Curcuma longa has well-known insecticidal and repellent effects on insect pests, but its impact on Trichoplusia ni is unknown. In this study, the compound ar-turmerone, extracted and purified from C. longa rhizomes, was identified, and its insecticidal effects, along with turmeric powder, curcuminoid pigments and crude essential oil were evaluated against this important agricultural pest. The role of natural  and synthetic [piperonyl butoxide (PBO)] synergists under laboratory and greenhouse conditions were also evaluated. The concentration of ar-turmerone in C. longa rhizomes harvested was 0.32% (dwt). Turmeric powder and its derivatives caused 10–20% mortality in third instar T. ni at a very low dose (10 μg/larva). Addition of PBO increased toxicity of turmeric powder and its derivatives  in most binary combinations (5 μg of turmeric powder or its derivatives +5 μg of PBO), but neither piperonal nor sesamol were active as synergists. The compound ar-turmerone alone and the combination with PBO reduced larval weight on treated Brassica oleracea in the laboratory and in greenhouse experiments, compared with the negative control. The compound ar-turmerone could be used as a low cost botanical insecticide for integrated management of cabbage looper in vegetable production. Derris Lour. and Lonchocarpus Kunth , and ryania, derived from the South American shrub Ryania speciosa Vahl. , are used against horticultural and ornamental crop pests. Pyrethrum, extracted from flowers of Dalmatian chrysanthemum, Tanacetum cinerariifolium (Trevir.) Sch. Bip. , is the most important botanical insecticide on the market. Pyrethroids  are synthetic derivatives of the natural pyrethrins and have enjoyed enormous commercial success as crop protectants and in other areas of pest management. Both natural pyrethrin and synthetic pyrethroid-based products usually contain a synergist such as piperonyl butoxide (PBO) or piperonyl cyclonene that substantially increases the efficacy of the active ingredient and reduces its costPlants produce toxic substances as natural defenses against pests, such as insects and pathogens13The toxicity of natural substances to insect pests can sometimes be comparable to that of synthetic insecticides, but often with less environmental effects491113Curcuma longa L.  , an herbaceous perennial plant with long lateral ramifications, originated from Southeast Asia, probably in the Indian subcontinent15171921C. longa have demonstrated insecticidal effects against a number of insect pests, and also repelled mosquitoes132324Curcuma longa is harvested when the aerial part of this plant senesces after flowering and its rhizomes develop an intense yellow color indicating the presence of concentrated pigments26Turmeric, C. longa and other plant species, in low concentrations. For example, 0.32% and 0.50% (dwt) ar-turmerone was extracted from C. longa rhizomes in BrazilTagetes minuta L.  in KenyaC. longa produced 44.4% and 38.6% (dwt) ar-turmerone in Nigeriaar-turmerone was extracted from the essential oil of leaves in NigeriaInsecticidal substances can be obtained from C. longa3031C. longa essential oils determine the characteristic smell of turmeric, whereas fixed phenolic compounds, such as the pigment curcumin (a diarylheptanoid), its derivatives and other substances, are responsible for the intense yellow color of the rhizomesC. longa contain a mixture of ketones and sesquiterpene alcohols, the latter mainly based on germacrene and bisabolane skeletons34Climatic and genetic factors, harvesting time, soil type, fertilization, drying process, and period of storage can all affect the chemical composition of essential oils from Trichoplusia ni  (Lepidoptera: Noctuidae) , a serious pest of cruciferous crops, is found throughout the southern Palaearctic ecozone, including all of North America, parts of Africa, much of eastern Europe, and the Indo-Australian region37Trichoplusia ni is not restricted to cabbage, Brassica oleracea L. , but also damages cucumber , potato (Solanum tuberosum L.) and tomato (Solanum lycopersicum L.) T. ni has evolved resistance against many synthetic insecticides and the microbial insecticide Bacillus thuringiensis Berliner The cabbage looper, Hübner, 100–1803  and synthetic (PBO) synergists under laboratory and greenhouse conditions. Synergists were added to enhance the efficacy of turmeric powder and its derivatives against the cabagge looper. Natural and synthetic synergists such as sesamol, piperonal and PBO inhibit detoxifying enzymes in insects and other organisms3941Aedes aegypti  (Diptera: Culicidae) larvae subjected to a mixture of deltamethrin and PBO in the ratio of 1:5 for 20 generations® contains 0.02% pyrethrins as the active ingredient and 0.20% PBO as a synergist. Schultz Insect Spray® is recommended for use on indoor and outdoor plants, including edible vegetables, and is a broad-spectrum insecticide.The objective of the current study was to identify insect bioactive compounds, extracted and purified from C. longa essential oil with hexane was 0.39% (dwt) (1.93 g) and that of ar-turmerone from this essential oil was 82% (dwt) after the chromatographic separations from the initial material (1.58 g). A total of 3.2 g of ar-turmerone was present per kg of rhizomes of this plant grown in Catalão, Goiás, Brazil. Consistent with our results, high concentrations of ar-turmerone in non-polar extracts and essential oils of C. longa have been reported from China (Asia), India (Asia), Nigeria (Africa), Pakistan (Asia), and the islands of Sao Tome and Principe (Africa)294546C. longa occurring at different localities3031Curcuma longa can be cultivated at low cost and sustainably in Brazil using minimal labour, a range of growing seasons and spacing, and organic fertilization with 50 tons of cattle manure per haThe yield of ar-turmerone + piperonal). Sesamol did not enhance activity in any treatment. However, addition of PBO as a synergist increased toxicity of turmeric powder and all of its derivatives.The initial screening dose (10 μg/larva) demonstrated low toxicity through topical application to third instars, ranging from 10–20% at 24 h . Binary ar-turmerone + PBO were 90%, 94% and 96%, respectively, even though the dose of the active ingredient was reduced by one-half. Mortality caused by binary mixtures of curcuminoid pigments and PBO was almost twofold that of the individual compounds. The positive control (Schulz Insect Spray®), tested at 1%, produced 71.4% mortality (−1) acetonic solution of ar-turmerone mixed in an artificial diet produced 58.3% mortality of one-day-old larvae of the fall armyworm, Spodoptera frugiperda  (Lepidoptera: Noctuidae) after 10 days of feedingar-turmerone caused 100 and 64% mortality of adult brown planthopper, Nilaparvata lugens  (Hemiptera: Delphacidae) females at 1,000 and 500 ppm, respectively. Against larvae of the diamondback moth, Plutella xylostella  (Lepidoptera: Plutellidae), the compound produced 100 and 82% mortality at 1,000 and 500 ppm, respectively. Against green peach aphid, Myzus persicae  (Hemiptera: Aphididae) adult females and tropical armyworm, Spodoptera litura  (Lepidoptera: Noctuidae) larvae, ar-turmerone at 2,000 ppm was effective but demonstrated weak insecticidal activity at 1,000 ppm. At a dose of 2.1 mg · cm−2, ar-turmerone was almost ineffective  against rice weevil adults, Sitophilus oryzae  (Coleoptera: Curculionidae), cowpea bruchid, Callosobruchus chinensis  (Coleoptera: Chrysomelidae) and tobacco beetle, Lasioderma serricorne  (Coleoptera: Anobiidae) as well as larvae of the Indian mealmoth, Plodia interpunctella  C. longa rhizomes fed to the freshly emerged peach fruit flies, Bactrocera zonata  (Diptera: Tephritidae) for 16 days at 1,000, 500 and 250 ppm produced 84.7, 79.0 and 67.7% mortality, respectively. Eggs deposited by the surviving females on clean guava fruits  in separate cages demonstrated 67.9, 60.7 and 51.9% pupal inhibition for the flies fed on 1,000, 500 and 250 ppm of C. longa extracts respectivelyMortality caused by exposure of cabbage looper larvae to binary mixtures of turmeric crude essential oil + PBO, turmeric powder + PBO and ortality . Similar50 values (50 = 0.03 μg) and turmeric crude essential oil + PBO (LD50 = 0.05 μg) were both significantly more toxic than ar-turmerone + PBO (LD50 = 0.26 μg) which itself was more toxic than curcuminoid pigments + PBO (LD50 = 0.61 μg) against third instar cabbage loopers   Anopheles gambiae Giles, 1902 (Diptera: Culicidae)A. aegyptiBased on the LD0 values , binary tervals) . PBO syn13,105 = 5.5; p < 0.05). Weights of larvae were significantly lower than negative controls for all treatments . Larval weight was significantly lower on ar-turmerone (119.1 mg), ar-turmerone + PBO (81.8 mg) and ar-turmerone + sesamol (116.7 mg) treatments, compared to all other treatments including the negative control (297.8 mg) and the positive control (200.0 mg). Weight reduction for these treatments varied from 60 to 72% compared with the negative control  (Coleoptera: Tenebrionidae) adults, fed on wheat flour , which had been treated with turmeric oil at 200 ppm produced fewer and underweight larvae, pupae, and adults compared with those fed on untreated flourSchistocerca gregaria  (Orthoptera: Acrididae) and the red cotton bug, Dysdercus koenigii  (Hemiptera: Pyrrhocoridae) nymphs. At a dosage of 20 μg per S. gregaria fifth instar nymph, curcumins injected into the hemolymph, produced 40–50% growth inhibition and 10–15% mortality. Turmeric oil produced 10% growth inhibition and 60% nymphal mortality at the same dosage. Topical application of a dosage of 50 μg of curcuminoids  produced 45% growth inhibition of D. koenigii nymphsWeight of cabbage loopers reared on artificial diets incorporating turmeric powder or its derivatives and their binary mixtures with PBO after seven days in the laboratory showed a significant effect  followed by turmeric powder (765.3 ppm) and turmeric crude essential oil (844.4 ppm). The EC50 values for the binary mixtures of ar-turmerone + PBO were also the lowest (304.7 ppm), followed