Prostate cancer (PCa) is a major cause of cancer morbidity and mortality for men globally, and androgen signaling clearly drives its onset and progression. Androgen receptor (AR) regulation is complex and remains elusive, despite several studies tackling these issues. Therefore, elucidating the mechanism(s) underlying AR regulation is a potentially promising approach to suppressing PCa.We report that Par14, one isoform of the prolyl isomerases homologous to Pin1, is a critical regulator of AR transcriptional activity and is essential for PCa cell growth.Par14 was shown to be overexpressed in PCa, based on analyses of deposited data. Importantly, overexpression of Par14 significantly enhanced androgen‐sensitive LNCap cell growth. In contrast, silencing of Par14 dramatically decreased cell growth in LNCap cells by causing cell cycle arrest. Mechanistically, silencing of the Par14 gene dramatically induced cyclin‐dependent kinase inhibitor p21 at both the mRNA and the protein level through modulating the localization of p53. In addition, suppression of Par14 in LNCap cells was shown to downregulate the expressions of androgen response genes, at both the mRNA and the protein level, induced by dihydrotestosterone. Par14 was shown to directly associate with AR in nuclei via its DNA‐binding domain and augment AR transcriptional activity.Thus, Par14 plays a critical role in PCa progression, and its enhancing effects on AR signaling are likely to be involved in the underlying molecular mechanisms. These findings suggest Par14 to be a promising therapeutic target for PCa. Prolyl isomerase Par14 directly associates with the androgen receptor. Par14 is mandatory for cell proliferation and its transcriptional activity. Moreover, Par14 interacts with AR and enhances its transcriptional activity. These findings suggest Par14 to be an attractive target for the discovery of drugs targeting PCa.22.13 and antibiotics. They were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. For DHT stimulation, LNCap cells were incubated in the medium supplemented with 10% charcoal‐stripped serum (CSS) overnight, and were then exposed to 10 nM DHT for the indicated times. DHT was purchased from Selleck Chemicals.LNCaP cells were purchased from RIKEN. All cells were routinely cultured in RPMI1640 (Life Technologies) containing 10% fetal calf serum, glutamine, NaHCO2.2https://tnmplot.com/analysis/). This platform includes a dataset from GTex, TCGA and TARGET.http://gepia.cancer‐pku.cn/).The expressions were compared between normal and tumor tissues based on TNM plot data (2.3Plasmid transfection was performed by using X‐treme reagent (Roche). The plasmid and X‐treme were both placed in Opti‐MEM to allow formation of the complex for 15 min. Subsequently, these complexes were added to the cells.For Par14 knockdown, siRNA treatment was conducted by the reverse‐transfection method. Briefly, all stars Negative or Par14 siRNA was mixed with RNAiMAX in Opti‐MEM for 15 min. The mixture was applied to each well, and the cells were then seeded. The following siRNA sequences were used.Par14 si‐1: CCGCACAGTATAGTGAAGATAPar14 si‐2: GGCCGCACAGTATAGTGAAGATAAA2.4Cell proliferations were examined employing WST‐1 reagent (Takara Bio). Each cell was treated with Par14 plasmid or siRNAs. After 5 days, WST‐1 reagent was added to fresh medium and the cells were incubated for 3 h at 37°C. The absorbance of the supernatant was measured at 450 nm employing a Varioscan (Thermo Fisher Scientific).2.5http://n2t.net/addgene:171240; RRID: Addgene_171240). AR deletion mutants were inserted into a myc‐TEV‐Flag‐pcDNA vector. PSA5.8k‐Luc reporter vector, which includes the 5.8 kb PSA enhancer/promoter region, was kindly provided by Dr. A. Mizokami (Kanazawa University).The human Par14 gene was inserted into pcDNA3.1 (‐) with a Flag tag sequence in the N‐terminal. Flag‐M4‐AR was a generous gift from Steven