), 117.8 (Ar-C), 114.2 (Ar-C), 111.2 (Ar-C), 97.0 (C=CH), 55.7 (OCH3); HRMS (ESI) calcd. for C17H12N2O6 [M+H]+: 341.0695; found 341.0692.Yellowish solid; yield: 58.51 mg (86%); Rf  = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 240–242°C; FT-IR  3436, 1763, 1622, 1596, 1268; 1H NMR  δ 8.06 – 8.00 , 7.83–7.81 , 7.62 , 7.54 , 6.99 ; 13C NMR  δ 190.7 (C=O), 156.1 (O=C-O), 142.2 (Ar-C-N), 141.1 (Ar-C-O), 139.2 (Ar-C-NO2), 138.3 (C=CH), 133.6 (Ar-C), 131.3 (Ar-C), 129.6 (Ar-C), 128.0 (Ar-C), 121.4 (Ar-C), 117.4 (Ar-C), 112.6 (Ar-C), 96.0 (C=CH); HRMS (ESI) calcd. for C16H10N2O5 [M+H]+: 311.0590; found 311.0595.Yellowish solid; yield: 55.22 mg (89%); Rf  = 0.70; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 235–237°C; FT-IR  3411, 3090, 1773, 1626, 1516, 1473; 1H NMR  δ 8.18 - 8.08 , 7.88 – 7.86 , 7.42–7.38 , 7.02 ; 13C NMR  188.7 (C=O), 163.7 (Ar-C-F), 155.5 (O=C-O), 141.8 (Ar-C-N), 140.6 (Ar-C-O), 138.7 (Ar-C-NO2), 134.4 (C=CH), 130.7 (Ar-C), 130.6 (Ar-C), 130.5 (Ar-C), 120.9 (Ar-C), 116.9 (Ar-C), 116.2 (Ar-C), 115.9 (Ar-C), 112.0 (Ar-C), 95.3 (C=CH); HRMS (ESI) calcd. for C16H9FN2O5 [M+H]+: 329.0495; found 329.0499.Yellowish solid; yield: 52.7 mg (80%); Rf  = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 205–207°C; FT-IR  3432, 2925, 2860, 1633, 1525, 1776, 1075; 1H NMR  δ 8.08 , 7.89-7.87 , 7.63 , 7.01 ; 13C NMR  δ 188.9 (C=O), 155.4 (O=C-O), 141.9 (Ar-C-N), 140.7 (Ar-C-Cl), 138.9 (Ar-C-O), 136.5 (Ar-C-NO2), 130.7 (C=CH), 129.8 (Ar-C), 129.5 (Ar-C), 129.2 (Ar-C), 120.9 (Ar-C), 117.0 (Ar-C), 112.1 (Ar-C), 95.2 (C=CH); HRMS (ESI) calcd. for C16H9ClN2O5 [M+H]+: 345.0200; found 345.0207.Yellowish solid; yield: 59.7 mg (87%); Rf  = 0.70; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 208–210°C; FT-IR  3433, 3088, 1770, 1620, 1522, 1470, 1072; 1H NMR  δ 12.39 , 8.09–8.08 , 7.95–7.93 , 7.78–7.58 , 6.62 ; 13C NMR  δ 190.3 (C=O), 155.3 (O=C-O), 142.2 (Ar-C-N), 140.8 (Ar-C-Cl), 138.5 (Ar-C-O), 137.7 (Ar-C-NO2), 136.2 (C=CH), 131.2 (Ar-C-Cl), 131.0 (Ar-C), 130.5 (Ar-C), 130.1 (Ar-C), 127.9 (Ar-C), 120.8 (Ar-C), 117.3 (Ar-C), 112.1 (Ar-C), 98.8 (C=CH); HRMS (ESI) calcd. for C16H8Cl2N2O5 [M+H]+: 378.9810; found 378.9818.Yellowish solid; yield: 64.6 mg (85%); Rf  = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 230–232°C; FT-IR  3435, 3093, 1769, 1619, 1521; 1H NMR  δ 8.09-7.96 , 7.83-7.44 , 7.01; 13C NMR  δ 188.9 (C=O), 154.7 (O=C-O), 141.8 (Ar-C-N), 140.1 (Ar-C-O), 137.9 (C=CH), 136.6 (Ar-C-NO2), 131.5 (Ar-C), 130.0 (Ar-C), 129.1 (Ar-C-Br), 126.6 (Ar-C), 120.7 (Ar-C), 116.5 (Ar-C), 111.4 (Ar-C), 95.7 (C=CH); HRMS (ESI) calcd. for C16H9BrN2O5 [M+2]+: 389.9695; found 389.9691.Yellowish solid; yield: 67.7 mg (87%); Rf  = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7:3) as an eluent; m.p. 218–220°C; FT-IR  3437, 2927, 2854, 1632, 1517; 1H NMR  δ 8.07-8.02 , 7.73 , 7.09 , 7.02 , 3.89 ; 13C NMR  δ 189.1 (C=O), 163.4 (Ar-C-OCH3), 155.8 (O=C-O), 141.7 (Ar-C-N), 140.6 (Ar-C-O), 138.1 (C=CH), 131.1 (Ar-C-NO2), 130.7 (Ar-C), 130.1 (Ar-C), 121.2 (Ar-C), 116.8 (Ar-C), 114.5 (Ar-C), 112.2 (Ar-C), 95.9 (C=CH), 55.8 (OCH3); HRMS (ESI) calcd. for C17H12N2O6 [M+H]+: 341.0695; found 341.0699.Yellowish solid; yield: 55.4 mg (81%); R−1 was mixed with 1.5 mL of a DPPH methanolic solution (20 mg L−1). Pure methanol was taken as control and ascorbic acid (vitamin C), vitamins A and E were used as a reference compounds. The absorbance was measured at 517 nm after 20 min of reaction. The % of DPPH decolouration of the sample was calculated according to the formula  at different concentrations ranging from 10 to 100 μg mL50. The results are expressed as antiradical efficiency (AE), which is 1000-fold inverse of the IC50 value AE=1000/ IC50.The decolouration was plotted against the sample extract concentration and a logarithmic regression curve was established in order to calculate the IC3.6H2O solution, 10.0 mM of ferric-tripyridyltriazine (TPTZ) solution and 300 mM sodium acetate buffer (pH 3.6) in a ratio of 1:1:10 (v/v/v). After that, Sample  was added to 3 ml of freshly prepared FRAP reagent and this reaction mixture was incubated at 37°C temperature for 30 min. and the absorbance was measured at 593 nm. It is also noted that, a freshly prepared solution of FeSO4 was used for calibration of standard curve. The FRAP antioxidant capability were evaluated in terms of C0.5FRAP  . Drawing of structures and simple geometry optimization were performed with Chem Bio-Office suite Ultra v12.0 (2012) . Docking of all compounds was carried out on the human antioxidant enzyme in complex (PDB ID: 3MNG)  were taken from the Protein Data Bank (http://www.rcsb.org/pdb). Program automatically docks ligand into binding pocket of a target protein by using protomol-based algorithm and empirically produced scoring function. The protomol is very important and necessary factor for docking algorithm and works as a computational representation of proposed ligand that interacts into binding site. Surflex-Dock's scoring function have several factors that play an important role in the ligand-receptor interaction, in terms of hydrophobic, polar, repulsive, entropic and solvation, and it is a worldwide well-established and recognized method. The most standard docking protocols have ligand flexibility into the docking process, while counts the protein as a rigid structure. Present molecular docking study involves the several steps viz., import of protein structure into Surflex and addition of hydrogen atoms; generation of protomol using a ligand-based strategy. During second step, two parameters first called protomol_bloat, which determines how far the site should extend from a potential ligand; and another called protomol_threshold, which determines deepness of the atomic probes, used to define the protomol penetration into the protein) were specified to form the appropriate binding pocket. Therefore, protomol_bloat and protomol_threshold was set to 0 and 0.50, respectively. In reasonable binding pocket, all the compounds were docked into the binding pocket and 20 possible active docking conformations with different scores were obtained for each compound. During the docking process, all of the other parameters were assigned their default values.To find the possible bioactive conformations of C-3 tethered 2-oxo-benzooxazine derivatives, molecular modeling studies were performed using the Sybyl X 2.0 interfaced for the synthesized compounds, which exhibited promising and lower antioxidant activity 20a-20ab using our reported procedure  solvent. The reaction of nitro/alkyl/halide-substituted 2, 4-dioxo-4-phenylbutanoic acid 18a-h with nitro/alkyl/halide-substituted 2-aminophenol 19a-f in water furnished C-3 tethered 2-oxo-benzooxazines 20a-20ab in 74-98% yields after purification either by flash column chromatography or by recrystallization method  by all the compounds 20a-20ab. The DPPH radical scavenging assay is generally utilized as a quick and reliable parameter to investigate the antioxidant activities of diverse heterocycles  mechanism and the second one is the single electron transfer (SET) mechanism  and/or electron-donating group (EDG) either at ring A or at C. The active group -CO-C=C-NH- enables resonance between the ring B and C, leading to multiple resonance structure, which may be further initiated by the attached substituents of ring A and C and in situ enhances the radical scavenging activity through the removal of hydrogen atom from NH of ring B via HAT mechanism. It has been found that electron withdrawing substituent NO2 at ring A or C increase the antioxidant activity which may be due to resonance based stabilizing effects. Therefore, based on the structures and their antioxidant activities, it was found that the compounds have either no substitution at ring A and C or have EWG/EDG at ring A and C plays a very important role in deciding their DPPH radical scavenging activities. Hence, based on the substituent's (either EWG or EDG) at ring A and C of 2-oxo-benzooxazines 20a-20ab, and their antioxidant activities, their structure-activity relationship can be explained by grouping all compounds into two groups:The generic scaffold of the newly synthesized C-3 tethered 2-oxo-benzo1,4]oxazines, as illustrated in Figure oxazines,No substitution or EDG at ring A or ring C: In the first group of compounds having no