by turmeric powder + PBO (471.2 ppm) and the binary mixture of crude essential oil and PBO (598.9 ppm)  against third/fifth instar of the Bihar hairy caterpillar, Spilosoma obliqua  (Lepidoptera: Arctiidae). Among the three different ratios  of azadirachtin-turmeric oil mixture, 1:1 mixture exhibited pronounced insect growth regulator (IGR) activity (EC50 = 1.26 × 10−2/2.16 × 10−2%) and considerable antifeedant activity (EC50 = 2.90 × 10−2/1.0 × 10−2%) against S. obliqua larvaeOur study demonstrated that dietary EC8.9 ppm) . Similar7,122 = 4.9; p < 0.05). Larval weight was significantly lower on ar-turmerone (24.3 mg) and ar-turmerone + PBO (14.5 mg) treatments compared with the negative control (46.1 mg) . Larval weights were reduced by 47 and 69% respectively in the ar-turmerone and ar-turmerone + PBO treatments, compared with the control. Similarly, the number of larvae recovered from the leaves was also the lowest on these two treatments  (Lepidoptera: Noctuidae) larvae. First instar H. armigera were reared on a semi-synthetic diet treated with 5% of C. longa rhizome powder for 7–10 days. Larval and pupal weights, survival, development time, and adult emergence rate were adversely affected by C. longa treatments. There was 69% growth inhibition in larvae and the adult emergence period was prolonged by 8 days compared with the controlSitotroga cerealella  (Lepidoptera: Gelechiidae) when treated with 1,000 ppm of turmeric extracts prepared in acetone, ethanol or petroleum etherWeight of cabbage loopers reared on cabbage leaves treated with turmeric powder or its derivatives and their binary mixtures with PBO for four days in the laboratory showed a significant effect  and ar-turmerone + PBO (14.6 mg) treatments compared with the negative control (49.5 mg). Weight of cabbage loopers reared on cabbage leaves treated with turmeric powder, its derivatives and their binary mixtures with PBO for four days showed a significant effect . There was a 69.5 and 70.0% reduction in weight of the larvae on ar-turmerone and ar-turmerone + PBO treated plants, respectively compared with the negative control . Although larval weights were lowest on ar-turmerone and ar-turmerone + PBO, they did not differ significantly from turmeric crude essential oil + PBO (22.6 mg). Larval weight was significantly lower on turmeric crude essential oil + PBO (22.6 mg) compared with the crude essential oil (36.9 mg) alone. Numbers of larvae recovered from these treatments were also the lowest (ar-turmerone + PBO was only 38% relative to the control. Results in the greenhouse confirm the toxicity of ar-turmerone (+/−PBO) and although not insecticidal in some cases, it can suppress larval growth and reduce feeding damage caused by this pest. Larvae from the ar-turmerone treatment (+/−PBO) were significantly lighter (~70% reduction in weight) and weaker than the control, increasing their probability of being preyed upon by natural enemies, as suggested for S. frugiperda larvae fed on an artificial diet treated with an acetonic solution of ar-turmeroneGreenhouse results , Fig. 1 e lowest . Larval ar-turmerone, could potentially control the cabbage looper larvae. In contrast, curcuminoid pigments were not active. Addition of PBO increased efficacy of turmeric solutions in most combinations, whereas the natural products, piperonal and sesamol, were not synergistic. The ar-turmerone could be a low-cost and sustainable alternative for IPM of cabbage looper larvae and the addition of PBO can improve its efficacy. Since the treatments exhibit more than one mode of action, it is believed that this will delay resistance development in cabbage looper and other insects. Plant defense chemicals that attack pests at multiple levels are especially suitable for crop protection.An insecticide based on turmeric powder or some of its derivatives, especially the sesquiterpene C. longa were collected from a commercial crop grown on the Macaúba farm in Catalão, Goiás, Brazil . Five large plants, free of pests or diseases, were chosen within the crop. The soil was dug with a hoe until rhizomes became visible. Entire plants were harvested and the rhizomes were cut from the plants with a knife. The rhizomes were placed in a polystyrene box lined with ice and brought to the laboratory, where they were washed in running water, dried and stored at 2 °C. This farm uses no synthetic agrochemicals.Rhizomes of ®  was used as the positive control. It contains 0.02% pyrethrins as the active ingredient and 0.20% PBO as a synergist (8H6O3) (aka heliotropin) and sesamol (C7H6O3)  were used as natural synergists. Piperonal is a compound commonly found in fragrances and flavors. It is structurally related to other aromatic aldehydes such as benzaldehyde and vanillin. Piperonal naturally occurs in various plants, including dill , violet flowers  and black pepper  . It can also be produced via synthesis from heliotropine  . Sesamolotropine 5657. PBO1H, 13C, Heteronuclear Single Quantum Coherence (HSQC), and Heteronuclear Multiple Bond Correlation (HMBC) Nuclear Magnetic Resonance (NMR) measurements were carried out on a Bruker Avance III 500 instrument (operating at 500.13 MHz for 1H) equipped with a 5 mm triple Resonance broadband inverse probehead (TBI) with Z-gradient. Deuterated chloroform was used as solvent and tetramethylsilane as the internal standard. Mass spectra were obtained by gas chromatography coupled to a mass spectrometry (GC-MS). The GC-MS analyses were performed using a gas chromatograph , with ionization by electronic impact, under the following conditions: 60 °C for 3 min; 5 °C · min−1 to 240 °C, for 8 min; with an injector temperature of 180 °C, a detector temperature of 260 °C and an injection volume of 1-L. Mass spectra were compared with the National Institute of Standards and Technology database 62 (NIST-62).C. longa to T. ni was initially evaluated in the laboratory  and later in a greenhouse .The toxicity of ® . Cabbage plants (B. oleraceae var. Stonehead) used in the greenhouse bioassays were grown in plastic pots with a mixture of sandy loam soil and peat moss (4:1).Cabbage loopers used in bioassays were obtained from the Great Lakes Forestry Centre (GLFC), Canadian Forest Service (CFS), in Sault Ste. Marie, Canada. Approximately 25 eggs were introduced per plastic cup (50 mL) with an artificial diet McMorranC. longa were air-dried at 40 °C for three days and ground into a fine reddish-yellow powder (turmeric powder). The major chemical constituents of turmeric powder are curcuminoids, including bisdemethoxycurcumin . Some volatile compounds in turmeric crude essential oil include atlantone , turmeroar-turmerone , eluted with hexane:ethyl acetate (9:1). The fractions of interest, containing urmerone , were anThe other portion of the turmeric powder was separated by column chromatography on silica gel Vetec, 60–270 mesh), eluted with hexane:ethyl acetate (1:1), to obtain curcuminoid pigments. These consisted of a mixture of bisdemethoxycurcumin , curcumi–270 meshT. ni following previous methodology with slight modifications8rd instar cabbage looper) or a 1:1 binary mixture with one of the synergists (5 μg of turmeric powder or its derivatives +5 μg of PBO or other synergists), on the dorsum with a repeating dispenser attached to a 50 μL syringe. Acetone and Shultz Insect Spray®, alone, were used as negative and positive controls, respectively. After the compounds were applied, the larvae were transferred to Petri dishes (90 mm diameter × 15 mm height) in groups of 10 along with a small piece of artificial diet (1.12 g). There were three replicates of 10 larvae each per treatment. Treatment groups were placed in sealed plastic boxes lined with moistened paper towels and held for 48 h in a growth chamber . The mortality of cabbage loopers was determined after 24 and 48 h. Larvae were considered dead if they did not respond to prodding with forceps according to methodology described for velvetbean caterpillar, Anticarsia gemmatalis  (Lepidoptera: Noctuidae) larvae treated with neem oil50  value was determined for treatments demonstrating >50% mortality at the initial screening concentration of 10 μg/larva. Mixtures were tested in a 1:1 ratio (dose = 5 μg of turmeric powder or its derivatives +5 μg of PBO or other synergists). LD50 values were calculated using the software EPA Probit Analysis Program, version 1.5Contact toxicity  of turmeric powder and its derivatives with or without synergists was determined by topical application to early third instar ®. Each 20 g piece of artificial diet was cut into 20 equal sized pieces and placed into individual compartments (3.81 cm length × 4.44 cm width × 2.54 cm depth) of plastic molded transparent assay trays  and covered with perforated lids . One freshly hatched neonate larva (24 h old) was placed into each compartment (n = 20). The plastic trays with larvae were transferred into a clear plastic box (39.0 cm length × 27.0 cm width × 14.0 cm height) lined with a moistened paper towel and the box was placed in a growth chamber .The effect of turmeric powder and its derivatives was assessed following modified methodology50  was determined using four concentrations of each sample . Synergists were added at 1:1,000 part of the mixture (v/v).After seven days, all larvae were removed from the trays and individually weighed. The mean larval weight from each treatment was expressed as a percentage of the controls. The ECOur contact toxicity experiments demonstrated high mortality in binary mixtures (turmeric powder or its derivatives + synergist in a 1:1 ratio) and, therefore, we could not use this ratio for growth inhibition experiments. Effect of an insecticide on the growth of larvae is normally assessed at 1,000 ppm. Synergists tested alone at this concentration did not cause any growth inhibition or mortality of the larvae and were therefore not included in statistical analysis.