C) into the pathologic isoform PrPSc is a key feature in prion pathogenesis. AR-12 is an IND-approved derivative of celecoxib that demonstrated preclinical activity against several microbial diseases. Recently, AR-12 has been shown to facilitate clearance of misfolded proteins. The latter proposes AR-12 to be a potential therapeutic agent for neurodegenerative disorders. In this study, we investigated the role of AR-12 and its derivatives in controlling prion infection. We tested AR-12 in prion infected neuronal and non-neuronal cell lines. Immunoblotting and confocal microscopy results showed that AR-12 and its analogue AR-14 reduced PrPSc levels after only 72 hours of treatment. Furthermore, infected cells were cured of PrPSc after exposure of AR-12 or AR-14 for only two weeks. We partially attribute the influence of the AR compounds on prion propagation to autophagy stimulation, in line with our previous findings that drug-induced stimulation of autophagy has anti-prion effects in vitro and in vivo. Taken together, this study demonstrates that AR-12 and the AR-14 analogue are potential new therapeutic agents for prion diseases and possibly protein misfolding disorders involving prion-like mechanisms.Prion diseases are fatal infectious neurodegenerative disorders that affect both humans and animals. The autocatalytic conversion of the cellular prion protein (PrP PRNP/Prnp gene encodes the cellular prion protein (PrPC), a protein highly expressed in the central nervous system in neurons and glial cells, and present in non-brain cells. The exact physiological role of PrPC is a matter of debate4. In prion diseases, PrPC is converted into the pathological isoform PrPSc that is infectious in the absence of encoding nucleic acid6. Subsequent accumulation of PrPSc leads to a series of fatal neurodegenerative diseases in humans and animals. Human prion diseases include the various forms of Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). Animal prion diseases are scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle and other species, and chronic wasting disease (CWD) in cervids10. Loss of neurons, astrogliosis and mild microglia activation are the main pathological features of prion diseases. This results in a progressive spongiform degeneration of the central nervous system (CNS), leading to ataxia, behavioral changes and, in humans, highly progressive loss of intellectual abilities13. In the last two decades, great efforts have been made to establish treatment options for prion diseases. These included testing existing drugs for anti-prion activity in experimental models21 with only a few agents progressing to human studies of patients with prion diseases25. Investigations to date have not resulted in a recognized/proven treatment for prion diseases.The highly conserved 30. Interestingly, it shows activity against a number of infectious agents including bacteria, fungi and viruses35. It is an orally available small molecule with human safety data and is known to cross effectively the blood-brain barrier36. Mechanistic studies suggest that AR-12 down-regulates the host cell chaperone machinery, preventing proper folding of viral proteins and efficient viral assembly37. Additionally, AR-12 has been shown to down-regulate GRP78, resulting in up-regulation of Atg13 and PERK, which induces autophagy and facilitates the clearance of intracellular viruses and/or unfolded proteins38. We have reported that drug-induced autophagy stimulation has anti-prion effects in vitro and in vivo39. In our search for compounds which induce autophagy and reduce prions, we tested AR-12 and several analogues in two prion-infected neuronal cell lines (N2a and CAD5) and prion-infected mouse embryonic fibroblasts (MEFs). AR-12 significantly decreased PrPSc levels in all three prion-infected cell lines after three days of treatment with a single dose of the compound. Our findings were confirmed by PrPSc-specific immunofluorescence analysis and real-time quaking-induced conversion (RT-QuIC), where application of AR-12 and its analogue AR-14 to infected cells resulted in a robust decrease in prion conversion activity. This very efficient anti-prion effect upon short-term treatment suggests that the compound may have multiple effects on prion propagation and/or acts on PrPSc clearance. In line with this, PrPSc decrease was accompanied by up-regulation of autophagy markers like LC3-II. Using prion-infected N2a cells with CRISPR/Cas9-based knock-out of ATG5 gene, resulting in a loss of autophagy function, proved that autophagy is involved in the mode of anti-prion action of AR-12 and AR-14. Importantly, prolonged treatment with AR-12 and AR-14 for two weeks substantially cleared prion infection from ScN2a and ScMEF cells.AR-12 (a.k.a. OSU-03012) is an antitumor celecoxib-derivative that lacks cyclooxygenase-2 (COX-2) inhibitor activity. It inhibits phosphoinositide-dependent kinase-1 (PDK1) activity in different cell models and a first human clinical trial has been completedTo our knowledge, this is the first report to investigate the role of AR-12 and AR-14 in prion-infected cells. Our data show that AR-12 and its derivatives could be promising therapeutic tools for the treatment of prion diseases and protein misfolding diseases.40, the murine catecholaminergic/neuronal cell line ScCAD5 (infected with prion strain 22 L) of CNS origin41, and prion infected immortalized mouse embryonic fibroblasts ScMEF (22 L infected) as non-neuronal cells. In order to analyze whether AR-12 is affecting the level of PrPSc in ScN2a cells, we treated cells for 72 h with increasing concentrations of AR-12, from 0.5 to 3 µM, in a single application. A dose-dependent reduction of PrPSc was observed upon treatment. The effective dose 50% (EC50) was 1.5 µM . Concentrations of 2, 2.5 and 3 µM of AR-12 significantly reduced PrPSc levels (p < 0.001). Of note, toxic effects were not observed when cells were treated with AR-12 under these conditions. Median lethal dose 50% (LD50) was 5 µM . Next, we investigated the effect of AR-12 in the catecholaminergic/neuronal cell line ScCAD5, using a range of concentrations from 1 to 5 µM for 72 h. A concentration of 5 µM significantly enhanced the clearance of PrPSc (p < 0.01), with an EC50 of 4 µM . Yet, the effect seemed less pronounced compared to the one in ScN2a cells. To exclude that the observed decrease in PrPSc was related to drug toxicity, cytotoxicity assays were conducted. The results showed a safety margin up to 5 µM of AR-12 for 72 h of treatment in ScCAD5 cells. LD50 was 9 µM . Next, we tested whether the effect of AR-12 on PrPSc is limited to neuronal cell lines. We treated ScMEFs cells (infected with prion strain 22 L) with AR-12 at a range of concentrations from 0.5 to 3 µM for 72 h. A substantial decrease in PrPSc was observed at concentrations of 2.5 and 3 µM (p < 0.001). EC50 was 2 µM . AR-12 treatment was non-toxic to ScMEF cells up to a concentration of 4 µM, with a LD50 of 5 µM . Similar results were obtained with ScMEFs infected with strains prions RML and Me7 (data not shown).To address the effect of AR-12 in prion infected cells, we used three different cell lines. The murine neuroblastoma cell line ScN2a (infected with prion strain 22 L) of peripheral nervous system (PNS) origin µM Fig. . Concen µM Fig. . Next,  µM Fig. . Yet, t µM Fig. . Next,  µM Fig. . AR-12  µM Fig. . SimilaSc in neuronal (ScN2a/ScCAD5) and non-neuronal cells (ScMEF).Taken together, our data show that treatment with AR-12 enhances degradation of PrP50 was 4 µM . For ScMEF cells, AR-14 did not show a significant toxicity up to concentration of 3 µM. LD50 was 5 µM . AR-15 was safe to use in a concentration of only 1 µM . Unfortunately, AR-13 and AR-16 were toxic at the tested concentrations. Consequently, they were excluded from further analysis . To test the effect of AR-14 on PrPSc, ScN2a cells were treated with variable concentrations from 0.1 to 2 µM of AR-14 for 72 h. A significant decrease of PrPSc was observed after treatment with either 1.5 or 2 µM of AR-14 for 72 h (p < 0.01 and 0.001 respectively). EC50 was 1.5 µM . Treatment with 1 µM of AR-15 for 72 h did not show an effect on PrPSc. As additional read-out we used PrPSc-specific confocal laser microscopy analysis . In this immunofluorescence analysis, treatment with AR-12 and AR-14 resulted in a significant decrease in PrPSc compared to vehicle treated cells (p < 0.001). Additionally, a co-staining with the lysosomal marker lamp-1 showed more intense and condensed lysosomes after treatment with AR-12 or AR-14 compared to the diffused staining for lysosomes in cells that received solvent only (DMSO) .Next, we tested the effect of various available AR-12 analogues on controlling prion infection  Fig. .Figure Sc, at even slightly lower concentrations than AR-12.Collectively, these results indicate that AR-14 has the potential to reduce PrP42, thereby, for example, reducing neuritic degeneration caused by amyloid-β peptide43. Upon autophagy stimulation, microtubule-associated protein 1 light chain 3 (LC3-I) is post-translationally converted to the lipidated form LC3-II, which is considered as a marker for autophagy. LC3-II is associated with the formation of autophagosomes44. To examine the effect of AR-12 and AR-14 on autophagy in our cell culture models, we treated N2a, CAD5 or MEF cells with AR-12 (3 µM) or AR-14 (2 µM) for 2, 4 and 6 hours. An increased level of LC3-II was observed after treatment with AR-12 and AR-14 in all 3 cell lines compared to the vehicle (DMSO) treated cell population . To exclude that the observed increase in LC3-II upon treatment with AR-12 and AR-14 was a result of impaired autophagic flux, cells were treated with AR-12 or AR-14 plus bafilomycin A1 (BA1). Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and endosomes/lysosomes and consequently results in accumulation of LC3-II45. N2a, CAD5 or MEF cells treated with BA1 together with AR compounds showed higher signals of LC3-II compared to cells treated with AR compounds alone .There is growing evidence that the AR-12 mode of action is related to induction of autophagyion Fig. . To excone Fig. .These data show that AR-12 and AR-14 treatment does not block autophagic flux, but stimulates autophagy in these cell lines.Sc, we tested the effect of AR compounds on PrPSc in autophagy-compromised cells. Using CRISPR/Cas9 technology, we had generated Atg5-deficient ScN2a cells (KO) which are defective in autophagy. KO cells do not show any signal for Atg5 or LC3-II in contrast to wild-type (WT) cells . To test the hypothesis that the intrinsic toxicity of AR-12 and AR-14 was related to autophagy, we performed XTT cytotoxicity assays for WT and KO cells treated with AR-12 and AR-14 for 72 h. We did not observe any remarkable differences in the viability pattern of the treated cells . These data exclude that intrinsic toxicity of AR-12 and AR-14 was related to autophagy. Next, we treated prion-infected WT and KO cells with different concentrations of AR-12 and AR-14 for 72 h . Our immunoblot results showed that AR-12 treatment significantly reduced PrPSc in both cell populations at concentrations of 2 and 3 µM (P < 0.001). Data were represented as a percentage of control (DMSO) treated cells .To further shed light on the mechanisms underlying the reduction of PrPlls Fig. . To teslls Fig. . These 2 h Fig. . Our imlls Fig. .Figure Sc significantly in WT and KO cells at 1 and 2 µM concentrations . The anti-prion effect of AR-14 was slightly higher in WT cells compared to KO cells . In wild-type cells, AR-14 (2 µM) was able to significantly decrease PrPSc to 7.75% (p < 0.001). In KO cells this reduction was only down to 45.7% (p < 0.05), which is a statistically significant difference relative to the 7.75% in WT cells (p < 0.05) . Data were represented as a percentage of control (DMSO) treated cells.AR-14 treatment reduced PrPons Fig. . The anlls Fig. . In wil05) Fig. . Data wIn conclusion, these results indicate an at least partial involvement of autophagy competency in AR-12 and AR-14 mediated anti-prion effects.Sc in different prion infected cell lines. Next, we investigated the effect of long-term treatment with AR compounds. We treated ScN2a cells with 3 µM of AR-12 or 2 µM of AR-14 for 20 days, adding the drug with every media change. Cells were passaged five times in the continuous presence of AR-12 or AR-14. Then, treatment was stopped and cells were passaged for another five times, without the test compounds. In addition to immunoblotting as read-out, real-time quaking-induced conversion (RT-QuIC) was