Balk  was conducted, as described in a previous report.2.7LNCap cells were treated with siRNAs. After 3 days, the cells were collected. Subsequently, they were fixed with ice‐cold ethanol for 2 h at 4°C. After washing with PBS, the cells were mixed with propidium iodide/RNase staining buffer (BD Biosciences) for 15 min. Samples were washed again and analysis of the cell cycle was carried out employing the LSR Fortessa X‐20 (BD Biosciences).2.8Western blotting was performed, as described in previous reports.Primary antibodies were diluted as follows: AR , GAPDH , PSA , NKX3.1 , KLK2 , p21 , Par14 , and Flag antibody . Par14 antibody was also raised by the injection of GST‐Par14 into rabbits. Primary and secondary antibodies were diluted in 3% skim milk or Can Get Signal (Toyobo).2.9http://www.heatmapper.ca/). The primers used were as follows.Total RNA was extracted using a NucleoSpin RNA kit, according to the manufacturer's protocol. After RNA concentrations had been measured using Nanodrop, the same amount of cDNA was then created using reverse‐transcriptase with a verso cDNA kit. Then, real time PCR was conducted employing SYBR Green and each specific primer. Heatmap was created by Heatmapper (hPSA (KLK3) F: atgacgtgtgtgcgcaagtt R: atggttcactgccccatgachKLK2 F: aggcacacacacagcaagga R: gggggacctgaacaaacctchNKX3.1 F: cttccaaggcttccccaaac R: agctccgaggagagctgctthPUMA F: tggactcagcatcggaaggt R: tgaaggagcaccgagaggaghBAX F: gcgtccaccaagaagctgag R: gccttgagcaccagtttgcthGADD45 F: atggataaggtgggggatgc R: gcctggatcagggtgaagtgh p21 F: tcaaatcgtccagcgacctt R: tggaaggtgtttggggtcagh p27 F: gggcaagtacgagtggcaag R: gtccaccaaatgcgtgtccthPar14 F: aaggggcttgtacggcaact R: actgcattgccaccaccttthGAPDH F: ggtgaaggtcggagtcaacg R: agggatctcgctcctggaag2.10LNCaP cells were cultivated on a 48‐well plate overnight, and then transfected with both PSA‐Luc and TK‐rluc vector, using X‐treme reagent. One day thereafter, the cell medium was switched to medium containing CSS. The next day, the cells were stimulated with DHT for 24 h, and the luciferase assay was performed using a Dual‐Glo Luciferase assay kit (Promega), according to the manufacturer's protocol.2.11The indicated plasmids were transfected into LNCap cells. After 2 days, the cells were solubilized by lysis buffer containing Tris–HCl, NaCl, ethylenediaminetetraacetic acid, 0.1% Triton, phenylmethylsulfonyl fluoride, NaF and orthovanadate. After centrifugation, the cell lysates were transferred into new tubes and flag beads were added. Samples were rotated for 2 h at 4°C, and the beads were then washed with lysis buffer four times. Finally, 2DB was added to each tube and the samples were heated at 95°C for 10 min.2.12Cells were cultivated in an 8‐well chamber. They were fixed with 10% formalin for 10 min, then for 5 min in 0.1% Triton. For blocking, 3% goat serum was added to each well for 30 min. Thereafter, cells were reacted with primary antibody (1:300) at 4°C overnight. Subsequently, the cells were mixed with Alexa488 (1:400) at room temperature for 1 h. Finally, cells on the slide were mounted with 4′,6‐diamidino‐2‐phenylindole and observed by BZ8000. After each process, the chamber slide was washed with PBS three times.2.13All data are presented as means + standard error of mean. Statistical significance was calculated employing the BellCurve for Excel.t test was used for the analysis of WST‐1, flow cytometry, real time PCR  which pools the previously deposited RNA‐seq data.Although both Pin1 and Par14 belong to the Parvulin family, the WW domain in the N‐terminal of Pin1 is replaced with a Basic domain in Par14, which accounts for the difference in their binding motifs Figure . As Pin1http://gepia.cancer‐pku.cn/). The patients with PCa expressing high levels of Par14 had shorter overall survival (OS) periods as well as disease‐free survival (DFS) periods than those expressing low levels of Par14  Figure  