® (diluted to 1%) and acetone were used as positive and negative controls, respectively.Five cabbage leaves were treated with 160 μL of acetonic solution of turmeric powder or one of its derivatives with or without synergists . Solutions were applied and carefully dispersed over the entire area of the leaves using a pipette. Larvae (third-instar) were introduced onto each leaf and allowed to feed for four days. There were five replicates per treatment with six insects per replicate. Larvae collected from each leaf were counted and weighed on day four. Individual treatments were tested at 1% and mixtures in a 1:1 ratio of each constituent. Shultz Insect Spray® was used as a positive control because it is based on pyrethrins that are both toxic and inhibit growth of cabbage looper larvae. It was tested at 1,000 ppm in the artificial diet and 10 μg/larva in contact toxicity bioassays.Shultz Insect Spray® (1%) as a positive control.We modified previous methodology8The plants were air-dried, and five third instar cabbage loopers were introduced onto each plant (n = 75 larvae per treatment). The plants were randomly placed on a table under grow lights (400-Watt light bulbs per square meter) in the greenhouse . On day four, larvae were removed from the plants, placed in separate polystyrene cups, and weighed in the laboratory. Experiments were repeated twice.50 values  and their corresponding 95% confidence intervals using the EPA Probit Analysis Program version 1.5. LD50’s are considered significantly different from one another if their 95% confidence intervals (C.I.) do not overlap. EC50  values were calculated by using linear regression analyses in Microsoft Excel. Growth inhibition data were analyzed using the Statistics 7 program for analysis of variance (ANOVA). When significant F values were found, Tukey’s HSD multiple-comparison tests were used to test for significant differences between individual treatments. Experiments were repeated at least twice.Mortality data were subjected to Probit analysis to determine LDHow to cite this article: de Souza Tavares, W. et al. Turmeric powder and its derivatives from Curcuma longa rhizomes: Insecticidal effects on cabbage looper and the role of synergists. Sci. Rep.6, 34093; doi: 10.1038/srep34093 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


AR activity has been shown to be preserved in spatially distinct metastatic tumors from the same patient suggesting the requirement for lineage-specific dependencies for metastatic castration resistant prostate cancer (mCRPC). Amplification of the AR gene is a common mechanism by which mCRPC increase AR activity. To determine whether AR amplification in circulating tumor cells (CTC) could complement metastatic tissue biopsies in men undergoing treatment for mCRPC, we developed a novel two-step assay to isolate CTCs and subsequently analyzed AR amplification status in CTCs and matched biopsy tissue from the same patient by fluorescence in situ hybridization (FISH). AR gene status in CTCs showed strong concordance with AR gene status in matched tissue samples in 24 of 25 patients . Our work demonstrates that AR amplification is conserved between CTCs and biopsies and that CTCs can serve as non-invasive surrogate to document AR amplification in mCRPC.Increased  AR signaling despite castrate levels of testosterone is responsible for CRPC in the majority of men. Importantly, the majority of spatially distinct metastases within individual patients with mCRPC continue to exhibit increased AR signaling [AR signaling in CRPC is through amplification of the AR gene [AR amplification status in CTCs from men with mCRPC undergoing treatment, and we evaluated the concordance of AR amplification status with matched metastatic biopsies.AR signaling is the primary driver of prostate cancer, and subsequently, medical castration with androgen deprivation therapy (ADT) is the backbone of all treatments in men with metastatic prostate cancer –4. Howevignaling . Hematogignaling , 7. Therignaling –10. One  AR gene . We deveAR gene status using FISH. Slides were re-imaged. LNCAP cells possessed 2 copies of AR and 2 copies X CEP gene and were identified by relocating the CK18 positive cells. White blood cells contained zero or one copy of AR gene and had zero or one copy of X CEP control. No white blood cells expressed greater than 1 copy of X CEP control, consistent with prior reported results [To demonstrate the validity of our CTC isolation method, we first spiked 10, 250, 500 and 1000 LNCAP cells into 15 mls of normal whole blood. The blood/cell mixture was diluted 1:1 with PBS. Fifteen mls of Ficoll Paque Plus  was added to a 50 ml Leucosep filter tube (Greiner Bio-One #227290P) and the blood/cell mixture was added to Leucosep tube and spun at 1600 g for 20 min without brake. After centrifugation, the PBMC layer containing spiked LNCAP cells was removed using a transfer pipet and placed in a clean 15 ml conical and pelleted at 400 g for 4 min. Cell pellet was resuspended in 1 ml of donor blood set aside at the start of the assay. Fifty microliters of RosetteSep CTC enrichment cocktail (Stemcell # 15137C) was added to each sample and incubated at room temperature for 20 minutes. After incubation, 4 ml of Ficoll Paque Plus was added to a new 15 ml conical for each sample. Enriched blood samples were brought to a total volume of 6 ml with PBS and was gently layered onto the Ficoll layer. Samples were spun at 1200 rpm for 20 min without brake. Enriched cell layer was removed and placed in a new 15 ml conical. PBS was added to enriched cells to bring volume to 9 ml. One ml of red lysis buffer was added and conicals were incubated on a rocker for 15 min. Cell samples were pelleted at 400 g for 4 min and supernatant was decanted. Remaining cells were resuspended in 200 ul of 4% paraformaldehyde. The re-suspended cells were cytospun using a Thermo Scientific Cytospin 4 to create 2 slides per sample, 100 ul of suspension per slide. Funnel and slide assembly was constructed and 100 ul of cell suspension was added to each funnel. Slides were spun at 700 g for 3 min. Funnel and slide were disassembled; slides were allowed to air dry and then were stored at −80C for up to six months. To quantify the capture rate of this method, slides were stained with CK18 (green) and CD45 (pink) markers to differentiate epithelial cells from WBCs. Slides were scanned using a Panoramic Midi slide scanner (Perkin Elmer) and quantified by visual inspection. Our recovery assays were performed by spiking between 10 and 1000 LNCAP cells into donor blood samples. Multiple recovery assays were performed with an average yield of 38%. Despite the low recovery rate, we were able to capture cells in our samples that contained only 10 spiked cells providing confidence that CTC capture would be attainable in patient samples. After capturing full slide images using Panoramic MIDI scanner, the slides were reprocessed for  results . . Fifteen subjects had abiraterone or enzalutamide treatment prior to biopsy compared to ten subjects that did not receive these drugs. No statistical differences were observed between treatment and AR signaling activity. We also compared AR signaling to PSA, Gleason score and LDH and found no significant differences between treatment groups or those that responded to treatment and those that progressed.We compared AR amplification . For amplified, CTCs, we assigned G/C 2.5 as weak AR amplification, G/C > 2.5 and < 4 medium amplification and G/C > 4 defined strong AR amplification. Twelve of twenty patients (60%) exhibited different levels of AR amplification and points to multiple temporal and spatial tumor clones in CTCs from patients with CRPC, that may be the result of selective pressures from treatment or evolution of the cancer as it progresses from hormone sensitive to CRPC.We observed inter- and intra-patient heterogeneity for n Figure . ThirteeAR amplification in three patients with sequential samples collected before starting treatment and at progression  developed AR amplification at progression and this was picked up both in his metastatic liver biopsy and CTCs drawn at the same time.We also investigated n Figure . The CTCHematogenous dissemination of cancer cells is a critical step in the development of metastases . ConsequPTEN, ERG and AR gene status and mRNA profiling in single circulating tumor cells [Circulating tumor cells offer a non-invasive method of determining the biological status of individual tumors, treatment evaluation and progression. The shedding of CTCs from the primary tumor is known to play a role in mCRPC. Studies have shown that the number of CTCs decreases with response to treatment and increases with disease progression. The study of genomic makeup of CTCs has been vastly increasing to include genomic and RNA profiling to identify of key drivers in CRPC. Recent studies have included or cells , 15. MolAR amplification is a major driver of CRPC and persists despite newer Androgen Signaling Inhibitors such as abiraterone and enzalutamide, we evaluated AR amplification status by FISH. Simultaneously, we collected metastatic CRPC biopsies and performed AR FISH on FFPE tissues. AR gene status detected in CTCs showed strong concordance with AR gene status in matched tissue samples in 24 of 25 patients (96%).Since AR amplification status, both binary (i.e. amplified and non-amplified CTCs in the same patient) and the degree of amplification (low to high number of AR gene copies). This may reflect selective pressures brought on by different treatments and which may ultimately define the viability of metastatic clones. Alternatively, these CTCs may represent tumor cells shed from different metastatic sites and therefore reflect tumor heterogeneity. However, despite treatment with more potent androgen signaling inhibitors, AR amplification continues to function as a potential resistance mechanism. Accordingly, the opportunity to perform serial blood draws for CTCs can be used to monitor the emergence of AR amplification.Intriguingly, this study through our ability to identify and quantify single CTCs revealed that there is striking intra-patient heterogeneity in AR gene status can be accurately determined in CPRC patients using a