used to assess prion seeding activity in AR-treated cells compared to vehicle (DMSO) treated cells. RT-QuIC is a very sensitive in vitro prion amplification technique and likely able to detect signs of prion infection in situations where immunoblotting is negative. Uninfected N2a cells were used at every passage as negative control and for calculating the cut-off values . At passage 1 with AR compounds, immunoblot analysis showed that PrPSc drastically decreased after treatment with AR-12 or AR-14 . RT-QuIC analysis showed that in DMSO-treated control cells seeding activity was detected up to a dilution of 10−3. Cells treated with AR-12 were positive up to a dilution of 10−2, paralleled by a strong decrease in seeding activity as evidenced by decrease in relative fluorescence units (RFU) . Cells treated with AR-14 also were positive up to a dilution of 10−2 and showed a decrease in seeding activity for the positive dilutions . Interestingly, RT-QuIC analysis of postnuclear lysates treated after lysis with AR-12 or AR-14 for 72 h showed less pronounced effects on the inhibition of seeding activity  compared to living cells that were treated with AR-12 and AR-14 , confirming that that AR-12 and AR-14 work mainly through an intracellular pathway in living cells. After 20 days (5 passages) of treatment with the AR compounds, PrPSc was not detectable by immunoblot analysis . AR-12 and AR-14 treated cells did not show any prion conversion activity in RT-QuIC , whereas DMSO treated cells were strongly positive . After 20 days of treatment, we passaged cells for another 20 days without any treatment. The first passage showed no prion seeding activity in cells that underwent AR-12 or AR-14 treatment . Similarly, immunoblot analysis did not show any PrPSc, whereas DMSO-treated cells were clearly positive . Five passages (20 days) after treatment was discontinued, AR-12-treated cells continued to demonstrate no prion seeding activity , while DMSO-treated cells did . However, in AR-14 treated cells prion seeding activity re-appeared, with dilutions 10−1 and 10−2 positive . Of note, immunoblot analysis did not show detectable PrPSc in both AR-12 and AR-14 treated cells compared to DMSO-treated cells .Our short-term treatment with AR compounds revealed a potent reduction of PrPues Fig. . At pas-14 Fig. . RT-QuI0−3Fig. . Cells FU) Fig. . Cells ons Fig. . Intereity Fig.  compare-14 Fig. , confirsis Fig. . AR-12 uIC Fig. , whereaive Fig. . After ent Fig. , in contig. Fig. . Similaive Fig. . Five pity Fig. , while did Fig. . Howeveive Fig. . Of notlls Fig. .Figure Taken together, these data demonstrate that AR-12, but not so much AR-14, is capable of completely clearing prion infection in ScN2a cells infected with 22 L prions., using immunoblotting and RT-QuIC analysis as read-outs. For RT-QuIC, uninfected cells were used at every passage as negative control and for calculating cut-off values . At passage 1 with AR compounds, PrPSc was undetectable after treatment with AR-12 or AR-14 by immunoblot analysis . Interestingly, in RT-QuIC, these cells continued to show a pronounced prion seeding activity , although their RFUs were lower than the ones of DMSO-treated cells , with AR-14 being more effective. Twenty days (5 passages) after treatment with AR compounds, PrPSc was negative in immunoblot analysis . Additionally, AR-12 and AR-14 treated cells did not show any prion conversion activity in RT-QuIC , while DMSO-treated cells did . As before, after 20 days of treatment, we passaged cells for another 20 days without any treatment. The first passage showed no prion seeding activity in cells that had received AR-12 or AR-14 treatment , compared to DMSO-treated cells . In line with this, immunoblot analysis did not reveal any PrPSc, in contrast to DMSO-treated cells . Five passages (20 days) after the treatment was discontinued, AR-12 and AR-14 treated cells continued to be negative for prion seeding activity , whereas DMSO-treated cells were positive . Similarly, immunoblot analysis did not show reappearance of PrPSc in previously AR-12 or AR-14 treated cells .Subsequently, we investigated the effect of long-term treatment with AR compounds on ScMEF cells. We used the same experimental design as for ScN2a cells Fig. , using ues Fig. . At passis Fig. . Intereity Fig. , althoulls Fig. , with Asis Fig. . AdditiuIC Fig. , while did Fig. . As befent Fig. , comparlls Fig. . In linlls Fig. . Five pity Fig. , whereaive Fig. . Similalls Fig. .Figure Taken together, our data show that both AR-12 and AR-14 were able to clear prion infection in ScMEF cells infected with prion strain Me7.6. In humans, manifestation of disease can be sporadic, genetic or acquired by infection. Human prion diseases are very rare46. Nevertheless, sporadic CJD as the main form is found worldwide at a constant incidence, resulting in up to 8,000 fatalities every year. Familial forms are much less frequent, but can be diagnosed long before clinical disease onset, providing a long window for intervention47. It will be crucial to understand the molecular requirements for prion propagation, to devise rational strategies for controlling these events, and in the long term, to establish means for treatment to effectively assist individuals incubating these diseases.Prion diseases are fatal infectious neurodegenerative disorders that affect man and animalsin vitro systems, with a substantial number still effective in animal models, usually prion-infected rodents, but only four agents were tested in human patients or small-scale human trials48.An ideal drug candidate should be bioavailable, able to effectively cross the blood-brain-barrier (bbb), have no adverse side effects, and achieve effective therapeutic concentrations in the CNS. As for many other neurodegenerative disorders, such a drug has not been found for prion diseases. Interestingly, a huge variety of compounds with often promising anti-prion activities has been described in 18. Most anti-prion compounds interfere in the process of prion conversion, by blocking either PrPc substrate or PrPSc template. Many strategies target the pool of PrPc amenable to prion conversion, e.g. by shutting-off or decreasing PrPc expression, preventing PrPc cell surface localization, extracting PrPc from the plasma membrane, or by inducing aggregation of PrPc at the cell membrane18. All these approaches result in a net lack of PrPc eligible for prion propagation. We and others have used the strategy to enhance the intrinsic cellular clearance of PrPSc49. We have reported that the pharmacological induction of autophagy has anti-prion effects in vitro and in vivo through enhancing the delivery of PrPSc for lysosomal degradation. Autophagy stimulators such as rapamycin, lithium, trehalose and imatinib have shown anti-prion effects in vitro and in vivo50. A less used strategy, testable only in animal models, is to block or reverse down-stream effects of prion-induced neurodegeneration, usually summarized under the term neuroprotection. More recently, restoring unfolded protein response has received great attention15. It was shown that re-establishing the translational machinery by inhibition of eIF2α-P activity restored memory and prevented features of neurodegeneration. Interestingly, although not entirely new for the prion field, the tested compounds were repurposed drugs, potentially accelerating their investigation in human clinical studies51.Conceptually, various molecular targets can be selected48. Pentosan polysulfate (PPS), a large polyglycoside molecule, has been shown to inhibit the formation of PrPSc and had some moderate effects in patients upon intraventricular application53. Acridine and phenothiazine derivatives like quinacrine and chlorpromazine inhibit prion replication in vitro and were tested in human patients, although without clear evidence of efficacy56. Flupirtine, a nonopioid analgesic with cytoprotective effects and without direct anti-prion activity, showed beneficial effects on cognitive function in some CJD patients57. Tetracycline and doxycycline have been shown to interact with PrPSc and to destabilize the structure of amyloid fibrils58, however, effects in humans are questionable59.The mode of action of the four compounds tested in human subjects is not completely understoodin vivo. AR-12/OSU-03012 is a celecoxib derivative with anti-cancer and anti-microbial activity but unlike celecoxib, AR-12 does not inhibit cyclo-oxygenase (COX)60, but inhibits several enzymes which may be useful in the treatment of some forms of cancer62. Additionally, AR-12 has antifungal activity via disruption of phosphoinositide-dependent kinase-1 activity63. Previous studies indicated that AR-12 is an autophagy stimulator64 and recently, it has been shown that AR-12 inhibits amyloid-β peptide-induced neuritic degeneration through autophagy stimulation43.Given all this, there remains a great need for anti-prion drugs which are effective Sc in ScN2a and ScMEF cells infected with different prion strains. Longer application completely eradicated PrPSc with a complete loss of prion conversion activity in a sensitive RT-QuIC analysis even after discontinuation of the treatment. Short-term treatment with AR-12 significantly decreased PrPSc in another neuronal cell line, ScCAD5 cells. Synergistic combination therapies might even increase the anti-prion efficacy and circumvent the potential problem of drug resistance, as reported previously66. Combining AR-12 with phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil or tadalafil, resulted in enhanced anti-tumor activity67. Similarly, combining two or more anti-prion agents may enhance their anti-prion effects and decrease the risk for drug-resistance and drug-induced toxicity. Our future direction is to combine AR-12 with other anti-prion agents and to investigate the effect on PrPSc and prion seeding activity.In the present study, we evaluated the effect of AR-12 and its analogues on controlling prion infection in neuronal and non-neuronal cell culture models of prion infection. Short-term application of AR-12 showed a robust decrease of PrPSc clearance mediated by induction of autophagy. We did not observe any effect of AR-12 treatment on quantity or subcellular distribution of PrPc in non-infected cells . The very pronounced effect after a 3-day treatment indicates activity on preexisting PrPSc and cannot be explained with effects on newly formed PrPSc only. Our data show that AR-12 treatment increased LC3-II in all tested cell lines, in line with induction of autophagy. Additionally, the anti-prion effect of AR-12 was less pronounced in autophagy compromised Atg5-KO cells compared to effects in wild-type cells. Interestingly, ScCAD5 cells showed the lowest induction of the autophagy marker protein LC3-II, which might explain why effects in ScCAD5 cells were weaker. Together, these results demonstrate that induction of autophagy is involved in the anti-prion activity of AR-12.The most likely molecular mechanism underlying the observed anti-prion effect is increased PrPlls Fig. . The ve36. A phase I clinical trial has been completed and AR-12 has received recently FDA IND-approval for cancer treatment and orphan drug status in Europe for selected indications. This underlines the potential of AR-12 as a drug candidate for counteracting human prion diseases.One of the biggest obstacles in developing treatments for prion diseases is that the compound has to cross the blood-brain barrier, at concentrations that are effective without inducing unwanted side effects. Importantly, AR-12 has been shown to cross the bbb effectivelySc in immunoblot and prion seeding activity in RT-QuIC analysis. In ScN2a cells, there was reappearance of prion conversion activity at the end of the drug-free period at passage 5. This indicates that although the treated ScN2a cells were negative in both immunoblot and RT-QuIC analysis over a period of almost 40 days, prion infectivity was not completely removed and had the potential to reinitiate prion propagation. Several conclusions can be made from these findings. First, the anti-prion effect of drugs in cell culture is dependent on the chosen cell line model and used prion strain. Second, negativity in immunoblot analysis is easily achieved and does not guarantee absence of PrPSc/prions. This is indicated, for example, by strong signals for RT-QuIC with entirely negative read-outs for immunoblot in passage 1 of AR-treated MEFs in Fig. Sc in immunoblot as parameter for treatment efficacy. Our data clearly show that such an approach can be misleading. Whereas the gold