This newly synthesized nanocomposite was chosen to act as a green, efficient, and recyclable Lewis acid for the multicomponent synthesis of new derivatives of pyranochromene-diones through the reaction of aromatic aldehydes, indandione, and 4-hydroxycoumarin in water. All of the synthesized compounds are new and are recognized by FT-IR, NMR, and elemental analysis; this avenue is new and has advantages such as short reaction times, high productivity, economical synthesis, and use of green solvent, H2O, as a medium. The catalyst is magnetically recoverable and can be used after six runs without a decrease in the efficiency.Nanomagnetic dapsone-Cu supported on the silica-coated Fe Multicomponent reactions (MCRs) are useful avenue for the synthesis of organic compounds. This reaction is a combination of at least three components in a one-pot domino . MCRs haPyranochromenes have various biological activities such as antifungal , antibacMagnetic nanoparticles (MNPs) as catalysts are interesting materials because of their high surface area, unique magnetic properties, and high catalytic activity. They were used as magnetic fluids catalysis and magnetic resonance imaging (MRI) data storage and environmental remediation ; 2020d.To the best of our knowledge, there are no reports on the use of Fe3O4@SiO2-propyl-loaded dapsone-copper as a catalyst for the synthesis of pyranochromenes via multicomponent reactions of aldehydes, indandione, and 4-hydroxycoumarin results.The X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscope (SEM), thermogravimetric analysis (TGA), and zeta potential analysis for synthesized MNPs were analyzed on X-PRTPRO (Netherlands) XRD, TEM Jeol model 3,010, Philips XL 30 scanning electron microscope , Q600 (made in America), and ZEN3600 (England) instrument, respectively. Fourier transform infrared (FT-IR) spectra of organic compounds were carried out by Shimadzu Fourier transform infrared spectrophotometer . 1H and 13C NMR spectra were determined using a Bruker DRX Avance instrument at 500 or 300 and 125 or 75 MHz.3O4 were synthesized by Zare Fekri  were irradiated under ultrasonic bath for 30 min. Then, 20 ml aqueous solution of copper chloride (I)  was added to the Fe3O4@SiO2@dapsone and stirred for 48 h. The Fe3O4@SiO2@dapsone-Cu MNPs were filtered in the presence of a magnetic bar and washed using ethanol and water subsequently, to separate the nanoparticles.500 mg Fe3O4@SiO2@dapsone-Cu MNPs was stirred at room temperature in 10 ml distilled water for the required reaction time as indicated by TLC . After completion of the reaction, the resulting mixture was filtered in the presence of an efficient magnetic bar to separate the catalyst. The catalyst was washed with 10 ml ethanol and reused. The crude products were collected and dried.A mixture of aldehyde (1.0 mmol), indan-1,3-dione (2.0 mmol), 4-hydroxycoumarin (1 mmol), and 0.05 g Fe−1): 1739 (C=O str), 1701 (C=O str), 1,561 (Asymmetric NO2 str or aromatic C=C str), 1,510, 1,456 (aromatic C=C str), 1,348 (symmetric NO2 str), 1,257 (C-O str), 1,099.1H NMR : δH 5.31 , 6.84–6.88 , 7.00–7.04 , 7.18 , 7.23 , 7.31 , 7.35 , 7.55 , 7.85–7.95 , 8.06–8.14  ppm. 13C NMR : δC 57.50 , 111.60 (Ar), 113.45 (Ar), 117.89 (Ar), 121.98 , 123.56 (Ar), 123.98 (Ar), 125.01 (Ar), 126.23 (Ar), 127.09 (Ar), 131.56 (Ar), 131.87 (Ar), 132.33 (Ar), 137.89 (Ar), 133.98 (Ar), 139.09 , 141.23 (Ar), 145.81 (Ar), 149.87 (Ar), 198.76 (C=O), 201.17 (C=O)ppm. Anal. Calcd. for C25H13NO6: C, 70.92; H, 3.09; N, 3.31. Found: C, 70.93; H, 3.05; N, 3.32.7-(4-Nitrophenyl)-6H-indeno pyrano chromene-6,8(7H)-dione 4a: m. p. 203–205°C, FT-IR : 1736 (C=O str), 1700 (C=O str), 1,660, 1,580 (asymmetric NO2 or aromatic C=C str), 1,430 (aromatic C=C str), 1,330 (symmetric NO2 str), 1,275 (C-O str). 1H NMR : δH 5.33 ), 6.85 , 7.00 , 7.22 , 7.31 , 7.35 , 7.52 , 7.78 , 7.85–7.94 , 8.00–8.02 , 8.18  ppm. 13C NMR : δC 58.30 , 112.00 , 113.54 (Ar), 114.00 (Ar), 118.96 (Ar), 119.18 (Ar), 121.80 (Ar), 122.03 (Ar), 123.29 (Ar), 123.33 (Ar), 124.75 (Ar), 126.61 (Ar), 129.98 , 135.84 (Ar), 136.36 (Ar), 136.66 (Ar), 142.40 (Ar), 142.44 (Ar), 144.60 (Ar), 147.91 (Ar), 198.90 (C=O), 200.16 (C=O) ppm. Anal. Calcd. for C25H13NO6: C, 70.92; H, 3.09; N, 3.31. Found: C, 70.95; H, 3.07; N, 3.36.7-(3-Nitrophenyl)-6H-indeno pyrano chromene-6,8(7H)-dione 4b: m. p. 176–177°C, FT-IR : 1735 (C=O str), 1700 (C=O str), 1,650, 1,457 (aromatic C=C str), 1,215 (C-O str). 1H NMR : δH 5.13 , 6.85 , 7.01 , 7.13 , 7.17 , 7.31 , 7.35 , 7.84–7.93  ppm. 13C NMR : δC 58.48 , 111.91 (Ar), 114.32 (Ar), 118.95 (Ar), 119.00 (Ar), 119.99 (Ar), 121.60 (Ar), 123.25 (Ar), 124.76 (Ar), 126.71 , 131.06 (Ar), 131.21 (Ar), 131.27 (Ar), 136.44 (Ar), 136.54 (Ar), 136.58 (Ar), 141.35 (Ar), 142.38 (Ar), 142.61 (Ar), 144.32 (Ar), 197.56 (C=O), 200.26 (C=O) ppm. Anal. Calcd. for C25H13BrO4: C, 65.66; H, 2.87. Found: C, 65.65; H, 3.07; N, 2.86.7-(4-Bromophenyl)-6H-indeno pyrano chromene-6,8(7H)-dione 4c: m. p. 276–278°C, FT-IR : 1737 (C=O str), 1705 (C=O str), 1,650, 1,608 (aromatic C=C str), 1,538, 1,224 (C-O str). 1H NMR : δH 5.04 , 6.41–6.44 , 6.57 , 6.92 , 6.84 , 6.90 , 7.00–7.04 , 7.14 , 7.32 , 7.41 , 7.83–7.92 , 9.13  ppm. 13C NMR : δC 58.71 , 111.83 (Ar), 113.91 , 115.11 (Ar), 116.07 (Ar), 118.86 , 118.99 (Ar), 119.63 (Ar), 121.48 (Ar), 123.15 , 124.78 (Ar), 126.94 (Ar), 129.23 (Ar), 136.36 , 136.43 (Ar), 142.45 (Ar), 142.85 , 143.06 (Ar), 157.23 (Ar), 197.83 (C=O), 200.51 (C=O) ppm. Anal. Calcd. for C25H14O5: C, 76.14; H, 3.58. Found: C, 76.11; H, 3.07.7-(3-Hydroxyphenyl)-6H-indeno pyrano chromene-6,8(7H)-dione 4d: m. p. 289–290°C, FT-IR : 2,921 , 1735 (C=O str), 1701 (C=O str), 1,593 (aromatic C = c str), 1,488 (aromatic C=C str), 1,423, 1,344, 1,261 (C-O str). 1H NMR : δH 2.43 , 5.10 , 6.84 , 7.01 , 7.13 , 7.31 , 7.36 , 7.86–7.92  ppm. 13C NMR : δC 51.63 (SCH3), 58.75 , 112.98 (Ar), 113.93 , 114.01 (Ar), 115.08 (Ar), 116.81 (Ar), 117.23 (Ar), 119.53 (Ar), 120.56 (Ar), 122.05 , 124.89 (Ar), 125.34 (Ar), 125.87 (Ar), 134.21 (Ar), 136.42 (Ar), 143.21 , 145.17 (Ar), 158.23 (Ar), 196.23 (C=O), 201.01 (C=O) ppm. Anal. Calcd. for C26H16O4S: C, 73.57; H, 3.80. Found: C, 73.51; H, 3.87.7-(4-(Methylthio)phenyl)-6H-indeno pyrano chromene-6,8(7H)-dione 4e: m. p. 269–270°C, FT-IR : 3,034 (aromatic C-H str), 2,910 , 1743 (C=O str), 1703 (C=O str), 1,565 (aromatic C=C str), 1,425 (aromatic C=C str), 1,265 (C-O str), 1,168.1H NMR : δH 5.21 , 6.86 , 7.02 , 7.18–7.22 , 7.30 , 7.65 , 7.87–7.94 , 8.31  ppm. 13C NMR : δC 56.78 , 113.09 , 114.56 (Ar), 116.12 (Ar), 116.91 (Ar), 116.99 (Ar), 118.13 (Ar), 118.76 (Ar), 121.43 , 122.16 , 123.54 (Ar), 125.57 (Ar), 126.47 (Ar), 128.46 (Ar), 134.09 (Ar), 138.11 , 147.87 (Ar), 155.08 (Ar), 197.34 (C=O), 200.07 (C=O) ppm. Anal. Calcd. for C24H13NO4: C, 75.98; H, 3.45; N, 3.69. Found: C, 75.93; H, 3.43, N, 3.65.7-(Pyridin-3-yl)-6H-indeno pyrano chromene-6,8(7H)-dione 4f: m. p. >300°C, FT-IR : 3,098 (aromatic C-H str), 1735 (C=O str), 1702 (C=O str), 1,603 (aromatic C=C str), 1,408, 1,365, 1,323, 1,211 (C-O str). 1H NMR : δH 5.10 , 6.35–6.56 , 6.68 , 6.86 , 6.98–7.11 , 7.28 , 7.34  ppm. 13C NMR : δC 58.52 , 114.23 (Ar), 114.38 (Ar), 116.01 (Ar), 116.98 (Ar), 117.45 (Ar), 117.67 (Ar), 117.89 (Ar), 121.32 (Ar), 121.67 (Ar), 123.55 (Ar), 124.78 (Ar), 126.98 (Ar), 127.46 (Ar), 129.00 (Ar), 132.65 , 136.89 (Ar), 138.91 (Ar), 141.24 (Ar), 143.67 (Ar), 196.34 (C=O), 200.32 (C=O) ppm. Anal. Calcd. for C25H14O4: C, 79.36; H, 3.73. Found: C, 79.31; H, 3.77.7-Phenyl-6H-indeno pyrano chromene-6,8(7H)-dione 4 g: m. p. 203–205 °C, FT-IR -6H-indeno pyrano chromene-6,8(7H)-dione 4h: m. p. 234–235°C, FT-IR : 1734 (C=O str), 1701 (C=O str), 1,576 (aromatic C=C str), 1,435 (aromatic C=C str), 1,387, 1,234 (C-O str), 1,114.1H NMR : δH 2.35 , 5.31 , 6.87 , 6.87–7.09 , 7.23 , 7.56 , 7.67–7.83 , 8.01–8.09  ppm. 13C NMR : δC 34.23 (benzylic carbon), 58.65 , 112.67 (Ar), 114.00 (Ar), 115.34 (Ar), 116.46 (Ar), 118.87 , 119.45 (Ar), 121.33 (Ar), 125.67 (Ar), 126.23 (Ar), 128.98 (Ar), 129.00 , 132.46 (Ar), 135.47 (Ar), 135.88 (Ar), 137.98 (Ar), 141.12 (Ar), 143.34 (Ar), 146.54 (Ar), 198.08 (C=O), 200.23 (C=O) ppm. Anal. Calcd. for C26H16O4: C, 79.58; H, 4.11. Found: C, 79.53; H, 4.07.7-, 1,628 (aromatic C=C stretching), 3,371 (N-H stretching), and 1,343 cm−1 (C-N stretching) in Fe3O4@SiO2-propyl@dapsone-Cu.3O4 (JCPD 19–0629). This confirmed the stability of the crystalline phase of the magnetite core in the structure after silica coating, condensation, and complexation process. The absence of an amorphous peak in pattern confirmed the crystalline structure. Using Debye–Scherrer equation, the mean size of crystallite was calculated as 12.1 nm from the XRD pattern . This value is lower than the size obtained by FE-SEM and TEM due to the fact that some crystallite forms a particle. Also, the d-spacing and full width at half maximum (FWHM) of the highest XRD peak (2θ = 35.71o) were obtained as 2.514 Å and 0.6888 (2θo).The XRD pattern of synthesized nanoparticles showed the sharp diffraction peaks with 2θ at 18° (111), 30.4° (220), 35.7° (311), 43.3° (400), 53.6° (422), 57.7° (511), 63.0° (440), and 74.6 (533) , which iAs shown in The presence of iron, oxygen, nitrogen, carbon, silica, sulfur, and copper, in EDX, revealed the successful synthesis of these nanoparticles.The magnetic properties of synthesized nanoparticles are shown in 3O4@SiO2-propyl@dapsone-Cu nanoparticles, for the multicomponent synthesis of novel derivatives