non-invasive procedure that utilizes peripheral blood.In summary, our results indicate the potential to expand CTC research through use of a simple, low cost assay to detect protein and gene expression in CTCs. Our research has shown that AR amplification results were blindly compared to the current gold standard tumor biopsy result for AR gene status. (R1 Q1) Four men were eliminated from the results because a paired tumor biopsy and CTC sample were not attained for various reasons. Two subjects were not tested for AR amplification in their tumor biopsy, one sample failed the tumor biopsy AR amplification assay and one sample failed the CTC AR amplification assay. Fifteen mls of blood for CTC studies were drawn for analysis at time of biopsy. Complete patient demographics are shown in Table Patients treated at Oregon Health & Science University with mCRPC were recruited under an institutional review board-approved protocol with informed consent. Specifically, men undergoing pretreatment and disease progression tumor biopsies for mCRPC were recruited under the SU2C/PCF/AACR West Coast Cancer Dream Team biopsy program. This study focuses on the patients recruited between Jan 2014 and June 2015. During that timeframe, 29 men were recruited. Here we report on the results of 25 of those men. Patients were selected based on having a tumor biopsy performed. CTC Please see results section for detailed protocol. Blood samples were collected using two EDTA collection tubes. Blood was processed within 24 hours of collection in accordance with the above described protocol. Please see Figure Immunofluorescent staining was performed on CTC samples. Slides were removed from −80C storage and allowed to reach room temperature. Once slides were room temperature they were placed in PBS. Within 20 minutes, permeabilization was performed for 20 minutes using cold methanol stored at −20C. Slides were rinsed in PBS. Nonspecific binding was blocked using Dako protein block (Dako X090930) for 30 minutes.AR FISH probe.Primary antibodies were stained sequentially. First, CK18 1:100 ( Santa Cruz Bio # sc-31700) was incubated for 1 hour at room temperature. Slides were rinsed in PBS 2 × 5 minutes. Secondary antibody Alexa 488 (Life Tech A11034) 1:1000 was diluted in PBS and was incubated at room temperature for 30 minutes. Slides were washed in PBS for 5 minutes. Slides were then incubated for one hour at room temperature with anti- CD45 1:00 (Abcam #ab8216). Slides were rinsed in PBS for 2 × 5 min. Alexa 647 (Life Tech #A31571) 1:1000 diluted in PBS was incubated for 30 minutes at room temperature. Slides were rinsed in PBS for 5 minutes. Slides were air dried and immediately coverslipped using mounting media with Dapi (Vector #H-1200). Slides were immediately scanned or stored at 4C overnight and scanned the next day using Perkin Elmer Midi scanner. Slide image was visually reviewed to determine the number of positive CK18 negative CD45 cells. Following completion of scanning and image review, coverslips were removed from slides by soaking slides in PBS to allow coverslips to float off. Slides were then immediately processed for AR (Xq12) probe (Vysis #30-190040) and Spectrum Green labeled ChrX centromere (Xp11.1-q11.1) X CEP (Vysis# 32-112023). Patient slides were permeabilized at 80C for 5 min in a 2xSCC/formamide solution. Slides were rinsed in PBS and dehydrated through a series of alcohols. Probes were prepared according to manufacturer's instructions and denatured at 72C for 5 min and hybridized overnight at 32C using Vysis hybridizer. The next day slides were washed in 2xSCC +0.1% tween solution at 42C for 2 min then rinsed in room temp PBS, dehydrated and coverslipped using Dako mounting media with Dapi (# H-1200). CTCs were identified by cells that expressed 2 or more copies of AR and X CEP probe, positive CK18 expression and negative CD45 expression. White blood cells were identified as having 1 copy of AR and 1 copy of X CEP, negative CK 18 expression and positive CD45 expression.A two color FISH assay was performed using Spectrum Orange in situ hybridization signals were assessed using FishQuant software. AR amplification was considered to be present when the AR gene to X chromosome ratio was greater than 2. Some leukocytes remained following isolation process and were used as internal controls.Slides were scanned on a 3D Histech Panoramic scanner. Fluorescent AR and centromere signals. Subsequently, fifty interphase cells were scored. A ratio of AR:X centromere signals > 2.0 was considered amplified.Five μm FFPE tissue sections were mounted on positively charged glass slides. One section was stained with H&E and examined by a pathologist, followed by marking of tumor region(s). Slides for FISH were baked in a dry oven at 60 degrees C for 2 hours. They were then cooled briefly and loaded into a Vysis VP2000 slide processor and were then deparaffinized and pretreated. Slides were dried and 5–10 μl of an androgen receptor (AR)/ X centromere probe solution were added to each slide. Slides were covered with glass coverslips, sealed with rubber cement, and placed into a Vysis Thermobrite machine for denaturation/renaturation. Coverslips were soaked off, slides post-washed and air dried before mounting with DAPI I using new glass coverslips. 100 cells were scored for the number of AR status from CTCs and archival tissue. Twenty-nine patients had matched biopsies and CTCs. Of these, 1 biopsy failed AR FISH testing, 1 CTC sample failed AR FISH testing, and 2 biopsies were inadequate for AR FISH. Subsequently, 25 patients were included in the final analysis as they had conclusive FISH results in both CTCs and tumor tissue. Descriptive statistical analyses, including Cohen's kappa testing were performed using GraphPad Prism software.Positive predictive value, specificity and sensitivity were calculated based on the classification of patients for


Epidemiologic, pathophysiologic, and clinical evidences recently revealed the link between upper and lower airways, changing the global pathogenic view of respiratory allergy. The aim of this review is to highlight the strong interaction between the upper and lower respiratory tract diseases, in particular allergic rhinitis and asthma. During the second century, Claudius Galenus identified the effect of the upper airway on the lower airway and defined the nose as a “respiratory instrument” in his work “De usu partium.” Nevertheless, the concept of the upper and lower respiratory passages being a continuum and forming a single unified airway has been highlighted only over the last 10–15 years, starting from the Allergic Rhinitis and its Impact on Asthma (ARIA) World Health Organization workshop .The mechanisms of nose and lung interaction are complex, not entirely understood, and they have been long investigated . Dating The aim of this review is to underline the strong anatomical, epidemiologic, pathophysiologic, clinical, and therapeutic evidences  and lower respiratory tract , separated by the larynx. Functionally, it is divided between conductive airways and gas-exchange region of the lungs, consisting of the respiratory bronchioles with the air cells . AlthougAsthma and rhinitis are a major public health problem because of their frequency, their impact on quality of life, school performance, and economic burden . The infAn immune response to external antigens induces the production of antibodies that are typically, but not exclusively, IgE antibodies. Allergic airway disease is caused by hypersensitivity or IgE-mediated reactions when inhaled allergen reacts with mast cells and basophils, the major effector cells, bearing IgE antibodies. The following cross-linking of allergen-specific IgE molecules bound to cells by allergen particles, results in the release of granule-associated mediators , membrane lipid-derived mediators , and cytokines , 20. Ovevia postnasal drip and systemic circulation. Although the nose is usually the first site of exposure to allergens or other noxious substances, despite a minimal nasal epithelial damage, a marked bronchial epithelial disruption may be present. Hence, researches hypothesized that the nasal mucosa has developed protective mechanisms that minimize remodeling and enhance epithelial regeneration , and autoimmunity  , toxins in utero .genus Moraxella. The linking of asthma and Moraxella, however, was restricted to children not living on farms. In contrast to the nasal samples, the throat microbiota characteristics were not related to asthma  pathway, which is involved in the rapid initial inflammation response. LTRAs block the cysteinyl-leukotriene receptor, which are peptide-conjugated lipids produced by activated basophils, eosinophils, mast cells, and macrophages, to relieve the symptom of AR (Some authors reported a decrease in asthma symptoms and AR after intranasal corticosteroid treatment of rhinitis and a recent meta-analysis confirmed the beneficial effect of intranasal steroids in AR . LTRAs aom of AR . Recent om of AR . The recom of AR . Omalizuom of AR . In patiom of AR .The only treatment potentially able to interfere with the natural history of respiratory allergy is AIT, specifically aimed at modifying the response to sensitizing allergens . CurrentThe link between upper and lower airways, the so-called UAD, has been revealed by several epidemiologic, pathophysiologic, and clinical evidences, changing the global pathogenic view of respiratory allergy. AR and asthma are both manifestations of a single inflammatory process and require an integrated diagnostic and therapeutic approach in order to get global disease control.All authors made substantial contribution to the conception of the work, reviewed the literature on the subject, and drafted the final version of the manuscript; AL and GM revised it critically for important intellectual content. All authors finally approved the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that written informed consent was obtained by the patients for the use of the images.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.