standard, testing for specific prion infectivity of cell lysates in rodent bioassays, is complicated and cannot be done by many laboratories, our study suggests RT-QuIC analysis as viable and easy to perform surrogate methodology.AR-14 is a chemical analogue of AR-12. Our study shows that AR-14 could be another potential candidate for drug-induced interference in prion disease. We tested AR-14 in various cell culture models, infected with different prion strains, and we found AR-14 has a robust anti-prion effect in the short-term treatments, even at lower concentrations as AR-12. Of note, treatment with AR-14 increased LC3-II levels and its anti-prion effect was less pronounced in Atg5-KO cells, indicating a role for autophagy in the anti-prion effect of AR-14. Upon extending the treatment with AR-14 in ScMEF cells, AR-14 cleared PrP69. Applying this approach in the future may provide further opportunities for AR-13, 15 and AR-16 in the prion field.Due to toxicity concerns in the tested cell lines, AR-13 and AR-16 were excluded in the very early stages from our study. As well, AR-15 could only be used in concentrations that were not sufficient to affect markers of prion infection. Interestingly, it has been published that encapsulation of AR-12 in acetalated dextran (Ace-Dex) microparticles reduced cytotoxicity while retaining the efficiency of the drug, allowing higher drug concentrations for treatmentSc and can clear prion seeding activity in cell line models infected with various prion strains. Therefore, they might represent novel therapeutic tools against prion diseases and possibly other protein misfolding diseases. Further work is necessary to establish whether these drugs can provide effective anti-prion effects in rodent models of prion infection, alone or in combination with other drugs.In summary, our study shows that the autophagy stimulator AR-12 and its analogue AR-14 enhance the degradation of PrP70. Peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch/USA . AR-12 and its analogues were obtained from ARNO therapeutics.Reagents used were: Proteinase K (PK) , Pefabloc inhibitor , and Lipofectamine Plus LTX reagent . Other reagents and chemicals were obtained from Sigma Aldrich, if not otherwise stated. Sources of primary antibodies were as follows: anti-Atg-5 , anti-β-actin , anti-lamp1  and anti-LC3 . The anti-PrP monoclonal antibody (mAb) 4H11 has been previously described2 atmosphere. CAD5 cells are a central nervous system catecholaminergic cell line71. CAD5 cells were subjected to various rounds of single cell sub-cloning and optimized for prion infection41. Cells were a generous gift of Dr. S. Mahal (The Scripps Research Institute Florida) and were cultured in OptiMEM Glutamax medium containing 10% bovine growth serum , and penicillin/streptomycin in a 5% CO2 atmosphere. Cells were persistently infected with a 1% brain homogenate from terminally-ill mice infected with mouse-adapted scrapie prion strains 22 L or RML. Immortalized mouse embryonic fibroblasts (MEFs) were maintained in DMEM Glutamax medium  containing 10% fetal bovine serum .The mouse neuroblastoma cell line N2a was obtained from ATCC (CCL-131) and was cultured in OptiMEM Glutamax medium  containing 10% fetal bovine serum , and penicillin/streptomycin in a 5% COATG5 in N2a cells was done using CRISPR/Cas9 technology as described previously72. Briefly, vectors for guide RNA (sgRNA) targeting exons 5 and 6 of the Atg5 gene were purchased from Sigma. The following constructs were used: exon 5: target ID: MM0000476061, vector: U6gRNA-Cas9-2A-GFP, target sequence: CCTCAACCGCATCCTTGGATGG; exon 6, target ID: MM0000476062, vector: U6gRNA-Cas9-2A-RFP, target sequence: GCCATCAACCGGAAACTCATGG. N2a-K21 cells were co-transfected with sgRNA and Cas9 expression vectors by Lipofectamine Plus LTX reagent according to the manufacturer’s protocol. We targeted exons 5 and 6 together in double-transfection. Transient GFP/RFP expression was used as control for transfection efficacy. Single cell clones were obtained and analysed by immunoblotting for Atg5 and LC3-II and by DNA sequencing of individual plasmid clones upon PCR amplification of genomic DNA. Characterization of cells will be described elsewhere (manuscript submitted for publication).Gene-targeting of 40. Briefly, confluent cells were lysed in cold lysis buffer ) for 10 min. Aliquots of lysates were incubated with PK (20 µg/ml) for 30 min at 37 °C. PK was stopped by addition of proteinase inhibitors (0.5 mM Pefabloc) and directly precipitated with methanol. For samples without PK treatment, proteinase inhibitors were added directly and precipitated with methanol. Precipitated proteins were re-suspended in TNE buffer . Samples were run on 12.5% SDS-PAGE, electro-blotted on Amersham Hybond P 0.45 PVDF membranes  and analyzed in immunoblot, using Luminata Western Chemiluminescent HRP Substrates .Immunoblot analysis was done as previously describedCells were seeded in 96-well cell culture plates at a density of 1,000 cells per well, then incubated overnight to allow attaching to the plate. The next day, cells were treated with AR-12 and its analogues, or treated with corresponding vehicle solution (DMSO), and incubated for 72 h. Treatment with 1% Triton X-100 was used as a positive control. Cell viability was assessed by TACS® XTT cell proliferation assay  according to the manufacturer’s instructions. Absorbance was measured at 490 nm using BioTek Synergy HT. Data were expressed as percentage of viability compared with the corresponding control (100%). Concentrations that showed a statistically significant difference from DMSO-treated control cells were considered toxic and consequently were excluded from further analysis.73. Briefly, mouse PrP (aa 23-231) was cloned into pET-41 plasmids, transformed into E. coli Rosetta, and bacteria cultured in LB media supplemented with kanamycin (0.05 mg/ml) and chloramphenicol (0.034 mg/ml). The Overnight Express Autoinduction System  was used to induce protein expression. Inclusion bodies were isolated from pelleted cells using Bug Buster Master Mix  and stored at −20 °C. For purification of recombinant PrP, inclusion bodies were solubilised in guanidine buffer  and incubated on the rocker for 1 h at RT. Ni-NTA Superflow resin beads  were incubated in denaturing buffer  for 1 h at RT. Solubilized inclusion bodies were centrifuged at 16,000 × g for 5 min, the supernatant added to the beads and incubated for 1 h with gentle rocking. Beads were then packed into a XK16 glass column . Using an Amersham ÄKTA Explorer FPLC unit running with Unicorn software , protein was refolded by a gradient from 100% denaturing buffer to 100% refolding buffer  over 4 h. The column was washed for 30 min with refolding buffer and proteins eluted using a linear gradient from 100% refolding buffer to 100% elution buffer . The central portions of the A280 UV peak were collected into dialysis buffer . Purified protein was filtered using a 0.22 µm filter, transferred into a Slide-A-Lyzer dialysis cassette , placed into a 4 liter beaker with dialysis buffer overnight at 4 °C with continuous stirring. Following dialysis, the protein solution was filtered again with a prewashed 0.22 µm Argos syringe filter. Protein concentration was measured using BCA protein assay , the solution aliquoted and kept in −80 °C until use.Preparation of recombinant prion protein was performed as described74. Briefly, reactions were set up in assay buffer containing 20 mM Na-phosphate, pH7.4, 300 mM NaCl, 1 mM EDTA, 10 μM Thioflavin T and 0.1 mg/ml rPrP substrate. Ninety-eight μl aliquots were added to the wells of a black-walled 96-well optical bottom plate . Tenfold serial dilutions of brain homogenate or cell homogenate were prepared in 0.5 ml microtubes. Quadruplicate reactions were seeded with 2 µl of cell lysate or brain homogenate for a final reaction volume of 100 µl. Reactions contained a final concentration of 0.002% SDS. Plates were sealed with Nunc Amplification Tape  and incubated in a FLUOstar Omega  plate reader for 30 h. Reactions were incubated at 42 °C, with cycles of 60 s shaking (700 revolutions per minute) and 60 s of rest throughout the incubation. ThT fluorescence measurements (450 nm excitation and 480 nm emission) were taken every 15 min. RT-QuIC data were averaged from four replicate wells and average values plotted against reaction time. Samples were scored positive if at least 50% of replicates reached a ThT fluorescence cut-off, which was calculated based on the average ThT fluorescence plus 5 x standard deviation.The Real-time Quaking Induced Conversion was performed as described4Cl, 20 mM glycine at RT for 10 min. Cells were then permeablized in PBS containing 5% FBS and 0.5% Triton X-100 (PBSST) for 30 min before incubation in 6 M GndHCl at RT for 7 min to denature PrPC and generating conditions specific for PrPSc . Cells were incubated with anti-PrP mAb 4H11 at 1:100 and anti-lamp1 at 1:100  diluted into PBSST for overnight at 4 °C, Alexa Fluor 488/Cy3 and 594 goat anti-mouse secondary antibody (Jackson Immunoresearch/USA) was used at 1:200 to visualize the immunostaining signal. All quantitative images were captured under the 63x oil lens at the same acquisition settings from a Zeiss LSM 700 confocal microscope. The overall immunofluorescence intensity of PrPSc from an image was measured from ImageJ software after the background was filtered by the same threshold applied to the same series images. Overall intensity was divided by the cell number obtained from the same image quantified by ImageJ “analyze particle” command to calculate the averaged immunofluorescence intensity per cell.ScN2a cells at 80–90% confluency were fixed in 4% paraformaldehyde for 30 min at RT, followed by quenching in 50 mM NHt-test for pair-wise comparisons or the One-way ANOVA analysis with Tukey post test for multiple comparisons. Statistical significance for all the experiments was expressed as (Mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001 considered significant. All the western blot figure were cropped using PowerPoint 2013 program.Statistical analysis was performed using GraphPad Instat  using either the unpaired two-tailed Supplementary Figures


The aim of the present study was to explore the views of professional rugby union players during the early rehabilitation, late rehabilitation and return to play stages, following anterior cruciate ligament (ACL) injury.A qualitative dominant, mixed methodological approach was utilized with five players who had suffered an ACL injury requiring reconstructive surgery. A longitudinal approach, concurrent with each player’s rehabilitation, consisting of twice monthly interviews, a self-report diary and three established questionnaires  were completed.Theoretical thematic analysis was conducted on three distinct phases  and coded relating to autonomy, competence and relatedness.The findings suggest that increased autonomy and control assist emotional and behavioral responses during rehabilitation and return to play, while development of competence increases self-confidence. Injury rates have increased significantly in rugby union over the last decade . The incCognitive appraisal models , 7 and sTwo main findings have been identified relating SDT to sports injury rehabilitation : (i) morExpanding on current practice within injury rehabilitation, this research utilizes a mixed methodological approach concurrent with the athlete’s rehabilitation through the physical rehabilitation process, to return to competition. The crucial aspect in justifying a mixed methodology is that the combination of methods focuses on the strengths of each single method. By using a combination of methods at various points in the research process, the researcher can build on the strength of each and minimise the weaknesses of a single method approach. A mixed-method can increase both the validity and the reliability of the data . In partFive professional rugby union players, who had suffered ACL injury that required surgical intervention committed to this study. Their age range was between 18 and 27 years, and the total time in rehabilitation was between 6 and 12 months. All players were playing in top tier professional rugby teams. See Table Initial contact was made with each player prior to them undergoing reconstructive surgery and within 2 weeks of the ACL injury being diagnosed. Following institutional ethics approval guidelines, each player completed informed consent at this initial contact time. The methodological procedure was split into three distinct phases of the