of pyranochromene-diones via the multicomponent reaction between various aldehydes, indan-1,3-dione, and 4-hydroxycoumarine (In continuation of our study to synthesize pharmaceutical compounds , we trigoumarine .3O4@SiO2-propyl@dapsone-Cu in aqueous media under stirring (To complete our assessment, we checked the effect of different conditions in the sample reaction. For example, we treated 4-nitrobenzaldehyde, indandione, and 4-hydroxycoumarin under stirring at room temperature and refluxing in EtOH. The satisfactory results were obtained via the reaction of 4-nitrobenzaldehyde, indandione, and 4-hydroxycoumarin in the presence of 0.05 g of Festirring .To expand the generality and efficiency of this avenue, some aldehydes with electron-donating or electron-withdrawing substituents were treated with indan-1,3-dione and 4-hydroxycoumarin. The results are summarized in As a proposed mechanistic pathway, initially, aldehyde was activated by the nanocatalyst, followed by nucleophilic attack of C-H acid of indan-1,3-diones, together with the departure of water, and chalcone was produced. Nucleophilic attack of 4-hydroxycoumarin to chalcone as Michael addition and then intramolecular cyclization followed by elimination of water lead to product 4 .Furthermore, the magnetic nanoparticles are magnetically recoverable and can be reused for six runs. Appearance features of the catalyst were not changed after several uses .To better understand the stability of catalyst after five cycles under these reaction conditions, FE-SEM and TEM analyses were carried out. The results are summarized in In conclusion, a new catalytic method for the synthesis of pyranochromene-diones has been developed. This method offers several advantages, such as simple workup and purification procedure without the use of any chromatographic method, mild reaction conditions, use of inexpensive and commercially available starting materials, recyclability and reusability of the catalyst, high product yields, and short reaction time. So we think that this procedure could be considered a new and useful addition to the present methodologies in this area.


Plasma free fatty acid (FFA) concentration is elevated in obesity, insulin resistance (IR), non-alcoholic fatty liver disease (NAFLD), type 2 diabetes (T2D), and related comorbidities such as cardiovascular disease (CVD). Furthermore, experimentally manipulating plasma FFA in the laboratory setting modulates metabolic markers of these disease processes. In this article, evidence is presented indicating that plasma FFA is a disease risk factor. Elevations of plasma FFA can promote ectopic lipid deposition, IR, as well as vascular and cardiac dysfunction. Typically, elevated plasma FFA results from accelerated adipose tissue lipolysis, caused by a high adipose tissue mass, adrenal hormones, or other physiological stressors. Reducing an individual’s postabsorptive and postprandial plasma FFA concentration is expected to improve health. Lifestyle change could provide a significant opportunity for plasma FFA reduction. Various factors can impact plasma FFA concentration, such as chronic restriction of dietary energy intake and weight loss, as well as exercise, sleep quality and quantity, and cigarette smoking. In this review, consideration is given to multiple factors which lead to plasma FFA elevation and subsequent disruption of metabolic health. From considering a variety of medical conditions and lifestyle factors, it becomes clear that plasma FFA concentration is a modifiable risk factor for metabolic disease. Long-chain fatty acids have a chain length of 12 carbons or greater. The vast majority of stored fatty acids in humans and other animals are in the long-chain form, with the majority being 16 carbons in length or longer and stored through esterification into the triacylglycerol (TAG) pool of white adipose tissue ,4. AlterThere are clear indications that reducing plasma FFA chronically could reduce disease burden, and thus significant efforts have been made to develop pharmaceuticals to reduce plasma FFA. These efforts are ongoing but have not yet led to an approved drug aimed at suppressing plasma FFA concentration ,39. HoweIn people afflicted with metabolic disease, it is common to observe a clustering of various aspects of metabolic dysfunction within the same individual; the term ‘metabolic syndrome’ refers to this phenomenon, in which abdominal obesity, IR, hypertension, and other aspects of T2D and CVD risk coincide . The eleEvidence indicating plasma FFA concentration as a risk factor for disease is derived from observation of patients (presented above), as well as from laboratory research linking an experimental elevation of plasma FFA with indicators of worsened metabolic health. A common approach for experimentally elevating plasma FFA is to simultaneously infuse heparin and a lipid emulsion ; the FFA elevation achieved by heparin and lipid infusion leads to rapid induction of IR ,68,69,70The detriments of chronic over-nutrition upon plasma FFA concentration cannot be corrected simply by a single bout of negative energy balance, because acute fasting actually raises plasma FFA concentration and elevates ectopic lipid deposition ,16,17. H2max or higher) with variable total training volumes [Even though chronic exercise is known to lead to improved health, a single exercise bout alone is not sufficient to reduce plasma FFA concentration. Indeed, quite the opposite occurs after each individual bout of exercise. As a result of enhanced adipose tissue lipolysis, plasma FFA concentration is elevated during ,96,97,98 volumes ,101,102. volumes . The red volumes ,104,105.Insufficient sleep quantity and quality can lead to IR, heightened T2D risk, and NAFLD ,50,51,52OSA increases risk for developing IR, other metabolic syndrome factors, and vascular disease ,53,54,55When considering the detrimental impacts of cigarette smoking, the focus is typically upon lung cancer and chronic obstructive pulmonary disease (COPD). However, it is important to note that chronic smokers also exhibit IR and elevated T2D risk . In maleAdipose tissue lipolysis is considered to be a primary determinant of the plasma FFA concentration. Thus, any worsening of the ability to restrain lipolysis in adipose tissue could lead to elevated plasma FFA. The elevated adipose tissue mass in obesity is statistically associated with the accelerated release rate of FFA into circulation. While in obesity the rate of FFA release per gram of adipose tissue is somewhat reduced, because total adipose mass is substantially increased in obesity, the net results is that obese people exhibit an elevated total rate of whole body plasma FFA turnover . TherefoFollowing consideration of the physiological mechanisms that typically govern changes in plasma FFA concentration, next it is important to consider the mechanisms that may link those FFA abundances to pathology and disease risk. While the primary focus of this review article is to highlight evidence that plasma FFA concentration is a risk factor for disease at the organismal level, below a brief review is presented of potential cellular mechanisms by which plasma FFA concentration exerts its detrimental effects. Uptake of FFA into cells occurs down concentration gradients. When intracellular FFA concentrations are elevated, FFA can be lipotoxic and lead to cell death and dysfunction ,123,124.In conclusion, habitual elevation of plasma FFA concentration in the postabsorptive and postprandial states can lead to heightened risk for developing various diseases related to metabolic syndrome, IR, and cardiovascular function. Plasma FFA concentration should not replace other well-established risk factors for disease . Rather, FFA concentration should be potentially added to the panel of clinical factors that are considered as components of risk determination for disease. While there are established management plans for some risk factors such as low-density lipoprotein cholesterol , for pla