substitution at ring A and C i.e., the model compound 20a, exhibited promising antioxidant activity (IC50 = 10.20 ± 0.08 μg/mL) in comparison with standard reference Ascorbic acid (IC50 = 4.57 μg/mL) [entry 1]. Then, by putting EDG (OMe group) at ring C, as in compound 20b, further increases activity (IC50 = 6.89 ± 0.07 μg/mL) [entry 2]. Reversing the order i.e., halogen substitution at ring A and no substitution at ring C do not cause any further increase in antioxidant activity as shown by compound 20c (entry 3). Furthermore, when we incorporated halogen substituents  or CH3 substituent either at A or C as in the case of compounds 20c-l; a decrease in the antioxidant activity was observed due to high electron density in compounds 20d-g, 20j and 20k (entry 4-12). Furthermore, It was observed that two EDG at ring A  exhibited moderate antioxidant activity having IC50 value in the range of 18.86 ± 0.72 to 44.32 ± 0.45 μg/mL. In this series , ring C having Fluorine substituent i.e. compound 20n exhibited good antioxidant activity (IC50 = 16.86 ± 0.72 μg/mL) in comparison with other compounds (20m and 20o-q).2 group at ring C and EDG group i.e., CH3 group at ring A (compound 20r); the antioxidant activity was regained and shows IC50 value of 12.23 ± 0.05 μg/mL nearly equivalent to 20a (entry 18).To our surprise; when we incorporated EWG group i.e., NO20s , which, contrary to our expectations, displayed further decrease in antioxidant activity (IC50 = 21.27 ± 0.28 μg/mL) (entry 19).It is to be noted that EDG at ring A decreases antioxidant activity as shown in entries 3-17; so, we synthesized compound EWG (-NO2Group) at ring A: Since 20r having EWG (NO2) at ring C showed promising antioxidant activity; inspired by this observation, we prepared 20t-v having NO2 group at C-4 position of ring A. 2-oxo-benzooxazine 20t having no substitution at ring C, showed excellent antioxidant activity having IC50 value of 4.74 ± 0.08 μg/mL (entry 20). Since 20b having OMe substituent at ring C, was also found to show excellent antioxidant activity; thus, we synthesized 20u and 20v having Fluoro as well as OMe substituent, respectively at ring C. Unfortunately, antioxidant activity diminishes (entry 21 and 22). In addition, we also prepared 2-oxo-benzooxazines 20w-20ab having EWG group (NO2) at C-5 position of ring A further to investigate SAR study. Compound 20w having no substitution at ring C showed promising antioxidant activity having IC50 value of 12.53 ± 0.09 μg/mL (entry 23). On incorporating Fluoro group at ring C; activity of 20x increases . Furthermore, when we incorporated halogen substituents  at ring C as in the case of compounds 20y-20ab; a decrease in the antioxidant activity was observed (entry 19-23).20a-20ab. Whereas EDG either at ring A or C diminishes antioxidant activity. Our SAR results depict that 20b and 20t, the best compounds of the series, showed antioxidant activity comparable to standard reference Ascorbic acid.Overall, we can interpret that no substitution or EWG at ring A or ring C enhances antioxidant activity of all the synthesized 2-oxo-benzooxazines 3+−TPTZ] and develops a colored ferrous tripyridyltriazine [Fe2+-TPTZ]. The reducing nature of an antioxidant depends on their property to donate a hydrogen atom for the breaking of the free radical chain, which is responsible for oxidative stress etc.The FRAP assay was deliberated using the method as illustrated by Benzie and Strain  were found more potent than BHT (C0.5FRAP = 546.0 ± 13.6 μM).All the synthesized C-3 tethered 2-oxo-benzo1,4]oxazine analogs ,4oxazine20a-20ab) displayed good to moderate activity in comparison with BHT in the range of C0.5FRAP = 328.6 ± 25.8 μM to 916.8 ± 21.4 μM in in-vitro antioxidant FRAP assay except compounds 20e, 20f, 20l, 20q, 20v, and 20ab which showed C0.5FRAP greater than 1000 μM. Compounds having EWG i.e. NO2 substituent at ring C (20r and 20s) showed different potency than standard reference BHT. While 20r having CH3 at C-4 position of ring A displayed potent activity than BHT; 20s was found to be less active. Anomaly was observed in the case of compounds having no substitution at ring C. While 20c and 20m showed greater potency; compounds 20a, 20t and 20w were found less active than BHT.In summary, all the compounds . Since 20b has OMe group at ring C and 20t has NO2 group at ring A; it is worthwhile to compare the docking studies of active compounds with molecules having no substitution either at ring A or C. Therefore, we have also selected compound 20a for our in silico molecular docking simulation studies. For that purpose, Peroxiredoxins (Prdxs), a family of small human antioxidant enzyme, was selected as our target protein. Peroxiredoxins contain essential cysteine residues as catalyst and thioredoxin as an electron donor, which help in scavenging peroxide and are involved in the metabolic cellular response to ROS  .Compounds were docked into the active site of the known the human antioxidant enzyme target peroxiredoxins (Prxs) DTT complex (PDB ID: 3MNG) were taken from the Protein Data Bank  between the ligand and amino acids in the binding site of the protein were used for the ranking of compounds. The mode of interaction of the co-crystallized ligand dithiothreitol (DTT) within the crystal structure of enzyme in complex was used as a reference binding model. The root mean-square deviation (RMSD) of each docking pose was compared to the co-crystallized ligand and used for ranking and for RMSD calculation. The co-crystallized DTT molecule was re-docked onto the same binding site and the most probable binding mode was selected as that with the highest docking total score of 4.8921. An RMSD value 0.6772Å between the predicted and crystal binding mode indicates the high reliability of Surflex-Dock for this protein target.20a, 20b, and 20t against the antioxidant target protein Prxs showed a high binding affinity docking score indicated by a total score of 3.8470  of length 1.8Å to the backbone of hydrophobic aliphatic residue that is, Glycine-46. In the docking pose of the 20a, 20b, and 120t and Prxs complex, the chemical nature of binding site residues within a radius of 3Å with diverse properties was aromatic (hydrophobic), for example, Phe-120, ; hydrophobic, for example, Leu-116, Ile-119, Leu-149, Leu-112(Leucine), Gly-46, Gly-148(Glycine);  residues, for example, Arg-127 (Arginine); nucleophilic , for example, Thr-147 and Thr-44 (Threonine), nucleophilic (polar uncharged), for example Cys-47 (Cysteine); and hydrophobic  residues, for example, Pro-40 and Pro-45 (Proline) as a result, the bound compound showed a strong hydrophobic interaction with Prxs, thus leading to more stability and activity in this compound.On the other hand, docking results for 0 Figure , 3.6567 0 Figure  and 4.270 Figure  forms a The docking results for the ascorbic acid (standard compound) with the antioxidant target protein Prxs showed a low binding affinity docking score, indicated by a low total score of 3.4829 with three H-bond (hydrogen bond) formation of length 2.2, 1.8 and 2.1Å to the Thr-147, Gly-46 and Thr44 Figure . The asc20b and 20t having IC50 value of 6.89 ± 0.07 μg/mL and 4.74 ± 0.08 μg/mL, showed the binding affinity docking score of 3.6567 and 4.2709, respectively  i.e., 3.8470 . Whereas in FRAP assay, eight compounds  were found more potent than BHT (C0.5FRAP = 546.0 ± 13.6 μM). The active compounds were also found non-toxic in 3T3 fibroblast cell lines in MTT assay. Our in silico molecular docking results reveal that 20b and 20t showed excellent docking total scores against human antioxidant enzyme target as compared to ascorbic acid. Thus, the in silico docking simulation studies effectively validated the in vitro experimental results.In summary, we disclose C-3 tethered 2-oxo-benzo oxazine analogs in vitro activity evaluation of synthesized compounds. DKY and Saloni performed docking studies. SM and MHK provided molecular modeling facility. SC and PKJ drafted the manuscript. All authors read and approved the final manuscript.SC was responsible for the study of concept and design of the project. VS and PKJ were responsible for the performing synthetic reactions, acquisition and analysis of data. MS, MM, and AKS were responsible for the pharmacological 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.