injury and subsequent rehabilitation, expanding on research conducted by Shelley , to coveA mixed methodological approach was undertaken concurrent with each player’s rehabilitation, comprising of semi-structured interviews, a self-report diary and completing three established questionnaires related to SDT and social support. This approach consisted of a dominant  – less dominant (quantitative) design as suggested by Tashakkori and Teddle . The semEach interview was audiotaped and transcribed verbatim and a theoretical thematic analysis, as suggested by Braun and Clarke  was condNo statistical analysis was completed on the quantitative data rather it was utilized to triangulate with the qualitative data to assist with the confirmation or rejection of the themes identified. Combining data sources allows for a more holistic view of the main factors and reduces the possibility of inaccurate interpretations of the qualitative data being attained . This trThematic analysis was conducted separately on the early limited participation phase, late limited participation phase and return to play phase, and split into the general dimensions Autonomy, Competence or Relatedness. Full results from the MOS-SSS, Sport Climate Questionnaire, and Injury Rehabilitation Questionnaire are presented in Table Positive Self-Regulation, Positive Locus of Control, Negative Self-Regulation, Negative Locus of Control (Autonomy); Positive Physical Ability, Negative Physical Ability, Negative Performance Ability (Competence); Positive Team Interaction, Positive Medical Interaction, and Negative Team Interaction (Relatedness). Raw data themes, higher order themes and general dimensions for this phase are presented in Fig. In the early limited participation phase we identified 26 raw data themes that we combined into 10 higher order themes: I feel understood by my physiotherapist’ and ‘I am able to be open with my physiotherapist’ on the Injury Rehabilitation Questionnaire.Of principle importance to all players was the need to understand the rehabilitation process and become personally involved in it ; “I’m more patient knowing the process. Knowing what I can and can’t do” (P2); “I am clear on what I want to get out of the next few weeks” (P5)). The gathering of detailed information related to the rehabilitation program and increased personal control appears to be extremely beneficial to the control of emotions during this phase. The rehabilitation team can assist this process by providing a supportive setting. Each player commented on the need to trust their physiotherapist and the importance of having regular meetings with all involved in the rehabilitation ; “I like the way [physiotherapist] allows me lots of control of what I’m doing. It’s not that I go off on my own and just do what I want, but he involves me with almost everything. It really helps to keep my interest on the job in hand and stops me from worrying about my knee and how long I’m going to be out for” (P5)). All players stated they ‘strongly agreed’ with the statements ‘It is important to note that the restrictive nature of some ACL rehabilitation protocols may be debilitative for these injured players, as they limit the control each player has over the rehabilitation. Player 4 commented, “I would like some more control over what I’m doing. I lose some motivation when I’m constantly doing the same thing. The physio lets me decide on some of the exercises, but in terms of leg strengthening all the exercises are quite similar and a bit boring” and “It’s so frustrating. I feel so helpless. The rehab is just boring and repetitive”.Seeing developments and improvements in physical ability assisted in developing competence. Player 1 stated, “I have seen a gradual improvement in my range of movement, so I am positive I am improving all the time”. Similar comments were made by all players as they reach their rehabilitation goals, with each observed improvement appearing to have a cyclical effect by increasing self-confidence which in turn increased each player’s commitment to the rehabilitation program. However, frustration can become a factor when players are unable to reach the set targets ; “Although it’s good to be doing some activity again, it’s frustrating not being able to do as much as I want” (P4)). The impact of set ACL rehabilitation protocols may again have an impact at this early stage of rehabilitation. In many cases these protocols are highly structured stating weekly training targets, allowing little flexibility for individual performers ).Social support was important for each player during this phase, in particular developing a connection with their physiotherapist. Scores from the Injury Rehabilitation Questionnaire highlight the good relationship each player gained with their physiotherapist, with average scores ranging from 6.33 to 7 out of a maximum of seven. Player 5 commented, “[physiotherapist] was really good. He answered all my questions and gave me information that I could understand”. A vast amount of positive social support was received from teammates, which predominantly involved encouragement and relatedness ). However, some aspects of teammate support had a negative impact on the rehabilitating player’s emotions. Comments from players included, “My only negative is having to watch the other guys training outside. I still want to be part of the squad but it’s hard seeing it from the gym all the time” (P1) and “It’s a bit lonely really. It’s not like when you train with the boys, because most of the time I’m doing things on my own” (P2). Player 5 had a number of difficulties with the lack of support provided to him by his coach (“I haven’t heard much from the coach. It’s annoying but I wasn’t expecting to have a huge amount of contact with him. There’s some other stuff going on in the club that affects him more”). The Sport Climate Questionnaire was scored at the lowest possible value by this player, indicating he perceived no support from his coach. Although the coach may try to keep emotional distances from players to reduce the chances of favoritism being perceived, in this case the coach appears to be more self-concerned (the coach’s contract was terminated while this player was still injured), which had a negative impact on the injured player’s emotions and behavior.Positive Self-Regulation, Positive Locus of Control, Negative Self-Regulation, and Negative Locus of Control forming the general dimension Autonomy; Positive Physical Ability, Positive Performance Ability, Negative Physical Ability, and Negative Performance Ability comprising the general dimension Competence; and Positive Team Interaction, Positive Medical Interaction, and Negative Team Interaction forming Relatedness. Raw data themes, higher order themes and general dimensions for this phase are presented in Fig. We identified 25 raw data themes in the late limited participation phase, which we determined into the higher order themes: Increased autonomy in the rehabilitation was experienced by all players during the late limited participation phase, with elevations in both self-regulation and locus of control ; “I’ve suffered what I thought was a pretty big setback … I got some reassurance from the medical team. I was quite relieved. Now I am back on track, trying to do everything I can to help myself” (P4)). Again asking questions and bouncing ideas off the physiotherapist proved important to the rehabilitation. Player 3 noted, “I’m learning more and more about the injury and the best methods to rehabilitate. The medical team probably hate it because I’m asking so many questions”. This sense of control was further enhanced by the players having some input into the rehabilitation program. Comments from players included, “The step up in training is great and allowed for some variety. I got to choose whether I was on the bike or in the pool or whatever” (P1) and “I set some of my own training targets. Obviously the training staff oversees these and ensure they meet my needs, but I’m involved” (P2).Players 4 and 5 had a slightly different experience stating, “All [goals] set by the physio. I do have some input into the different exercises. Well that’s what they say, because I don’t think they take it into account” and “There’s no need for me to be involved in goal setting. [The physiotherapist] is a specialist and I trust him”. Although the lack of autonomy experienced by these players did not appear to negatively affect their emotions, both players spent the longest time in rehabilitation.Again competence was developed by seeing progressions in the rehabilitation and an increased confidence in the ability to achieve targets. The players in the present study with lower self-confidence stated they missed some training sessions or “didn’t give 100%”, while those with increased self-competence reported wanting to push themselves further (“I’ve done a few things that my physio shouldn’t know about to test my knee. I just needed that confidence in my own ability” (P2)). Players did comment that they were restricted by the ACL rehabilitation protocol ).Having a positive medical interaction was important for relatedness. Particularly the development of comradery between players within the rehabilitation environment was highlighted as facilitative (“Training with another player helps to keep me motivated. I don’t want to let him down” (P2); “Training as part of a rehab team is good. We constantly encourage each other and it boosts you’re confidence when one of the other guys is back playing. Although sometimes it’s a case of how long will I still be here for. Generally the support is really beneficial” (P3)). High average scores for all players on the MOS-SSS indicate the range of support available. Needing a sense of belonging encouraged three of the players to undertaken alternate work within the club (“to feel part of it again”). Player 1 became involved with video and game analysis, stating “It allowed me to feel part of the team, to be involved, even though I could not physically perform. I’m motivated by being involved, and not only does it take my mind off my knee, it allows me to help the team and even develop my own game”.A lack of relatedness had negative emotional impact on Player 5, who struggled initially with the change of coach at the club . However, as the new coach began to recognize him as part of the team, his perspective changed .Autonomy was formed by Positive Self-Regulation, Positive Locus of Control, and Negative Self-Regulation; Competence comprising of Positive Physical Ability, Positive Performance Ability, Negative Physical Ability, and Negative Performance Ability; and Relatedness including Positive Team Interaction and Positive Medical Interaction. Raw data themes, higher order themes and general dimensions for this phase are presented in Fig. Within the return to play phase we identified 24 raw data themes that we combined into nine higher order themes. A positive locus of control was experienced by all players during this phase, with none stating that they experienced any pressure to return to play (“[Coach] been good. He’s just spoken to me about being fully prepared to get back out there” (P1)). By having minimal pressure to return to play appears to greatly enhance each player’s confidence to return and reduce anxieties. All players noted the benefit of having goals set for their return ; “Having targets for my involvement meant I wasn’t concentrating on my knee, just trying to do what I needed to” (P3)). The goals set ranged from playing specific time periods to needing to be involved in play a set number of times. The only reported reduction in autonomy came from player 4 who reported being warned by his coach for trying to do too much and not concentrating on the game plan.Competence was developed by physical and performance achievements during this phase. All players stated they had confidence in their knee ; “I’ve been lifting some big weights in the gym … It’s just given me confidence. I know my knee’s good enough to go, so I don’t have to worry. I’d probably be shying out of tackles if I wasn’t 100%. I’ve just taken control of what I’m doing” (P1)). Greater concerns were related to a lack of physical fitness and overall performance (“I’m still nervous about how my fitness will hold up” (P1); “I am very excited about the prospect of playing a game. I’m nervous about how I will perform rather than how my knee will be” (P2)). With game time and by utilizing previous performance routines, each player was able to develop their physical competence enabling a successful return