Objective: To understand the reason for low implementation of clinical and home-based rehabilitation robots and their potential.Design: Online questionnaire (November 2020 and February 2021).Subjects: A total of 100 professionals in stroke rehabilitation area were involved .Interventions: Not applicable.Main Measures: Descriptive statistics and thematic content analysis were used to analyze the responses: 1. Participants' details, 2. Professionals' views and experience of using clinical rehabilitation robots, 3. Professionals' expectation and concerns of using home-based rehabilitation robots.Results: Of 100 responses, 37 had experience of rehabilitation robots. Professionals reported that patients enjoyed using them and they increased accessibility, autonomy, and convenience especially when used at home. The main emergent themes were: “aims and objectives for rehabilitation robotics,” “requirements” , “cost,” “patient factors” , and “staff issues” (concerns and benefits). The main benefits of rehabilitation robots were that they provided greater choice for therapy, increased the amount/intensity of treatment, and greater motivation to practice. Professionals perceived logistical issues , cost and limited adaptability to patients' needs to be significant barriers to tier use, whilst acknowledging they can reduce staff workload to a certain extent.Conclusion: The main reported benefit of rehabilitation robots were they increased the amount of therapy and practice after stroke. Ease of use and adaptability are the key requirements. High cost and staffing resources were the main barriers. Approximately two-thirds of stroke survivors are left with some degree of residual disability, particularly in limitations of the activities of daily living and mobility , 2. PhysIn this mixed method study, an online questionnaire was devised by the authors and optimized with the support of the wider research team (see section Acknowledgment). It included three sections using a mixture of open and closed questions, the latter answered with Likert scales . The firAll responses were anonymous and did not contain any sensitive data thus, according to the requirement for ethical approval of the authors' institution(s), it was not required.RRID:SCR_019096). Descriptive statistics were used to aggregate the collected data. Mann-Whitney and Kruskal-Wallis tests compared groups. We categorized groups based on following characteristics: (1) participants' occupation ; (2) career stage ; and (3) rehabilitation robot type (end-effector and exoskeleton).All quantitative data were analyzed using IBM SPSS  v25.0 . Then two of the authors (LT.L and ST) independently developed a coding matrix to categorize the respondents' composite statements. Our coding was shared and revised until a consensus was reached. The resulting coding framework was member checked with two respondents in the questionnaire (who had made themselves known to us—Dr. Verity Longley and Dr. Nick Preston), both experienced stroke professionals. Examples of the coding are shown in the For the qualitative data, an inductive thematic content analysis was undertaken using the questions in the questionnaire as a framework . FirstlyOne hundred professionals responded to the survey . Two-thip <0.05).Only 37% had experience of using rehabilitation robots, mainly in hospital and out-patient clinic environments with patients in the sub-acute stages of stroke recovery (1–3 months post stroke) . PoweredRespondents with and without experience of using rehabilitation robot had similar views regarding the type of patient for whom they were suitable; patients with moderate or severe lower or upper limb weakness were considered the most suitable .Five main themes emerged from the framework analysis: “aims and objectives for rehabilitation robotics,” “requirements” , “cost,” “patient factors” , and “staff issues” (concerns and benefits). These are detailed in Each theme is described, and the representative quotation for each theme is shown in Participants reported that the main objective for using robots in stroke rehabilitation was to increase the amount of therapy patients received. This was expressed in a variety of ways; the terms “therapy,” “practice,” and “movement,” were used interchangeably as was “intensity” and “amount.” This was important, as the amount of therapy is related to the degree of recovery. Additionally, using a rehabilitation robot was thought to improve patients' motivation and engagement with therapy as it was considered an enjoyable activity. An additional objective for some participants was that using the robot enabled the patient to practice movement independently or autonomously of the therapy. However, some respondents had concerns about patients using the robots without professional supervision.In addition, improving function and functional carry over were also important considerations, although some noted that the transfer from practicing movements with the robot to functional activity in everyday life could not be assumed. Others focused on the potential impact on impairments such as strength and range of movement. A final objective was that robotic therapy was useful to broaden the range of therapy options available to patients, which was also reported as one of the biggest advantages of using rehabilitation robots.Respondents identified many features which were needed to ensure that a stroke rehabilitation robot was feasible and acceptable for use in clinical practice. These were categorized into functional, software, and safety requirements.The participants most frequently noted that a rehabilitation robot needed to be user-friendly. It needed to be easy and intuitive to set up, administer, don and doff (put on and take off), maintain, transport, and store. The device needed to be useable as a game or to practice functional tasks/movements and be operable as either an active or an active-assisted device to eliminate the effects of gravity during three-dimensional movements. To achieve this, a robot needed to be highly adjustable to meet a wide range of patients' needs—in terms of size, level of assistance needed, cognitive demands of the games, and individual preferences. Furthermore, the appearance needed to be appealing so that it did not put patients off using it and it had to be comfortable to use.Not only was ease of use important, but it also needed to be quick to set up, program, and “put away” as the time taken for these activities impacted on staff resources and ultimately the cost. Compatibility with other systems such as virtual reality systems or other gaming devices was also important.The software supporting the robot's function was considered as important as the physical device. Again this needed to be easy and intuitive to use and suitable for a wide range of needs in terms of patients' aims of treatment; level of ability, and preferences.Participants with experience using rehabilitation robots reported that there was room for improvement with the games available. For example a greater choice and range of difficulty but also suitable for adults.The software needed to provide feedback on usage and performance to patients and therapists to facilitate motivation and progress treatment. It was also important that the application settings, dose of use , and amount of assistance provided by the device were recorded and easily transferred to clinical notes. The need for data security to protect patient confidentiality was noted.Safety is a paramount and non-negotiable issue for any clinical device, which was frequently raised by respondents. Their concerns and requirements went beyond the obvious need to ensure the robot did not make the patient move beyond their range of movement and was comfortable. The risk of pressure points on fragile skin or finger traps were raised, as was the need for the device to be easy to clean and to comply with infection control policies. Given the patients' movement limitations, an alarm system, emergency stop, auto shutdown, and simple/quick release mechanisms were needed to avoid incorrect or over use and to prevent secondary injury, especially for home or unsupervised use. Stability and reliability were also important issues, as was the restricting access to the settings of the robot.The high cost of rehabilitation robots and difficulty persuading hospitals to commit funds to pay for them were major barriers to their implementation into practice in both clinical and home-based settings. The cost of current rehabilitation robotics varies from $75,000 to $350,000 without any hidden cost such as taxes, installation or training, maintenance, and shipment . AccordiParticipants raised many concerns and issues regarding the type of patients for whom robot therapy would not be suitable. This included people with non-motor stroke related impairments such as behavioral, cognitive, perceptual, communication, or visual difficulties which could make them unable to use or understand how to use the robot. However, one participant noted that using rehabilitation robots could be beneficial for some of these impairments, as well as motor problems, for example encouraging visual scanning and practicing memory and problem solving tasks. Further problems which could limit the patients' ability to engage with robot therapy or safety were pain, fatigue, skin integrity, spasticity, or limited range of movement, particularly fixed joint contractures and should be carefully considered before use. Co-morbidities such as fractures, other musculoskeletal problems, or epilepsy also needed to be borne in mind when considering whether robot therapy was suitable for a patient.As well as patients' clinical condition, therapists also needed to consider the patients' and their families' willingness to try rehabilitation robots, particularly for home use. The “flip side” to this was concerns that patients could become “over-reliant” on the robots and disengage or refuse other aspects of therapy which may limit progress toward independent practice and activity in everyday life or could be a risk for overuse or secondary injuries.Participants highlighted the potential impact of rehabilitation robots on staff as well as patients. A common concern was that using rehabilitation robots to deliver greater amounts of therapy could replace, disempower, or deskill therapists. Respondents were careful to point out that robotic therapy should be an adjunct, but not a substitute to “traditional” care.Other respondents raised concerns about the resources needed to deliver robotic therapy. As therapists' working days were already full, if they were to incorporate robotic therapy into practice, either more staff were needed or they needed to stop providing some other aspect of care.While some participants were confident