MS). However, many people with MS do not make use of rehabilitation. We do not know much about the barriers to the use of rehabilitation in MS, but in other patient groups, illness representations have proven to be predictors of service utilization. Therefore, the aim of our study was to explore whether, in patients with MS, illness representations are associated with self‐reports of rehabilitation use in the past and the intention to use rehabilitation in the future, beyond sociodemographic and illness‐related factors.Multidisciplinary rehabilitation improves illness outcomes and is recommended in clinical guidelines for multiple sclerosis (Patients were recruited in a cross‐sectional nationwide online survey in Germany. Hierarchical binary logistic regression analysis was used to analyze whether illness representations are associated with the use of rehabilitation in the past and the intention to use rehabilitation in the future, over and above socio‐demographic and illness‐related variables.MS, participating in the study. Illness representations were correlated to both outcome variables beyond sociodemographic and illness‐related factors: The probabilities of having the intention to use rehabilitation and of making using of rehabilitation were higher in patients who believed that their MS was controllable by treatment and perceived that their MS would have severe consequences.There were 590 patients, who had Our data suggest that addressing patients’ illness representations may facilitate the intention to use and the use of multimodal rehabilitation, contributing to better illness outcomes. Furthermore, the link to the online survey was distributed via study flyers sent to 27 clinics and specialized medical practices (nationwide) which are listed on the DMSG website and which treat a minimum of 40% patients with MS per year. The study was approved by the Ethics Committee of the University of Freiburg .2.2Illness representations were measured using the German version of the Brief Illness Perception Questionnaire  and the intention to use rehabilitation in the future were assessed via the two items “Have you ever made use of rehabilitation because of your MS?” and “Would you make use of rehabilitation because of MS if necessary?” Both items had to be answered dichotomously (“yes” or “no”).Furthermore, patients completed a questionnaire covering sociodemographic variables  and self‐reported information about illness‐related factors .2.3p < .05 with the outcome variables. The following coefficients were interpreted: Nagelkerke R2 as an index of the quality of the overall model and the percentage of the variance explained by stages 1–3. Values >.20 can be defined as acceptable, values >.40 are interpreted as good, and values >.50 are interpreted as very good  using Pearson correlation coefficients. Correlations of .1 are interpreted as small, correlations ≥.3 as medium, and correlations ≥.5 as strong Cohen, . SecondlAll data analyses were performed using IBM SPSS Statistics 23  were recruited via the DMSG , while only 2% of participants were recruited via MS clinics. Women comprised 72.4% of the sample. The mean age was 45.6 years (SD = 10.3). Among the patients, 51.7% had relapsing‐remitting MS, and the mean time since the MS diagnosis was 11.0 years (SD = 8.5). Two‐thirds of the sample had made use of rehabilitation because of MS in the past and would make use of rehabilitation in the future because of MS if indicated . Table A total of ars SD = 0.3. AmonThe baseline associations between the B‐IPQ items are shown in Table p‐values <.05. However, with the exception of the correlation between the time since the first symptoms and the perceived consequences (r = .323), the correlations are all in the small range.Table The results concerning the use of rehabilitation are shown in Table Table p < .001), treatment control  and concern  were also significantly associated with the intention to use rehabilitation.The same procedure also controlling for use of rehabilitation in the past (in the first step) showed that perceived past behavior explained 56% of the variance of intention to use rehabilitation. The explained variance was slightly higher when including the sociodemographic and illness‐related variables (+ 1.5%) and including illness representations yielded a further increase in the explained variance, by four percentage points. Besides use of rehabilitation, with the highest Wald coefficient  was controlled for the intention‐related outcome variable.With respect to the outcome domain, our results are largely in line with studies in the context of cardiac rehabilitation. The illness representation domains “cure/control,” “consequences,” “identity,” and “coherence” were significantly associated with attendance at cardiac rehabilitation in patients with acute myocardial infarction  is influenced by patients’ attitudes and (adherence) expectations, treatment beliefs such as the perceived benefits of DMT therapy, and self‐efficacy  epidemiological data found in the literature . The authors have no further competing interests to report.