to play, at previous performance standards (“The more I played the less I focused on the knee” (P3)). Players 3 and 4 both stated they tried to protect their injured knee, by using some form of strapping or protective sleeve, and both had concerns about contact situations. This could be a predisposed trait or may be the influence of having less prior injury experience.Relatedness within the return to play phase was positive for these players (“The coaches and the boys were excellent. The team atmosphere was good and I knew this is where I was supposed to be” (P1)). The physiotherapist was also a valuable source of support during the return to play phase. Prior to competition the physiotherapist reassured each player that their injured knee was strong, and post-game they ensured each player cared for their injured knee correctly ; “[physiotherapist] came straight up to me after the first game to check how my knee was. He told me to make sure that I iced it to stop any soreness” (P3)).The aim of the present study was to explore the views of professional rugby union players during the early rehabilitation, late rehabilitation and return to play stages, following ACL injury. The discussions are presented to support and enhance critical understanding of the psychological factors associated with return to sport following ACL injury, and are provided to compliment the physical rehabilitation process. The results are discussed independently for the each of the three phases identified.Within general health psychology increased personal control has led to more adaptive coping responses being utilized  and gathIncreases in physical competence were achieved by the injured players through achievement of training targets and perceived improvement in performance. These increases in confidence could lead to better rehabilitation  by focusAthletes with serious injury are more likely to seek social support  and deveHealth psychology literature has identified asking questions as an active coping method, which increases perceived control of the rehabilitation process . SimilarHaving realistic short-term goals can develop physical and performance competence , with thRelatedness is enhanced by the involvement of the injured player with significant others  and consAll players in the present study stated they sensed no pressure to return and felt that they had control over the rehabilitation process, which may have facilitated a positive return experience. Gagné, Ryan and Bergmann  acknowlePhysical competence is an important concern when returning to play  with allWithin the literature sports medicine practitioners are encouraged to provide players returning to play with the required level of relatedness . In addiThe generalization of these findings is limited by the small sample size, a characteristic that is common within qualitative research in general, however five participants is consistent with similar qualitative research in this area , 45. StiDeveloping autonomy, competence and relatedness during rehabilitation appears to facilitate effective return to competition. Self-determination can produce more adaptive coping strategies and increase adherence to the process . Specifi


Based largely on retrospective analyses and a handful of prospective case reports, pharmacological inhibition of the beta adrenergic receptors using beta blockers has shown clinical anti-cancer efficacy in reproductive cancers, as well as angiosarcoma and multiple myeloma. Because of the potential promise of beta blockers as an adjunct to standard anti-cancer therapy, it is imperative to identify other tumor types expressing beta adrenergic (β-AR) receptors so future preclinical and clinical studies can be directed at the most promising tumor targets. We performed immunohistochemical detection of β1-AR, β2-AR, and β3-AR across 29 of the most common human cancer types  and 19 matching non-diseased controls . Our analysis revealed all three β-AR receptors were expressed most strongly in melanoma relative to other cancer types. Other malignancies that revealed relatively higher levels of β-AR receptors were esophagus, pancreas, kidney, and lung cancers. Moreover, particular β-AR receptors exhibited significant overexpression in tumor tissue relative to their matching normal tissue in urogenital/reproductive malignancies including breast, endometrium, ovarian, and urothelial cancer, as well as colon, lung, and thyroid cancer. This study identifies several cancer types expressing the β-AR receptors which should be evaluated in future studies for susceptibility to beta blockade. Beta adrenergic (β-AR) receptors are G-protein coupled receptors responsible for mediating vasodilation following stimulation by the catecholamines epinephrine and norepinephrine , 2. WhenThree different β-AR receptors have been identified in humans, and are widely expressed in vascular tissues –5. β-AR1The β-AR signaling pathway drives the pathogenesis of the benign vascular tumor infantile hemangioma , 11 and To accomplish this, we determined the expression of β-AR1, β-AR2, and β-AR3 receptors across 29 tumor types representing the most common cancers found in humans and 19 matching normal tissues using high density tissue microarrays. The knowledge gained from this study will allow researchers and clinicians to efficiently target the most promising cancer types that express the β-AR receptors.www.gtexportal.org), BioGPS (www.biogps.org), and the Human Protein Atlas (www.proteinatlas.org) reveal that β-AR mRNA and protein are surprisingly expressed at variable levels across a large number of normal and diseased tissues.Numerous studies have examined the expression of the β-AR receptors primarily in the heart, lung, and vascular tissues due to the effects of beta blockers on these tissues . InteresWe analyzed the expression of β-AR proteins across 19 normal tissues comprising epithelial, mesenchymal, and hematopoietic lineages Figure ; β2-AR wWithin cancer tissues, β1-AR was detected most highly in pancreas adenocarcinoma, melanoma, lung adenocarcinoma, clear cell carcinoma, and esophagus adenocarcinoma Figure . β2-AR wTo evaluate if the β-AR receptors are overexpressed in any of the cancers analyzed in this study, we performed the Mann-Whitney rank sum test to compare IHC scores between normal and cancer tissues. Using a very strict statistical cutoff p<0.01), we identified a number of cancers that exhibited overexpression for individual β-AR receptors  was performed on a clinically annotated tissue microarray (US Biomax #MC5003b) containing formalin-fixed paraffin-embedded tumor (N=389) and normal (N=100) tissues from human patients. Hematoxylin and eosin staining was used to confirm the classification of all tumors. The clinicopathological features of these tissues are shown in Table Expression of the three β-AR receptors was evaluated using IHC for each tissue section. The primary antibodies used were anti-β1-AR , β2-AR , or β3-AR  under conditions previously reported . The β-A


The debilitating effects on hand function from a number of a neurologic disorders has given rise to the development of rehabilitative robotic devices aimed at restoring hand function in these patients. To combat the shortcomings of previous traditional robotics, soft robotics are rapidly emerging as an alternative due to their inherent safety, less complex designs, and increased potential for portability and efficacy. While several groups have begun designing devices, there are few devices that have progressed enough to provide clinical evidence of their design’s therapeutic abilities. Therefore, a global review of devices that have been previously attempted could facilitate the development of new and improved devices in the next step towards obtaining clinical proof of the rehabilitative effects of soft robotics in hand dysfunction.A literature search was performed in SportDiscus, Pubmed, Scopus, and Web of Science for articles related to the design of soft robotic devices for hand rehabilitation. A framework of the key design elements of the devices was developed to ease the comparison of the various approaches to building them. This framework includes an analysis of the trends in portability, safety features, user intent detection methods, actuation systems, total DOF, number of independent actuators, device weight, evaluation metrics, and modes of rehabilitation.In this study, a total of 62 articles representing 44 unique devices were identified and summarized according to the framework we developed to compare different design aspects. By far, the most common type of device was that which used a pneumatic actuator to guide finger flexion/extension. However, the remainder of our framework elements yielded more heterogeneous results. Consequently, those results are summarized and the advantages and disadvantages of many design choices as well as their rationales were highlighted.The past 3 years has seen a rapid increase in the development of soft robotic devices for hand rehabilitative applications. These mostly preclinical research prototypes display a wide range of technical solutions which have been highlighted in the framework developed in this analysis. More work needs to be done in actuator design, safety, and implementation in order for these devices to progress to clinical trials. It is our goal that this review will guide future developers through the various design considerations in order to develop better devices for patients with hand impairments. Imagine tying your shoes or putting on a pair of pants while having limited use of your hands. Now imagine the impact on your daily life if that limitation was permanent. The ability to perform activities of daily living (ADL) is highly dependent on hand function, leaving those suffering with hand impairments less capable of executing ADLs and with a reduced quality of life. Unfortunately, the hand is often the last part of the body to receive rehabilitation.According to a 2015 National Health Interview Survey, there were approximately 4.7 million adults in the United States that found it “Very difficult to or cannot grasp or handle small objects” . AdditioIt is thought that the benefits of a robotic device for rehabilitation purposes include: more intense and longer therapy sessions, feedback mechanisms to amplify movements, automated sessions to reduce therapy hours, automation of patient-specific therapy based on degree of motor impairment, and more precise measurements of motor function . DespiteIn contrast, soft robotics are fabricated from easily deformable materials such as fluids, gels, and soft polymers that have better biomimetic qualities due to their increased compliance and versatility while conforming to the contours of the human body. The lack of rigid components removes constraints on non-actuated degrees of freedom and also reduces joint alignment issues, which could prevent joint damage . AdditioSoft robotic devices have been developed for rehabilitative applications for most major joints of the body, including the ankle, knees, shoulder, elbows, and wrists –14. TherThis narrative review was conducted by performing a literature search in SportDiscus, Pubmed, Scopus, and Web of Science using their respective controlled vocabularies for terms related to soft robotics and rehabilitation of the hand. Appropriate syntax using Boolean operators and wildcard symbols was used for each database to include a wider range of articles that may have used alternate spelling or synonyms Table .Table 2DAfter obtaining primary search results from each database, the following inclusion and exclusion criteria were used to further narrow the literature search:The device described was a soft robot: No rigid components on the robot-human interface or minimal rigid components that will not impose physical restraints on joint motionsThe device intended to facilitate the movement of at least one finger joint in the handThe paper was a scientific article written in the English language and accessible to the authorsInclusion criteria:The device contained rigid components on the robot-human interface that could reasonably impose physical restraints on joint motionsThe device focused on other joints without including the fingersThere was insufficient information about the device’s design such that analysis was not clearThe device was intended for use as a prostheticExclusion criteria:While there are a number of devices aimed at rehabilitation of other major joints of the body, they were not included in this review because the kinematics of the hand are complex and as