Experiments using T47D and ZR75-1 demonstrated AR-overexpressing cell lines decreased in cell proliferation through downregulation of ER, but FOXA1 did not change. Knockdown of FOXA1 resulted in a significantly reduced cell viability. Patients with immunohistochemically AR(-)/FOXA1(-) tumor frequently showed node metastasis, high grade, and high Ki-67 proliferation, therefore, significantly worse survival in ER-positive disease. AR and FOXA1 mRNA levels were significantly higher in ER-positive than in ER-negative tumors and AR-low/FOXA1-low tumors showed high grade, frequent basal-like subtype and worse disease-free survival in ER-positive cancers of public gene dataset, similarly to patient cohort results. The Kaplan-Meier Plotter analysis independently validated patients with both low AR/FOXA1 tumor were significantly associated with worse relapse-free survival in ER-positive cancers. This study suggests that distinctive clinicopathological features according to AR and FOXA1 are determined and a lack of both biomarkers is an independent poor prognostic factor in ER-positive tumors.The present study aimed to examine the associations between androgen receptor (AR) and forkhead box A1 (FOXA1) and to investigate clinicopathological features and survival according to both biomarker status in estrogen receptor (ER)-positive breast cancers using  DFS and OS curves according to AR and FOXA1 status showed no statistically significant prognostic value in all patients . HoweverIn patients with ER-positive tumors, multivariate analysis revealed that AR(-)/FOXA1(-) tumors were independently poor prognostic factors for DFS and OS when age at diagnosis, tumor and node stage, histologic grade, HER2, Ki-67, and use of chemotherapy and endocrine therapy were adjusted  in ER-positive cancers and strongly positive  in ER-negative cancers. Since the SD range in consideration with mean value was relatively wider in ER-negative than in ER-positive tumors, and most samples with high AR mRNA levels exhibited high FOXA1 expression in ER-positive cancers; therefore, the correlation coefficient was higher in ER-negative tumors.Using data of mRNA levels and clinical attributes from the METABRIC dataset, AR and FOXA1 mRNA expression patterns and clinicopathological characteristics according to AR/FOXA1 status were investigated. The median values of AR and FOXA1 mRNA were 7.57  and 11.37 , respectively. The frequencies of AR and FOXA1 mRNA levels are shown in Figure The lower quartile cutoff values for defining high versus low mRNA expression of biomarkers were determined 6.93 for AR and 10.81 for FOXA1 from the METABRIC dataset. By these cutoff points, the number of AR-high/FOXA1-high, AR-high/FOXA1-low, AR-low/FOXA1-high, and AR-low/FOXA1-low cases was 1,303 (66.5%), 170 (8.7%), 168 (8.6%), and 317 (16.2%), respectively. In the whole population of the METABRIC dataset, AR-low/FOXA1-low tumors were significantly associated with age at diagnosis ≤ 50 years, high grade, ER-negativity, PR-negativity, HER2-negativity, and the basal-like subtype . AR-highp = 0.020) and the AR-low/FOXA1-high group exhibited the best statistically significant 5-year OS (p = 0.002). However, the AR-low/FOXA1-high group presented the worst 5-year DFS (p = 0.011) and 5-year OS (p = 0.002) in ER-negative tumors  and recurrent or progressive events and deaths were in 32.6% and 57.8% of patients, respectively. To investigate the prognostic roles of AR and FOXA1 status in ER-positive breast cancers, multivariate analysis was performed using clinical variables of the METABRIC dataset Table . The AR-p < 0.001). This statistical significance was maintained in only 1,172 ER-positive tumors /n] is computed for survival analysis of the KM Plotter. Figure p = 0.002), but not in other subtypes .The KM Plotter provides subgroup analyses according to the intrinsic subtype based on the 2013 St. Gallen criteria using the expression of ESR1, HER2, and MKI67 as follows; luminal A (ESR1+/HER2–/MKI67 low), luminal B (ESR1+/HER2–/MKI67 high and ESR1+/HER2+), HER2-enriched (ESR1–/ HER2+), and basal subtype (ESR1–/HER2–) , 20. Strin vitro model presenting no significant change in FOXA1 by AR overexpression and vice versa and multivariate analysis demonstrating statistical significance of AR and FOXA1 status suggested that combined biomarker was independently significant in ER-positive cancers, although the number of cases with alteration in AR and FOXA1 was very small.An exploration of the web-based genetic analysis in the present study showed that approximately 10% of breast cancers were altered in ESR1, AR, or FOXA1 genes and generally changes in genes concurrently occurred even though the frequency was low. The roles of changes in the AR or FOXA1 genes have not been much studied in female breast cancers. However, this study showed a close correlation between AR and FOXA1 expression levels. Clinically undetermined genetic networks between these markers have been proposed in ER-negative cancers, suggesting combined biomarker studies may be critical , 21. Ourin vitro study also confirmed that overexpression of AR induced downregulation of ER expression and cellular proliferation. A subsequent cistrome study demonstrated that AR signaling was less likely to rely on FOXA1 than ER colocalization in ZR75-1 cells [Peters et al.  provided-1 cells . Approxi-1 cells , 22. The-1 cells , 23. Rec-1 cells , 12, 24.Approximately 10–20% of all cases showed negative protein expression or low mRNA levels of both AR and FOXA1 in our study. These cases were significantly associated with aggressive tumor features such as ER-negative, PR-negative, TNBC, basal subtype, high grade, and high Ki-67 labelling index. These features were maintained in patients with ER-positive cancer. Habashy et al.  demonstrp < 0.001). In ER-negative samples of the METABRIC dataset, positive HER2 was 57.9% of AR-high/FOXA1-high, 47.1% of AR-high/FOXA1-low, 44.4% of AR-low/FOXA1-high, and 6.8% of AR-low/FOXA1-low tumors (p < 0.001). Although the proportion of HER2- positive cases in ER-negative tumors was different among datasets, AR- or FOXA1-positive patients showed higher HER2-positive tumors. Therefore, further studies are necessary to understand the clinical implications of these networks in ER-negative breast cancers.Interestingly, a half of the apocrine carcinomas overexpressed the HER2 protein . RegardiAs a prognostic marker, AR is consistently reported to be associated with better survival outcomes , 28, 29.Interestingly, according to analyses of ER-negative tumors using the METABRIC dataset, 18 (4.1%) cases with AR-low/FOXA1-high tumor showed the worst survival and 114 (26.0%) with AR-high/FOXA1-high presented worse DFS with statistical significance. In the TMA study, AR(-)/FOXA1(-) or AR(-)/FOXA1(+) subgroups demonstrated a trend of better survival in ER-negative patients, although no statistical significance was noted. Additional studies with larger sample sizes should be required to understand the different clinical impact of AR and FOXA1 status on survival in ER-negative breast cancers.in vitro study could not find out details of subcellular molecular mechanisms between AR and FOXA1 and many other ER-positive, luminal subtype breast cancer cell lines were not investigated. In addition, methodological problems were key issues. The evaluation and interpretation of immunohistochemical AR and FOXA1 expression were not standardized and the use of TMA tumor blocks with small sized cores may not have been able to represent the results of whole sections. Detection methods and cutoff values of public datasets were varied and arbitrary. Among the genomic profiles on the bioinformatics analysis website, the number of mutations, copy-number alterations, or methylations was not incorporated into the present study and only mRNA expression data were used. Results from the independent datasets could not be used to calculate the associations between protein and mRNA expression levels of biomarkers. Nevertheless, the present study had strengths to explore and validate the undisclosed role of combined AR and FOXA1 status in ER-positive breast cancers using the genetic and clinical datasets with in vitro cell lines study.A potential limitation of the present study was inevitably the nonrandomized and retrospective nature of the clinical dataset. Difficulty in handling and manipulation of an In conclusion, the present results indicate that AR and FOXA1 are closely associated in breast cancers, and distinctive clinicopathological features are presented in ER-positive tumors according to AR and FOXA1 status. More importantly, loss of or decrease in both AR and FOXA1 expression is an independently significant poor prognostic factor in ER-positive tumors. Since different molecular mechanisms between AR and FOXA1 signaling pathways have been suggested in ER-negative breast cancers, the clinical implications of AR and FOXA1 status on patient prognosis should be further investigated to improve the survival of patients with heterogeneous breast cancers. Therefore, possible therapeutic strategies such as anti-androgens should be examined considering the AR, FOXA1, and ER status in breast cancer patients.http://www.cbioportal.org), which provides web-based visualization and access to large-scale cancer genomic datasets [Genomic analysis was performed for investigating the associations between ESR1, AR, and FOXA1 through the cBioPortal for Cancer Genomics  or stage IV disease (n = 10) were excluded from analysis. Upon exclusion, 1,958 samples with stage I–III disease were analyzed for survival in this study.http://kmplot.com/analysis) [p-value were calculated, and survival curves were displayed on the webpage.The probability of relapse-free survival according to AR and FOXA1 status including subgroup analyses was calculated using the KM Plotter  . It is a2 humidified environment. Cells (1×104 cells/well) were plated on 12-well plates and counted every 24 hours for 5 days using the ADAM-MC automatic cell counter .Human breast cancer cell lines (T47D and ZR75-1) were obtained from the American Type Culture Collection . All reagents related to animal cell culture were purchased from Life Technologies . Cells were cultured in Dulbecco’s modified Eagle’s medium. All media contained 10% fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Cells were cultured at 37°C in a 5%-COTotal RNA was isolated from cultured cells using TRIzol  according to the manufacturer’s instructions. For quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), cDNAs were synthesized from 4 μg of total RNA using random hexamer primers and SuperScript reverse transcriptase II (Invitrogen) following the manufacturer’s instructions . DilutedFor the stable overexpression of AR, the fragment encoding the full-length cDNA of AR was cloned into the pLL-CMV-puro lentiviral vector. Plasmid DNAs and a lentiviral packaging mix containing an envelope and packaging vector were transfected into human embryonic kidney (HEK293T) cells according to the manufacturer’s instructions to produce lentiviruses packed with AR cDNA cassettes. Positive cells harboring AR cDNA cassette were selected by 1 μg/ml puromycin  selection after infection. For the knockdown assay, targeting small interfering RNA (siRNA) and non-targeting control siRNA were transfected into cells utilizing Lipofectamine RNAiMax reagent (Invitrogen) following the manufacturer’s protocols. The sequences of targeting oligo duplex against FOXA1 were as follows: 5`–GAGAGAAAAAATCAACAGCTT–3`(sense) and 5`–GCTGTTGATTTTTTCTCTCTT–3`(antisense) .3 cells/well) followed by medium changed the next day. After 48 hours, Ez-Cytox reagent (10 ul/well) was added and absorbance (OD450) was detected at 450 nm after 4 hours.Cell viability was determined by EZ-Cytox Cell Viability Assay Kit  based on the cleavage of the tetrazolium salt to water-soluble formazan by succinate-tetrazolium reductase. Cells in suspension with siRNA mixtures were transferred to 96-well plate . Protein concentrations were measured by the bicinchoninic acid assay. Equal amounts of protein extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto nitrocellulose transfer membranes . The membranes were blocked in 5% (w/v) non-fat Difco™ skimmed milk , followed by incubation with the primary antibodies in 1% bovine serum albumin. The following antibodies were used: anti-AR (custom-made), anti-ERα , α-tubulin , and FOXA1 .