MAOA) and androgen receptor (AR) genes are important candidates associated with mammal personality. In our analysis, MAOA exon 15 showed no polymorphism but a novel polymorphism was seen in the camel AR exon 1; 16, 17, 18, and 19 glutamine repeats were detected. We genotyped 138 camels belonging to four Egyptian breeds: Maghrabi (n = 90), Sudani (n = 15), Somali (n = 23), and Baladi (n = 10) for AR. Out of the 90 genotyped Maghrabi camels, we evaluated responses of 33 and 32 mature females to a novel object and exposure to an unfamiliar person, respectively. AR gene showed a significant association based on the principal component (PC) score, which indicated the fear of human touch, and the PC score indicates fear during interaction with novel objects. Individuals carrying a shorter genotype in homozygote (S/S) were found to be more fearful. Furthermore, we found that Sudani and Somali breeds had a higher frequency of shorter genotype (S/S), which was associated with increased fearfulness. These findings reflect the behavioral tendency and consequently, affect the use of this breed. This is the first report showing the role of AR glutamine repeats influencing a behavioral trait in dromedary camels and leading to inter-breed differences. Fear-related traits reported here are important because camels cope with various types of stresses and fear, resulting from the demands of intensive production systems and racing events. However, further studies, employing functional genomics and linkage analysis are necessary for confirming the relationship between fearfulness and genetic variation.Temperament traits such as fearfulness are important as they define an animal’s responses to its environment and handling. The increasing automation of daily tasks and growing population limits contact between camels and humans. Such limitations contribute to fear of humans and changes in physical environment. Monoamine oxidase A ( Animal temperament influences individual behavioral changes in regard to stressors or environmental challenges, and impacts the adaptability of the animal to new environments, handling, animal welfare, and productivity . TemperaBehavioral traits are defined by a combination of environmental and genetic factors. A genetic basis of animal behavior was indicated by breed differences , and by Over the past few decades, new management systems for livestock production have been introduced worldwide. Camel management has been changing in recent years from an extensive to a semi-intensive or intensive system, and has presented new challenges for both the animal and farmer . IncreasMAOA) codes for an enzyme that plays an important role in the breakdown of neurotransmitters, such as serotonin, dopamine, and norepinephrine, and maintains a constant level of these chemicals in the brain [MAOA is one of the candidate genes associated with the personality of mammals, such as generalized anxiety and panic disorders, which are characterized by feelings of anxiety, fear, excessive worrying, and tension in the face of life experiences [AR) is located on the X chromosome, and has eight exons. It contains a highly polymorphic tri-nucleotide (CAG) repeat encoding glutamine in exon 1, and the length of this poly-glutamine repeat affects the level of transactivation [AR, the dromedary camel AR is composed of nine exons [AR exon 1 and various behavioral traits have been documented in a number of animal species [The Monoamine oxidase A gene  is a multipurpose animal. It provides milk, meat and wool, and can be used for agricultural work, transportation, racing, and beauty contests [The dromedary camel  to a novel object and an unfamiliar person; second, to estimate the polymorphism of the above-mentioned candidate genes among the four Egyptian camel breeds , and third, to investigate the possible association between genetic polymorphism and personality. To our knowledge, there is no existing study that has evaluated the genetic basis for the personality of dromedary camels.n = 90), Sudani (n = 15), Somali (n = 23), and Baladi (n = 10). The Maghrabi individuals were reared at the Camel Studies and Production Development Center in Matrouh Governorate, belonging to the Animal Production Research Institute (APRI), Agricultural Research Center, Egypt. All animals were identified by ear tags. Concentrate feed mixture (CFM) was offered each morning. The CFM was composed of 25% wheat bran, 25% yellow corn, 9% decorticated cotton seed meal, 20% barley, 15% rice bran, 3% molasses, 2% premix, and 1% common salt. Rice straw forage was offered to the animals in the afternoon. The Camel Studies and Production Development Center in Matrouh Governorate has a yard measuring about 50 × 60 m2 and 20 shaded rooms (6 × 6 m2) constructed with iron bars for housing six camels per room. The other three breeds  were collected from five breeders in four governorates  located in the Nile Delta in the northern part of Egypt.We used 138 dromedary camels  belonging to four Egyptian breeds: Maghrabi  and the approach of unfamiliar person (UF.P.) behavioral tests, respectively. The 32 individuals used in the UF.P. and 33 individuals in the N.O. partially overlapped. The assessed fear responses towards both, novel object and unfamiliar person, were video-recorded for three minutes from the start of the test. The age of tested individuals ranged from 6 to 13 years. All individuals were sexually matured and under similar management and feeding systems. They were not pregnant nor in their breeding season (November to March).The novel object (N.O.) test was conducted in order to investigate reactions related to fearfulness. The object used was a red pilates ball (60 cm diameter). The test was based on the novel object test for dairy cattle , which wIn the same arena but on a different day, camels were evaluated for their reactions to an unfamiliar person. The test was based on studies on dairy cows and dogs , 34, whiMAOA exon 15, the primer pair F: 5′-AAATCACCCACACCTTCTGG-3′ & R: 5′-CTGTTTCTTATGAGCACGTCAA-3′, and for AR exon 1, the following primer pair F: 5′-ACTTTCCCGGGCTTAAGCAG-3′ & R: 5′-GAAGCTGTTCCCCAGG-ACTC-3′, were designed using Primer 3 plus Version 2.3.6 software [MAOA, and a 316 bp fragment of camel AR exon 1. PCR was performed in a 15 μl reaction mix containing 20 ng of genomic DNA, 2x PCR buffer, all four dNTPs at 400 μM, each primer at 0.3 μM, and 0.5 U of LA-Taq DNA polymerase . The PCR cycle conditions for MAOA were as follows: initial incubation at 95°C for 2 min, followed by amplification for 35 cycles with 95°C for 30 s, 62°C for 45 s, 74°C for 60 s, and for AR, cycling at 95°C for 30 s, 61°C for 30 s, 74°C for 15 s, with a final extension at 74°C for 10 min was used. The amplified products were purified using a PCR purification kit , and the resultant products were sequenced using the same primers with Big Dye Terminator ver. 3.1 Cycle sequencing kit  according to the standard protocol, and electrophoresed on an ABI PRISM 3130xl sequencer . BLAST software [http://www.geospiza.com), MEGA 6 [AR labeled with 6-FAM, and genotyped the 138 samples on an ABI 3130xl DNA Sequencer , and the sizes of the fragments were estimated based on 400 HD ROX size marker using the GENEMAPPER software .Genomic DNA from hair samples were extracted for the above-mentioned 138 camels using a QIAGEN DNeasy tissue kit . For genotyping of software , based osoftware . We amplsoftware  was used, MEGA 6 , and BioWe conducted principal component analysis (PCA) to contract the behavioral data obtained from the novel object and unfamiliar person tests, using the ‘prcomp’ function in the R software ver. 3. 1. 2 . Correlation matrices were used for analysis. The number of components to be extracted was determined by parallel analysis. Principal component (PC) scores were calculated using the default steps of the ‘prcomp’ function. We genotyped the male and female camels, but because the small number of males, they were not included in the behavioral analysis. The association between genotypes and PC scores was tested by one-way analysis of variance (ANOVA) using the SAS software ver 9.1.3 All aspects of the study were performed according to the guidelines established by the Ministry of Education, Culture, Sports, Science and Technology in Japan (Notice No. 71). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Wildlife Research Center, Kyoto University (Permit No. WRC-2017-005A). No special permission for behavioral research, such as this study, is requiredin Egypt.We constructed three principal components (PCs) for both the novel object and approach by unfamiliar person tests, which explained 89% and 83% of the total variance of the data for the two tests, respectively. The loading matrix of behavioral items on each PC is shown in Tables In the novel object test, PC1 highlighted the fear of novelty because the seven behavioral items involved had high loading values. It indicates that individuals with high PC1 scores are more fearful when exposed to a novel object. PC2 explained the exploratory behavior, because it positively correlated with latency to approaching a novel object and negatively correlated with the number of sniffs. It indicates that individuals with high PC2 scores are less inquisitive. PC3 contained only one item depicting the fear response when crossing over a novel object. It indicates that individuals with high PC3 scores are more fearful when passing a novel object.For the approach by unfamiliar person test, PC1 indicates the fear and avoidance of human touch, because it positively correlated with items involving human avoidance, and negatively correlated with items involving acceptance of human interactions. This indicates individuals with high PC1 scores are more fearful of humans. PC2 showed common signs of fear and anxiety, as it positively correlated with behavior related to fearfulness like urination, teeth grinding, and vocalization. Individuals having high PC2 scores are intimidated by approaching humans. PC3 indicated signs of fear similar to PC2, except for vocalization item that was not included in PC3.MAOA (accession number: MF787280), but failed to detect any polymorphism in this region. However, we detected a novel