such the design considerations are more unique. Additionally, prosthetic devices which replace human anatomy were deemed to have design considerations that differ from the focus on rehabilitation and so were similarly excluded. Finally, after narrowing our search results, the reference sections of each article were also screened for any devices that fit the search criteria but were not identified previously. These references were the final source of devices for this review.In order to guide the analysis of a relatively large number of devices, a general framework was developed Fig.  that conControl Unit and the Wearable Orthosis. Beginning with the Control Unit, there are many design considerations that go into developing this part of the device and they can be quite complicated with subtleties that are difficult to summarize across many devices. Therefore, the analysis of the Control Unit focuses on a few essential characteristics which will help these devices advance to the next steps in development. These characteristics include portability, safety mechanisms, and user intent detection modalities. To further clarify, portability in this context is defined as the ability for the device to be used in a patient’s home without the aid of a clinician or technician. This is because home rehabilitation has been shown to be beneficial for functional and psychological performance [Control Unit to power down the device, is important for these robots in order to provide the patient with some form of protection should the device begin to malfunction. Finally, the user intent detection modality is any signal that monitors the patient’s attempt to move their hand and is sent to the device’s Control Unit to augment that movement. Not only does this feedback control facilitate neuroplasticity in the rehabilitation process, but it can also allow for objective evaluation of robot motion and clinical outcome recovery. Thus, a comparison between the different modalities could drive future rehabilitation strategies.The first part of the framework which deals with the design of these devices is further broken down into two basic functional units: the formance  and woulWearable Orthosis, which is considered the minimal equipment that must be in contact with the hand in order to operate. This part of the discussion was divided into two related parts: the actuation system and the human-robot interface. The actuation system is defined as the mechanical system in place that is used to guide finger joint movement. The various approaches to designing these systems have inherent advantages and disadvantages that need to be understood as they directly influence the human-robot interface. This interface will cover each device’s total degrees of freedom (DOF), number of independently controlled actuators, and weight of the hand orthosis. For these devices, the DOF will be counted as 1 DOF for flexion/extension of each of the distal interphalangeal joints, proximal interphalangeal joints, and metacarpophalangeal joints. The thumb will be counted as 1 DOF each for flexion/extension of the interphalangeal joint, metacarpophalangeal joint, and carpometacarpal joint. Abduction/adduction of joints will also be counted when mentioned in their design. The weight of the device will include only the portion of the device that is worn on the hand and should weigh less than 0.5 kg [The other half of this framework concerned with device design is the n 0.5 kg  in orderInteraction with the Environment. The first half of this section focuses on the different metrics that have been used to evaluate these devices. All of these devices are uniquely built and have been evaluated in many ways, but there is no real standard by which to compare them. Therefore, an analysis of the studies that have been completed can help guide future developers in their experiments by understanding the most commonly used metrics. This would facilitate an ease of comparison between these devices and provide a way to monitor the progression of these devices. The second half of this section is dedicated to observing the trends in the types of rehabilitation these devices intend to implement. This includes Active Resistance (AR), Continuous Passive Motion (CPM), Task Specific Training (TST), and Virtual Reality (VR). A glossary of these terms can be seen in Table The final component of this framework is focused on these devices’ While this framework does not address the exact technology to build a soft robotic device, its utility lies in its compartmentalization of various aspects of device design which facilitates an ease of comparison between the different approaches to building these devices.The final search queries for this review were completed on November 10th, 2017. A total of 62 articles, representing 44 unique devices were identified for this overview. A summary of the article selection procedure can be seen in Fig. A summary of all the devices used for this analysis, grouped according to their type of actuation, is shown in Table The data are grouped according to their type of actuation in order to ease their comparison. The types of actuators identified include: Cable systems, Pneumatic systems, and Hydraulic systems. A distribution of the prevalence of each type of actuator is seen in Fig. Twenty out of the 44 (45%) devices analyzed were designed with portability in mind. Of these portable devices, 85% were published in the last 3 years which shows that this trend towards home rehabilitation is a recent one. Given the multitude of benefits potentially attainable through home rehabilitation, we expect that there will be an increasing trend towards developing devices that are fully capable of being operated in a patient’s own home without the need of a clinician or technician. In addition to portability, several devices were observed to be mobile due to compact control units that were battery powered. To clarify, a portable device was not necessarily mobile since some portable devices were powered by wall outlets but could still be used in a patient’s home. Mobility enabled users to power the device while freely moving about their environment, which permitted more assistance with their ADL’s compared to if they were tethered to a wall outlet. This allowed devices to have further applications as assistive devices. Currently, however, it is unclear what the benefit having this mobility grants in terms of rehabilitative success and so it should be elucidated to determine whether it is a desirable trait in these rehabilitative devices. On the other hand, it is much clearer how portability could influence outcomes and should be strived for in as many devices as possible for future evaluations to validate those claims.In terms of safety measures, only 12 of the 44 devices (27%) had implemented clear forms of safety mechanisms, which was lower than expected given the importance of safety in robotics. Additional devices may have safety precautions built into their device’s control algorithms, but the details of those algorithms were beyond the scope of this analysis. Instead, this discussion focuses on those additional safety measures that have been implemented and reported in the literature. A description of each safety mechanism as well as their foreseeable advantages and disadvantages are shown in Table The ability to detect a user’s intent to engage hand movement is a major advantage these robotic devices have over traditional rehabilitation yet currently, only about 30% 13/44) of the devices reviewed reported this functionality. A distribution of the feedback modalities that have been investigated is shown in Fig. 4 of the A distribution of the total DOF per device in this review may be seen in Fig. In terms of the weight of the wearable portion of these devices, all devices with reported weights fell within the 0.5 kg requirement previously established. One exception included the RoboGlove, however, since this device was not designed as a rehabilitative device, it was excluded in this section. Groups that did not report a weight likely were early in development and still focusing on actuator design and did not have a wearable prototype of their systems yet. A comparison of the average weight of each of the categories of devices can be seen in Fig. An index of the various metrics observed with brief descriptions is seen in Table Many of the devices used in this analysis were developed primarily as assistive devices or still early in development as rehabilitative devices and there was little quantitative data to report. However, there is still a general trend in the direction of modes of rehabilitation these devices intend to implement. Currently, Task Specific Training (TST) was the most common form discussed as it aims to allow patients to begin recovering function in their hand. TST is often one of the first rehabilitation exercises and is likely to continue to be a staple of robotic rehabilitation. The next most common training modality was Continuous Passive Motion (CPM), which is likely to continue to rise in popularity because of its ease of implementation and functional efficacy. CPM is focused on improving range of motion and recovering strength, a logical next step in the rehabilitative process. Finally, Active Resistance (AR) was the least common exercise implemented, probably because it represents one of the final stages of rehabilitation: strength training. As more devices progress to their evaluation phases, AR exercises are expected to increase in popularity. Ideally, these robotic devices should incorporate all of these exercises in order to have a more complete rehabilitation regimen. This need for an all inclusive rehabilitation program has likely partially inspired the development of Virtual Reality (VR) systems , 20, whiSince the emergence of soft robotic devices for hand rehabilitation roughly 10 years ago, the field has progressed rapidly. Significant progress has been made in establishing proof-of-concept of designs of preclinical research prototypes, with clinical trials being the next logical goal. But for many devices, more work needs to be done to perfect actuator design and feedback to maximize patient safety and rehabilitation outcomes.Control Unit, we have seen only a number of devices beginning to consider portability in mind, but we expect this to steadily increase as the field advances due to the perceived benefits of at home rehabilitation. Likewise, safety did not appear to be a major concern for many devices, but as more devices move to testing on human subjects, we expect it to become more relevant. This analysis may serve as a reference for those looking to incorporate safety features (or any other elements of our framework) into their devices. We hope that it may also serve as a guide for the different feedback mechanisms that have been implemented, which are gaining ground as ways of facilitating rehabilitation that are unique to robotics. On our conceptualized Wearable Orthosis, we have noticed a trend towards the production of Pneumatic Systems as well as devices that strive for increased total DOF and number of independent actuators. For the latter, this may allow for the ability to assist specific hand motions. This section also included a brief investigation of the weight of these devices, which were well within the limits for all devices presented. Additionally, our framework includes a discussion of the devices’ Interaction with Environment. This includes an index of some of the more common or more relevant metrics that have been used to evaluate these devices, which we hope may guide future experimentation and facilitate an ease of comparison between the devices’ functionality. We also include a brief discussion on the roles of the different modes of rehabilitation that have been considered in these devices, which we hope future developers will consider as they move onto clinical trials.A framework was developed for comparison of these devices based on critical features of the devices and the availability of these details in the literature. Beginning with the The list of design criteria that was analyzed is far from exhaustive in terms of all of the considerations needed. Nevertheless, different solutions to developing these devices were presented by highlighting key design features as well as some of their advantages and disadvantages. We hope that this review of the current approaches in designing soft robotic devices for hand rehabilitation will serve as a useful resource for future developers and facilitate the evolution of the field.


