Three interrelated themes formed an understanding of rehabilitation practice in an urban green rehabilitation context: “ambivalence due to contextual change”, “negotiating rehabilitation assumptions” and “expanding the frame of rehabilitation”. Expanding the frame of rehabilitation to an urban green context may provide a basis for enhancing compatibility to everyday life for people with disabilities and still accommodate structural quality standards of professional rehabilitation practice.Research points to the health benefits of rehabilitation in urban green spaces. Nevertheless, more studies indicate complexity of utilising urban green spaces in an established health system context. An understanding of challenges related to rehabilitation in urban green spaces remains unaddressed. Therefore, the aim was to describe and analyse people with disabilities’ and health professionals’ perceptions on combining traditional indoor rehabilitation practice with an urban green rehabilitation context. The interpretive description methodology was applied supplemented by Edgar Schein’s Model of Organisational Culture. Three online focus group interviews were conducted with people with disabilities ( Rehabilitation provides support to people with disabilities in obtaining optimal functioning in everyday life and improving life quality ,2. TradiTo address contemporary needs for developing urban green spaces for rehabilitation activities ,19 and eBy offering an additional avenue to rehabilitation, urban green spaces like the SPARK park may lead to a considerable change of longstanding practices, extending rehabilitation from the field of medicine to encompass urban green space interventions ,32 and hThis qualitative study used interpretive description  as the m1) artefacts and behaviours, the visible elements that one can see and feel thus marking the surface of the culture; (2) espoused values, the stated values, norms and rules of behaviour which are a less visible part of the culture; and (3) basic assumptions, the unwritten rules which are taken for granted as an acceptable way of perceiving the world, representing an underlying unconscious level of culture . Mo. Mo25]. The physical transformation of the park has run parallel to this study (2020–2021). In this period, the outdoor surroundings were temporarily turned into a construction site with less attractive conditions for rehabilitation activities. The opportunities for developing specific new solutions were thus temporarily limited, and alternative areas were used as temporary sites for rehabilitation in an urban green context. This study was conducted in collaboration with three future key users of the SPARK park: The Orthopaedic Rehabilitation Centre, the Neurological Rehabilitation Centre and the Dementia Activity and Rehabilitation Centre .n = 4) and health professionals (n = 10). A purposive sampling strategy  played a secondary role. There was a bunch of other benefits … And seeing how people improved…it made me change my attitude, you see.”“Physiotherapist, 40–50 yearsThe ambivalence expressed in this quotation between the meaningfulness of the individual and the quality of the rehabilitation practice appeared to cause critical perceptions among the health professionals. Although health professionals stressed practical and structural barriers  as the main challenges, these arguments seemed to be rooted in an underlying uncertainty related to the possible compromised quality of rehabilitation practices. A health professional with more than 15 years of professional experience shared an experience of how deep scepticism about rehabilitation in green spaces was gradually turned to reflective action:Although the quotation does not refer specifically to the SPARK park, the health professional used this retrospective experience to exemplify how building practice experiences over time may reduce professional uncertainty.I hadn’t thought about going outside would be considered exercise. I thought it was a bit special in the beginning. But I could see that after the first five minutes you feel ready because there’s a good atmosphere and it’s another way of exercising. It was definitely an eye-opener to me.”“Participant recovering from a back injury, <40 yearsParallel to the development process of the park, first movers among the health professionals initiated external adaptions to current practices, although scepticism among their colleagues persisted. For instance, they used the varied surfaces in the park for balance training, walked around the park to follow the transformation process or temporarily moved rehabilitation activities to other green spaces. Based on these first-mover initiatives, the persons who had previously attended rehabilitation—and thus shared their experiences from a retrospective point of view—expressed how they had been reluctant at first, doubting the effectiveness and overall purpose of including green spaces in their rehabilitation. Nevertheless, opposite the health professionals, the perceptions of people with disabilities reflected an immediate change in their experiences of rehabilitation. A participant who attended rehabilitation 1.5 years ago explained having changed the perception already after the first outdoor experience:This quotation illustrates how a change of context represented new opportunities to people attending rehabilitation. In general, people with disabilities expressed great confidence in the health professionals and the activities initiated and appeared to build their own experiences in continuation hereof.The arguments both for and against changes in the rehabilitation context appeared to be based on an artefact level of reasoning such as providing fresh air and light or an opportunity to get people outside and participate in organised activities involving physical artefacts such as a bonfire, a play field or a cross-fit area. As this artefact level of reasoning was not explicitly grounded in professional evaluation, it did not seem to constitute an evident imperative for change. To move beyond ambivalence and contrasting perceptions, negotiation of rehabilitation assumptions among people with disabilities and health professionals appeared a crucial step, bringing all three levels of culture into play.The group discussions exposed an underlying divide in rehabilitation assumptions. The people with disabilities related to everyday experiences and complex life situations, whereas the health professionals seemed to relate to health structures and traditional disciplinary reasoning. It appeared central to establish a common ground of rehabilitation assumptions.We are in a health professional world where measuring things is a top priority…our municipality is very much in favour of data-documented work. Well, it is not a lot of what we do outside that can be measured just like that. We are really stuck in this way of thinking!”“Occupational therapist, 40–50 yearsIn the negotiations taking place through discussing what is at stake combining traditional indoor rehabilitation practice with an urban green rehabilitation context, more health professionals defended the traditions of the current rehabilitation practice culture and longstanding disciplinary assumptions. As an example, physiotherapists work with physical activity and body functions and occupational therapists work with daily activities one-to-one. The rehabilitation assumptions reflected a test culture with a focus on measuring body functions and documenting progression. A health professional with more than 15 years of professional experience expressed being stuck in the current practice values:You have a lot of methods and so on in your work. But right outside the door, there will be a park where you can re-think your way of managing your practice. It’s a new opportunity of making the person in rehabilitation re-start, you know, after being ill … You have to go in front as professionals. It really means a lot that you take the first step. And that you dare to! To cross the barrier, I can hear you have. You all have a kind of barrier—is this beneficial or what? Will we be a little ridiculed for this? NO you won’t [be ridiculed]!”“Participant recovering from stroke, >50 yearsThis quotation illustrates how the health professionals continued to argue their case based on structural and disciplinary reasoning. In response, one of the participants who attended rehabilitation one year ago challenged the assumptions of the health professionals:There are many things I was not aware of…especially the part about challenging the established testing approach—that we have to document a result of the rehabilitation period they [the people in rehabilitation] have been through. Usually we make a 6-min walk test or a mini balance evaluation system test, and then think…we can’t really do that outside, yet.”“Physiotherapist, 40–50 yearsThis quotation exemplifies how rehabilitation assumptions of people with disabilities and health professionals were negotiated through critical reflection during group discussions. In contrast to the structured healthcare systems, people with disabilities related to their everyday experiences and complex life situations. Reflecting on what was (and still is) meaningful to the rehabilitation process, they capitalised on the importance of being guided on how to proceed with their daily life in addition to the support of the specific injury or illness. To negotiate and develop a common ground for rehabilitation in an urban green space, sharing reflections and exchanging knowledge between and across diverse contextual understandings appeared relevant. A health professional with more than 10 years of professional experience explained having developed a new consciousness about their own rehabilitation assumptions, based on the research presented and the shared discussion with the people attending rehabilitation:This quotation exemplifies how exposing underlying assumptions enabled health professionals to critically reflect upon perceived barriers to expanding rehabilitation practices to urban green spaces. By negotiating underlying assumptions, the perceptions of rehabilitation practices among people with disabilities and health professionals seemed to expand, and new possibilities of combining traditional indoor rehabilitation practice with an urban green rehabilitation context were discussed.Well, it shouldn’t be either or; it should be a combination of the two. Because we get something positive from both worlds … Sometimes we as physiotherapists want to focus and only think about the bodily functions and the physical part. But it is also about daring to come out and bring the whole person [attending rehabilitation] into play, you know. It may not just be about the body, but you get a lot of other things. And I believe, that is also what rehabilitation is about.”