length polymorphism in AR; at 316 bp, 319 bp, 322 bp and 325 bp of the alleles , respectively. For AR, the frequency of major (316 bp) and minor (322 bp) alleles were 0.45 and 0.015, respectively. We designated the major allele (316 bp) as short (S), and the other three alleles  as long (L). Sequencing data suggested that the 316 bp, 319 bp, 321 bp, and 325 bp alleles contained 16, 17, 18, and 19 glutamine repeats, respectively. We divided the subjects into separate groups based on the possible interactions between S and L alleles; for instance, in the case of additive interaction, we designated them as S/S, S/L, and L/L groups. However, in the case of dominance of L over S or S over L alleles, we used two groups; with/without L where S/L and L/L were L+, S/S was L-, or with/without S where S/S and S/L were S+, L/L was S-, respectively. The AR genotype frequency among the four camel breeds was higher for the short genotype (S/S) in Sudani (0.692) and Somali (0.579) compared to the Maghrabi (0.192) and Baladi (0.286) breeds (AR among the four camel breeds studied are shown in We sequenced a 406 bp fragment of exon 15 for ) breeds . The allAR , and a marginal significant association with PC1, reflecting fear from and during interaction with the novel object  in case of additive interaction between S and L alleles. Highest score was observed for S/S genotype . The S/S genotype displayed a higher score than the other genotypes  reacted more to fear, from either the novel object or an unfamiliar person. In the case of fearfulness toward an unfamiliar person, S/L heterozygote individuals behaved differently and were less fearful than the L/L homozygote individuals. This result implies that additive interactions between S and L alleles are unclear, especially in the case of fearfulness in the unfamiliar person test. As a result, we assumed dominance of one allele (L) over another (S), and found a significant association with PC1 for both behavioral tests. In the case of dominance of S over L allele, there was no significant association detected with any of the PCs.We identified a significant association between fearfulness towards a novel object and unfamiliar person, as assessed from video recordings, to the AR glutamine repeat tend to show more aggressiveness and impulsiveness [AR allele exerts greater androgen activity [S/S) reacted intensely with fear upon exposure to a novel object and an unfamiliar person. As shorter AR alleles exert greater androgen activity, it might be expected that individuals carrying shorter genotypes show less fearfulness, and this trend does not seem to straightforwardly correspond to the previous patterns of associations in humans and dogs. Fearfulness is not the opposite of aggressiveness; fearfulness and aggressiveness are both negative reactions to novel objects and unfamiliar people. Although we did not measure the camels’ aggression in this study, fear response is able to be explained in accordance with aggression. On the other hand, some camels reacted positively to the previous two behavioral tests, and they scored low in their fear response.The association between androgens and various traits  has been documented in a number of animal species , 39. Humsiveness , 40. Preactivity . Our resAR glutamine repeat polymorphism and AR function are somewhat contradictory. In vitro studies have shown that medium-length glutamine repeats had high transcriptional activity of AR compared with both shorter and longer glutamine repeats [AR transcriptional activity was positively affected by an increased AR glutamine repeat length in skeletal muscle cells. Moreover, a positive association between the AR glutamine repeat and free testosterone in men has been observed in two large and independent population samples with more than 2,500 individuals [AR glutamine repeats in three zebra species had longer alleles than those in horses. As zebras have not been domesticated it might be expected that they are more aggressive than horses and are expected to have shorter AR glutamine repeats. Finally, the low variation in camel AR glutamine repeats (16–19 repeats) compared to human (10–37 repeats) may cause minor variations in the AR activity, and there might be another locus in this gene which causes the major effect on the AR activity. Thus, the associations between AR glutamine repeat polymorphism and AR function are somewhat conflicting, and demand a more thorough hormonal investigation, such as measuring the circulating steroid of the tested individuals before conducting any behavioral tests. Moreover, these results should be tested in more camel populations with the help of functional genomics to confirm the trend of the association between AR activity and gene polymorphisms.The recent data on the associations between  repeats . Sheppar repeats  found thividuals . Ito et ividuals  found a AR, we found that both Sudani and Somali camels contained a higher frequency of shorter genotype (S/S), which was associated with higher fearfulness. The Maghrabi and Baladi camels contained lower frequency of shorter genotype (S/S). This indicates that Sudani and Somali camels respond strongly to fear either due to a novel object, or an unfamiliar person than the responses of Maghrabi and Baladi. This could be attributed to the fact that Baladi breeds are mainly used for agricultural work, whereas Maghrabi are bred for meat and milk production. During breeding, continuous contact between camels, humans, and inanimate objects exists. These conditions might accustom these two breeds to many activities and objects, and hence, less fearful. On the other hand, Sudani and Somali breeds were imported from Sudan and Somali African countries, and originally raised in large groups foraging natural pasture . Such isolation minimizes contact with different objects and people, thus causing anxiety and more fear upon exposure to strange events or environments.By genotyping other Egyptian camel breeds for AR glutamine repeat affects fear response towards novelty and unfamiliar person. Such polymorphism might be useful for a marker-assisted selection program for improvement of camel personality. We conclude that Sudani and Somali breeds show higher degrees of fear to novelty than the Maghrabi and Baladi breeds. This is the first evidence correlating polymorphism in androgen receptor gene and individual differences in behavioral traits in dromedary camels. Fear-related traits assessed in this study are important, because camels have to cope with various types of stress and fear, compounded by the demands of intensive production systems and racing events. However, further studies employing functional genomics and linkage analyses are necessary for validating the relationship between fearfulness and genetic polymorphism.Our results suggest that a genetic polymorphism in S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table*Numbers inside brackets = numbers of males.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Marked up-regulation of aldose reductase (AR) is reportedly associated with the development of hepatocellular carcinoma (HCC). We investigated how aberrantly overexpressed AR might promote oncogenic transformation in liver cells and tissues. We found that overexpressed AR interacted with the kinase domain of AKT1 to increase AKT/mTOR signaling. In both cultured liver cancer cells and liver tissues in DEN-induced transgenic HCC model mice, we observed that AR overexpression-induced AKT/mTOR signaling tended to enhance lactate formation and hepatic inflammation to enhance hepatocarcinogenesis. Conversely, AR knockdown suppressed lactate formation and inflammation. Using cultured liver cancer cells, we also demonstrated that AKT1 was essential for AR-induced dysregulation of AKT/mTOR signaling, metabolic reprogramming, antioxidant defense, and inflammatory responses. These findings suggest that aberrantly overexpressed/over-activated hepatic AR promotes HCC development at least in part by interacting with oncogenic AKT1 to augment AKT/mTOR signaling. Inhibition of AR and/or AKT1 might serve as an effective strategy for the prevention and therapy of liver cancer. In adulr absent , 4. A numa (HCC) . Consistma (HCC) , 8, 12. ma (HCC) . In spitPotential mechanisms are aberrant overexpression/activation of hepatic AR and/or Polyol Pathway (PP)-associated overt oxidative stress and inflammation, which are believed to contribute significantly to the development of cancers , 13, 14.Aberrant overexpression/activation of hepatic AR/PP may also contribute to lactate over-production, as in the well-known “Warburg effect” or aerobic glycolysis, whereby cancer cells exhibit increased conversion of glucose to lactate, even in the presence of sufficient oxygen . Aberranin vitro in cultured liver cancer cells and in vivo in the livers of DEN-induced transgenic HCC model mice.In the present study, we investigated the potential roles of AR in the development of HCC. The effects of AR overexpression and AR knockdown/knockout on lactate formation, the expression of inflammatory cytokines, and the most important Warburg effect regulating pathway, the AKT/mTOR signaling pathway, were evaluated AR and AKR1B10 mRNA up-regulated in the development of hepatitis C virus (HCV)-associated HCC [AR mRNA ranked at the top 3% and 7% of the significantly altered genes in HCV-positive HCC and HCV-positive cirrhosis respectively, in comparison with the HCV-negative normal subjects . Meanwhile, AKR1B10 mRNA ranked at the top 2% and 9% of the significantly altered genes in HCC and cirrhosis respectively (p < 0.001).In humans, microarray analyses identified ated HCC , 27. AR AR overexpression or knockdown on hepatocarcinogenesis, we performed transfection studies using HepG2 or SMMC-7721 liver cancer cells with a plasmid overexpressing AR or three plasmids overexpressing shRNAs against AR  and the control cells (pLV-ctrl-transfected), knockdown of AR suppressed wound-healing in EGF-stimulated cells (pLV-ctrl+EGF versus pLV-shAR-1+EGF) [AR overexpression or knockdown on cell proliferation, colony formation, migration and invasion, and wound-healing observed in SMMC-7721 liver cancer cells  . Similarer cells . TogetheAR-induced