accountsIn this study, we began by demonstrating Par14 to be overexpressed in PCa as compared with normal tissues, based on analyses of deposited data, and this overexpression was more evident than that of Pin1 Figure . ImportaA direct association between Par14 and AR was demonstrated. As to PCa, there are studies indicating Pin1 to be a critical regulator for both PCa proliferation and AR signaling.However, as is well known, ADT eventually leads to ligand‐independent AR activation, so‐called CRPC. This phenomenon is partially explained by the manifestation of AR‐V7 which is LBD lacking a receptor.https://thebiogrid.org/111320/table/homo‐sapiens/pin4.html), which is a database of protein interactions, showed the presence of numerous Par14 binding partners. These findings raise the possibility that Par14 has divergent roles in cell growth and other physiological functions.We cannot exclude the possibility that Par14 promotes growth through an AR‐independent pathway, in addition to AR‐dependent signaling. Par14 is known to be expressed in both the nucleus and the cytosol according to the Human Protein Atlas database. We assume that Par14, like Pin1, interacts with a number of substrates to modulate cellular functions. In fact, both Pin1 and Par14 associate with insulin receptor substrate‐1 (IRS‐1), enhancing its insulin‐induced phosphorylation.As to the limitations of this study, first, we used only single AR‐positive cells, because we cannot obtain others from cell banks in Japan. As is well known, androgen ablation therapy causes the development of CRPC which was, at least partially, triggered by the expression of AR‐V7. Cancer tissues are of course heterogonous, and Par14 might have distinct roles, depending on cell types. Accordingly, future studies are required to fully clarify the roles of Par14 in the functions of AR and its splicing variants, such as AR‐V7. In addition, unfortunately, no Par14 inhibitors have as yet been developed and we thus cannot examine their effects on the in vivo growth of PCa. Creating Par14 inhibitors and conducting in vivo trials might facilitate understanding the effects of Par14 on the survival rate and/or proliferation under physiological conditions.In conclusion, the findings of our current study reveal that Par14 plays a critical role in PCa progression, and its enhancing effect on AR signaling may well contribute to the underlying molecular mechanisms. Accordingly, Par14 is a key molecule for suppressing PCa and its inhibitors are promising targets for novel therapies.Miki Naito: Data curation (lead); investigation (lead). Kenichiro Ikeda: Data curation (supporting); investigation (supporting). Shunya Aoyama: Investigation (supporting). Mayu Kanamoto: Investigation (supporting). Yasuyuki Akasaka: Investigation (supporting). Yuri Kido: Investigation (supporting). Mikako Nakanishi: Investigation (supporting). Machi Kanna: Supervision (supporting). Takeshi Yamamotoya: Supervision (supporting). Akio Matsubara: Project administration (supporting); supervision . Nobuyuki Hinata: Project administration (supporting); supervision (supporting); writing – original draft (supporting). Tomoichiro Asano: Conceptualization (supporting); funding acquisition (lead); project administration ; supervision ; validation ; writing – original draft ; writing – review and editing . Yusuke Nakatsu: Conceptualization (lead); data curation (lead); funding acquisition ; investigation (lead); project administration ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing .This work was partially supported by Grants‐in‐Aid for Scientific Research (C)  and (B) (To T.A. 20H03732). This research was also supported by the Nozomi H Foundation, Nagao Takeshi Orphan Disease Research Foundation and the Institute for Adult Diseases Asahi Life Foundation.The authors have no conflicts of interest.N/A.Figure S1.Click here for additional data file.