“Physiotherapist, 40–50 yearsExpanding the way of rehabilitation practices to encompass urban green spaces may pave the way for accommodating structural quality standards and still enhance the everyday compatibility to the lives of people with disabilities. A health professional with more than 10 years of professional experience highlighted the possibilities of adopting a combined approach:Across the participants, a combined approach was suggested: capitalising on the “best practices” of traditional indoor rehabilitation and building on the capacities in urban green spaces. During the construction phase of the SPARK park, basic elements influencing culture change could be extracted from the practice experiences of the first-mover initiatives already taking place. Four basic elements expanding the frame of the current rehabilitation practice culture were apparent: (1) engaging the whole life situation, (2) influencing role development, (3) enhancing everyday compatibility and (4) building inclusive social communities.The outdoor environment is everybody’s space. We work with the whole person and not just the body … And it may seem that we just play outside in the green area, but it all has a deeper meaning, and it also takes a lot to both be able to see—what is actually needed—and to take a step back and know when to assist and when you have to step back and allow the persons [in rehabilitation] to do it their own way; or to allow the participants to interact.”“Physiotherapist, >50 yearsEngaging the whole life situation was widely considered a key possibility of expanding current rehabilitation practices to urban green spaces. Both health professionals and the people attending rehabilitation highlighted how focus on the specific injury or body part was supplemented by a wider perspective. The roles of people with disabilities and health professionals seemed to develop in an empowering sense with influence from the urban green context. A first-mover health professional with more than 20 years of professional experience outlined how the green space could be used as a conscious strategy to support people with disabilities in taking on an active role in their rehabilitation:The transformation from rehabilitation or training to your everyday life has been really easy…in this way it has meant that we [he and his family] use the green areas in our neighbourhood more than ever before (laughing) … If you had only trained in a fitness centre or a gym, then you would probably have thought about the fitness centre when transferring it to your everyday life. And this can be difficult if you live far away [from a fitness centre or gym] or you, as it has been mentioned, don’t want to be part of a formal sports community or fitness centre.”“Participant recovering from a back injury, <40 yearsThis quotation exemplifies how the green space supported health professionals in targeting the rehabilitation to the individual needs of people with disabilities. By creating identifiable situations, challenges and feelings, rehabilitation in an urban green context may be a powerful means for the individual development of people with disabilities. The urban green space was perceived to be a neutral arena, enhancing the compatibility to everyday contexts of people with disabilities, and thus appeared a central element for changing the culture of rehabilitation practices. A participant who had just ended rehabilitation explained how the outdoor experiences enhanced compatibility to everyday life and provided additional opportunities for including one’s family in the sustained rehabilitation:The context and variety of opportunities introduced during rehabilitation seemed to have a considerable impact on what people with disabilities experienced as available and validated options for their sustained rehabilitation. The people attending rehabilitation perceived the future SPARK park as a possibility for expanding rehabilitation to being more supportive of the transitional phase in the rehabilitation process. By providing a basis for an inclusive environment, the SPARK park was highlighted as a future arena for building inclusive social communities operating in the intersection between formal rehabilitation centres, organised sports clubs and conventional community networks.In summary, the ambivalence due to contextual change, specifically expressed by health professionals, appeared to be rooted in an artefact level of reasoning. By negotiating rehabilitation assumptions of people with disabilities and health professionals, all three levels of culture  were brought into play and enabled shared perceptions to be formed. Expanding the frame of rehabilitation to urban green spaces seemed to provide a basis for enhancing compatibility to the everyday lives of people with disabilities and still comply with structural quality standards for rehabilitation.Expanding the frame of rehabilitation to urban green spaces is not simply a matter of moving the recreational activities from an exercise room to outdoor contexts. Based on the results of this research, rehabilitation in an urban green context may induce a shift in professional rehabilitation culture in order to progress.n = 231) employment of the restorative capacity of nature showed that “overstepping therapist–client boundaries” was perceived as a main barrier [In relation to the prospects of combining traditional indoor rehabilitation practice with an urban green rehabilitation context, role identity appeared both as a barrier and motive for action. The negotiation of rehabilitation assumptions of people with disabilities and health professionals may be grounded in contemporary discussions regarding professional–consumer relationships in rehabilitation ,3,46. In barrier . This ba barrier . In the In this research, the people with disabilities perceived rehabilitation in urban green spaces as a possibility for being guided on how to proceed with their daily life, in addition to the specific support of their injury or illness. Still, concerns of compromising the quality of rehabilitation practices were raised by the health professionals. Expanding rehabilitation to urban green spaces thus seems to give rise to reconsidering how the quality of rehabilitation may be obtained. To help people regain loss in functioning, or maintain functioning, it is essential to build empowering experiences that teach how to cope with and overcome possible barriers in public settings somehow comparable to the individuals’ everyday context ,48. BesiThe future work of expanding the frame of rehabilitation to urban green spaces may involve a fundamental change in or expansion of professional norms, values and practice culture. To understand change processes within health practices, the consideration of different levels of cultural influence on the organisation and groups involved has been suggested as beneficial . OrganisMoreover, it is critical to be able to document, assess and evaluate initiatives to promote sustainable change and ensure political and financial support . CentralThe credibility and trustworthiness of the findings were enhanced by providing a clear description of the analytical process elaborated in four iterative steps . All autThe application of the cultural analytical approach by Schein  providedThe fact that the first author had previously conducted an ethnographic fieldwork in the same practice field meant that pre-understandings were inevitable . HoweverThe composition of people with disabilities and health professionals in the FGIs created an interesting dynamic in the discussions, challenging each other’s perceptions and creating new reflections. The sample size is a limitation, as twice as many health professionals as people with disabilities participated. Due to the COVID-19 pandemic, we unsuccessfully tried to recruit a higher number of people attending rehabilitation. The imbalance in the sample may have impacted the results, as the reflections of health professionals may have dominated the topics discussed. Health professionals seemed to have more restrictive attitudes than people attending rehabilitation regarding future possibilities  within the SPARK park. Further, conducting online FGIs may have limited interaction between participants ,43. To eTo expand the frame of rehabilitation, future research would benefit from examining and developing current documentation practices and interventions adjusted to urban green spaces. Further, examining the perspective of leaders and managers regarding the expansion of the frame of rehabilitation is relevant in future research.This study takes the first steps to understand people with disabilities’ and health professionals’ perceptions on combining traditional indoor rehabilitation practice with an urban green rehabilitation context. Expanding the frame of rehabilitation to an urban green space context is not simply a matter of moving the recreational activities from an exercise room to outdoor contexts. In the present research, the health professionals expressed being stuck in current health structures and traditional disciplinary frameworks, which caused ambivalence. The expansion of rehabilitation to urban green spaces seems to potentially support health professionals in enabling people with disabilities to take an active part in rehabilitation. Based on the results, a combined approach is suggested: capitalising on the “best practices” of conventional indoor rehabilitation and building on the capacities in urban green spaces. Expanding the frame of rehabilitation to urban green spaces may provide a basis for enhancing compatibility to everyday life for people with disabilities and still accommodate structural quality standards of professional rehabilitation practice.Linking research results with the perceptions of people with disabilities and health professionals enabled the building of a mutual understanding and meaningful collaborations between researchers and the practice field to generate new forms of impact. The perceptions of people with disabilities and health professionals provided insights into their shared views on what needs to be considered as part of the future work of implementation.