to delegate the supervision of robot rehabilitation to therapy assistants , family members or, for patients to use the robots unsupervised, others felt that direct supervision from qualified staff was necessary. Furthermore, several pointed out that qualified staff's time was needed to prescribe and set up the robot, problem solve, and oversee progression whether the patient was supervised or not.Time and staffing were not the only important resourcing issue. Staff also needed adequate training and on-going support to develop clinical skills to use the robots effectively in every-day practice. As many staff regularly change work areas (referred to as “rotation”), this input needed to be on-going to accommodate staff changes. All of which increased the cost of robotic therapy.Finally, given the professional duty to provide evidence-based care, some respondents raised the limited evidence to support its use as a barrier to implementation.This research identified the benefits, requirements, concerns, and barriers to implementation of rehabilitation robots by analyzing professionals' experience and views of their use in both clinical and home-based environments. The majority (71%) of the survey responses were from UK and as such are representative of this country, it cannot be assumed that these results are necessarily representative of other geographical regions. Participants were generally positive about rehabilitation robots. Their biggest advantages was that they facilitated high repetition task-oriented training, which was consistent with the principle of efficient rehabilitation –30. ThisPowered upper limb robots (whether exo-skeleton or end-effector) were the most commonly used rehabilitation robot in clinical practice. This may not only be because active robots can provide a wide range of support and functions, but also because upper limb robots are relatively small, which is more convenient, than lower limb robots.Home-based rehabilitation robots were identified as having great potential to facilitate accessibility, autonomy, and increase choice for therapy. Home-based systems can be convenient for patients as they eliminate the need to travel for access and enable autonomous usage. Professionals would be more confident in prescribing home-based robot therapy with well-developed interaction systems, such as remote monitoring and supervision.Our findings have provided a wealth of detail regarding professionals' requirements for stroke rehabilitation robots to be fit for purpose and to be adopted into practice. In addition to reducing the costs, rehabilitation robots need to be “user friendly,” safe, and suitable for a wide range of needs. All aspects of use needed to be quick, easy, intuitive, and adaptable. Safety is, of course, a non-negotiable issue especially if the robot is to be used unsupervised. Participants recognized that although there were few absolute contraindications, robots needed to be considered cautiously for people with non-motor stroke related impairments and co-morbidities which could limit their ability to understand or to use a robot safely. Furthermore, patients' preferences also need to be taken into account—not everyone wants to play games, or use technology. However, accommodating this range of needs is likely to increase costs. Alternatively if costs are minimized, then the range of users is likely to be limited. In reality, commercialization of rehabilitation robots needs to balance these competing demands with a priority to ease of use as the mismatch between the demands of operating devices and other aspects of clinical practice has rendered many promising health technologies unfit for purpose –33. FutuThe main limitations of this study include the convenience of the recruitment strategies, which may be biased toward professionals with positive views or experiences of using rehabilitation robots. Although we received responses from all over the world, most were from the UK. The findings may differ if census or probability style recruitment techniques were used or if participants from other countries were involved. However, we suggest that need for low cost, user-friendly, adaptable devices is probably universal. We have also, to date, only involved professionals. Stroke survivors and their carers/families may have other views, cautions or requirements. For the further research, the involvement of these important stakeholders should be considered.The original contributions presented in the study are included in the article/LL, ST, and AW conceptualized the paper. LL and ST analyzed the collected data. All authors contributed to the evaluation study itself, the manuscript, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The growth of prostate cancer is dependent on the androgen receptor (AR), which serves as a ligand‐specific transcription factor. Although two immunophilins, FKBP51 and FKBP52, are known to regulate AR activity, the precise mechanism remains unclear. We found that depletion of either FKBP51 or FKBP52 reduced AR dimer formation, chromatin binding, and phosphorylation, suggesting defective AR signaling. Furthermore, the peptidyl‐prolyl cis/trans isomerase activity of FKBP51 was found to be required for AR dimer formation and cancer cell growth. Treatment of prostate cancer cells with FK506, which binds to the FK1 domain of FKBPs, or with MJC13, an inhibitor of FKBP52–AR signaling, also inhibited AR dimer formation. Finally, elevated expression of FKBP52 was associated with a higher rate of prostate‐specific antigen recurrence in patients with prostate cancer. Collectively, these results suggest that FKBP51 and FKBP52 might be promising targets for prostate cancer treatment through the inhibition of AR dimer formation. We demonstrated that the two immunophilins, FKBP51 and FKBP52, are required for dimer formation and chromatin binding of AR. The PPIase activity of FKBP51 is essential for cancer cell proliferation and AR dimer formation. We conclude that both FKBPs promote dimer formation in AR and activate AR‐dependent transcription, which is associated with the etiology of prostate cancer. ARandrogen receptorCo‐IPco‐immunoprecipitationFKBP51FK506‐binding protein 5FKBP52FK506‐binding protein 4Hsp90heat shock protein 90PPIasepeptidyl‐prolyl cis/trans isomerasePSAprostate‐specific antigenTCGAThe Cancer Genome Atlas1The androgen receptor (AR) is a ligand‐dependent nuclear receptor that is required for the expression of androgen‐regulated genes. AR is key to normal prostate development as well as oncogenesis of prostate cancer, especially the development of castration‐resistant prostate cancer . AndrogeAndrogen receptor activity is regulated in a highly coordinated dynamic manner at multiple levels such as expression, androgen binding, nuclear translocation, homodimer formation, and DNA binding of AR. Androgen binding induces conformational changes, including intramolecular interactions in the AR, which facilitates its translocation to the nucleus . In the Androgen receptor is localized to the cytosol in the absence of androgen, as a complex with the molecular chaperone heat shock protein 90 (Hsp90), and various other cochaperones, including the small acidic protein p23, FKBP (FK506‐binding protein) 51, and FKBP52 . FKBPs a2‐terminal FK1 domain that is responsible for its PPIase activity and an FK1‐like FK2 domain that lacks PPIase activity. FK506 binds to the FK1 domain and inhibits its enzymatic activity, but does not bind to the FK2 domain. The COOH‐terminal region contains three tandem repeats known as the tetratricopeptide repeat (TPR) domain, which mediates interactions with other proteins, including the COOH terminus of Hsp90  were prepared by directional cloning the full ORF region of these genes into NanoBiT vectors , according to the manufacturer's instructions.Using pcDNA3 as the parental vector, the coding region of the AR gene was fused to ‐HA or V5‐ using the In‐Fusion2.10m HEPES, 150 mm NaCl, 2.5 mm EGTA, 1 mm EDTA, 1 mm DTT, 0.1% Tween‐20, 10% glycerol, 50 mm NaF, 0.1 mm Na3Vo4, 15 mm PNPP, 80 mm β‐glycerophosphate) at least three times, centrifuged, and subjected to SDS/PAGE, followed by immunoblotting.Total proteins (500 μg) were precleared with protein‐G Sepharose  for 1 h at 4 °C and immunoprecipitated with 0.02 μg of anti‐FKBP51, anti‐FKBP52, or immunoglobulin G  overnight at 4 °C. For co‐immunoprecipitation (Co‐IP), 1.1 μg anti‐V5 was used against 300 μg of protein. The immune complexes were recovered with protein‐G Sepharose for 1 h and then washed with IP Kinase buffer  and an StepOnePlus™ real‐time PCR system (Applied Biosystems). Expression levels were normalized to those of β‐Actin. Primers used for the real‐time PCR included AR‐F: AGCAGCAGGGTGAGGATG and AR‐R: GACTGCGGCTGTGAAGGT.Total RNA extraction was performed as described previously . A total2.12−1 Dox in a medium containing charcoal‐stripped FBS for 2 days. After treatment, the cells were fixed in ice‐cold MeOH, followed by incubation with 3% acetone for 10 min at −20 °C. Cells were then washed three times with PBS and incubated with 0.5% Triton/PBS for 10 min, followed by washing and blocking in 5% goat serum for 30 min, before incubation with antibodies (at a dilution of 1 : 200) for an hour to detect AR. Antigens were visualized using anti‐rabbit antibodies coupled to FITC . Photomicrographs were taken at a magnification of 400× using DeltaVision Elite .LNCaP cells were grown on coverslips and treated with DHT or MeOH for 20 h. Cells were pretreated with 1 μg·mL2.134 cells/well in a 96‐well plate containing MEM‐α with charcoal‐stripped FBS. On the following day, expression vectors and PEI were diluted in 16 μL of Opti‐MEM (Gibco). Of note, 6.25 ng·μL−1 of LgBiT‐AR and AR‐SmBiT or LgBiT‐AR P767A and AR P767A‐SmBiT expression vectors were used for the AR dimer formation assay. Further, AR‐LgBiT and FKBP‐SmBiT; LgBiT‐HSP90 and SmBiT‐FKBP52; AR‐LgBiT; and LgBiT‐FKBP51, AR‐LgBiT, and SmBiT‐HSP90 expression vectors were used for the protein–protein interaction assays. Diluted vectors and PEI were combined and vortexed and then incubated for 30 min at room temperature. After incubation, the solution mixture was directly added to the cells drop‐wise. Two days after transfection, the luminescence was measured using a Nano‐Glo® Live Cell Assay System (Promega) and Nivo  or ARVO X4 (PerkinElmer) or GloMax® Navigator (Promega). DHT or MeOH was added 4 h before or 15 min after the addition of the luminescence reagent.One day before transfection, HEK293T cells were seeded at a density of 2 × 102.144 cells/well in 96‐well plates for 24 h in MEM Alpha  containing 5% charcoal‐stripped FBS and 1 μg·mL−1 Dox. PC3 cells were cultured at a density of 1 × 104 cells/well in 96‐well plates for 24 h in RPMI Medium 1640  containing 5% charcoal‐stripped FBS and 1 μg·mL−1 Dox. The cells were transfected with PSA‐430‐pGL3 [m DHT or DMSO for 24 h. The luminescence caused by firefly luciferase and Renilla luciferase was measured using the Dual‐Glo Luciferase Assay System  and Nivo (PerkinElmer). Fold induction was calculated as the ratio of the luminescence of firefly luciferase to that of Renilla luciferase.The pcDNA3 AR and pCMV Rluc (Renilla luciferase) constructs were provided by M. Okada. 293T cells were cultured at a density of 2 × 10430‐pGL3 , pcDNA3 2.15http://xena.ucsc.edu/). We selected only primary tumor samples with expression data (n = 9858). Batch effect‐normalized mRNA data were used as the expression value. Box plots were generated using graphpad prism 6 .Transcriptome data were obtained from the TCGA Pan‐Cancer dataset using the University of California at Santa Cruz Cancer Genomics Browser  analysis with antibodies against FKBP52 for specimens obtained from prostate cancer patients as reported previously . A total2.17m citrate‐buffered solution (pH 6.0) for 15 min and covered in blocking solution  for 30 min at room temperature. Following that, the sections were incubated overnight at 4 °C with a primary antibody , according to the manufacturer's instructions, followed by incubation with the respective secondary antibody (N‑Histofine Simple Stain™ MAX PO (MULTI); cat. no. 414152F; Nichirei Biosciences Inc., Tokyo, Japan) for 30 min at room temperature. The H‑score was used to evaluate IHC staining in the present study. Briefly, > 500 tumor cells were counted in five different fields of vision in each section (×100 magnification) and the H‑score was calculated by multiplying the percentage of positive cells by the intensity , yielding a possible range of 0–300. Two independent examiners (HM and HM) judged the scores, and the mean score obtained was set as the representative score. Cutoff for the H‑score was determined based on the receiver operating characteristic curve.Formalin‐fixed and paraffin‐embedded tissue specimens were subjected to H&E staining and IHC staining as described in previous study . For eac2.18t‐test with graphpad prism 6 (GraphPad Software). A P‐value of < 0.05 was considered statistically significant.Association with PSA failure‐free probability was determined using Kaplan–Meier curves, while a log‐rank test was used to determine the level of significance. The relationship between the two data groups was compared using Tukey's multiple comparison test or Student's 33.1Steroid receptors are known to form large oligomeric structures with chaperones and cochaperones, including Hsp90, p23, and TPR‐domain proteins, such as FKBP51 and FKBP52. FKBPs, a large family of immunophilins that are conserved in eukaryotes, mediate diverse cellular functions including protein folding, cellular signaling, and transcription , we examIt has been reported that the PPIase activity of FKBP52 is dispensable for AR activity . We next3.2Although FKBP51 and FKBP52 have been shown to contribute to the regulation of AR signaling, the detailed underlying mechanisms still remain unclear. We examined the expression of AR in 22Rv1 cells depleted of FKBP51 or FKBP52 at 0, 4, and 12 h after the onset of dihydrotestosterone (DHT) treatment. While the expression of AR increased upon DHT binding in control cells, consistent with previous studies that have demonstrated that AR protein stability increases upon DHT binding , 27, dep81), which is associated with AR activity  system and found that AR forms dimers immediately after the addition of DHT Fig. . To veri81) upon DHT treatment in shControl 22Rv1 cells, but the extent was significantly decreased in FKBP51‐ or/and FKBP52‐depleted cells  Pan‐Cancer dataset (9858 samples) and found that the expression of FKBP51 was highest in prostate cancer, among various cancers Fig. . SimilarSpecimens obtained from prostate cancer patients were subjected to immunohistochemical (IHC) analysis with antibodies against FKBP52, following which the patients were classified into two categories based on FKBP52 expression. These groups of patients were tested for the blood level of prostate‐specific antigen (PSA), elevation of which is indicative of metastasis or recurrence after castration. PSA recurrence occurred in 75% of patients in the FKBP52‐positive group within 100 months, a significantly shorter time to recurrence than in the negative group Fig. . We perf4In this study, we showed that the two immunophilins, FKBP51 and FKBP52, are known to be regulators of AR and are required for prostate cancer proliferation. The growth of prostate cancer is largely dependent on activation of AR. AR activity is regulated in a highly coordinated dynamic manner at multiple levels such as expression, androgen binding, nuclear translocation, homodimer formation, and DNA binding of AR. In the nucleus, an AR dimer binds to androgen‐responsive elements in the regulatory regions of its target genes . MultiplOf note, the depletion of FKBP51 or FKBP52 reduced dimer formation, chromatin binding, phosphorylation, and transcriptional activity of AR, suggestive of defective AR signaling. Furthermore, the FKBP51 PPIase inhibitor and SAFit1/2 suppressed cell proliferation and colony‐forming ability, and inhibited ligand‐induced AR dimer formation. Treatment of prostate cancer cells with FK506, which binds to the FK1 domain of FKBPs, or with MJC13, an inhibitor of FKBP52‐AR signaling, also inhibited AR dimer formation.767 mutated to Ala, remain as monomers even in the presence of DHT [767, which may trigger dimer formation. The enzymatic activity of FKBP52 is not involved in AR dimerization, suggesting that effects other than isomerization are important for AR dimerization. Dissociation of AR from the HSP90‐FKBP51/52 complex leads to nuclear translocation of AR, subsequent dimerization, and transcriptional activation of target genes (Fig. FKBP51 was previously shown to attenuate the activity of Cdk4 by inhibiting the phosphorylation of Thr172 through the isomerization of Cdk4‐Pro173 in myocytes . Based oe of DHT . Consistnes Fig. .FKBP51 and FKBP52 might be linked to the etiology of prostate cancer, given that the levels of FKBP51 and FKBP52 have been found to be elevated in human prostate cancer, compared to the noncancerous part of the prostate gland , 38, 39.Collectively, our results suggest that inhibition of FKBP51 and FKBP52 activity might have a therapeutic effect on prostate cancer by abrogating AR dimer formation. In addition, dysregulation of FKBP51 or FKBP52 has been implicated in a variety of diseases, including stress‐related diseases and neurodegenerative disorders . A bette5Two immunophilins, FKBP51 and FKBP52, are known to be positive regulators of AR; however, the precise mechanism by which they control AR activity remains unclear. We found that both FKBPs were important for dimer formation and chromatin binding of AR. Furthermore, the PPIase activity of FKBP51 was found to be required for AR dimer formation and cancer cell growth. Of note, elevated expression of FKBP52 was associated with the PSA recurrence rate of prostate cancer. Collectively, these results suggest that FKBP51 and FKBP52 might be promising targets for prostate cancer treatment through the inhibition of AR dimer formation.The authors declare no conflict of interest.MS designed and coordinated this research. MS drafted the manuscript. KM, MH, MK, HM, SH, TM, YS, HM, KK, and HN carried out the experiments and data analysis. The authors have read and approved the final manuscript.The studies involving human participants were reviewed and approved by Institute Research Ethics Committee of Yamaguchi University. Written informed consent was obtained from all the patients. The IRB approval number is H29‐132.https://publons.com/publon/10.1002/1878‐0261.13030.The peer review history for this article is available at Fig. S1. Synthesis of compound 6.Fig. S2. Synthesis of FKBP inhibitor.Fig. S3. FKBP51 and FKBP52 are essential for AR dimer formation.Click here for additional data file.