Targeting the activation function-1 (AF-1) domain located in the N-terminus of the androgen receptor (AR) is an attractive therapeutic alternative to the current approaches to inhibit AR action in prostate cancer (PCa). Here we show that the AR AF-1 is bound by the cochaperone Bag-1L. Mutations in the AR interaction domain or loss of Bag-1L abrogate AR signaling and reduce PCa growth. Clinically, Bag-1L protein levels increase with progression to castration-resistant PCa (CRPC) and high levels of Bag-1L in primary PCa associate with a reduced clinical benefit from abiraterone when these tumors progress. Intriguingly, residues in Bag-1L important for its interaction with the AR AF-1 are within a potentially druggable pocket, implicating Bag-1L as a potential therapeutic target in PCa. Prostate cancer is the second most common cancer in men around the world. The cancer relies on a protein called the androgen receptor in order to develop and grow. Currently, some of the most common treatments for prostate cancer, especially in its advanced stages, are drugs that block the activity of this receptor. However, such treatments are only successful for a limited period of time, and so alternative methods to inhibit this receptor are still needed.The androgen receptor must bind to a number of proteins to carry out its activity. These proteins include one called Bag-1L, which is also important for the development of prostate cancer. Stopping such a protein from binding with the androgen receptor might represent a new way to treat prostate cancer; but first it will be important to understand how this interaction actually regulates the activity of the receptor.Now, Cato et al. have analyzed samples of cancer cells that had been collected from 43 patients with prostate cancer and found that Bag-1L levels increase as the disease progresses. Looking at the patients’ medical records then revealed that therapies targeting the androgen receptor were less effective in people with high levels of Bag-1L. Conversely, altering, removing or inhibiting Bag-1L in prostate cancer cells grown in the laboratory made the receptor less active and made the cells grow slower.Further experiments went on to reveal that Bag-1L interacts with a regulatory region of the androgen receptor. Cato et al. note that this region remains largely unexplored therapeutically, because it has some unique structural properties that restrict how much it can interact with drug molecules. Targeting Bag-1L and stopping it from binding to this region of the androgen receptor would represent a different approach to inhibiting the androgen receptor and treating patients with prostate cancer. Together these new findings should provide pharmaceutical companies with much of the information they would require to immediately start screening for therapies that target Bag-1L. Ultimately, Cato et al. hope that any follow-up findings will benefit prostate cancer patients by improving the currently available treatments. The androgen receptor (AR) plays a central role in the development of prostate cancer (PCa). Current approaches aimed at reducing persistent AR signaling either inhibit the production of androgens, or compete with endogenous ligands for binding to the AR C-terminal ligand-binding domain (LBD) . While tBag-1 (Bcl-2-associated athanogene-1) is a multifunctional protein involved in a number of key cellular processes including proliferation, differentiation, cell cycle, transcription and apoptosis . Its maiHere we show that the conserved BAG domain within the C-terminus of Bag-1L selectively interacts with the intrinsically disordered, but partially folded N-terminal AR tau-5 domain. Disrupting this interaction by knocking out or mutating the BAG domain of Bag-1L alters the structural properties of the AR NTD and receptor folding, and reduces the ability of AR to bind to chromatin and regulate transcription.Bag-1L has been reported to upregulate the activity of AR . HoweverSince LNCaP cell growth is primarily driven by AR activity, we next tested whether Bag-1L has any effect on the AR cistrome or transcriptome. We performed ChIP-seq in hormone-depleted control, Bag-1L KO and the rescue LNCaP cell lines, treated for 4 hr with vehicle (ETOH) or 10 nM DHT. We observed only limited binding of AR in response to vehicle treatment, regardless of the presence or absence of Bag-1L . This is−31; q = 1.7×10−29)  and direct AR target genes were defined as genes with DHT-induced differential expression (p(FDR)≤0.05, fold change ≥1.5) that harbor a DHT-responsive AR binding site within 50 kb of their transcription start site (TSS). Using this approach, we determined 599 direct AR target genes in the control and 306 in the Bag-1L KO cells . Using t7×10−29) , supportOne of the consequences of the loss of a chaperone or cochaperone is an alteration of the folding properties of its client proteins . The impWe have previously shown that Bag-1L enhances AR transactivation via direct interaction of the two proteins . We demoTo test if mutations in the BAG domain would disrupt the integrity of associated biochemical complexes, we next employed quantitative, stable isotope labeling with amino acids in cell culture (SILAC) combined with rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME)  of LNCaP13C correlation nuclear magnetic resonance (NMR) spectra to compare Cα and Cβ shifts  could indeed function as a substitute for Bag-1L inhibition or Bag-1L loss, we measured cell growth of Bag-1L KO cells stably expressing an empty vector control, or the wild-type or CMut Bag-1L . Equal pGiven our hypothesis that Bag-1L is a driver of castration resistance, we next investigated if Bag-1 (and AR) expression levels change with PCa progression. H-scores were determined by immunohistochemistry (IHC) of matched diagnostic  hormone-sensitive PCa (HSPC) and CRPC biopsies  of 43 paGiven our finding that Bag-1L is a key regulator of AR action , we nextBag-1L is a known regulator of AR action implicated in PCa progression, but its mechanism of action is poorly understood. We show here that Bag-1L is essential for AR transactivation and function, and PCa growth. This function is mediated through the direct interaction of Bag-1L and the AR. Mutations that disrupt this interaction, inhibition of the Bag-1L:AR interaction or loss of Bag-1L altogether, alter the structural properties of the receptor and result in changes in the AR cistrome and transcriptome . We showHuman Bag-1L is one of four polypeptides translated from a single mRNA by a leaky scanning mechanism  and as aWe have previously reported that Bag-1L binds the AR through a novel GARRPR motif, found at its N-terminus, and a BF-3 pocket at the AR LBD . HoweverAberrant expression of Bag-1 has been described in a variety of human malignancies, such as breast, lung, cervical, colorectal and hepatocellular carcinoma . Here weIn conclusion, we demonstrate here the importance of Bag-1L for AR activity and function in PCa. Combined, our data support targeting the BAG-1L BAG domain:AR tau-5 interaction therapeutically in the treatment of PCa and CRPC.TALEN Bag-1L KO and vector controls were created as described . In brieAll animal experiments were performed per European and German statutory regulations. Animal protocols were approved by the ‘Regierungspräsidium’ Karlsruhe, Germany (AZ 35–9185.81/G-43/14 ‘Bag-1L-Prostastakarzinomprojekt’). LNCaP xenograft tumor studies were carried out as previously described for intact  or castrPatients were identified from a population of men with metastatic CRPC treated at the Royal Marsden NHS Foundation Trust. All study participants had given written, informed consent and were enrolled in institutional protocols approved by a multicenter research ethics committee . Forty-three patients with sufficient formalin-fixed, paraffin embedded (FFPE), matched diagnostic  HSPC and CRPC tissue were included in our study. HSPC tissue demonstrated adenocarcinoma and was obtained from either prostate needle biopsy (35), transurethral resection of the prostate , prostatectomy (4) or bone biopsy (1). CRPC tissue was obtained from the same patients through biopsies of bone (25), lymph node (10), liver (5), prostate , bladder (1) or chest wall (1). All tissue blocks were freshly sectioned and only considered for IHC analyses if adequate material was present (≥50 tumor cells). For Bag-1 IHC , HSPC and CRPC FFPE biopsies were first deparafinised, followed by antigen retrieval (microwaving in citrate buffer (pH 6.0) for 18 min at 800 W). The Bag-1 antibody was diluted (1:250) in Dako REAL diluent  and tissue was incubated for 1 hr. After washes, the bound antibody was visualized using the Dako REAL EnVision Detection System (Agilent Technologies). Sections were counterstained with hematoxylin. AR protein expression was determined using the AR mouse monoclonal antibody , as previously described . Nuclear2, 0.2 mM EDTA, 25% Glycerol, 1 mM DTT, protease inhibitors) for total, cytoplasmic and nuclear proteins respectively. Western blotting was carried out using standard protocols with following antibodies: Bag-1 , Bag-1L  and were sequenced on the Illumina NextSeq 500 platform at the Molecular Biology Core Facility (Dana-Farber Cancer Institute). ChIP-seq data was processed using ChiLin2 . All ChITotal RNA was extracted from cells using innuPREP RNA Mini , following the manufacturer’s instruction. mRNA libraries were generated using the Illumina TruSeq stranded mRNA sample kit and 1 µg of total RNA per sample. Library preparation, sequencing on a HiSeq1500 Illumina platform, and data analysis were carried out at the NGS facility of the Institute of Toxicology and Genetics (KIT). Fastq files were processed with CASAVA and mapped against the human reference genome GRCh37 using TopHat 2.0.11 . Reads wcorr. FRET= IFRET – 0.17 ICFP. Nuclear regions were identified manually based on YFP staining. Acquired images were analyzed using ImageJ (Cells were transiently transfected using Lipofectin (Thermo Fisher Scientific) with plasmid CFP-AR-YFP , generoug ImageJ .Short overlapping peptides (21 amino acids each) spanning the entire human Bag-1L BAG domain and its flanking regions were synthesized and spotted in duplicates onto amino-PEG cellulose membranes  using an automated SPOT synthesizer  . The memGST pull-down was performed as previously described . Mammali13C6L-lysine HCl, 13C615N4L-arginine-HCl) and mixed at a 1:1 ratio (30 million cells per treatment arm) prior to IP. Proteins were immunoprecipitated using 20 μg anti HA-tag (Abcam) or rabbit IgG (Santa Cruz Biotechnology) antibody-coupled Dynabeads (Thermo Fisher Scientific). Immunoprecipitated proteins were digested directly on beads as described (SILAC-labeled rapid IP-mass spectrometry of endogenous protein (RIME) was carried out essentially as previously described . Bag-1L escribed . Recoverescribed . After tescribed  (30 um Iescribed . The masescribed  against 15N-labeled thrombin protease-cleavable GST-fused BAG domain (wild-type or K231/232/279A mutant) was cloned into pGEX-6P vector  and expressed in E. coli strain BL21 (DE3), grown in M9 minimal medium supplemented with 0.5 g/l 15NH4Cl. The 15N-BAG domains were cleaved off by rhinovirus 3C protease . To remove residual GST, the protein solution was purified using a glutathione sepharose column and subjected to size exclusion chromatography . 