G-1, a GPER1 agonist, was shown to inhibit the growth of castration-resistant mouse xenografts but not their parental androgen-dependent tumors. It is currently unknown how the androgen receptor (AR) represses GPER1 expression. Here, we found that two GPER1 mRNA variants (GPER1v2 and GPER1v4) were transcriptionally repressed, not via transcript destabilization, by the androgen-activated AR. Although no AR binding was found in all active promoters near GPER1, data from promoter assays suggested that both variants’ promoters were inhibited by androgen treatment. Site-directed mutagenesis on Sp1/Sp3 binding sites revealed their role in supporting the basal expression of GPER1. Knockdown of Sp1 and Sp3 together but not separately repressed GPER1 expression whereas overexpression of both Sp1 and Sp3 together was required to alleviate AR repression of GPER1. Based on the chromatin immunoprecipitation data, Sp3 was found to bind to the promoters prior to the binding of Sp1 and RNA polymerase II. However, the binding of all three transcription factors was inhibited by DHT treatment. Concordantly, DHT treatment induced nuclear interactions between AR and Sp1 or Sp3. Taken together, these results indicate that AR represses transcription of GPER1 by binding to Sp1 and Sp3 independently to prevent their transactivation of the GPER1 promoters. Therapies that target the activity of the androgen receptor (AR) remain central to the treatment of prostate cancer (PCa), as AR signaling is known to be a primary factor for driving PCa growth, progression, and metastasis . AlthougIn addition to targeting AR activity, it has long been known that estrogens are highly effective at treating advanced PCa . DespiteAR mediated gene repression includes both non-genomic and genomic mechanisms. Non-genomic mechanisms occur seconds to minutes after activation and include the modulation of protein kinase pathways (such as activation of Src ) and redIn the presence of androgens, the AR binds free floating (but not DNA bound) Sp1 as shown in gel shift assays, and this interaction requires the AR DNA binding domain , 29. SimThis study aimed to determine the mechanism of how AR represses GPER1 and thereby by-passes the tumor suppressive action of G-1 treatment in CRPC. Understanding this underlying mechanism would allow us to better design more effective strategies for targeting the AR in both primary PCa and CRPC patients.Since the GPER1 promoter has not been fully characterized yet, Rapid Amplification of cDNA Ends (RACE) experiment was performed on LNCaP cells to determine the transcription start sites (TSSs) . We found 1 TSS corresponding with GPER1 mRNA variants 2 and 3 (which share the same promoter) and 2 TSSs corresponding with variant 4. Variant specific primers (Supplementary Table 1) were only able to amplify variants 2 (GPER1v2) and 4 (GPER1v4), and both variants are encoded for the same protein. PCR reactions against all other GPER1 variants found no detectable expression (data not shown). To determine whether GPER1v2 or GPER1v4 were selectively repressed by DHT, two variant specific primers overlapping intronic regions were compared to a primer pair internal to the GPER1 exon which would effectively amplify all GPER1 variants . When trLNCaP, C4-2, and VCaP, all AR+ cell lines, were treated with 10 nM DHT for 24–48 hours to compare relative expression of all GPER1 transcripts. Androgen-mediated AR activity significantly repressed GPER1 mRNA levels in LNCaP (57%), C4-2 (46%), and VCaP cells (85%) after 24 hrs and even further by 78%, 63% and 95% respectively after 48 hrs . FurtherNuclear run-on assays confirmed that 10 nM DHT inhibited the rate of GPER1v2 and GPER1v4 transcription . In thisOur previous data  along wiTo determine if androgen treatment modulates the expression of Sp1 or Sp3, western blot analysis was performed on LNCaP, C4-2, and VCaP cells treated with DHT. We found that VCaP cells (which repress GPER1 mRNA expression to the highest degree of the tested cell types ) show a p = 0.0012). Of note, knockdown of Sp3 alone caused a reciprocal 92% increase in Sp1 protein levels  and/or Sp3 (pN3-Sp3). Overexpression of CMV-Sp1 or CMV-Sp3 led to a slight rise in basal GPER1 mRNA levels (12% and 13% respectively), although this was not statistically significant . No chanSeven putative Sp1/Sp3 binding sites were identified in the GPER1 proximal promoter regions . To see As there is no evidence of direct or indirect AR binding to the promoter of GPER1, we sought to determine whether the AR-mediated repression of GPER1 transcription occurred through AR interactions with Sp1 or Sp3 to reduce nuclear localization and/or decrease DNA binding potential. Nuclear and cytoplasmic co-Immunoprecipitation (CoIP) experiments were performed in LNCaP cells using the same treatment conditions as for the ChIP assay. Sp1  and Sp3 Here we report for the first time an instance of AR gene repression being mediated through both Sp1 and Sp3. In this study, the androgen bound AR directly represses GPER1 transcription, but not mRNAstability, by interacting with two essential transcription factors of GPER1, Sp1 and Sp3. The nuclear interactions between AR with Sp1 and Sp3 prevent them from binding to and activating transcription at the GPER1 promoters. As Sp1 and Sp3 are overexpressed in most cancer types , have beThe relationship between Sp1 and Sp3 is still in debate. While Sp3 has been shown to repress the transactivation potential of Sp1 depending on gene context , Sp3 hasSeveral studies have suggested that Sp3 is involved in promoting transcription in response hormones and growth factors; whereas a primary functional role of Sp1 is to drive basal transcription –55. Our AR mediated gene repression without DNA binding has previously been demonstrated to be mediated through Sp1 –29. To oExpanding our understanding of AR mediated gene repression will be paramount to developing future therapies against PCa. Newer generation ADT drugs such as enzalutamide and abiraterone have led to increased reports highlighting the essential roles of AR repressed genes in the development of treatment resistance. It appears that these drugs at least partly contribute to the development of hormone independent PCa by abolishing the AR mediated repression of oncogenes. For example, TGF-β, Cyclin B1, and Cyclin D1 are all repressed by the AR , 9, 28. As therapies that target the AR signaling axis remain central to PCa treatment, more details will emerge regarding the mechanisms of AR-mediated gene repression and their contribution to patient outcomes. With both oncogenes and tumor suppressors being repressed by the androgen activated AR, careful delineation of the impacted target genes will lead to more potent combination therapies that prevent CRPC development. Therefore, we examined AR-mediated repression of the newly described targetable tumor suppressor, GPER1. Our novel findings add Sp3 to the list of AR repressed TFs and highlight the importance of examining both Sp1 and Sp3 together to fully capture the scope of AR repressed genes. These results provide rationale for targeting GPER1 under ADT and provide the groundwork for discovery of additional AR repressed target gene.2. For androgen treatment, cells were switched to phenol-red-free media supplemented with 2.5% charcoal-stripped FBS (CSS)  unless indicated otherwise. DHT and R1881 were dissolved in DMSO as a vehicle and treatment was performed at a 1:10,000 dilution. 24–48 hrs after switching to CSS media, cells were treated with DHT daily or R1881 every other day. Cells were seeded onto poly-l-lysine coated plates for experiments as CSS media reduced cell adhesion.Human PC cell lines LNCaP, C4-2, and VCaP were obtained from the American Type Culture Collection  and recently authenticated . LNCaP c® Select Negative Control No. 1 Cat# 4390843, Silencer® Select siSp1 Cat# 4392420 ID: s13318, Silencer® Select siSp3 Cat# 4392420 ID: s13326, and Silencer® Select siSp4 Cat# 4392420 ID: s13329. The following plasmids were used from Addgene: pN3 Control Cat# 24544, pN3-Sp1 FL Cat# 24543, and pN3-Sp3 FL Cat# 24541.Manufacturer protocols were followed for each transfection reagent used. X-tremeGENE™ HP DNA Transfection Reagent (Cat# 6366236001) was used for plasmid transfections and X-tremeGENE 360 Transfection Reagent (Cat# 8724121001) was used for siRNA transfections. For all transfection assays, cells were transfected and switched to CSS media 6-hrs post transfection. After 48 hrs of incubation, the cells were treated with DHT or vehicle each day for 48 hrs. The following siRNAs were used from Invitrogen: Silencer2O RT-qPCR reactions for SYBR Green/primer contaminations. Calculation of relative fold changes was performed as in [All RNA extraction was performed with RNAzol RT (MRC Cat. # RN 190) following the manufacturer’ recommended protocol. Genomic DNA (gDNA)was removed with Promega’s RQ1 RNase-Free DNase (Cat. # M6101) following the manufacturer’s protocol. Reverse transcription was performed with Invitrogen’s SuperScript III following the manufacturer’s protocol with 1 μg of RNA input. RT-qPCR was performed on the ViiA 7 Real-Time PCR System in 384 well plates with SYBR SELECT Master Mix (Cat.#4472919) using primers validated by sequencing amplicons. All primer set reactions contained the following controls for each lot of RT-qPCR reactions:1) RNA input with no reverse transcriptase (gDNA contamination test), 2) no RNA input with reverse transcriptase (RNA contamination of buffers/reagents), and 3) Hed as in .After a 48 hr incubation CSS media, cells were treated with 100 nM R1881 or vehicle for 12 hrs before co-treatment with vehicle or R1881 and 10 μM actinomycin D. RNA was collected at various time points up to 24 hrs. Genes of interest were analyzed by RT-qPCR and normalized to the vehicle treated group before the addition of actinomycin D.Nuclear run-on RT-qPCR was performed as previously described . Newly sCells were rinsed 2 times with cold PBS. Protein was isolated with RIPA buffer (Invitrogen) supplemented with 5 μL of Protease Inhibitor Cocktail Set III, EDTA-Free – Calbiochem (Cat. # 539134) per 1 mL of RIPA buffer. Protein isolates were incubated on ice for 10 minutes, and then spun at 16,000 g’s for 10 minutes at 4°C to remove cell debris. Protein concentration was calculated by BCA assays, and 20–40 μg of protein lysates were run onto 10% polyacrylamide gels with SDS and transferred onto polyvinylidene fluoride (PVDF) membranes. Blocking and probing were performed in 5% milk dissolved in 0.2% Tween-20 PBS at 4°C ON. Blots were imaged by Odyssey CLx Imaging System by LI-COR Biosciences and analyzed in the Image Studio software Ver 5.2.The following Co-Immunoprecipitation protocol  was follChromatin immunoprecipitation (ChIP) was carried out as previously described . Antibod® Site-Directed Mutagenesis Kit. Transfections were 10% pCMV-β-Gal for normalization. 6 hrs post transfection, cells were to CSS media for 18 hrs before DHT treatment for 48 hrs. Cells were lysed with 100 μL 1X passive lysis buffer. Luciferase assay (Cat. # E2620) and β-Galactosidase Enzyme assay (Cat. # E2000) were performed per the manufacturer’s protocol using a Perkin Elmer Wallac 1420 VICTOR³™ and Biotek μQuant™ 96-well Microplate Spectrophotometer.gDNA regions of interest were amplified using primers referenced in Supplementary Table 1. Gene specific primers included both MluI and BglII sites on flanking sides for cloning. Amplicons were cloned into Promega’s pGL3 basic vector. Site-directed mutagenesis was carried out using primers indicated in Supplementary Table 1 with the Q5t test where applicable, and standard deviation was plotted as error bars. Levels of probability were noted. *=p < 0.05; **=p < 0.01; ***=p < 0.005; ****=p < 0.0001.Statistical significance of differences between the treatment groups was determined by an analysis of variance (ANOVA) or Student’s