wound-healing was largely suppressed by LY294002 treatment, we investigated how AR might affect the activity or expression of AKT1 and AKT2. Our qPCR analyses revealed that both AR overexpression and AR knockdown in HepG2 cells did not affect the mRNA expression of AKT1 and AKT2 (S473-AKT1), AKT1, AKT2, and several AKT1 and AKT2 down-stream signaling proteins, including serine-256 phosphoryalted-FOXO1 (pS256-FOXO1) and the key pathway of Warburg Effects [AR, with the exception of FOXO1 , dose-d1 Figure . ConsistAR suppressed EGF-induced up-regulation of AKT1 and AKT2 in HepG2 cells   Figure .AR overexpression markedly suppressed the binding of MYC-ubiquitin to AKT1, whereas this effect was not significantly in AR knockdown cells  assays were performed. In HEK293T cells, Flag-tagged AR co-precipitated with HA-tagged AKT1, using either anti-Flag or anti-HA antibody Figure . FurtherT Figure .T308D/S473D) than either the WT AKT1 or a constitutively-inactive AKT1 (AKT1T308A/S473A)  had a much weaker affinity  was significantly diminished in cells co-treated with siAKT1 (pFlag-AR+siCtrl transfected versus pFlag-AR+siAKT1 co-transfected)  was significantly restored in AKT1 co-overexpression cells (pLV-shAR-1+pcDNA3.3-HA transfected versus pLV-shAR-1+pHA-AKT1 co-transfected), although HIF1a were not altered significantly. Additionally, inactivation of AKT1 by LY294002 also reversed AR-induced mTOR, HIF1a and PKM2 up-regulation  Figure . Conversn Figure .AR overexpression increased lactate formation  or inactivation by LY294002 treatment significantly diminished AR overexpression-induced lactate formation and LDH activity increasing. Meanwhile, AR knockdown suppressed lactate formation and LDH activity, but AKT1 overexpression significantly restored AR knockdown-induced lactate formation and LDH activity decreasing.In comparison with the control cells, n Figure  and LDH n Figure , AKT1 knTNFα/IL-6 mRNA expression increasing  had significantly higher tumor incidence (% mice with tumors > 1 μm), visible tumor number per mouse, maximal tumor size and accumulated tumor size  than the DEN-treated control mice (WT/FVB)  as compared to the control mice (WT/B6)  Figure , but not) Figure . Convers) Figure , with a AR overexpression significantly increased hepatic Akt1 expression, whereas Ar deficiency significantly reduced hepatic Akt1 expression in DEN-treated mice  analyses, liver-specific e Figure . Moreovee Figure . In AR od Figure . Conversin vitro studies, AR overexpression increased serum and hepatic lactate concentration, liver LDH and ALT activities, and hepatic Tnfα/Il-6 mRNA expression  with the following parameters “clinical specimen” in sample type, “mRNA” in Data type, “Liver cancer” in cancer type.Clinical AR-overexpressing transgenic FVB (TG1/FVB and TG2/FVB) mice were generated by Dan Song at Yun-qing Yang's lab of Xiamen University. The liver-specific human AR-overexpressing transgenic FVB (TG1/FVB and TG2/FVB) mice, Ar knockout C57BL/6 mice (KO/B6) and their controls (WT/FVB or WT/B6) were also kind gifts from Prof. Yun-qing Yang and only used for this project.The liver-specific human Mice were bred and maintained under a standard 12–12 h light-dark cycle, and were fed standard rodent chow and water ad libitum, and housed in the barrier facility of the Laboratory Animal Center, Xiamen University. All animal experiments were performed according to the protocols approved by the Institutional Animal Use and Care Committee of Xiamen University, China.AR-overexpressing transgenic FVB (TG1/FVB and TG2/FVB), Ar knockout C57BL/6 (KO/B6) and their controls (WT/FVB or WT/B6) were injected intraperitoneally with diethylnitrosamine (DEN) at 25 mg/kg body weight at the age of 2 wk  [For the induction of HCC, the male mice of liver-specific human MO, USA) . DEN-treAll of the other procedures are established standard techniques and are described in the t-test (two-tailed) for pair-wise comparisons. A probability value (p) < 0.05 was considered to be significant, those < 0.01 or < 0.001 more so.All statistical analyses were performed with the GraphPad Prism 5.0 software. Values are expressed as the means ± SEM. The Student's


Neurological patients after stroke usually present cognitive deficits that cause dependencies in their daily living. These deficits mainly affect the performance of some of their daily activities. For that reason, stroke patients need long-term processes for their cognitive rehabilitation. Considering that classical techniques are focused on acting as guides and are dependent on help from therapists, significant efforts are being made to improve current methodologies and to use eHealth and Web-based architectures to implement information and communication technology (ICT) systems that achieve reliable, personalized, and home-based platforms to increase efficiency and level of attractiveness for patients and carers.The goal of this work was to provide an overview of the practices implemented for the assessment of stroke patients and cognitive rehabilitation. This study puts together traditional methods and the most recent personalized platforms based on ICT technologies and Internet of Things.Journal of Medical Internet Research, and 15 European research projects from Community Research and Development Information Service from the last 15 years were reviewed for classification and selection regarding their relevance.A literature review has been distributed to a multidisciplinary team of researchers from engineering, psychology, and sport science fields. The systematic review has been focused on published scientific research, other European projects, and the most current innovative large-scale initiatives in the area. A total of 3469 results were retrieved from Web of Science, 284 studies from A total of 7 relevant studies on the screening of stroke patients have been presented with 6 additional methods for the analysis of kinematics and 9 studies on the execution of goal-oriented activities. Meanwhile, the classical methods to provide cognitive rehabilitation have been classified in the 5 main techniques implemented. Finally, the review has been finalized with the selection of 8 different ICT–based approaches found in scientific-technical studies, 9 European projects funded by the European Commission that offer eHealth architectures, and other large-scale activities such as smart houses and the initiative City4Age.Stroke is one of the main causes that most negatively affect countries in the socioeconomic aspect. The design of new ICT-based systems should provide 4 main features for an efficient and personalized cognitive rehabilitation: support in the execution of complex daily tasks, automatic error detection, home-based performance, and accessibility. Only 33% of the European projects presented fulfilled those requirements at the same time. For this reason, current and future large-scale initiatives focused on eHealth and smart environments should try to solve this situation by providing more complete and sophisticated platforms. Imagine if people rejuvenated as time went by, such as Benjamin Button in the F Scott Fitzgerald's tale—a man who was an elder at birth and died with the appearance of a baby. Instead, human abilities deteriorate as they become older. Life expectancy at birth in Europe is increasing steadily , and by Although aging involves several components, deterioration in cognitive abilities does not always appear due to aging, but rather, it often appears following a stroke. The risk of suffering a stroke duplicates in people aged above 65 years . AccordiRegarding those stroke survivors, as many as 68% of stroke patients meet the criteria for apraxia and action disorganization syndrome . This deWith the objective of assisting the cognitive rehabilitation of stroke survivors, the traditional methodologies used to support the movements of the user act as a guide to be followed, and generally, they depend on the help of therapists. Currently, health care services are using centrally directed general guidelines which have very limited effect.To address this issue at the cutting edge, some research and commercial home-based information and communication technology (ICT) systems , mobile apps , and Web-based strategies  have intThe objective of this study was to provide an identification of the effective assessment of rehabilitation practices for cognitive disorders from a traditional perspective to a more technological one by presenting the most recent advanced eHealth systems and projects to not only maintain but also improve the cognitive status of both elderly people and stroke patients in their daily living.Moreover, this study encompasses the concepts and manifestations in the execution of daily tasks of the main and most common stroke-related syndromes mentioned before, which can cause deficits in the execution of daily activities. They are presented in a broader context considering their influence in a high percentage of stroke survivors.It is thought that Hugo Liepmann presented the first description of apraxia as a distinct neuropsychological syndrome in the 19th century . LiepmanOne of the most accepted definitions of apraxia is as follows: apraxia is a “disorder of skilled movement not caused by weakness, akinesia, differentiation, abnormal tone or posture, movement disorders (such as tremors or chorea), intellectual deterioration, poor comprehension, or uncooperativeness” . This deThe consequences of cognitive disorders in these domains have been studied mainly in specific activities which involve the use of tools and multistep tasks .One of the main consequences of apraxia is the misuse of common everyday objects, for example, forks to eat soap, cutting paper with closed scissors, biting on the toothbrush when cleaning teeth, pressing knives into the loaf without moving it, or closing a paper punch on top of the sheet without inserting it. Although some apraxic patients present right-sided hemiplegia and their errors may be associated with the paralysis of the nondominant left hand, there is evidence of their pathological nature when observing the behavior of healthy people when manipulating objects with the nondominant hand ,11.The errors that appear when