15N-HSQC spectra for wild-type and mutant BAG domains (at approximate 500 μM) and standard triple resonance backbone experiments NH) for peak assignment were acquired at 23° C on a Bruker Avance I 600 spectrometer. The spectrometer was equipped with a broadband triple resonance probe head with 4 scans per increment and a total of 128 increments in the indirect dimension. For chemical shift calibration and to compare relative signal intensities, 0.2 mM DSS  was added. Data were processed with NMRPipe , at a 1:5 molar ratio with GST (as a control) or with unlabeled GST-BAG (wild-type or mutant) were acquired at 278K on a Bruker 800 MHz spectrometer. Data was processed using NMRPipe and NMRDraw, and analyzed using CcpNmr Analysis .Thank you for submitting your article "Transactivation by the intrinsically disordered androgen receptor N-terminal domain is enabled by the cochaperone Bag-1L" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a response to the critiques, which are quite significant.Summary:The manuscript by Cato et al. provides an analysis of the interaction of the androgen receptor (AR) transactivation domain (AF-1) with the chaperone BAG1L, which is demonstrated to be required for AR's full activity. As such, BAG1L may play a role in castrate-resistant prostate cancer (CRPC). The authors document that BAG1L is necessary for proper conformation of AR through FRET reporters and for DNA binding of AR to target sites. Using a peptide screen and mutational analysis a BAG1L domain required for docking with AR was identified. Further structural analysis and reconstitution of BAG1L KO cells with WT or mutant BAG1L demonstrated the necessity of this interaction domain of BAG1L or AR function in cells. Structural analysis further uncover a potentially druggable BAG1L pocket flanking this interaction domain. Key findings are supported by data to demonstrate: that loss of Bag-1L inhibits AR activity (as monitored by transcriptome profiling and ChIP-seq), prostate cancer growth using hormone-dependent model systems; that the Bag-1L/AR interaction is mediated by discrete domains in Bag-1L within a potentially targetable Bag domain; that Bag-1L enhances prostate cancer growth; and that in clinical specimens, nuclear Bag-1L levels increase during disease progression, predicting a reduced clinical benefit from the ligand-targeting agent abiraterone.Essential revisions:1) The concept that targeting Bag-1L to suppress AR activity and prostate cancer growth is novel, but additional experimentation that would enhance confidence in these findings would be to assess the Bag-1L requirement in additional model systems. The present study largely utilized a single model of hormone-therapy sensitive disease (LNCaP). Demonstration that the Bag-1L is maintained in other models, especially in castrate-resistant disease, should be included.2) The model in 3) The authors take advantage of previous AR-NTD assignments to demonstrate clear loss of signal in tau-5 of AR  that is 4) The identification of the triple mutant (CMut) has experimental utility, however it does not provide information about the actual BAG-AR interaction. As the substantial changes in the NMR make clear  and the 5) Bag-1L has multiple functions outside AR regulation that may contribute to the anti-tumor effects observed in the xenograft studies. Investigation of the impact of Bag-1L alteration in the xenograft tumors should be explored . Given the role of Bag-1L as a cochaperone, and nucleotide exchange factor for Hsp70, it would be important to determine the role of Hsp70 in the observed effects. Essential revisions:1) The concept that targeting Bag-1L to suppress AR activity and prostate cancer growth is novel, but additional experimentation that would enhance confidence in these findings would be to assess the Bag-1L requirement in additional model systems. The present study largely utilized a single model of hormone-therapy sensitive disease (LNCaP). Demonstration that the Bag-1L is maintained in other models, especially in castrate-resistant disease, should be included.We have investigated the function of Bag-1L in LNCaP95 and LNCaP-abl cells, two castrate-resistant prostate cancer (CRPC) models derived from the hormone-sensitive LNCaP cell line. Overexpression of wild-type Bag-1L did not affect CRPC cell line growth, presumably due to endogenously elevated levels of Bag-1L in these lines compared to the parental LNCaP cells. However, we observed a dominant negative effect on cell growth when overexpressing the Bag-1L CMut, which is defective in AR interaction. This result is in agreement with findings in LNCaP cells and suggests that Bag-1L remains important for AR function at CRPC status. The described data has been included in 2) The model in The novel findings in this study are (1) the discovery of specific residues in the BAG domain of BAG-1L required for the interaction with the AR tau-5 domain; (2) the impact of Bag-1L on the AR cistrome; (3) demonstration that the effect of Bag-1L on AR function is through a change in AR conformation; (4) comprehensive analysis of the Bag-1L interactome and its alterations in response to BAG domain mutation; (5) identification of a potential druggable pocket in Bag-1L. In addition, in the revised manuscript we have included data utilizing a tool Bag-1 inhibitor that prevents the Bag-1L:AR interaction. We agree with the reviewers that the present title does not highlight the novelty of the present manuscript. To address this concern, we have changed the title of the current manuscript to: “Development of Bag-1L as a Therapeutic Target in Androgen Receptor-Dependent Prostate Cancer”. Moreover, we have updated the model  and have3) The authors take advantage of previous AR-NTD assignments to demonstrate clear loss of signal in tau-5 of AR  that is We agree with the reviewers that we cannot confirm that the interaction between the Bag-domain and AR tau-5 occurs in a specific conformation. Although we observe a decrease of about 50% intensity in the resonances of AR (between residues 375 and 448) in the presence of Bag-1L, no chemical shifts changes were observed. Based on this data, we conclude that the AR tau-5 domain is the main interaction site, because the decreases in residue intensities that we observe in the rest of the AR-AF-1 domain are smaller. As previously shown by our characterization of the structural properties of AR-AF-1, the region of sequence that is involved in the interaction with Bag-1L has substantial secondary structure when unbound. We think that it is plausible that these structural properties play a role in the described interaction with Bag-1L, but since we did not measure the chemical shifts of the bound state of AR, we cannot substantiate this claim. For this reason, we agree with the reviewers that we should rephrase the sentence: “suggesting that wild-type BAG domain interacts with a specific conformation of this sub-domain”, which has now been updated in the manuscript . The proposed experiment of BAG domain NMR in response to the addition of AR tau-5 suggested by this reviewer may provide additional information on binding of these proteins, but these are technically very challenging studies due to the nature of the two proteins. We therefore propose to attempt these experiments in a separate study.In response to the comments regarding the use of tool compounds, we have now added some novel data on the function of the BAG domain inhibitor Thio-2 . We have4) The identification of the triple mutant (CMut) has experimental utility, however it does not provide information about the actual BAG-AR interaction. As the substantial changes in the NMR make clear  and the 13C correlation nuclear magnetic resonance (NMR) spectra to compare Cα and Cβ shifts  Bag-1L has multiple functions outside AR regulation that may contribute to the anti-tumor effects observed in the xenograft studies. Investigation of the impact of Bag-1L alteration in the xenograft tumors should be explored . Given the role of Bag-1L as a cochaperone, and nucleotide exchange factor for Hsp70, it would be important to determine the role of Hsp70 in the observed effects.We agree with the reviewers that Bag-1L has multiple functions outside AR regulation that may contribute to its anti-tumor effects in the xenograft studies. The many functions of Bag-1L originate mainly from its BAG domain, a protein binding surface for many cellular factors and signaling molecules. To explore the impact of Bag-1L alteration in AR signaling, apoptosis or proliferation, as suggested by the reviewers, we performed extensive in vitro mutagenesis studies of the BAG domain. This allowed us to separate the different function of Bag-1L and to specifically focus on its (Hsp70-independent) role on AR action. In addition to extensively testing the effect of Bag-1L alterations (through the study of CMut compared to wild-type Bag-1L) on AR-mediated cell growth in vitro and in xenograft studies , we haveOn the issue of Bag-1L functioning as a cochaperone of Hsp70, we have generated additional mutations, including a mutant that is defective in binding both AR and Hsp70 (D293A). Although such a mutation may implicate Hsp70 in the Bag-1L-mediated action on AR function, our main aim in this work was to identify mutations that would inhibit the BAG domain:AR interaction, independent of Hsp70. In line with recent findings of heat shock protein-independent functions of cochaperones , we believe Bag-1L has a unique ability in regulating AR tau-5 activity, independent on Hsp70. In this connection, it is important to note that the residues of the Bag-1L CMut protein, which disrupt the BAG domain:AR tau-5 interaction, lie within helices 1 and 2 and are therefore distinct from the Hsp70 interaction sites in helix 3 .