Xanthine dehydrogenase (XDH) is a critical enzyme involved in the oxidative metabolism of purines, pterin and aldehydes and a central component of the innate immune system. However, the prognostic value of XDH in predicting tumor-infiltrating lymphocyte abundance, the immune response, and survival in different cancers, including hepatocellular carcinoma (HCC), is still unclear.XDH expression was analyzed in multiple databases, including Oncomine, the Tumor Immune Estimation Resource (TIMER), the Kaplan–Meier plotter database, the Gene Expression Profiling Interactive Analysis (GEPIA) database, and The Cancer Genome Atlas (TCGA). XDH-associated transcriptional profiles were detected with an mRNA array, and the levels of infiltrating immune cells were validated by immunohistochemistry (IHC) of HCC tissues. A predictive signature containing multiple XDH-associated immune genes was established using the Cox regression model.XDH mRNA expression was detected in human cancers originating from the liver, bladder, breast, colon, bile duct, kidney, and hematolymphoid system. The prognostic potential of XDH mRNA expression was also significant in certain other cancers, including HCC, breast cancer, kidney or bladder carcinoma, gastric cancer, mesothelioma, lung cancer, and ovarian cancer. In HCC, a low XDH mRNA level predicted poorer overall survival, disease-specific survival, disease-free survival, and progression-free survival. The prognostic value of XDH was independent of the clinical features of HCC patients. Indeed, XDH expression in HCC activated several immune-related pathways, including the T cell receptor, PI3K-AKT, and MAPK signaling pathways, which induced a cytotoxic immune response. Importantly, the microenvironment of XDHhigh HCC tumors contained abundant infiltrating CD8 + T cells but not exhausted T cells. A risk prediction signature based on multiple XDH-associated immune genes was revealed as an independent predictor in the TCGA liver cancer cohort.Decreased These findings suggest that XDH is a valuable prognostic biomarker in HCC and other cancers and indicate that it may function in tumor immunology. Loss of XDH expression may be an immune evasion mechanism for HCC.The online version contains supplementary material available at 10.1186/s12935-021-02173-7. Hepatocellular carcinoma (HCC) is the seventh most common tumor and the third most common cause of cancer-related death worldwide . PatientThe major risk factors for developing HCC include chronic hepatitis B or C virus infections, nonalcoholic steatohepatitis, and alcohol-related liver diseases . Indeed,Xanthine dehydrogenase (XDH) functions as a key regulator of purine metabolism , 13, infIn this study, we found that the expression of XDH has significant prognostic implications in different types of tumors, including HCC. Moreover, the XDH-associated signaling pathway may regulate the immune response and tumor-infiltrating immune cells in HCC. Importantly, the prognostic value of the XDH-associated immune signature in HCC shed light on XDH might modulate tumor immunity in HCC.XDH gene in various types of cancers (https://www.oncomine.org/resource/login.html) [http://kmplot.com/analysis/) [https://cistrome.shinyapps.io/timer/) [http://gepia.cancer-pku.cn/index.html) is an interactive website that includes data for 9736 tumors and 8587 normal samples from the TCGA and Genotype-Tissue Expression (GTEx) projects and analyzes RNA sequencing expression [The Oncomine database was used to determine the expression level of the in.html) . The scralysis/) . Hazard /timer/) . TIMER a/timer/)  to infer/timer/) . In addipression . GEPIA wAs in our previous report , MHCC-97The preparation of cell lysates and the western blotting procedure were carried out as previously indicated , 37. Bri2. Two pathologists who were blinded to the patient outcomes independently evaluated the immunohistochemistry (IHC) staining of each sample.The immunohistochemical staining procedure was conducted as previously described , 37, 38.https://immport.niaid.nih.gov). The Pearson coefficient between XDH and these immune genes was calculated, and an absolute value of coefficient > 0.3 with P < 0.05 was set as the identification criterion. Then, stepwise variable selection was performed with the Akaike information criterion in the Cox model [1x1 + β2x2 + … + βixi. In this formula, xi is the expression level of each gene, while βi is the risk coefficient of each gene derived from the Cox model [To develop an XDH-associated prognostic signature including multiple immune genes, a comprehensive list of immune-related genes was downloaded from the Immunology Database and Analysis Portal (ImmPort) database  and lung cancer in some datasets compared to that in normal tissues. The detailed results of XDH expression in different cancer types are summarized in Additional file XDH mRNA expression was significantly associated with breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), and rectum adenocarcinoma (READ), while XDH mRNA expression was higher in HNSC, lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC) tissues than in adjacent normal tissues  and cholangiocarcinoma (CHOL) tumor tissues and higher in esophageal carcinoma (ESCA) and uterine corpus endometrial carcinoma (UCEC) than in adjacent normal tissues. However, XDH mRNA expression was higher in bladder urothelial carcinoma (BLCA). In summary, a decrease in the level of XDH mRNA was detected in human cancers originating from the liver, bladder, breast, colon, bile duct, kidney, and hematolymphoid system.To profile oma Fig. A, while ues Fig. B. AdditiXDH mRNA expression on outcomes in patients with cancer, the correlations between XDH expression and OS prognosis and disease progression were evaluated using Kaplan–Meier plotter data based on Affymetrix microarrays. In HCC, patients with a high XDH mRNA expression level had significantly prolonged OS (hazard ratio (HR) 0.55, 95% confidence interval (CI) 0.38 to 0.78, P = 0.00072) and slower disease progression  HR 0.68, 95% CI 0.49 to 0.95, P = 0.024; progression-free survival (PFS) HR 0.69, 95% CI 0.5 to 0.0.94, P = 0.017; disease-specific survival (DSS) HR 0.53, 95% CI 0.33 to 0.83, P = 0.0045) than patients with a low XDH mRNA expression level   and in terms of DFS in LUSC. Together, these findings suggest that the level of expression of XDH mRNA has a significant impact on the prognosis of specific human cancers.To investigate the impact of XDH mRNA expression is associated with aggressive HCC phenotypes [XDH mRNA expression correlated with poorer OS and PFS in males , Asians , alcohol consumers , nonconsumers of alcohol , patients without vascular invasion  and patients without hepatitis viral infection . Additionally, low XDH mRNA expression correlated with worse OS and PFS in HCC patients with grade 3 disease . However, XDH mRNA expression was not significantly correlated with OS and PFS in stage 1 or stage 2 patients or patients with vascular invasion. Taken together, these results show the prognostic significance of XDH expression in HCC patients based on their clinical characteristics, particularly in those with advanced-stage HCC.We previously reported that decreased enotypes . In humaenotypes . TherefoRecently, several clinical trial results showed that immunotherapy is a promising therapy in HCC , 45. In To understand the mechanistic role of XDH, we performed in-depth analysis of the relationship between XDH expression and tumor-infiltrating immune cells. To profile XDH-related gene expression, XDH was overexpressed in an HCC cell line (MHCC-97H), and the cells were subjected to RNA array analysis. Differential gene expression analysis (overexpression group versus control group) was performed with the limma package. GO enrichment analysis showed that the immune response was upregulated by XDH Fig. A. GSEA cTo further study the prognostic value of XDH, 218 immune-related genes were identified to be significantly associated with XDH expression by Pearson correlation analysis. The protein–protein network generated with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) online server showed a tight correlation , LUAD, and thyroid carcinoma (THCA).In the present study, we first comprehensively analyzed the mRNA levels of We hypothesized that XDH could play a significant role in regulating tumor immunology and therefore influence the outcomes of cancer patients. Analysis of the TIMER database revealed that XDH expression correlated with the infiltration status of immune cells in several cancer types, including HCC. In HCC, a positive correlation was found between XDH expression and CD8 + T-cell infiltration in the analysis of both the TIMER database and IHC staining of immune cell markers in HCC tumor tissues. Although there was a significant positive correlation between XDH expression and macrophage infiltration in the TIMER database, only a positive trend was found between XDH expression and CD68 + macrophage infiltration in this small group of HCC tissues. Indeed, Saidak et al. indicated that XDH expression is linked to an immune infiltrate in tumors . XDH expThe development of well-verified signatures for cancer prognosis evaluation represents a critical step for the implementation of stratification strategies and personalized immunotherapy for cancer –61. In tOur study has some limitations to be improved. First, this study is mainly based on data retrieved from public databases. Some in vitro or in vivo experiments are necessary to validate the immune evasion mechanism by which XDH contributes to the progression of HCC. Second, the sample size for some specific tumor types including HCC was too small to form a solid conclusion. Hence, further studies are necessary to verify the role of XDH in the regulation of immune-related pathways in HCC.In summary, our results suggest that XDH is a potential independent prognostic biomarker for HCC. The XDH-associated cell signaling pathway may affect immune cell infiltration into the tumor microenvironment. In HCC, decreased XDH expression correlates with a reduced cytotoxic immune response, and our prognostic XDH-associated immune signature provides a valuable tool for precision therapy. These data may indicate that XDH plays a role in HCC tumor immunology via an immune evasion mechanism.Additional file 1:Figure S1. Correlation of xanthine dehydrogenase (XDH) expression with prognosis in 25 diverse types of cancer. Overall survival (OS) and disease-free survival (DFS) curves comparing the high and low XDH expression groups in the BLCA (A-B), BRCA (C-D), CESC (E-F), CHOL (G-H), COAD (I-J), ESCA (K-L), GBM (M-N), HNSC (O-P), KIRC (Q-R), KIRP (S-T), LIHC (U-V), LUSC (W-X), MESO (Y-Z), OV (AA-AB), PAAD (AC-AD), PRAD (AE-AF), READ (AG-AH), SARC (AI-AJ), SKCM (AK-AL), STAD (AM-AN), TGCT (AO-AP), THCA (AQ-AR), THYM (AS-AT), UCEC (AU-AV), and UCS (AW-AX) cohorts. Figure S2. Quantification of western blotting results in Fig. Figure S3. Representative images of CD3 (A), CD4 (B), CD8 (C), CD20 (D), CD68 (E), PD1 (F) and XDH (G) staining. Figure S4. Protein-protein network of 128 XDH-associated immune genes in LIHC produced by the STRING online server. Table S1. List of the primary antibodies and dilutions used in the study. Table S2. Antibody sources and staining conditions. Table S3. XDH expression in cancer tissues versus normal tissues in the bladder cancer, breast cancer, colorectal cancer, head and neck cancer, leukemia, liver cancer, lung cancer, and lymphoma datasets in the Oncomine database. Table S4. Correlation of XDH expression with immune infiltration levels in diverse types of cancer in the Tumor IMmune Estimation Resource (TIMER) database. Table S5. Functions of the genes included in the prognostic signature.Additional file 2: Immunostaining score.Additional file 3: WB quantification.