manipulating single tools, and those which are detectable in explicit testing, are probable to arise in the execution of multistep tasks also. However, there is a classification of specific errors associated with multistep tasks. The most relevant are as follows  is a neuropsychological disorder after a brain injury. Schwartz and colleagues were the first in providing a description . The maiAccording to the studies presented in , 5 errorOn the basis of different case studies with ADS patients, some errors are found to be more frequent in specific patients than others. In addition, thanks to these studies, it can be revealed that those patients who present high error rates commit more omission errors -20.It is found that 80% of stroke survivors suffer from hemiparesis. The main consequence of this disease is usually the weakness or inability to execute movements in one side of the patient's body. This inability can be centered in the user’s hands, facial muscles, arms, or even legs, leading to significant difficulty when carrying out their ADL .When users present weaknesses in the body segments mentioned before, it is very probable that they will suffer from loss of balance due to muscle fatigue, coordination for walking, coordination when manipulating objects, and dexterity in achieving accuracy.Obviously, the part of the body that experiences the inability depends on the part of the brain where the stroke happens. So, when an injury occurs on the left side of the brain, it usually results in deficits in right-sided components and vice versa.Neglect is one of the consequences derived from a brain accident, typical of the left hemiplegic patient, that is, one who has suffered damage to the right hemisphere of the brain. It is often also recognized as unilateral spatial agnosia, hemi-inattention, or hemispatial neglect.Neglect is a disorder of attention to space, especially with respect to space on the left. Neglect often appears as a problem exclusively visual in nature, because the difficulties of the left hemiplegic patient in orienting his or her eyes toward the visual field of the left are evident, especially during the first weeks following the stroke.Neglect or hemi-inattention often represents an aspect that is not valued in terms of rehabilitation with due dedication but rather is defined as a phenomenon that in most cases resolves spontaneously after a few months following stroke.However, neglect is not attributed to a problem in the ability to see but it is a problem related to attention. Already in 1874, Hughlings Jackson hypothesized that the right hemisphere was directly involved in perception and in relation to the outside world, and almost 100 years later, the neuropsychologist AR Lurija confirmed his perceptive peculiarities . But eveADL comprises activities of basic self-care such as washing, grooming, and dressing, besides preparing drinks and food. The execution of these tasks involves sequences of more basic actions with manipulation of environmental objects directed at some desired end goal.Once these studies are analyzed, the main thought is that a strong correlation exists between those impairments in the execution of ADL and apraxia scores. The functional independence measure provides a rough evaluation of the performance of ADL at home or at hospitals, and this score was used to establish a correlation between outcome and apraxia . A similTaking into account the behavior of stroke patients and with the purpose of providing a complete report on the evolution of methodologies for their screening and techniques of cognitive rehabilitation, a literature review was carried out by a multidisciplinary team of researchers from engineering, sports science, and psychology fields.First of all, regarding the traditional methods for the individual evaluation of stroke patients as well as for providing cognitive rehabilitation after the screening, some material has been used from the results generated in some European projects that the authors collaborated in. In addition, a deep review of additional bibliography has been carried out. This additional review was designed using a systematic protocol to interpret and analyze the most relevant research ,34. For Are the current methodologies for cognitive rehabilitation effective enough to improve health status and independence during the rehabilitation phase?Is the workload of the therapists in current rehabilitation techniques worthwhile based on the slow improvement of the cognitive abilities of patients?Second, taking into account the negative answers to those questions, 2 additional ones were proposed:What types of technology are adequate and used nowadays to design a smart ICT–based system that would interact with stroke patients?How well could those technologies be received by end users, their families and carers, as well as health professionals?Journal of Medical Internet Research) for research studies and Community Research and Development Information Service (CORDIS) [Journal of Medical Internet Research, and 15 research projects from CORDIS were reviewed for classification and selection.Then, a new search of those actual ICT–based approaches or research projects that provide cognitive rehabilitation was done. For that purpose, using relevant keywords such as “cognitive rehabilitation,” “personalized health care,” and “stroke,” an extensive review of Web of Science and other sources   for reseFinally, special attention was paid to the following features to classify the ICT–based approaches:Do they provide a home-based rehabilitation?Do they assist in the execution of complex ADL tasks?Do they present automatic error recognition?Are they accessible?Taking into account the review methodology described above, the material presented in this section focuses on (1) relevant classical techniques for the individual assessment of patients who suffer from cognitive disorders; (2) traditional methods to provide cognitive rehabilitation after screening; and, finally, (3) the most recent proof of concepts, projects, and innovative actions that provide smart interactive ICT systems, which implement the concept of eHealth to maintain the cognitive status of users while providing novel ways of rehabilitation.Some of the most important aspects taking into account the traditional techniques for assessment of stroke patients and cognitive rehabilitation are related to the following:The analysis of the manipulation of single tools, which demonstrates the existence of performance deficits even in the execution of simple activitiesThe importance of the use of kinematics as a quantitative approach to analyze the performance of an action with tool useThe fact that many goal-direct movements  are impaired even in the hand ipsilateral to the lesionTesting the use of actual tools with real target objects has been mandatory to analyze the ability of stroke patients in interacting with objects in their daily living. The temporal and spatial features of the movements are subaspects in some of the scoring methods, but the assessment is very rough. Nevertheless, there are other tests focused on these specific aspects, which monitored user movements in 3D space by using the adequate methodology for analyzing kinematics .Bearing in mind that the studies that consider the recording of user movements during tool use are relatively rare in patients who suffer from left brain damage (LBD) or right brain damage (RBD), there is a great number of research on the kinematics of more basic goal-directed movements such as pointing, aiming to targets, or grasping neutral objects.As indicated in The implementation of intelligent environments to provide a rehabilitation platform is something relatively new, which many researchers are focusing their efforts on. In fact, later in the study, the most important work exploring this topic will be presented.Once a general view of how to assess the level of severity of stroke patients, as well as to screen them, has been presented, the main traditional techniques to provide cognitive rehabilitation in their daily living are summarized. As derived from the reading of this subsection, classical techniques are not especially based on the use of smart technology, which means high workload for therapists and clinicians along with long and frequent patient visits to the hospitals or rehabilitation centers.Considering the limitations presented in traditional techniques, these methodologies do not offer many benefits during the rehabilitation stage. For that reason, efforts have been taken in the previous years to develop systems that monitor the performance of a task and provide feedback, making it familiar, personalized, and attractive for the user. Successful execution of rehabilitation tasks would increase and the use of a smart environment (even at patients’ house) would improve independence in daily living and would alleviate occupational therapists' workload.This subsection aims at describing the main research approaches and projects in the last 15 years related to providing smart platforms or environments that support cognitive rehabilitation and even maintain the cognitive status of the elderly, empowering their active aging.There are 9 main European research projects focused on the development of prototypes based on new technologies for providing advanced cognitive rehabilitation, excluding those centered in the rehabilitation of the movement of body segments. These are explained in detail in Elderly people wish to live at home and independently as long as possible. Different multidisciplinary research groups are nowadays working on achieving the “smart house of the future” to suit the needs of people. For example, the Orcatech project is focused on carrying out a pilot study to determine adherence to treatment of an internet-based platform that provides mindfulness meditation . The plaThe Gloucester “Smart House” project for people with dementia supports this population in their ADL and focused on the cognitive aspects . The smaMeanwhile, “UTA’s MavHome Smart House” project physically assists the elderly and individuals with cognitive disabilities by providing home capabilities that will monitor health trends and assist