Objective: To extend life expectancy after surgery, patients with hip fractures need to improve their mobility quickly through postoperative rehabilitation. Voluntary hip joint motion supported by the hybrid assistive limb (HAL) lumbar type, an exoskeleton robot suit characterized by its ability to detect the wearer’s intentions through the bioelectrical signals and assist hip extension motions at an optimal timing, may be effective to improve mobility in patients with hip joint dysfunction after surgery. We aimed to introduce rehabilitation using the HAL lumbar type in the early period after hip fracture surgery.Methods: Patients who underwent internal fixation for hip fracture at a single institution were prospectively enrolled. They received early postoperative rehabilitation  using the HAL lumbar type (six times a week for 15 min per session). Five-times-sit-to-stand (FTSS) and timed-up-and-go (TUG) tests were conducted at baseline before HAL rehabilitation (pre-HAL) and after the HAL rehabilitation (post-HAL) intervention.Results: We enrolled 14 patients  in this study. There were no adverse events, and all patients were able to complete the entire rehabilitation program. Post-HAL FTSS showed significant improvement compared with pre-HAL and had a large effect size of 1.81 (95% CI = 0.93 to 2.66) and sufficient power.Conclusions: Robotic rehabilitation with HAL lumbar type could be introduced without adverse events, even in the early postoperative period following surgery for hip fracture. Further study is needed to develop an appropriate rehabilitation protocol using the HAL lumbar type. Aging societies have led to increased hip fractures among the elderly worldwide . Hip fraLee et al.  found eaParticipantsThis study is a pilot, prospective, single-arm, inpatient postoperative rehabilitation study using the HAL lumbar type for patients following hip fracture surgery. Patients who underwent internal fixation for hip fracture at a single institution between April 2019 and June 2020 and satisfied the following inclusion criteria were enrolled: (1) able to walk independently or with a cane before hip fracture; (2) immediate full weight-bearing after surgery was allowed. We excluded the patients who met the following criteria: (1) severe cognitive impairment that makes it difficult to understand the rehabilitation protocol; (2) difficult to walk due to cerebrovascular disease before surgery; (3) implanted pacemakers; (4) underwent bipolar hip arthroplasty. We obtained written informed consent from all participants. This study was conducted following the Declaration of Helsinki with the approval of the Ethical Review Committee of HITO Hospital .Early postoperative rehabilitation with the HAL lumbar typeThe HAL lumbar type consists of a lumbar frame, thigh mold power units, and electrode detecting BES Figure .It is equipped with angle sensors in the power units located in the bilateral hip joint to detect the wearer’s hip joint angle. The electric motors in the power units can support hip joint extension when the wearer stands up. Based on BES detected from electrodes attached to the lumbar erector spinae muscles, the HAL lumbar type has the characteristic of providing hip joint motion support at the timing intended by the wearer .Participants underwent rehabilitation using the HAL lumbar type in addition to the usual conventional postoperative rehabilitation after hip fracture surgery. Once the patient’s general condition was stabilized after surgery, HAL rehabilitation was started as soon as possible. The following four types of HAL rehabilitation were performed: (1) forward and backward bending of the lumbar spine in a sitting position; 2) pelvic tilt forward and backward in a sitting position; 3) standing up from a sitting position with the support of the upper limbs  and the timed-up-and-go (TUG) tests were used to assess locomotion function. These tests were conducted at baseline before HAL rehabilitation (pre-HAL) and after the HAL rehabilitation intervention (post-HAL). The FTSS test was performed at maximum speed while holding onto a 40-cm high platform. The TUG test was performed using a walker both pre-HAL and post-HAL.A Wilcoxon signed-rank test was used to compare pre-HAL and post-HAL test results. Statistical analysis was performed using the JMP software package version 14.0.0 . Effect size and post-hoc statistical power were calculated using Gpower software . P < 0.05 was considered significant.Fourteen patients one man, 13 women) were enrolled in this study. No patient met pre-set exclusion criteria. The demographic data of the participants are summarized in Table 3 women wThere were no adverse events associated with rehabilitation using the HAL lumbar type, and all participants were able to complete the entire rehabilitation program. The clinical characteristics of each case are summarized in Table Post-HAL FTSS showed significant improvement compared to pre-HAL with a large effect size of 1.81 (95% CI = 0.93 to 2.66) and sufficient power (0.99). In pre-HAL tests, seven out of 14 patients could not walk sufficiently to complete the TUG test, but all patients could achieve it in post-HAL. In all other cases, the TUG time decreased.In the present study, we aimed to introduce rehabilitation using the HAL lumbar type in the early postoperative period following surgery for a hip fracture. All 14 patients were able to complete the HAL rehabilitation without adverse events. Furthermore, post-HAL FTSS test results showed improvement compared to pre-HAL.Various rehabilitation procedures after hip fracture surgery have been reported. In a review by Lee et al. , they foIn the present study, we attempted early postoperative rehabilitation intervention using the HAL lumbar type. To date, several reports have shown that exercise therapy using the HAL lumbar type improved physical functions in the elderly. Kotani et al.  reportedSeveral limitations of the present study should be considered. The effect of the HAL lumbar type on physical function could not be clarified because there was no comparison with the control group, which received only conventional rehabilitation. It was impossible to prove that the HAL lumbar type reduced the physical load because there was no evaluation of pain and EMG data changes during rehabilitation. Future comparative studies with large sample sizes will be needed to resolve these limitations.Robotic rehabilitation with HAL lumbar type could be introduced without adverse events, even in the early postoperative period following internal fixation for hip fracture. Post-HAL FTSS test results showed improvement compared to pre-HAL FTSS test results. These findings suggest that HAL lumbar type may be an option for early postoperative rehabilitation after hip fracture surgery. Further study is needed to demonstrate the effect of HAL lumbar type on physical function and to develop an appropriate rehabilitation protocol.


Inhibition of the androgen receptor (AR) remains the mainstay treatment for prostate cancer. All current therapies involving AR inhibition either directly or indirectly target its ligand-binding domain (LBD). We have developed the first novel compounds which target the N-terminal domain, (NTD) a region which is essential for AR transcriptional activity. First-generation ralaniten (NCT02606123), and second-generation EPI-7386 (NCT04421222) remain the only AR-NTD inhibitors to progress to clinical trials. Here we aim to characterize differences between different classes of AR antagonists targeting the AR-LBD and the AR-NTD, as well as next generation AR-NTD inhibitors. An incidental finding was that ralaniten was uniquely associated with increased metallothionein expression which was independent of AR activity. Instead, expression of metallothionein genes was driven by MTF-1 indicating a potential off-target effect. Neither AR-LBD inhibitor enzalutamide nor second-generation AR-NTD inhibitor EPI-7170 had this effect. This work has important implications for the development of novel AR-NTD inhibitors.Hormonal therapies for prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD). Clinical development for inhibitors that bind to the N-terminal domain (NTD) of AR has yielded ralaniten and its analogues. Ralaniten acetate is well tolerated in patients at 3600 mgs/day. Clinical trials are ongoing with a second-generation analogue of ralaniten. Binding sites on different AR domains could result in differential effects on AR-regulated gene expression. Here, we provide the first comparison between AR-NTD inhibitors and AR-LBD inhibitors on androgen-regulated gene expression in prostate cancer cells using cDNA arrays, GSEA, and RT-PCR. LBD inhibitors and NTD inhibitors largely overlapped in the profile of androgen-induced genes that they each inhibited. However, androgen also represses gene expression by various mechanisms, many of which involve protein–protein interactions. De-repression of the transcriptome of androgen-repressed genes showed profound variance between these two classes of inhibitors. In addition, these studies revealed a unique and strong induction of expression of the metallothionein family of genes by ralaniten by a mechanism independent of AR and dependent on MTF1, thereby suggesting this may be an off-target. Due to the relatively high doses that may be encountered clinically with AR-NTD inhibitors, identification of off-targets may provide insight into potential adverse events, contraindications, or poor efficacy. Between 20 and 30% of patients with localized prostate cancer will experience recurrence and require systemic therapy. Androgen deprivation therapy (ADT) remains the first-line treatment; however, patients with metastatic disease will inevitably progress to castration-resistant prostate cancer (CRPC) despite initial response . FollowiAR functions as a master regulator of thousands of genes and directly influences a myriad of cellular responses. Compounds that target the AR-NTD versus the AR-LBD have a markedly different mechanism of action, and potentially disrupt different downstream cellular pathways. Indeed, differences between ralaniten and SINT1 have been shown despite both targeting the AF-1 region within the AR-NTD . Here, wMetallothioneins are small (~6 kDa) cysteine-rich proteins that are capable of binding numerous essential as well as toxic metal ions; however, they preferentially form complexes with zinc ,26. Metan = 456) or repressed (n = 209) ≥2-fold following stimulation with synthetic androgen R1881 were identified. Of these genes, the ability of each inhibitor to attenuate expression in the induced gene set, or de-repress expression in the repressed gene set by ≥2-fold was determined  has a high degree of structural similarity to ralaniten, yet does not bind AR and has no antagonistic effect against the AR [To determine if the induction of metallothionein isoforms was specific to