There are four subtypes of AR: A1, A2A, A2B, and A3. Positive allosteric modulators of the A1 AR have been proposed for the cure of pain. A3 positive allosteric modulators are thought to be beneficial during inflammatory processes. More recently, A2A and A2B AR allosteric modulators have also been disclosed. The A2B AR displays the lowest affinity for its endogenous ligand adenosine and is mainly activated as a consequence of tissue damage. The A2B AR activation has been found to play a crucial role in chronic obstructive pulmonary disease, in the protection of the heart from ischemic injury, and in the process of bone formation. In this context, allosteric modulators of the A2B AR may represent pharmacological tools useful to develop new therapeutic agents. Herein, we provide an up-to-date highlight of the recent findings and future perspectives in the field of orthosteric and allosteric A2B AR ligands. Furthermore, we compare the use of orthosteric ligands with positive and negative allosteric modulators for the management of different pathological conditions.The development of GPCR (G-coupled protein receptor) allosteric modulators has attracted increasing interest in the last decades. The use of allosteric modulators in therapy offers several advantages with respect to orthosteric ones, as they can fine-tune the tissue responses to the endogenous agonist. Since the discovery of the first A Thus, several efforts have been made to discover molecules acting on GPCRs that represent about one-fourth of marketed drugs approved in 2019 by the FDA . Among ttressors . Adenositressors .2B AR has emerged as a possible therapeutic target in several physio-pathological conditions, including asthma  site of the endogenous agonist adenosine challenging the design of selective agonists. The design and development of positive (PAMs) as well as negative (NAMs) allosteric modulators binding to a less conserved, and topographically distinct site have emerged as an attractive strategy. Allosteric modulators can modulate the effects of the endogenous ligand in its site of production evidencing their ability to be spatially and temporally more selective than the orthosteric ones  and Epac leading to the phosphorylation of cAMP response element-binding protein (CREB) and extracellular signal-regulated kinases (ERK) (2B AR is coupled with Gq11 that mediates the activation of Phospholipase C (PLC) to increase the levels of 1,4,5-inositol triphosphate (IP3)/diacylglycerol (DAG), leading to activation of Protein kinase C (PKC) and increase of Ca2+ levels (The coupling of the Aes (ERK) . Further+ levels   and pulmonary fibrosis : in fact, the administration of aerosolized BAY-60-6583 1 attenuates pulmonary edema and diminishes lung inflammation  as well as brown adipose tissue (BAT), and its gene deletion in SKM cells causes sarcopenia, diminishes muscle strength, and reduces energy expenditure  differentiation has been the focus of several researches  compounds to 1) offer a reliable overview of those drugs involved in both preclinical and clinical studies, and 2) briefly recapitulate the effects produced by a specific orthosteric or allosteric binding to the A2B AR.It is beyond the scope of this perspective to review the whole available literature concerning A2B AR agonists developed is the most limited among the ARs agonists, and no agonists for this AR subtype have yet entered clinical trials; nevertheless, some relevant A2B AR agonists have been proposed and subjected to preclinical studies. Among them, the most promising compound, the non-nucleoside agonist BAY-60-6583 1 . Quite surprisingly, the new compounds act as selective human A2B AR modulators in a stably transfected cell line. In particular, 6a,b and 7a behave as PAMs of the A2B AR in functional assay by increasing the efficacy but not the potency of the A2B AR agonists  in stimulating cAMP accumulation. These compounds have been deeply investigated using competitive and kinetic binding experiments resulting in the hypothesis that they favor the receptor active state without altering the orthosteric site. Compounds 7b,c and 8a,b act as NAMs of the A2B AR by decreasing both efficacy and potency of agonists. Similar to the PAMs, the activity of these compounds have been also investigated with binding experiments. They probably interfer with receptor-Gs protein coupling and, consequently, with the agonist functional response favoring the receptor uncoupled state  in promoting MSC differentiation to osteoblasts in vitro  suggest that the indole nucleus and N1-arylalkyl group do not represent key pharmacophoric elements for a compound acting as A2B AR PAM. In this study compound 9  promoting undesired side effects. Conversely, allosteric modulators may represent a more physiologic alternative to orthosteric agonist and antagonist as they promote site-specific and event-specific responses mainly in damaged tissues, where adenosine is massively released. Thus, the development of potent and selective A2B AR PAMs can open the way to a new application of AR ligand in clinical practice likely reducing the side effects with respect to those potentially caused by orthosteric agonists.Among the GPCRs, ARs have attracted considerable attention in drug developmemt. During the last fifty decades, several efforts have been made to develop small molecules acting as agonists, antagonists or allosteric enhancers of ARs. Most of these molecules failed in clinical trials and only three are currently approved for human use: adenosine, Regadenoson and Istradefylline. However, increasing evidence demonstrates the biological role played by ARs, particularly by the A


Stroke is a worldwide cause of disability; 40% of stroke survivors sustain cognitive impairments, most of them following inpatient rehabilitation at specialized clinical centers. Web-based cognitive rehabilitation tasks are extensively used in clinical settings. The impact of task execution depends on the ratio between the skills of the treated patient and the challenges imposed by the task itself. Thus, treatment personalization requires a trade-off between patients’ skills and task difficulties, which is still an open issue. In this study, we propose Elo ratings to support clinicians in tasks assignations and representing patients’ skills to optimize rehabilitation outcomes.This study aims to stratify patients with ischemic stroke at an early stage of rehabilitation into three levels according to their Elo rating; to show the relationships between the Elo rating levels, task difficulty levels, and rehabilitation outcomes; and to determine if the Elo rating obtained at early stages of rehabilitation is a significant predictor of rehabilitation outcomes.2 for linear regressions.The PlayerRatings R library was used to obtain the Elo rating for each patient. Working memory was assessed using the DIGITS subtest of the Barcelona test, and the Rey Auditory Verbal Memory Test (RAVLT) was used to assess verbal memory. Three subtests of RAVLT were used: RAVLT learning (RAVLT075), free-recall memory (RAVLT015), and recognition (RAVLT015R). Memory predictors were identified using forward stepwise selection to add covariates to the models, which were evaluated by assessing discrimination using the area under the receiver operating characteristic curve (AUC) for logistic regressions and adjusted RP<.001). The proportions of patients from the mid-Elo level that performed tasks at difficulty levels 1, 2, and 3 were 32.1% , 31.% , and 36.9% , respectively (P<.001), showing the highest match between skills (represented by Elo level) and task difficulties, considering the set of 38,177 task executions. Elo ratings were significant predictors in three of the four models and quasi-significant in the fourth. When predicting RAVLT075 and DIGITS at discharge, we obtained R2=0.54 and 0.43, respectively; meanwhile, we obtained AUC=0.73 (95% CI 0.64-0.82) and AUC=0.81 (95% CI 0.72-0.89) in RAVLT075 and DIGITS improvement predictions, respectively.Three Elo levels  with the same number of patients (n=96) in each Elo group were obtained using the 50 initial task executions  for N=288 adult patients consecutively admitted for inpatient rehabilitation in a clinical setting. The mid-Elo level showed the highest proportions of patients that improved in all four memory items: 56% (54/96) of them improved in DIGITS, 67% (64/96) in RAVLT075, 58% (56/96) in RAVLT015, and 53% (51/96) in RAVLT015R (Elo ratings can support clinicians in early rehabilitation stages in identifying cognitive profiles to be used for assigning task difficulty levels. Recent Cognitive rehabilitation  relies on brain plasticity to induce neuroplastic changes to compensate for cognitive impairments . Brain iOne of the most frequent cognitive problems reported by poststroke patients in their daily lives is related to memory loss ,6. To daNew strategies for providing cognitive rehabilitation services are constantly required and are continuously being integrated into clinical practice . One sucA typical cognitive rehabilitation program mainly provides exercises that require repetitive use of the impaired cognitive system in a progressively more demanding  sequenceFurthermore, prediction of specific outcomes after stroke rehabilitation is used by clinicians to improve the accuracy of prognoses, set attainable goals, reach shared decisions, personalize rehabilitation plans, and inform patients and relatives .In this study, we propose the application of Elo ratings to provide clinicians with a ranking of patients at an early stage of cognitive rehabilitation by using the results of web-based cognitive rehabilitation tasks. We hypothesize that (1) such ranking of patients will allow clinicians to match patient’s skills with task difficulties, thereby enabling better treatment personalization, and (2) such a rating will be a significant predictor of patients’ outcomes for memory cognitive function. The original proposal of the Elo rating system was designed to rate chess players, and the rating system was named after its creator Arpad Elo .The Elo system works as follows: an initial rating is assigned to each player every time a player plays a match. This rating is updated for both players depending on the result of the match. If the winner is the player with the higher rating, the update is small, and it is larger depending on how unexpected the victory is, according to their previous ratings .The basic Elo rating system is used in several types of contests beyond chess, for example, football ; howeverNevertheless, to the best of our knowledge, Elo ratings have not been applied in cognitive rehabilitation in general or in the specific use–case of a web-based application where patients perform web-based cognitive tasks during their rehabilitation period.In this study, we propose that instead of considering matches between, for example, chess players, we consider matches between patients and web-based cognitive rehabilitation tasks.The aims of this study are (1) to demonstrate the feasibility of the approach by presenting a synthetic data set where we obtain an Elo rating for each patient by considering each execution of a cognitive rehabilitation task by the patient as a match between the patient and the task; (2) to obtain the Elo rating of each patient in a real rehabilitation setting where adult patients with ischemic stroke follow cognitive rehabilitation by executing web-based rehabilitation tasks and use these Elo ratings to perform a stratification of patients into 3 groups according to their Elo rating ; (3) to analyze the relationship among the three Elo rating levels and the proportion of tasks executed at three increasing difficulty levels  with the rehabilitation outcomes in the memory cognitive function; and (4) to develop and internally validate four predictive models for auditory verbal learning memory and working memory outcomes using Elo ratings obtained at early stages of rehabilitation as independent variables and state-of-the-art variables . The first two models are developed for predicting auditory verbal learning memory and working memory at discharge and the other two for predicting improvements in auditory verbal learning memory and working memory at discharge.The setting was the inpatient acquired brain injury rehabilitation unit of the