in the inhabitant's day-to-day activities in their own homes .Finally, City4Age aims at enabling “Ambient Assisted Cities” by defining elderly-friendly city services for the active and healthy aging. The core idea is to demonstrate that “smart cities” can play a pivotal role in the early detection of mild cognitive impairment (MCI) and frailty risks and subsequent interventions by collecting data about individual behaviors in an unobtrusive way . Data coThe City4Age project lead by Politecnico di Milano makes use of current city services (open data) and collected large amount of data about citizens, such as usage of public transports, services, and shopping, to develop useful interventions. Interventions could include ecological momentary interventions to improve the quality of life in daily living, dynamic interventions providing positive patterns of behavior change, and just-in-time interventions to assist individuals in case a higher priority intervention is required.The material presented in this study addressed different topics relevant for the cognitive rehabilitation of stroke patients and the importance of ICT technologies as well as the implementation of eHealth and tele-rehabilitation concepts for the evolution of classical methodologies. Moreover, the concept and consequences of the most common syndromes after stroke were described along with an introduction.From the review of the literature and regarding the assessment of the performance of stroke patients during the use of single tools for their individual screening, 7 main traditional relevant works have been presented as well as 6 examples of methods that include analysis of kinematics, and 9 focused on the performance of goal-oriented tasks.First of all, the analysis of single tool use has emphasized the role of the left hemisphere of the brain in this type of tasks  and also showed that actual task performance is generally less compromised than out-of-context performance. However, most of the tests demonstrated that a high number of patients present deficits even in the performance of seemingly simple activities. Second, the analysis of kinematics has also been considered as useful in the analysis of actual tool use. As derived from the tables presented, this kind of analysis is more sensitive than scoring systems. Finally, the results of some studies noted that the processing and interpretation of the data acquired from patients’ behavior for the recognition of movements, which makes possible the determination of the success of an action, can be affected by more elementary deficits of the patients during the performance of goal-directed movements.Meanwhile, the classical methods to provide cognitive rehabilitation once the screening of the corresponding stroke patients is done have been classified in 5 main techniques. On the basis of the analysis of the results, there is evidence that new rehabilitation technologies are needed. Among the main reasons why new ICT systems are necessary, the following ones can be derived:There are a high number of stroke survivors who need long-term cognitive rehabilitation.Consequences and traditional rehabilitation techniques of apraxia and action disorganization syndrome reduce independence.The economic costs of health care are significant when dealing with rehabilitation after stroke.Stroke patients show improvements very slowly by using current hospital-based rehabilitation methods.One of the main consequences of the inadequacy of current techniques is the fact that many patients present lifelong disabilities and suffer from social exclusion.These statements are directly related to the questions proposed and indicated in the methodology. For that reason, the review has covered the main current instances of ICT–based cognitive rehabilitation systems or approaches. The study has been completed with 8 different technology-based approaches from scientific-technical literature; 9 European projects from past Framework Programs to the current Horizon 2020; and other actions such as the use of virtual coaching, 3 examples of smart houses, and the large-scale initiative City4Age. This compilation of projects has been limited to the last 15 years approximately.Although nowadays ICT–based platforms are the main interest of research for many professionals and institutions to provide cognitive rehabilitation after stroke, current research initiatives focused on providing such smart systems for coaching and maintenance of cognitive well-being should be well aware of some barriers and take them into account when designing and developing the smart environments.In terms of user acceptance, older adults are mainly using the internet and mobile phones to keep in contact with family and friends. Although the proportion of older adults using these technologies is less than in any other age, the numbers are steadily increasing ,87. TherTo deal with this issue, focus groups should be involved in the user requirements and throughout the development of the prototypes. In addition, user groups should be recruited to validate the components developed in different cycles. A user-centered approach would ensure that the usability and perception of the usefulness of the systems are maximized. Moreover, cost barriers must also be addressed by aiming to use affordable, off-the-shelf technologies.Stroke has significant negative effects on the socioeconomics of countries. Considering the health budget allocated for stroke in Europe and with the goal of improving the efficacy of current rehabilitation techniques and the personal life of stroke patients, the future implementation of systems that are able to provide cognitive rehabilitation at home will have a positive impact on daily living.Increasing the independence of stroke patients can improve their personal lives because self-confidence of patients will improve as well as their socialization with other family members and friends. It is quite important to ensure proper emotional status in patients which will make the acceptance of rehabilitation easier. Moreover, increasing the personal independence of stroke patients will have a direct implication for health care services.From the review of current state-of-the-art ICT–based approaches for cognitive self-rehabilitation and tele-rehabilitation, the most relevant ones have been presented in this document. Only home-based performanceHome-based performance + accessibilityHome-based performance + accessibility + support in the execution of complex daily living tasksHome-based performance + accessibility + support in the execution of complex daily living tasks + automatic error detectionBy analyzing the data, it can be derived that none of the approaches found in the literature provide all the 4 features at the same time, and only 50% of them are accessible. Meanwhile, although 77% of the European projects presented are conscious of the importance of making new technologies accessible, bearing in mind the limitations of some patients, only 33% of them fulfill the 4 requisites considered as essential.For this reason, current and future large-scale initiatives focused on smart environments should try to present all these features to users. The design and use of personalized and eHealth rehabilitation systems, which could be used for the assessment of a wide range of neurological disorders including those syndromes not presented in this study, will reduce hospitalization rates as well as the frequency of home visits by health professionals, which means a reduction in costs for the national health care services.


Speech production impairment is a frequent deficit observed in aphasic patients and rehabilitation programs have been extensively developed. Nevertheless, there is still no agreement on the type of rehabilitation that yields the most successful outcomes. Here, we ran a detailed meta-analysis of 39 studies of word production rehabilitation involving 124 patients. We used a model-driven approach for analyzing each rehabilitation task by identifying which levels of our model each task tapped into. We found that (1) all rehabilitation tasks are not equally efficient and the most efficient ones involved the activation of the two levels of the word production system: the phonological output lexicon and the phonological output, and (2) the activation of the speech perception system as it occurs in many tasks used in rehabilitation is not successful in rehabilitating word production. In this meta-analysis, the effect of the activation of the phonological output lexicon and the phonological output cannot be assessed separately. We further conducted a rehabilitation study with DPI, a patient who suffers from a damage of the phonological output lexicon. Our results confirm that rehabilitation is more efficient, in terms of time and performance, when specifically addressing the impaired level of word production.


Reason for Corrigendum:Due to an oversight, there were errors in the list of authors, correspondence author, acknowledgments, and author's contributions. These things have been fixed in all respective sections, as agreed with the rest of the authors and the research topic editor. The authors would like to apologize for any inconvenience caused and this change does not affect the scientific conclusions of the article in any way. The original article has been updated.JL explored different ways for emotion recognition, including sources of data from different signal measurement devices. He also helped defining the experimental protocol and providing ways to appropriately analyse gathered data. JV performed the device calibration and synchronization, data collection and facilitated the study. RGil coordinated the analysis of gathered information. She has found ways to expand it to medical environments. RGarcía participated in the definition of the protocol and explored ways to improve it. TG, IB, and JS recruited the medicine students that participated in the study and dealt with the required authorization by the ethics committee of the Arnau de Vilanova University Hospital. They also obtained the images and videos used in the study and supervised the experiments.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.