ralaniten or common to inhibitors of the AR-NTD, we employed a second-generation ralaniten analogue EPI-7170) and two sintokamide analogues (SINT1 and LPY-26), which are structurally and mechanistically unique A,B. Sint0 and twot the AR , and wasWe first compared the AR-NTD inhibitors that bind to unique regions in the NTD to determine if the induction of expression of metallothionein genes was specific to ralaniten or common to NTD inhibitors. LNCaP human prostate cancer cells were transfected with luciferase reporter constructs fused to the promoter regions of metallothionein isoforms  found to be upregulated in the array data, and treated with 35 μM of each compound. Only ralaniten induced these metallothionein reporters C, suggesMT1F mRNA slightly but significantly compared to the vehicle control (CMC), but had no significant effect on levels of MT1X, MT1G, or MT2A.To confirm if induction of metallothionein genes by ralaniten occurred in vivo, castrated mice bearing LNCaP xenografts were randomized into three treatment groups: ralaniten (233 mg/kg), EPI-7170 (56.6 mg/kg), or vehicle control (CMC). Consistent with previous reports ,22, bothTo determine whether the induction of metallothionein expression was dependent upon concentration of ralaniten, MT1F and MT2 reporter gene constructs were transiently transfected into LNCaP cells. A dose-dependent response was observed, with significant induction occurring at 25 μM concentrations of ralaniten for both reporters A. Our neExpression of metallothionein genes are regulated through a number of response elements within its regulatory regions. These include binding sites for glucocorticoid receptor (GR), NRF2, and MTF-1 ,34,35, wTo more definitively address the requirement for AR in the mechanism of induction of metallothionein isoforms by ralaniten, levels of AR were knocked down in LNCaP and LN95 cells, and subsequently treated with DMSO vehicle (in the presence and absence of androgen), enzalutamide (5 μM), ralaniten (35 μM), or EPI-7170 (5 μM), all in the absence of androgen stimulation. While AR protein levels were robustly silenced after 24 h in LNCaP cells A, the efThe genes for metallothionein contain antioxidant response elements (ARE) to which NRF2 binds to increase levels of expression in response to oxidative stress ,38,39. Btert-butyl hydroperoxide (tBHP). Neither enzalutamide nor the AR-NTD inhibitors mediated a positive signal for mitoSOX  were measured. Targeted knockdown of MTF-1 was repeated in LNCaP and LN95 cells treated with increasing concentrations of ralaniten and stimulated with R1881. Ralaniten inhibited the levels of expression of androgen-regulated genes in a dose-dependent manner regardless of knockdown of MTF-1 C,D. Howe2O, were purchased from Sigma Aldrich  unless stated otherwise.Metribolone (R1881) was purchased from AK Scientific ; the antiandrogen bicalutamide was a kind gift from Mark Zarenda (AstraZeneca) and the antiandrogen enzalutamide was purchased from Omega Chem . Ralaniten was provided by Naeja-RGM . SINT1 is a natural compound, and EPI-7170 and LPY-26 were synthesized by Raymond Andersen at UBC . All other chemicals, including BADGE-2HLNCaP cells were from Leland Chung  and maintained in phenol-red-free RPMI 1640 supplemented with 5% FBS (VWR). LN95 cells were provided by Stephen Plymate (University of Washington). LN95 cells were maintained in RPMI 1640 medium supplemented with 10% dextran-coated charcoal-stripped FBS. DU145 cells were from Victor Ling  in October 1998. Cells were maintained in DMEM with 10% FBS and supplemented with 2 mM L-glutamine and 1 mM of sodium pyruvate. PC3 cells were purchased from ATCC and maintained in DMEM with 5% FBS and supplemented with 2 mM L-glutamine and 1 mM of sodium pyruvate. LN95 cells were not authenticated in our laboratory, but were regularly tested to ensure that they were mycoplasma-free . LNCaP, PC3, and DU145 cells were authenticated by short tandem repeat analysis and tested to ensure that they were mycoplasma-free by DDC Medical. All cells used in the experiments were passaged in our laboratory for fewer than 3 months after resurrection.www.cdrd.ca (14 October 2014)). Analysis of raw signal output was done using GeneSpring software (version 13.1). Clustered data was generated by conducting a two-way ANOVA on data with the significance threshold set at 0.05. The Benjamini–Hochberg correction was applied to reduce the false discovery rate (FDR).Total RNA was extracted from LNCaP cells treated with ralaniten (35 µM), enzalutamide (5 µM), bicalutamide (10 μM), or DMSO vehicle and stimulated with either 1 nM R1881 or EtOH vehicle. RNA was reverse-transcribed, and cDNA was hybridized to the GeneChip Human Transcriptome Array 2.0 from Affymetrix. RT-PCR, cDNA hybridization, and chip reading was carried out at CDRD’s Target Validation Division at the University of British Columbia  was used, and the difference in the expression levels between vehicle and drug treatment for each gene was analyzed based on the Molecular Signatures Database C2 sets . The permutation number was set to 1000. Those enrichment gene sets revealed by GSEA as exhibiting a nominal p < 0.05 and FDR < 0.05 were considered to indicate a statistically significant difference. The default parameters were used in the GSEA software.Data generated from the microarray analysis was used to identify differences between AR antagonists. GSEA version 7.0 software (2O (35 μM), ralaniten (35 μM), or DMSO vehicle. After 48 h of treatment, cells were lysed and analyzed for luciferase activity and normalized to protein expression. For dose-escalation experiments, LNCaP cells were transfected with the reporter plasmid pMT1F-luciferase, pMT1G-luciferase, or pMT2-luciferase in serum-free, phenol-red-free media using Lipofectin (Invitrogen). After 16 h, cells were treated with increasing concentrations of ralaniten or DMSO vehicle. After 48 h of treatment, cells were lysed and analyzed for luciferase activity and normalized to protein expression.LNCaP cells were transfected with reporter plasmids pMT1F-luciferase, pMT1G-luciferase, or pMT2-luciferase (1 μg/well) in serum-free, phenol-red-free media using Lipofectin . After 16 h, cells were treated with SINT1 (35 μM), LPY-26 (35 μM), BADGE-2H7 cells/site) using Matrigel (Becton Dickinson). Mice were castrated once tumors reached ~100 mm3 and randomized into treatment groups to receive ralaniten (233 mg/kg), EPI-7170 (56.6 mg/kg) or vehicle control (5% DMSO/1% CMC/0.1% Tween 80) once daily by oral gavage. Treatment began one week following castration. Mouse body weight and tumor volume (defined as volume = length × width × height × 0.5236) were regularly recorded, and tumors were excised 24 h after the last treatment. To analyze tumor gene expression, tumors were flash frozen, and ~100 mg was added to 1 mL TRIzol reagent (Invitrogen) and homogenized using a tissue homogenizer . RNA was extracted and reverse-transcribed as detailed below.All experiments involving animals were conducted in compliance with, and with the approval of, the Animal Care Committee of the University of British Columbia (A18-0077). Male NOD/SCID mice at 6 to 8 weeks of age were subcutaneously injected with LNCaP cells , enzalutamide (0.5–20 μM), EPI-7170 (0.5–20 μM), or DMSO vehicle in the absence of androgen stimulation. After 24 h of treatment, cells were harvested in 1 mL TRIzol reagent (Invitrogen). Total RNA was extracted using the RNeasy Micro Kit , cleaned using an amplification-grade DNase I Kit  and reverse-transcribed using the High-Capacity RNA-to-cDNA Kit . Diluted cDNA and Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen) were combined with gene-specific primers. Transcripts were measured by a qRT-PCR QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems by Life Technology), and gene expression was normalized to the housekeeping gene SDHA. Gene-specific primer sequences are given in Pooled siRNA against AR (L-003400-00-0005), MTF-1 (L-020078-00-0005), NRF2 (L-003755-00-0005), and nontargeting control (D-001810-10-05) were purchased from Dharmacon Research . Lipofectamine RNAiMAX (Invitrogen) was used to transfect 10 nM  or 15 nM (MTF-1) of siRNA into cells in Opti-MEM serum-free media (Thermo Fisher Scientific). For experiments examining protein or mRNA expression, cells were plated on 10 cm or 6-well plates, respectively, in complete media for 24 h prior to transfection. After 24 h, media were removed and replaced with Opti-MEM (Gibco) containing 10 nM  or 15 nM (MTF1) siRNA/transfection reagent complexes . Cells were pretreated with ralaniten (5–35 µM), EPI-7170 (5 µM), enzalutamide (5 µM), or DMSO vehicle for 1 h, followed by stimulation with 1 nM R1881 or EtOH. Cells were harvested in 1 mL TRIzol, and RNA was extracted and reverse-transcribed as detailed above.Protein lysates were harvested 24 h after R1881/EtOH treatment in RIPA buffer and separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane . AR was probed using an antibody obtained from Abcam (Ab198394). MTF-1 was probed using an antibody obtained from Invitrogen (PA5-55945). NRF2 was probed using an antibody obtained from Abcam (Ab137550). β-actin was used as a loading control, and membranes were probed using the mouse monoclonal anti-β-actin antibody (a5316 from Sigma).For the proliferation assay, 5000 (LNCaP) or 7500 (LN95) cells were plated per well in 96-well plates in respective full media and incubated for 24 h to allow cells to attach. Media were removed and replaced with Opti-MEM containing 15 nM MTF1 siRNA or scrambled control for 24 h. Treatments were prepared by serial dilution, and cells were pretreated with DMSO or ralaniten (5–35 µM) for 1 h prior to stimulation with 0.1 nM R1881 (LNCaP) or 1.5% charcoal-stripped serum (LN95). After 72 h post-treatment, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) and incubated with 0.1% crystal violet solution (Sigma). Dye was solubilized using a 1% SDS solution, and absorbance was read using a VersaMax Microplate Reader (Molecular Devices) at 595 nm.Total cellular ROS in cells was detected using the DCFDA/H2DCFDA-Cellular ROS kit from Abcam (ab11351) according to the manufacturer’s instructions. LNCaP and LN95 cells plated on 96-well, black, clear-bottom plates were stained with DCFDA/H2DCFDA (20 μM) for 30 min at 37 °C. After washing, cells were treated with tBHP (75 μM), enzalutamide (5 μM), ralaniten (35 μM), EPI-7170 (5 μM), or DMSO vehicle for 4 h. Fluorescence was measured using the Infinite M1000 (Tecan) with excitation at 485 nm and emission at 535 nm.Mitochondrial superoxide was detected using MitoSOX Red from Sigma (M36008). LN95 cells were seeded on 8-well chamber slides and treated with tBHP, enzalutamide, ralaniten, EPI-7170, or DMSO vehicle as above for 4 h at 37 °C. Cells were then washed and stained with MitoSOX Red (5 μM) diluted in HBSS/Ca/Mg buffer  for 10 min at 37 °C. Cells were washed gently three times in warm buffer before counterstaining with Hoechst 33342 . Slides were mounted and visualized using a fluorescence microscope  at Ex/Em 350/461 (Hoechst) and 510/580 (MitoSOX Red).p-value corrections were applied for all multiple comparisons , and p < 0.05 was considered statistically significant.A one- or two-way ANOVA statistical test was used to determine significance for all comparisons unless specifically stated otherwise . The Despite initial response to targeted AR inhibition, most CRPC remains driven by continued AR transcriptional activity. Currently, there is no cure for CRPC, and patients will inevitably succumb to their disease. The existence of constitutively active AR-Vs, gain-of-function mutations within the AR-LBD, amplification of the AR gene, and intratumoral synthesis of androgens have been shown drive resistance to abiraterone and enzalutamide ,13,14. AFurthermore, the discovery and development of small-molecule inhibitors that target the AR-NTD have significant potential to improve outcomes of CRPC patients who have progressed following treatment with existing AR-targeted therapies ,18,19,42