Institut Guttmann hospital, a specialized clinical center certified in quality of care and patient safety . The initial study population consisted of 344 patients with ischemic stroke who were consecutively admitted for inpatient rehabilitation from March 2009 to September 2019. Patients were included in the study if they had been admitted within 180 days of the onset of an ischemic stroke. Patients who were admitted >180 days after a stroke , who had no cognitive assessment within a week after stroke rehabilitation admission , or had missing data  were excluded. Therefore, 83.7% (288/344) of the patients were available for analysis. Patients with aphasia were not included in the n=344 initial sample as they follow a different rehabilitation protocol involving a different set of cognitive assessments and, therefore, need to be analyzed separately (in future work).At admission, each patient was assigned a physician who coordinated the rehabilitation team . Therefore, admission and discharge cognitive assessments  were systematically recorded in the electronic health records of the hospital. The authors confirm that this study is compliant with the Helsinki Declaration of 1975, as revised in 2008, and it was approved by the Ethics Committee of Clinical Research of Institut Guttmann.The participants were anonymized and nonidentifiable. A specific written informed consent was not required for participants to be included in this study; nevertheless, at admission to Institut Guttmann, participants provided written informed consent to be included in research studies addressed by the Institut Guttmann hospital.The Guttmann, NeuroPersonalTrainer web-based cognitive rehabilitation platform used in this study comprises a set of 149 different web-based cognitive rehabilitation tasks. There is no established previous order in which patients should execute such tasks. Therefore, every patient executed  a different subset of them in a different order during their rehabilitation process, taking between 2 and 6 months, distributed over two to five sessions a week. During each session, the patient executed between 4 to 10 cognitive rehabilitation tasks, and the total duration of one session ranged between 45 minutes to 1 hour. Each task mainly addressed one of the following functions: memory, executive functioning, attention, gnosias, calculus, orientation, language, and social cognition. Immediately after each execution of a task, the patient received a feedback on performance , with 0% being the lowest level of compliance and 100% being the highest.Before starting web-based cognitive rehabilitation using the Guttmann, NeuroPersonalTrainer platform, every patient was assessed once using standardized tests specifically validated for the population under study. Specific linguistic abilities were assessed using three subtests of the Barcelona test ,33: (1) In this study , we assessed improvements in the memory cognitive function using the Rey Auditory Verbal Memory Test (RAVLT)  and the The DIGITS subtest (direct version) of the Barcelona test addresses working memory, and the patient was asked to repeat a series of numbers of variable lengths (3-9) until they failed in two consecutive series, reporting the largest series before failure .iθ for each player i, the result of a match between players i and j is represented by Rij∈{0,1}.The Elo rating system  is formai wins:The actual ratings of each player are used to estimate the probability that player which is used to update the ratings as follows, based on the Bradley-Terry model :iθ changes, with large K values resulting in iθ changing quickly and small K values resulting in iθ changing slowly. In this study, we considered three extensions to the original Elo system: Glicko [i, the patient, and player j, the cognitive rehabilitation task.where K is a constant parameter that controls how quickly : Glicko , Glicko-: Glicko , and Ste: Glicko . Glicko : Glicko  being in2 and adjusted R2 for linear regressions. The variance inflation factor and tolerance (1/variance inflation factor) were used to test the multicollinearity of independent variables (tolerance ≤0.40 indicates a multicollinearity problem) [P=.05.Demographic and clinical state-of-the-art variables such as age, gender, marital status, and variables related to the rehabilitation program, such as the time in between the onset of stroke and initiation of the rehabilitation program or length of stay, were considered as candidate predictors. Categorical variables were dichotomized: female=0, male=1; low level of education=0, high level of education=1 (depending on the number of years of education); married=1, not married=0. Forward stepwise selection was used to add covariates to the models, which were evaluated by assessing discrimination using area under the receiver operating characteristic curve (AUC), accuracy, sensitivity, and specificity for logistic regressions and to maximize Rproblem) . The Durproblem) ). The Elproblem) . R v3.5.In linear regressions, the dependent variables were RAVLT075 and DIGITS at discharge. In logistic regressions, the aim was to predict improvement in RAVLT075  and improvement in DIGITS .The mean age at the time of the lesion was 51 (SD 9) years. The proportion of participants aged <65 years was 93.8% (270/288) . In relation to sex, in our data set, the proportion was 67.7% (195/288) men and 32.3% (93/288) women, which seems to suggest a bias in favor of men. Nevertheless, it somehow reflects reality in the general population, where the proportion of men experiencing ischemic stroke is larger than that of women -45; howeWe initially ran the four different Elo rating approaches  in a reduced data set of 20 patients, each of whom executed the same task 20 times. Two screenshots of the selected task are presented in The included 20 patients were stratified into three categories according to their compliance in the maze task as follows:low compliance={id1, id2, id3, id4, id5, id6};mid compliance={id7, id8, id9, id10, id11, id12, id13};high compliance={id14, id15, id16, id17, id18, id19, id20}.We then ran the four Elo rating systems with default values for the initial ratings and K. We considered that when a patient gets a result >50%, they win the match against the maze; however, if their result is <50%, the maze wins. The ratings obtained using the Glicko approach are presented in Web-Based Cognitive Rehabilitation System, the Guttmann, NeuroPersonalTrainer cognitive platform includes 149 different web-based tasks. There is no established previous order or frequency in which patients should execute such tasks; therefore, in this section, we analyze task execution frequencies. As shown in Table S1 , according to their Elo ratings . When the Elo rating feature is excluded from model 1, it explains 52% of the variance, and when it is excluded from model 2, the resulting model explains 42%.Dependent Variables section; 50.6% (146/288) of patients improved in RAVLT075, and 34% (98/288) of patients improved in DIGITS. We used the same Elo ratings as described in the Ranking Patients Using the Initial 50 Task Executions: Elo Rating section. Model 3 yielded an AUC of 0.73 (95% CI 0.64-0.82) for improvement in RAVLT075, with an accuracy=0.64 (95% CI 0.54-0.72), specificity=0.55, and sensitivity=0.73. Model 4 yielded an AUC of 0.81 (95% CI 0.72-0.89) for improvement in DIGITS, with an accuracy=0.73 (95% CI 0.64-0.81), specificity=0.22 and sensitivity=0.97. Models 3 and 4 are detailed in To the best of our knowledge, in this study, Elo ratings were applied in the context of web-based cognitive rehabilitation tasks for the first time. We demonstrated the feasibility of using Elo ratings by using a publicly available R library (PlayerRatings)  on a synWe then obtained the Elo ratings for each patient in a real rehabilitation setting where 288 adult patients with ischemic stroke followed cognitive rehabilitation, executing 68 different web-based rehabilitation tasks 38,177 times. We then performed a stratification of the patients into 3 groups (96 patients each) according to their Elo rating . We have shown the relationships among the three Elo rating levels and the proportion of tasks executed at three increasing difficulty levels  with the rehabilitation outcomes in the memory cognitive function. We then developed four predictive models, where the Elo rating variables were significant in three of them (and quasi-significant in the fourth) for auditory verbal learning memory and working memory outcomes. We found that including Elo ratings as independent variables increased the model performance (for both linear and logistic regressions).Several web-based cognitive rehabilitation platforms integrate some kind of stratification of patients as an initial step for treatment personalization. The web-based platform used in this study integrates an automatic therapy planning functionality—the intelligent therapy assistant (ITA) . The ITAObtaining an initial Elo rating for each patient is a simple process (in terms of both implementation and interpretation of results). As remarked in previous research, for example, in the field of educational tutoring systems, Elo rating use is encouraged because of its simplicity . As showTherapists can then use the Elo rating to assign the patient to a skill level. In this study, in Another clinical implication was noted on in a recent systematic review on computerized cognitive training . The revSeveral limitations to the study need to be highlighted. First, we conducted a single-center study, an advantage of which is that data were obtained and included by clinicians trained in neurological rehabilitation, and all patients were managed under the same stroke rehabilitation protocols. The Guttmann, NeuroPersonalTrainer platform has already been integrated into the clinical practice of several acquired brain injury centers; nevertheless, their patients were not included in this analysis. A multicenter stroke study may include an initial preprocessing phase, wherein patients are grouped according to their initial National Institutes of Health Stroke Scale severity to avoid additional heterogeneity. Thereafter, Elo rating techniques, such as those proposed in this study, maybe applied within such groups. External validation assessments common to all participating centers are also an important aspect to be addressed in this future multicenter study. Second, the studied health area belongs mainly to the urban population, with a small rural population or populations from other regions. Third, our analysis lacked computerized tomography or magnetic resonance imaging examinations that describe the presence of contusion, hematoma, hemorrhage, ischemia, or other signs of parenchymal lesions in the frontal, temporal, parietal, occipital, and cerebellar lobes or diffuse axonal injury.Fourth, our sample did not include any patients with missing data. All data used as inputs were complete. Fifth, our analysis did not include indicators of mental health or other comorbidities. Persons who experience a stroke may have one or more preexisting medical comorbidities at the time of injury . Therefore, we plan to include comorbidity analyses in future research studies. Sixth, in all our Elo rating calculations, we used the default value for the K constant. Several approaches to K optimization have been reported, such as hill climbing, gradient descendent, or Bayesian , which cCluster analysis has been extensively proposed in previous research to address heterogeneity in patients with acquired brain injury -51 and aIn a recent study, Faria et al  presenteWe have shown the feasibility of Elo ratings for identifying patients’ profiles at the early stages of cognitive rehabilitation in a real clinical setting. Elo ratings can be used to match skills with task difficulties, aiming to maximize improvements in specific cognitive functions. Such Elo ratings are also significant in predicting cognitive outcomes. Elo ratings increased the models’ performance (for both linear and logistic regressions). Generalization of the use of Elo ratings beyond patients with stroke to any other population with acquired brain injury requiring cognitive rehabilitation in any web-based platform is straightforward because of the simplicity of existing open-access Elo rating implementations.