® . Using a pRL-SV40 construct (Promega Corp.), luciferase activity was normalized to each cell lysate’s Renilla luciferase activity levels.Luciferase activities in whole cell lysates were measured using the Dual-Luciferase Reporter Assay SystemA previous study cohort was selected to investigate the clinical implications of immunohistochemically determined AR and FOXA1 expression levels on breast cancer patients’ survival outcomes. Immunohistochemical AR expression was evaluated from TMA blocks of 931 patients treated between November 1999 and August 2005 . Using cPatient demographics, histopathology of primary tumor, treatment patterns and survival rates were retrospectively obtained from medical records. Tumor-node-metastasis (TNM) stage was determined from the 6th American Joint Committee on Cancer criteria. Histological grade was assessed by the modified Bloom-Richardson classification. DFS time was measured from the date of the first curative surgery to the date of the first local, regional, or distant recurrence or death without any type of relapse. OS time was measured from the date of the first operation to the date of the last follow-up or death from any cause.Immunohistochemical staining was performed using prior formalin-fixed, paraffin-embedded TMA tumor blocks as detailed in procedure descriptions from a previous study . Using cThis study was approved by the Institutional Review Board of Severance Hospital, Yonsei University Health System, Seoul, Republic of Korea (IRB No. 4-2016-0993). Written informed consent was waived and patient information was anonymized and deidentified prior to analysis.r), HR with 95% CI, and a log-rank p-value were automatically calculated in a website and the results were displayed. An independent, two-sample t-test was used to compare the means of continuous numerical datasets. Differences between the groups were evaluated by a chi-square test. In order to analyze the downloaded METABRIC dataset and the TMA study, survival curves were plotted using the Kaplan-Meier method and group differences in survival time were investigated by a log-rank test. A Cox’s proportional hazards model was used to identify the variables that were independently associated with survival. All statistical tests were two-tailed and a p-value < 0.05 was considered statistically significant. SPSS version 23.0  was used for all statistical analyses.Web-based bioinformatics statistics including mutual exclusivity, correlation coefficient (


Robot-mediated neurorehabilitation is a growing field that seeks to incorporate advances in robotics combined with neuroscience and rehabilitation to define new methods for treating problems related with neurological diseases. In this paper, a systematic literature review is conducted to identify the contribution of robotics for upper limb neurorehabilitation, highlighting its relation with the rehabilitation cycle, and to clarify the prospective research directions in the development of more autonomous rehabilitation processes. With this aim, first, a study and definition of a general rehabilitation process are made, and then, it is particularized for the case of neurorehabilitation, identifying the components involved in the cycle and their degree of interaction between them. Next, this generic process is compared with the current literature in robotics focused on upper limb treatment, analyzing which components of this rehabilitation cycle are being investigated. Finally, the challenges and opportunities to obtain more autonomous rehabilitation processes are discussed. In addition, based on this study, a series of technical requirements that should be taken into account when designing and implementing autonomous robotic systems for rehabilitation is presented and discussed. According to the findings obtained in the context of a Global Initiative on Neurology and Public Health carried out by the World Health Organization (WHO), many of the neurological disorders are chronic and progressive, constitute a global public health problem , and affThis situation, together with the need for rehabilitation and assistance for people with disabilities, means that robotic care and rehabilitation may play an important role in the years ahead.Nowadays, research on the use of robotic systems in different fields related to healthcare is widespread –7. In thIn this paper, a systematic literature review is conducted to identify the contribution of robotics for upper limb neurorehabilitation highlighting its relation with the rehabilitation cycle and to clarify the prospective research directions in the development of an autonomous rehabilitation process.The World Report on Disability by the WHO and World Bank  providesDespite this, the term rehabilitation covers a wide field of applications, being a subject to different connotations in a world characterized by a profound cultural diversity. Meyer et al.  providedThe health strategies can be different, but they can share a series of steps to improve the patient's health status throughout the rehabilitation process. This process involves the identification of a person's problems and needs, relating the problems to relevant factors of the person and the environment, defining rehabilitation goals, planning and implementing the measures, and assessing the effects . This apIn a simplified way, the rehabilitation cycle includes four steps: assessment, assignment, intervention, and evaluation. The process takes place on two levels: the first corresponds to the guidance provided along the continuum of care and the second refers to the provision of a specific service .From the point of view of the care guide, the assessment consists of the identification of the problems and needs of the person, the analysis of rehabilitation potential and prognosis, the definition of the long-term service, and the goals of the intervention program. Assignment refers to the inclusion of the person in a program of intervention in the most appropriate service for the treatment of their needs. For the guidance perspective, no specifications appear in the intervention. Evaluation refers to the service and the achievement of the intervention goal.From the perspective of providing a specific service, the assessment includes the identification of the problems, the review and potential modification of the service or goals of the intervention program, the definition of the first goals of the rehabilitation cycle, and the objectives of the intervention. The assignment step refers to the allocation of professionals and health interventions necessary to achieve the intervention objectives. The intervention consists in the specification of the techniques, measures, and the definition of target values that must be achieved within a predetermined period of time. Finally, the evaluation determines the achievements of the objectives with respect to the specific indicators, the goals of the rehabilitation cycle, and, ultimately, the goals of the intervention program. It also includes the decision regarding the need for another intervention cycle based on a new assessment.Rehabilitation requires the services of multiple healthcare providers who possess unique skills, training, and expertise that are employed for the full restoration of the patients' function and their optimal reintegration into all aspects of life . Rehabil(a) multidisciplinary team model—in which team members interact and communicate among themselves, knowing the work of all the components and offering an evaluation and parallel but independent work; (b) interdisciplinary team model—where the team members share a formal space in which information is exposed  and decisions are made around one or several common objectives ; and (c) transdisciplinary team model—which not only promotes communication among group members but also acquires knowledge from other related disciplines and incorporates them into the practice [The integration of the different medical means can be done through three working models , 28: (a)practice .Because the interdisciplinary model is designed to facilitate lateral communication, it is theoretically better suited for rehabilitation teams .Rehabilitation measures are a set of recovery actions that target body functions and structures, activities and participation, environmental factors, and personal factors.Rehabilitation outcomes are the benefits and changes in the functioning of an individual over time that are attributable to a single measure or set of measures . These oA particular case of rehabilitation is aimed at treating the problems caused by disorders affecting the nervous and neuromuscular system, known as neurorehabilitation. These types of disorders can produce mental or physical disabilities or both and are chronic and/or progressive.Neurological rehabilitation can be defined as a process that aims to optimize a person's participation in society and sense of well-being. This definition highlights several important features: rehabilitation is not a particular type of intervention; the focus is on the patient as a person; the goals relate to social functioning, as well as health or well-being; and it is not a process restricted to patients who may recover, partially or completely, but applies to all patients left with long-term problems . This wiThe complexity of the problems caused by a neurological damage highlights even more the need for a team to work on its treatment, the interdisciplinary model being the most used . The comThe rehabilitation cycle shown in The rehabilitation process starts with collecting data from the patient and others to establish: the problems; the causes of, and factors influencing, each problem; and the wishes and expectations of all interested parties. It is also important to consider the prognosis based on the diagnosis, natural history, distribution, and severity and type of the impairment, as well as other personal, social, and environmental factors .Impairments: the typical body functions that need to be assessed in the neurological patient are those related to the functions of the joints, muscles, movements, and sensation and cognitive functions. Thus, some constructs of relevance are muscle, ranges of movement, attention, memory, and balance. There are scales classically encompassed at this level such as Beck Depression Inventory, Behavioral Inattention Test, Canadian Neurological Scale, Clock Drawing Test, Frenchay Aphasia Screening Test, Fugl-Meyer Assessment of Motor Recovery after Stroke, General Health Questionnaire-28, Geriatric Depression Scale, Hospital Anxiety and Depression Scale, Mini-Mental State Examination, Modified Ashworth Scale, Montreal Cognitive Assessment, Motor-Free Visual Perception Test, National Institutes of Health Stroke Scale, and Orpington Prognostic Scale.Activity: when examining a patient's activities, the therapist will examine whether they can do not only the tasks but also the quality with which the task is performed. According to Lennon's study , one of Participation: this a more complex concept than impairments and activities, but it is fundamental to understand the patients and their life and help with planning treatment. Physiotherapy assessment of participation therefore focuses on those activities or roles in which patients take part in, patients are hindered in, and patients wish to work on and which could be improved and will inevitably deteriorate. Common scales used are the following: Canadian Occupational Performance Measure, EuroQol Quality of Life Scale, London Handicap Scale, Medical Outcomes Study Short Form 36, Nottingham Health Profile, Reintegration to Normal Living Index, Stroke-Adapted Sickness Impact Profile, Stroke Impact Scale, and Stroke Specific Quality of Life Scale.To this end, a series of objective scales have been developed to assess the level of independence of patients. The three main domains of the ICF can be used with this aim as a clinical tool , 38:ImpAccording to the pathology, the rehabilitation team designs a specific plan based on the diagnosis (problems identification) and disability of the patient. It is necessary to identify clear objectives related to the functional problems. Rehabilitation objectives normally follow the SMART rule because they must be specific, measurable, achievable, relevant, and time-limited .There are three key areas that the rehabilitation process is broken down: (1) approaches that reduce disability; (2) approaches designed to acquire new skills and strategies, which will maximize activity; and (3) approaches that help to alter the environment, both physical and social, so that a given disability carries with it minimal consequent handicap. The planning of a neurological rehabilitation program should consider the previous three approaches, in addition to the SMART rule.Specific rehabilitation interventions include those related to physical medicine, occupational therapy, speech and language therapy, dysphagia management, neurophysiological interventions, psychological assessment and interventions, nutritional therapy, and other interventions . A wide At present, the evidence suggests that to be effective, rehabilitation requires the practice of activities in the most relevant possible environments, rather than undertaking analytical exercises aimed at changing impairments . This isIn this phase, the physical condition of the patient