FOLH1 gene. The PSMA protein has become an important factor in metastatic PCa diagnosis and radioligand therapy. However, low PSMA expression is suggested to be a resistance mechanism to PSMA-based imaging and therapy. Clinical studies revealed that androgen receptor (AR) inhibition increases PSMA expression. The mechanism has not yet been elucidated. Therefore, this study investigated the effect of activation and inhibition of androgen signaling on PSMA expression levels in vitro and compared these findings with PSMA levels in PCa patients receiving systemic therapy. To this end, LAPC4, LNCaP, and C4-2 PCa cells were treated with various concentrations of the synthetic androgen R1881 and antiandrogens. Changes in FOLH1 mRNA were determined using qPCR. Open access databases were used for ChIP-Seq and tissue expression analysis. Changes in PSMA protein were determined using western blot. For PSMA staining in patients’ specimens, immunohistochemistry (IHC) was performed. Results revealed that treatment with the synthetic androgen R1881 led to decreased FOLH1 mRNA and PSMA protein. This effect was partially reversed by antiandrogen treatment. However, AR ChIP-Seq analysis revealed no canonical AR binding sites in the regulatory elements of the FOLH1 gene. IHC analysis indicated that androgen deprivation only resulted in increased PSMA expression in patients with low PSMA levels. The data demonstrate that AR activation and inhibition affects PSMA protein levels via a possible non-canonical mechanism. Moreover, analysis of PCa tissue reveals that low PSMA expression rates may be mandatory to increase PSMA by androgen deprivation.Prostate-specific membrane antigen (PSMA) is an essential molecular regulator of prostate cancer (PCa) progression coded by the  Prostate cancer (PCa) is the most common cancer in men. According to the “Cancer Today” project, PCa has the second-highest incidence of all cancers in men, with 1,414,259 newly diagnosed cases  and 375,304 deaths yearly related to PCa worldwide  ,7.FOLH1 gene located in the short arm of chromosome 11 [Prostate-specific membrane antigen (PSMA), also known as Folate Hydrolase 1 or Glutamate carboxypeptidase II, is a transmembrane type II glycoprotein coded by the osome 11 ,7. The posome 11 ,7. It isosome 11 ,7,8. Howosome 11 ,7,8. Theosome 11 ,7.18F)- and 68Gallium (68Ga)-labelled radiotracers targeting PSMA have demonstrated significantly higher sensitivity. They became part of the primary diagnostic in PCa guidelines for cases of PSA-recurrence after local therapy [177Lu), labelled PSMA in metastatic castration-resistant PCa (mCRPC) that delivers β radiation to PSMA expressing cells. Cellular uptake of 177Lu labelled PSMA led to a higher PSA response and fewer adverse events than cabazitaxel [177Lu -PSMA-617 to standard care significantly extended survival among patients with mCRPC [PSMA became one of the most promising targets for imaging and novel therapies in PCa ,7. Sever therapy ,4. Besidazitaxel . Moreoveth mCRPC .In vivo and in vitro studies suggested an influence of the androgen receptor (AR) on PSMA expression ,12. The The study aimed to investigate the influence of androgen activity manipulation by ADT and the antiandrogens bicalutamide, enzalutamide, apalutamide, or darolutamide on PSMA levels in vitro and to compare these findings with PSMA expression in patients receiving systemic therapy. To substantiate our investigation, three AR-positive cell lines  and one AR negative cell line (PC3) have been used . The LNCFOLH1 expression, the AR-positive castration sensitive cell lines LAPC4, LNCaP, and the castration-resistant LNCaP sub-cell line C4-2 was used [FOLH1 mRNA at which LAPC4 expressed the lowest and C4-2 the highest amount of FOHL1 mRNA. On protein level  have been found in all examined cell lines  datasets . No correlation could be shown between AR and FOLH1 mRNA expression  were analyzed  databases were used to analyze the of FOLH1 ,49,50. Hof FOLH1 . Therefoand CREB ,52,53,54silenced . Togethe sarcoma . As AR aFOLH1 mRNA is regulated by modulation of the AR activity in in vitro models [Furthermore, as the mechanism is not fully solved yet, the already published data has revealed that o models ,43,49,50o models . Low PSMo models . Phenotyo models . This hyo models . In contThe data in this study could not generalize the regulating effects of antiandrogens on PSMA protein levels. However, direct regulation of PSMA by the synthetic androgen R1881 could be shown in the cell lines LNCaP and C4-2. Therefore, tissue specimens of treatment-naive and ADT PCa patients were analyzed for PSMA levels. Interestingly, the suggested increase in PSMA level after ADT or ADT+NHT could not be confirmed here ,35,36,58FOLH1 in vivo and patients’ specimens was assessed. The data demonstrated that activation and inhibition of the AR influence PSMA protein levels by a possible non-canonical mechanism. Moreover, analysis of PCa tissue reveals that low PSMA expression may be mandatory to increase PSMA by ADT. However, more studies are required to decipher how the AR influences PSMA to recommend ADT before diagnostics or treatment with PSMA-ligands. Moreover, as the endocytosis transport function is mandatory for PSMA ligand-based methods, the relationship between AR signaling and the transport function of PSMA needs further investigation.In this study, the influence of the manipulation of the AR activity on the expression of 2. Mycoplasma testing was performed using the Mycoalert Detection Assay . STR profiling was used to verify cell line authentication.The human PCa cell lines LNCaP and PC3 were obtained from the American Type Culture Collection. C4-2 cells were kindly provided by Prof. Thalmann  . LAPC4 cFor AR ChIP-Seq analysis, the AR binding sites were extracted from the publicly available GEO dataset  and the data were visualized with the Integrative Genomics Viewer ,61,62.For PSMA expression analysis, the public datasets GSE11428, GSE13332, and GSE15091 have been used. GSE11428 investigated AR-induced gene expression in PCa cells LNCaP and LNCaP abl by transfecting siRNA against AR . GSE1333R1881 , bicalutamide , enzalutamide , apalutamide , and darolutamide  were dissolved in DMSO (100 mM stock solution), and aliquots were stored at −80 °C.Bicalutamide is a first-generation non-steroidal antiandrogen used to treat metastatic and local advanced PC combined with LHRH analogues or orchiectomy . While tThe second-generation antiandrogen enzalutamide is indicated for metastatic PC . EnzalutApalutamide is a second-generation antiandrogen indicated for metastatic PC treatment . Its mecDarolutamide is a non-steroidal AR antagonist used in metastatic PC and inhibits nuclear translocation of the AR, thereby decreasing the activation of genes required for the growth and survival of PC cells .The medium was changed to 5% dextran-coated charcoal treated FBS  24 h after seeding cells to deplete the steroid hormones and growth factors. After 24 h, for qPCR, cells were subsequently treated for 16 h with the synthetic androgen 1 nM R1881 or with a combination of 1 nM R1881 and 10 µM antiandrogens. For western blot analysis, cells were subsequently treated for 48 h with the synthetic androgen 1 nM R1881 or with a combination of 1 nM R1881 and 10 µM antiandrogens.As described earlier, subcellular fractionation, cell harvest, protein determination, and western blot were performed . SDS-gelGOI-CpHousekeeper values were calculated and expressed as 2−ΔCp. The following primer assays have been used : FOLH1 (Hs00379515_m1), HPRT1 (Hs02800695_m1), and TBP (Hs00427620_m1).Total RNA was isolated using the DIRECT-ZOL RNA MINIPREP  following the manufacturer’s instructions. cDNA synthesis was performed with 500 ng total RNA using the Superscript II RNase H Reverse Transcriptase kit . The quantitative real-time polymerase chain reaction (qPCR) was performed with GoTaq Probe qPCR Master Mix  and appropriate primers for 45 cycles on a LightCycler 480 instrument . The geometric mean of HPRT1 and TBP was used for normalization. The LightCycler® 480 Software, Version 1.5  was used for the determination of crossing point (Cp) values. ΔCp = CpCarl Gustav Carus Dresden database according to statutory provisions. The cohort contained 105 tissue specimens of 97 PCa patients undergoing TURP recruited from 2011 and 2020. Tissue blocks were cut in serial sections of 1–2 µm thickness; sections were deparaffinized with BenchMark XT  and then exposed to a heat-induced epitope retrieval. The PSMA  antibody  was used in a 1:50 dilution for staining, followed by counterstaining with hematoxylin, dehydration, and mounting of the slides. For PSMA, IHC was evaluated by the department of pathology Dresden using the following modified “quick-score” protocol: staining intensity was scored 0–4 . The percentage of positively stained cells was scored 0–4 . Both scores were multiplied to obtain an immunoreactivity score (IRS), ranging from 0–16.Patients’ samples were selected from the Tumor and Normal Tissue Bank of the University Cancer Center Dresden. The Ethics Committee approved the use of archived material of the Medical University of Dresden . Written consent was obtained from all patients and documented in the medical hospital t-test. Data are presented as mean ± s.e.m. to estimate the various means in multiple repeated experiments [p-values of ≤0.05 were considered statistically significant. All differences highlighted by asterisks were statistically significant as encoded in figure legends . All experiments have been performed in at least three biological replicates unless noted otherwisePrism 9.2  was used for statistical analyses. Differences between treatment groups were analyzed using ordinary a one-way ANOVA or Student’s eriments . p-value