Arabidopsis, which are involved in the occurrence and regulation of autophagy. Among these, 17 proteins are related to resistance against plant pathogens. The transcription coactivator non-expressor of pathogenesis-related genes 1 (NPR1) is involved in innate immunity and acquired resistance in plants, which regulates most salicylic acid (SA)-responsive genes. This paper mainly summarizes the role of ATGs and NPR1 in plant immunity and the advancement of research on ATGs in NPR1 metabolism, providing a new idea for exploring the relationship between ATGs and NPR1.Autophagy is an important pathway of degrading excess and abnormal proteins and organelles through their engulfment into autophagosomes that subsequently fuse with the vacuole. Autophagy-related genes (ATGs) are essential for the formation of autophagosomes. To date, about 35 ATGs have been identified in  R genes), these proteins can directly or indirectly recognize the effectors secreted by pathogenic microorganisms. This process is known as effector-triggered immunity (ETI) that usually leads to local programmed cell death (PCD) called hypersensitive response (HR) [R genes are highly expressed during pathogen infection, most of them encode the nucleotide-binding (NB) domain and Leu-rich repeat (LRR)-containing (NLR) proteins that recognize pathogen effectors and activate ETI, which usually leads to the accumulation of reactive oxygen species (ROS) and HR. Based on the N-terminal structures, NLR proteins can be classified into two categories. TIR-NLR (TNLs) contain the toll/interlcukin-1-reccptor (TIR) region and CC-NLR (CNLs) contain coiled coil (CC) domain [Plants have evolved a complex immune system to combat the threat from pathogenic microorganisms in nature, including innate and acquired immunity ,2,3. It nse (HR) ,8. R gen) domain ,13,14,15Arabidopsis mutants, fls2/efr/cerk1 (fec) and bak1/bkk1/cerk1 (bbc) mutants, ETI induced by Pst DC3000 (avrRpt2) was severely impaired [The latest studies have clarified the new mechanism of crosstalk and cooperation between PTI and ETI, they activate many pathways that are closely related to each other and activate plant immune signaling pathways ,17,18. Epathogenesis-related (PR) genes [Plant system acquired resistance (SAR) can be activated by local defense response, which emits chemical signals to alert neighboring cells and tissues and protect the whole organism ,21,22,23R) genes ,30,31. RR) genes ,33.ATGs) that strictly regulate this membrane trafficking process have been identified in yeast [Arabidopsis, many genes with sequence similarity to the yeast ATGs have been identified. Current information from Arabidopsis database TAIR  and related literature showed that about 35 ATGs have been identified. Except for ATG14/29/31, other homologous genes of ATGs have been found in yeast [Autophagy is an evolutionary conserved intracellular regulatory mechanism, involving the degradation and recycling of intracellular proteins, metabolites, and organelles. One of its main characteristics is the formation of double-membrane vesicles, known as autophagosomes, which engulf a portion of cytoplasm and transport it into vacuoles for degradation ,35,36,37in yeast . In Arabin yeast . The evoin yeast ,41,42,43Pto DC3000 hrcC, HopF3 targeted ATG8 to suppress autophagy. Although the interactions between ATG1, ATG7, ATG12, and several effectors were found in this study, the exact mechanism of these interactions in plant disease resistance is unclear [ATGs knockout mutations exhibited enhanced susceptibility to pathogen infection, such as atg2, atg5, atg6, atg7, atg9, atg10, and atg18 [atg2 mutants displayed less HR-PCD and ATG4, ATG5 inhibited the occurrence of HR-PCD, ATG6 antisense plants displayed enhanced HR-PCD during pathogen infection [In recent years, great progress has been made in the identification of ATGs and the study of autophagy pathways. Some of these gene knockout mutations revealed the physiological role of autophagy in nutritional stress (nitrogen and carbon deficiency) and senescence ,45,46. I unclear . Some ofnd atg18 ,58,59,60nfection ,58,59,61nfection . Here, wThe transcription coactivator non-expressor of pathogenesis-related genes 1 (NPR1) is a key regulatory factor of SAR, which regulates most SA-responsive genes ,64,65,66PR gene expression by interacting with TGA in the nucleus and plays an important role in regulating the PRs protein downstream [PR1 expression in the SA signaling pathway [NPR1 is a master regulator of plant resistance to pathogen stress, which confers immunity through multiple transcription factors ,76,77. Rwnstream ,80,81. Twnstream . Recent  pathway ,84.A new study found that the formation of SA-induced NPR1 condensates (SINCs) is mediated by conserved cysteine clusters in intrinsic disorder regions (IDRs) of NPR1 protein. SINCs are rich in stress-responsive proteins, including NB-NLR receptors, oxidative and DNA damage-responsive proteins, and ubiquitination-related proteins. In addition, SINCs are required to form functional NPR1-Cullin 3 RING E3 ligase (CRL3) complex in the cytoplasm. NPR1-CRL3 complex can ubiquitinate and degrade EDS1 and some important ETI regulatory factors such as WRKY transcription factors, thereby promoting cell survival in ETI .Arabidopsis, the NPR family consists of NPR1 and five NPR1-like genes, named NPR1-like 2 (NPR2), NPR3, NPR4, BLADE-ON-PETIOLE2 , and BOP1 (NPR6) [NPR family contains a set of highly conserved cysteine residues that are thought to be involved in redox control [d = 223.1 ± 38.85 nM) and NPR3 (Kd = 176.7 ± 28.31 nM) binding to SA with similar affinity. However, the affinity of NPR4 (Kd = 23.54 ± 2.743 nM) with SA is much higher [R-Avr) gene interaction [ATGs and the protein concentrations of ATG7 and ATG8a-PE were lower in npr3/npr4 mutants than in the wild-type. NPR3 and NPR4 may regulate the production of autophagosomes by promoting two ubiquitin-like conjugated systems [In 1 (NPR6) ,87,88,89 control . It was h higher . Under nh higher ,91,92. Dh higher ,92. At th higher . In addiPathogen infection causes accumulation of SA thus leads to post-translational modification of NPR1, allowing it to enter into the nucleus. NPR1 is recruited to Cullin3 (CUL3) for ubiquitination and subsequent degradation, this process requires phosphorylation of NPR1 at residues Ser11 and Ser15 ,94,95,96Some studies have found that the plant hormones abscisic acid (ABA) and SA antagonistically affect the level of NPR1 in cells. ABA promotes NPR1 degradation through the proteasome pathway mediated by the CUL3-NPR3/NPR4 complex, while SA protects NPR1 from ABA-induced degradation through phosphorylation ,99,100. ATGs expression. NPR1 inhibited the mRNA expression of ATG1, ATG6, and ATG8a during the early HR induced by Psm ES4326/AvrRpt2 [atg2 [atg5 mutants but could not induce senescence and cell death in atg5 npr1 double mutants, indicating that the cell death phenotype in atg5 mutants depended on NPR1 under SA induction [Studies have found that NPR1 regulates /AvrRpt2 . SA anal/AvrRpt2 . In addit2 [atg2 . Yoshimonduction . Our prenduction . In receATGs affect NPR1 transformation from dimer to monomer? What are the effects of different ATGs on NPR1 entering the nucleus? What is the relationship between ATGs and NPR1 regulation of the HR-PCD response? Does autophagy and 26S proteasome co-regulate NPR1 turnover? An in-depth study of these issues will help us to understand how the autophagy pathway participates in the regulation of NPR1 metabolism. A recent study showed that the protein expression of NPR1 was significantly higher in atg4a4b than that in wild type under normal condition and the expression of NPR1 in atg4a4b was higher than that in wild type under avrRpt2 treatment [Autophagy-mediated degradation of proteins and organelles is essential for plant growth, development, maintenance of cell homeostasis, and immune response ,49,50,51reatment . Based o