is reevaluated in order to determine the effectiveness of the treatment, based on the SMART objectives  initiallPrevious quantitative investigations and case studies have shown that the use of patient-centred goal planning with adults undergoing neurological rehabilitation can improve self-perceived and observed goal performance and satisfaction . A patieIn addition, one must take into account the underlying pathological process, the chronic nature of certain pathologies, the need for supervision and/or the continuity in the absence of an expressive face-to-face rehabilitation treatment, or the degenerative and progressive character of some neurological pathologies, such as Parkinson's disease, multiple sclerosis, or Alzheimer's disease.In this section, this review will highlight the particular aspects of the rehabilitation cycle applied to upper limb neurorehabilitation performed with the assistance of any kind of robotic system.The authors undertook a literature search in October 2017 about robot-assisted upper limb rehabilitation in neurological diseases, using keywords such as robot, neurological, rehabilitation, upper, limb, extremity, arm, hand, neurorehabilitation, intervention, assisted therapy, treatment design, and various combinations. The databases were Brain, Science Direct, PubMed/Medline, and IEEE. Only papers written in English were considered, and the search was extended to the whole database. Studies were included when (1) systems for upper limb training  were used; (2) systems are based on end-effector and exoskeleton devices ; (3) the clinical intervention was conducted; and (4) the effects of the robot-assisted therapy were investigated.According to the Strategic Research Agenda for Robotics in Europe (SPARC) , healthcThus, devices to train (robot-aided therapies), support (exoskeletons), or replace (prosthesis) impaired activities or impaired body functions and structures are covered in rehabilitation robotics. In this way, robots are presented as a useful tool in the recovery process in neurological treatment. Such systems participate actively and help the therapist to perform a better rehabilitation process. However, it is not clear in what way and to what extent robotic systems provide this help during the rehabilitation cycle. To improve the quality of help provided, it must be identified how and when the aid is administered.The summary presented in Rehabilitation, like many aspects of human behaviour, can be thought of as a purposive problem-solving activity . The folAs previously indicated, the starting and ending component of the rehabilitation cycle is the functional assessment. It is important to take into account that most of the assessments performed by robotic systems are not functional assessments (carried out in baseline and follow-up stages of treatment), and its provided outcomes are indicators of a patient's performance. Currently, functional assessment is still carried out by traditional tests and scales provided by therapists. The main features of the robot-aided systems reviewed related to the assessment phase of the rehabilitation cycle are described as follows:(1) Assessment Mode. Assessment of the patient's performance can be carried out in two modes: automatic or nonautomatic. The automatic mode corresponds with the online data analysis, that is, during the development or at the end of the session. On the contrary, the nonautomatic mode corresponds with the offline data analysis (after of the end of the session).(2) Assessment Method. Robotic rehabilitation systems present evaluation methods that are based on the biomechanical data they are able to acquire. Based on such data, a rapid report that could be performed in an online or offline mode is provided to the therapist. 74% of the reviewed systems have not specified assessment methods, but propose an evaluation method based on the offline analysis of the biomechanical data acquired during therapy. In these studies, a later analysis of the stored information is done, applying algorithms to obtain information on the patient's performance. However, besides having an automatic record of information, only 26% of the systems perform online processing of these parameters by using specific software .(3) Provided Outcome. Robot-assisted systems have the advantage of providing a reliable and objective quantitative rapid assessment, based on the comparison of the metrics acquired during therapy. However, this assessment is at the level of impairment but does not provide information on how such impairment influences the activities of the patient's daily life. The most automated are commercially available systems like INMOTION ROBOTS, ARMEOSPRING, AMADEO, REOGO, and DIEGO. They have an online processing that generates a report at the end of the therapy session. However, the reliability of these automatic assessments, although they are based on objective measures, has not been validated with respect to determining, on their own, whether the rehabilitation has been adequate or not. Also, robot-mediated measurements have even smaller dissemination. For this reason, most of the systems reviewed carry out additional clinical evaluation, using functionality scales that are of standardized use at the clinical level, such as those mentioned in (4) Functional Assessment. Given the importance of making a correct evaluation, it is necessary to highlight the need to use standardized tools and procedures. The classification of the ICF is very useful for this functional assessment. The use of these standard functional scales as the main output of the rehabilitation systems would provide a better and more collaborative way to determine the effectiveness of the therapy based on the metrics obtained by the rehabilitation systems themselves. Currently, this issue is addressed by INMOTION software (INMOTION EVAL) that, based on multiple regression models, calculates Fugl-Meyer Assessment (FMA), Motor Status Score (MSS), Motor Power (MP), and Modified Ashworth Scale (MAS) from the robot-based metrics. These measurements of motor control are highly correlated with the traditional scales [l scales .As previously mentioned, it is important to emphasize that the complexity of a neurorehabilitation treatment usually requires the participation of a work team. Therefore, it is important that the patient's progress information is available to the entire work team, according to the interdisciplinary model. The management of information is one of the more time-consuming tasks that facilitate the decision-making of the therapist. Currently, there are several electronic medical record (EMR) software for the management of the patient's data , includiThe results of the review show that 45% of the systems  also provide some kind of help in the elaboration of the therapy. The most common assistance is through offering a set of exercises, games , or therapy protocols (INMOTION system) that can be configured or combined by the therapist. One of the systems (REHAROB) also allows the option of selecting exercises that are based on the intervention methods most used in physical rehabilitation, such as the Bobath or Kabat method. On the other hand, in-depth analysis of the data recorded robot-aided therapy, as well as allowing rapid functional assessment, serves as a tool for decision support to determine the patient's discharge. The INMOTION system allows discharge plots to be generated based on the performance of 5 tests that register kinematics and kinetics data. To the authors' knowledge, there are no commercial systems able to automatically generate a complete rehabilitation strategy from the initial functional assessment data and thus the therapist still has to properly identify the patient's problems by means of a reliable diagnosis and the right choice of clinical measures to evaluate the effectiveness of the treatment.Typically, rehabilitation occurs for a specific period of time but can involve single or multiple interventions delivered by an individual or a team of rehabilitation workers and can be needed from the acute or initial phase immediately following recognition of a health condition through postacute and maintenance phases. Rehabilitation reduces the impact of a broad range of health conditions. Further, neurorehabilitation is often still based on therapists' expertise, with competition among different schools of thought, generating substantial uncertainty about what exactly a neurorehabilitation robot should do .Robot-aided systems allow the training of an impaired limb in multiple sessions and in a systematic way, without loss of efficiency. With respect to the target region of treatment, the number of joints that the same system is capable of treating has been identified. No devices covering the movement of all joints of the upper limb have been found, that is, the shoulder, elbow, wrist, and hand (including fingers joints). The ARMEOSPRING, INMOTION, and ARMEOPOWER systems manage to cover the shoulder and elbow joints and also to train the flexoextension of the wrist and the manual grip, excepting finger joints.The effectiveness of treatments based on task-specific training in robot-assisted interventions is demonstrated. So it is understandable that 86% of the review systems consider this approach. It is observed that the systems have more than one operating mode . This represents a great advantage when considering treatment measures in a flexible way and better adapted to the type of injury. Some systems describe the mechanisms of action of the robots, which can offer assistance to the movement or gravity compensation through cable-based transmissions or pneumatic actuator systems. The pneumatic actuator systems offer the advantage of producing large forces with low weight added to the device, while cable transmission systems have greater shock absorption, smoothness in movement, and greater versatility in their passage through the joints.Finally, all the robotics rehabilitation systems reviewed are able to acquire and automatically store biomechanical metrics during the therapy. Depending on each robotic system, it can measure the workspace, joint movement ranges, and force exerted, as well as the quality in terms of the precision and smoothness of the trajectories. Other measures derived from the previous ones for a certain interval of time are the speed of execution and completion of the tasks, as well as the reaction times. The acquisition and storage of these parameters are immediate due to the inherent sensorization of the robotic systems . These are objective records due to the robotic intrinsic sensory systems.The development of autonomous systems is an active line in robotics in general, and with increasing presence in healthcare applications, it is already generating beneficial results as it has done in industry . That isThe common understanding in the robotic community is that the goal of robotic rehabilitation devices should be to assist therapists in performing the types of activities and exercises they believe give their patients the best chance of a functional recovery. But several barriers have been identified, for the particular case of rehabilitation robotics. The first identified barrier is the lack of effective communication in the planning stage of designing robotics aids, between engineers and therapists. Second, many of the devices are incredibly complicated, from both an engineering and a usability point of view. In fact, “simple-to-use” devices are more likely to be adopted by the clinical community than those that have long set-up times or require multiple therapists and/or aids to use . AnotherTo apply this automation approach to the rehabilitation process, it is first necessary to identify how the process is developed and identify which are the most susceptible elements to be automated, as well as the requirements and limitations to achieve this purpose.Based on the review presented in this article, we have identified three main areas within the rehabilitation cycle where robotics is contributing to automation: planning treatment protocols, implementing interventions, and evaluating the treatment's effectiveness. This rehabilitation cycle, shown in the previous This paper proposes a framework for the development of the rehabilitation cycle that clearly identifies which parts of the process are more likely to be automated, as well as the actors and elements involved. The autonomous rehabilitation cycle would be composed in this way by five elements that are directly correlated with the blocks of the original cycle. According to this approach, three main actors have been identified: user, clinician (understood as the team), and automated systems. Although several automated systems could be available, as denoted in The interaction between these three participants during the course of an automated neurological rehabilitation process