Despite androgen dependence in a majority of castration-resistant prostate cancers, some cancer cells are independent of androgen receptor (AR) function, a feature of heterogeneity in prostate cancer. One of the aggressive variants of prostate cancer that are AR independent is neuroendocrine prostate cancer (NEPC). This manuscript will focus on the new finding of human one cut domain family member 2 (ONECUT2) transcription factor and its role in castration resistance, especially in NEPC. Given its dependence on the AR axis, prostate adenocarcinoma can respond to androgen deprivation therapy (ADT) for a variable duration, but eventually, it progresses to metastatic castration-resistant prostate cancer (mCRPC). Most recently, the newer generation of androgen receptor (AR) signaling inhibitors such as enzalutamide and apalutamide has improved outcomes in patients with mCRPC, but they inevitably develop resistance to these drugs as well. There are several proposed mechanisms of resistance to androgen deprivation or AR inhibitors, many of which are thought to be AR-dependent, including AR-activating mutations and constitutively active AR splice variants. However, there are also AR-independent mechanisms, with very low or absent AR expression found in tumor cells that render ADT ineffective and are associated with neuroendocrine (NE) differentiation. NEPC is an aggressive variant of prostate cancer that exhibits not only AR independence but also neuroendocrine (NE) differentiation and even distinct histological features such as small cell carcinoma instead of adenocarcinoma,3. Though NEPC can rarely arise de novo, more commonly, it arises from adenocarcinoma in response to selection pressure for AR-independent cells from treatment with AR signaling inhibitors via lineage plasticity and NE differentiation,4. Recent studies spearheaded by Rotinen et al. and Guo et al. showed that ONECUT2 is an important regulator of AR-mediated growth and a driver of NE differentiation in transition from adenocarcinoma to NEPC, and is being investigated as a new drug target with potential therapeutic implications. This manuscript explains how ONECUT2 leads to AR independence and NE differentiation observed in NEPC.Prostate cancer is the most common cancer diagnosed in men and the second leading cause of cancer-related deaths in the United States. In particular, ONECUT1 plays an important role in hepatobiliary tract disease by regulating the development, differentiation, and function of hepatocytes and cholangiocytes. Decreased expression of ONECUT1 and its target genes is associated with malformation of the liver, bile duct, gallbladder, and pancreas, as well as diabetes. Furthermore, ONECUT1 may also play a role in the prevention of hepatocellular carcinoma and pancreatic cancer by acting as a tumor suppressor. In addition, ONECUT1 also helps regulate the development of retina and motor neuron.ONECUT is a family of transcription factors related to hepatic nuclear factor 6, which has been shown to be involved in organogenesis, cell fate, and tumorigenesiset al. in 1999. ONECUT2 is found in various organs, including the liver, skin, brain, testis, and bladder. In contrast to ONECUT1, ONECUT2 expression has been shown to be elevated in multiple different cancers, including prostate cancer, ovarian cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, lung adenocarcinoma, and neuroendocrine tumors. Specifically in prostate cancer, increased ONECUT2 expression has been linked to the aggressiveness of the disease, disease progression, biochemical recurrence, and metastasis,6. ONECUT2 mRNA level was found to be elevated in mCRPC prior to any treatment and was higher than in non-metastatic tumors. ONECUT2 target genes are involved in the cell cycle, angiogenesis, and hypoxia, which in turn are implicated in tumor growth and metastasis in prostate cancer.ONECUT2 is another transcription factor that regulates cell proliferation, migration, and differentiation, which was first discovered by Jacquemin Both ONECUT1 and ONECUT2 are transcriptional activators of the ONECUT family with similar but distinct functions in development and pathogenesis in various organs. Similarities and differences between ONECUT1 and ONECUT 2 are outlined in et al. showed that ONECUT2 expression is negatively correlated to AR activity. AR activity was significantly lower in mCRPC tumors with high ONECUT2 expression, whereas in tumors with high AR activity, the ONECUT2 expression was suppressed. ONECUT2 directly suppressed genes regulated by AR, including kallikrein-related peptidase 3 (KLK3)/prostate-specific antigen, kallikrein-related peptidase 2 (KLK2), and ETS homologous factor. Altogether, these findings show that ONECUT2 leads to AR independence in prostate cancer. Tumors with high ONECUT2 expression such as NEPC represent a variant group of mCRPC that is independent of AR function.Rotinen . ONECUT2 has been shown to play a role in NE differentiation in prostate cancer. A significantly higher level of ONECUT2 expression was found in NEPC compared to adenocarcinoma, and conversely, a reduction in ONECUT2 expression was shown to decrease NE marker gene expression. Deletion of TP53 and Rb1, two of the most frequently mutated genes in NEPC, was shown to increase ONECUT2 expression and promote NE plasticity in prostate adenocarcinoma. ONECUT2 upregulates genes involved in NE differentiation, such as neuron-specific enolase, synaptophysin, and chromogranin A. Furthermore, ONECUT2 has complex interactions with modulators of NE differentiation such as RE1-silencing transcription factor (REST), forkhead box A1 (FOXA1), and paternally expressed gene 10 (PEG10),14,15. REST is an inhibitory regulator of NE differentiation that directly suppresses ONECUT2. A decrease in REST expression leads to upregulation of ONECUT2 mRNA and allows for the transition from adenocarcinoma to NEPC. FOXA1, another modulator that normally inhibits NE differentiation, is suppressed by ONECUT2 during transdifferentiation of adenocarcinoma into NEPC. PEG10 is different from REST and FOXA1 in that it promotes NE differentiation and is directly suppressed by AR,15. An increase in ONECUT2 expression corresponds to upregulation of PEG10 and transition from adenocarcinoma to NEPC. NE differentiation in prostate cancer is associated with a more aggressive phenotype, metastatic disease, and poor response to AR signaling inhibitorset al. demonstrated that hypoxia can induce NE differentiation and disease progression in prostate cancer, evidenced by an increase in NE marker gene expression in hypoxic conditions compared to normoxic ones. Knockdown of hypoxia-inducible factor 1α (HIF1α), a transcription factor that regulates the hypoxia signaling pathway, led to a reduction in NE marker gene expression. Guo et al. also showed that ONECUT2 is involved in regulating the hypoxia pathway, with ONECUT2 activity correlating with tumor hypoxia in the progression from adenocarcinoma to NEPC. ONECUT2 upregulates hypoxia-associated genes such as adrenomedullin and angiopoietin-like 4 by activating SMAD family member 3, which interacts with HIF1α in regulating hypoxia signaling , and metastatic growth.Following the discovery of ONECUT2 and its role in the transition from prostate adenocarcinoma to NEPC, new drugs targeting this pathway are being developed and studied in clinical trials. CSRM617, a small molecule inhibitor of ONECUT2 developed by Rotinen . TH-302 was shown to significantly inhibit tumor growth in NEPC, but less so in adenocarcinoma. There was a positive correlation between the level of ONECUT2 expression and the treatment response to TH-302. These findings show that NEPC and a higher level of ONECUT2 expression are both associated with a greater degree of hypoxia, which in turn lead to an enhanced response to TH-302. TH-302 is being studied in clinical trials, including a phase I/II clinical trial (NCT00743379) that focuses on the efficacy of TH-302 in combination with chemotherapy such as docetaxel in multiple solid tumors, including mCRPC that were not previously treated with chemotherapy. Another phase I study (NCT03098160) examines the safety and toxicity of TH-302 in combination with ipilimumab in advanced solid malignancies, including mCRPC.TH-302 (evofosfamide) is a hypoxia-activated prodrug with an alkylating agent moiety that is released only in hypoxic environments, such as those found in hypoxic tumors like NEPC. Treatment of prostate cancer with AR inhibition may lead to selection for cancer cells with elevated ONECUT2 expression, thereby contributing to resistance to AR-targeted therapy and development of NE features that lead to more aggressive phenotypes with worse clinical outcomes,3,5. It is, therefore, crucial to continue further research on the understanding of the master regulators of AR independence such as ONECUT2 and develop safe and efficacious drugs against such targets. We believe biomarker-driven correlative studies are key for the success of future drug development targeting ONECUT2.Given the rising use of newer generation AR signaling inhibitors, mCRPC exhibiting AR independence will likely grow in prevalence