will be described in The therapist discusses with the patient to review and adjust the objectives, deciding which treatment plans proposed by the DSS will be adopted. Then, the selected robotic rehabilitation systems (RRS) perform the intervention. After the intervention with the RRS, an assessment of functionality similar to the initial one is carried out again, in order to quantify the effectiveness of the therapeutic measures. For this, the AAS is used again. Finally, if all the problems identified are considered resolved or accepted by both the clinician and the patient, the rehab cycle is concluded. Otherwise, the necessary iterations will be made to try to solve the remaining problems.It can be deduced that the proposed automated systems operate separately and independently but that they are intrinsically connected and depend on each other for efficient operation, in coordination with the clinician and the patient. The methods to extract metrics and share them and their degree of acceptance by both users and health professionals should be rationalized and assessed, as a prerogative to achieve the automation. To design assistance rehabilitation systems, although the focus is on the subject to be treated, it is important to systematize the understanding of the requirements demanded by therapists in order to enable an easier integration of technology in their daily activities .By providing low-cost and easy to access tools for implementing this automated rehabilitation cycle, the viability of extending the rehabilitation cycle can be increased, not only as a temporary activity but also as a lifelong rehabilitation, as needed, for example, for affordable robotic therapy in maintaining function in degenerative disorders.Thus, in the opinion of the authors, the requirements that the components of a rehabilitation cycle must meet to be more autonomous are described below.As revealed by the analysis of assessment methods in neurorehabilitation, the use of traditional motor and functional scales is the main approach to determine the effectiveness of the rehabilitation process. For this reason, the development of methods based on traditional assessment scales that are widely used and known by specialists in rehabilitation is one of the lines of research that have been highlighted to achieve a more autonomous rehabilitation cycle.There are already oriented studies in this line of work, taking into account two premises: the method and metrics. Regarding the method, tests that are administered without direct contact of the professional are more suitable to be automated. Concerning metrics, it is essential to assess which ones give relevant information and are less invasive for the subject to be evaluated .It can be seen that the FMA is one of the most used scales employed for the motor assessment in the clinical trials that this review included. So it appears reasonable that the potential for the automation of these kinds of assessment methods is being studied. The application of RGB-D sensors, inertial measurement sensors, and other sensors has allowed the scoring of a part of the FMA to be automated . HoweverIn this respect, the literature also presents several projects that are focused on the automation of the traditional and still “gold standard” scales. As traditional scales are widely used in clinical trials in rehabilitation, as seen in this article, and because the administration of the evaluation is time-consuming, it appears reasonable that the automation of these kinds of assessment methods is being studied. There is an important difference in emphasis between clinical assessment and measurement. Traditional scales comprise several items. However, measurement concerns the quantification of an attribute and some studies  demonstrFollowing the model of the rehabilitation process, most of the systems reviewed (based on end-effector or exoskeletons) are clearly located within the intervention stages of the rehabilitation cycle. However, a percentage of them (46% end-effector and 43% exoskeletons) addresses the assessment stage, based on the metrics that are obtained from the use of systems in therapy. This assessment serves as a method of “rapid assessment” to support the therapist and inform the patient of the effectiveness of the rehabilitation process, but there are few works that report comparative studies or clinical trials to validate nonclinical metrics.Decision support systems based on artificial intelligence (AI-powered DSS) are one of the most active fields in recent years, and it is expected that they will soon contribute to the decision-making process. In healthcare, a variety of software for EMR management is already available see  to help Clinical decisions are an important component of the rehabilitation cycle, since they involve the determination of the objectives and design of the rehabilitation treatment. As can be seen in this review, the support provided by automated systems for this kind of task is by providing more reliable and objective information about the motor performance of the user during the intervention, as well as allowing the execution of different types of intervention procedures that can be configured by the clinician.Regarding the assignment stage of the rehabilitation cycle, there are two steps that could be automated by using artificial intelligence techniques: the planning of intervention treatments and the assignment of the appropriate RRS for intervention.Related to the planning of intervention treatments, the generation of these protocols is based on different factors that depend on the type of lesion and on how it affects the development of the patient's daily living activities. Many of the intervention measures are systematized in order to deal with a particular effect (concrete measures for specific problems), but there is no reason to believe that a “one-size-fits-all” optimal treatment exists. Instead, therapy should be tailored  to each patient's needs and abilities . In addiThus, we have identified some requirements that must be met to develop intelligent systems for treatment planning: (1) coherence between technological and traditional outcome measures, for the purpose of a therapeutic intervention based on technology and the problem-solving approach; (2) differentiating these measures according to the level of the effect ; (3), based on models, to identify the parameters that define an adequate physical condition according to the demographics of the patient and healthy profiles; (4) to be able to estimate the physical condition of the user to compare it with the welfare reference model; and (5) to generate a protocol that can be executed by the available intervention systems.These requirements imply that the integration of an AI-powered DSS in the automated cycle requires as input parameters the results of the evaluation systems (AAS) and, based on them, generates an optimized treatment protocol that can be executed by the systems of automatic intervention (RRS). This is why special attention is needed to the development of strategies that allow the integration and collaborative execution of these automated systems.The developments in medical robotics systems and RRS are fields that have awakened most interest for research in robotics. Due to the direct participation in the intervention phase, the different methods used in rehabilitation , and the understanding of what constitutes the most, appropriate therapy has the potential to become an intensively active topic of research .Two main issues have been highlighted: the ability of the RRS to acquire multiple information on patient performance during the development and the fact that from these data an assessment of patient functionality is obtained, even in the same type of score as the traditional scales.However, the type and amount of information that is obtained depend a lot on the type of robotic system (end-effector or exoskeleton) and the intrinsic sensory system. Also, the parameters derived from the measurements, as indicators of quality , can be very heterogeneous. Therefore, a critical issue is to unify the metrics acquired by the RRS, so that they provide as much information as possible for a rapid assessment by the therapist and not just raw data. Thus, among this type of metrics we have the following: range of movement, speed, precision, efficiency, percentage of work of the patient and percentage of work of the robot, and degree of attention in the task. All the works reviewed coincide in capturing the kinematic data; however, they do not address high-level indicators such as the percentages of robot and patient work (excepting NeReBot that gives it as a percentage) nor the degree of attention.Another important issue is to promote the adherence of the user to therapy. It is necessary to provide an adequate feedback that motivates the patient. Using virtual reality systems is the most widely used solution for this purpose. However, it is important not only the way in which the feedback is given but also the information provided to the user. In this sense, therapists agree that a visual feedback that tells the user if he has improved his score during the execution of the therapy would be beneficial. Other high-level indicators such as the percentages of robot and patient work, control signal, or kinematic data could be helpful to the user only if they help to show the relevance of the patient's progress.RRS-type systems are already integrated into the rehabilitation cycle, due to their imminent nature in the intervention; however, addressing the aforementioned questions would allow the rest of the automated components indicated in this paper (AAS and DSS) to take advantage of the objective information that is acquired with the RRS.A new automated rehabilitation framework has been proposed based on a literature review of robotic rehabilitation systems (RRS) for the upper limb treatment, highlighting its relation with the rehabilitation cycle. This framework has been presented regarding the implementation of more autonomous rehabilitation procedures. Three automated elements were described to make up the proposed framework: automated assessment systems (AAS), decision support systems (DSS), and robotic rehabilitation systems (RRS).The development of AAS should be based on the traditional assessment methods, since the traditional scales are still the “gold standard” for measuring outcomes and determine the effectiveness of treatment. In addition, the outcome provided by the AAS is obtained in an objective way, generating additional information about the user's performance.Those systems must be complemented with a novel DSS to help in clinical decision-making and treatment planning. The management of the patient's data (EMR) is currently addressed by using specific software based on high-level algorithms and also on artificial intelligence (AI). Optimized treatment protocols customized to the patient's condition are expected to be automatically generated by these DSS. For this purpose, AI is a promising tool. Dealing with multiple objectives in decision-theoretic planning and reinforcement learning algorithms  could coThe development of strategies for allowing the integration and collaborative execution of these automated systems is needed. It must be considered a proper data management in order to allow the AAS and DSS to use the objective information that is acquired with the RRS. In this way, a communication channel similar to the interdisciplinary team model will be enabled for the automated elements.In the case of the AAS development, the automatic administration of the assessment must be considered and not only the automation of the outcome. Knowledge of the user is as important as system functionality, since without the user's cooperation and acceptance, the system's functionality may be ineffective.The complexity of neurological disorders and its effect normally presents additional diseases concurrent with the primary disorder (comorbidity) that could limit the patient recovery.The feasibility of using AI to generate optimal treatment protocols is still unclear, but considering that AI is a mature science at present, the potential to contribute to the implementation of the proposed DSS is encouraging.Clinical protocols are validated through randomized control trials (RCT) where a large number of patients undergo the same treatment. In this regard, the most homogeneous samples must be recruited for RCTs that is challenging because of the inherent nature of neurological disorders.To conclude, the implementation of the proposed framework should consider some issues that are summarized as follows:Robots are currently viewed as advanced therapy tools under a therapist's guidance. However, the implementation of the above-mentioned systems could lead to more autonomous and intelligent processes in neurorehabilitation.