Following anterior cruciate ligament reconstruction (ACLR), many athletes do not return to their sport, often driven by concerns about re-injury. Psychological support strategies might help, but are not routinely included in rehabilitation after ACLR. The BAck iN the Game (BANG) intervention is a 24-week eHealth program delivered via smartphone application (app), beginning directly after ACLR, with a self-directed approach that aims to target the specific challenges athletes encounter in rehabilitation.To describe athletes’ experiences of using the BANG app during rehabilitation, to support returning to sport following ACLR.Participants were athletes, in contact and/or non-contact pivoting sports, who had ACLR with the goal to return to sports. Semi-structured, individual interviews were conducted 6–10 months after their ACLR; all had access to the BANG intervention. Verbatim transcripts were analysed with a qualitative content analysis.Interacting with the app illustrated how, when, or why the participants engaged with the app. The app was helpful because of its varying content, the notifications served as reminders and participants stopped using the app when no longer needing it. (B) Challenging experiences with the app illustrated that the app itself came with some difficulties e.g., content not appearing with the right timing and material not tailored to their sport. (C) Supportive experiences with the app reflected how the app facilitated the participants’ rehabilitation progress; it included positive aspects of the app content and navigation, boosting their confidence to return to sport, and motivated them to continue with rehabilitation.The 19 participants were 17–30 years, mean 21.6 years (SD 3.5); 7 men and 12 women. The analysis generated three main categories. (A) The analysis of the interviews illustrates athletes’ awareness in interacting with, and the challenging and supportive experiences of using the app. The BANG app might provide support for returning to sport, primarily psychological support, as an adjunct to regular physiotherapy-guided rehabilitation. Athletes’ experiences of the BANG app could be improved by healthcare professionals providing additional advice about when to use which content and why.ClinicalTrials.gov, NCT03959215. Registered 22 May 2019.The online version contains supplementary material available at 10.1186/s13102-023-00731-2. One of the most serious sports-related knee injuries is a rupture of the anterior cruciate ligament (ACL), which typically requires patients to engage in musculoskeletal rehabilitation to restore knee function . AthleteWith the goal of returning to sport, the focus of rehabilitation has traditionally been on recovering physical attributes . Yet, moTo meet the demand of a psychological support in ACL-injury rehabilitation, we designed an eHealth intervention called BAck iN the Game (BANG), to be available on-demand and delivered via a smartphone application (app) , 21. WhiThis paper presents an interview study analysed with a qualitative content analysis, employing an inductive manifest approach , 30. QuaThe BANG intervention is a 24-week eHealth program, beginning directly after ACLR, with a self-directed approach that aims to target the specific psychological challenges encountered by the individual, such as confidence for return to sport. The intervention has been described previously , 21. In The BANG trial is a randomised controlled trial testing the effectiveness of the BANG intervention , 21. ParFor this study, potential participants were identified from the experimental group of the ongoing BANG trial , 21. ParIndividual semi-structured interviews were conducted via telephone between October 2020 and June 2021 by the same interviewer (JK) and no interview was repeated. An interview guide (Appendix 1) was used as a foundation for the interviews and consisted of open-ended questions encouraging participants to describe their experiences. The interview guide originated from the previous feasibility study . (ID 16)Being supported by watching other athlete’s videos and/or practicing visualization exercises assisted them in knowing that others have had similar injuries and experiences. Learning about other athletes’ ups and downs in rehabilitation offered a realistic expectation for their own experience. Knowing that obstacles can occur in or during rehabilitation validated their worries about setbacks, as did knowing that a loss of motivation can also happen to others. When other athletes shared (via the app content) how they began their rehabilitation post-surgery on the couch and then later returned to sport, participants were inspired: they described how the stories gave hope that they too would succeed. Further, practicing visualization exercises was supportive to prepare for upcoming movement skills in their rehabilitation.It was a learning experience watching other athletes talking about the same type av injury as I have. (ID 6)The educational content of the app helped participants understand the process of being in rehabilitation; it was comforting to read and watch (text and videos) that fear is common during return to sport. Participants described that Enabling reflection, illustrated an inspiration to sit down, and think about how one feels in a particular moment. The participants described how the app helped them to reflect about returning to sport and whether they trusted their injured knee. While reflection could be scary, it helped maintain motivation. Yet, if the exercises with reflections occurred too often in the app, with not enough time in between, it was less helpful: one needed to have made some progress before one could take on the next perspective. Using the app for reflections was particularly helpful due to its format: the participant was safe and undisturbed in their private space.Reflections are helpful; I get a chance to ask myself ‘how do I really feel’? (ID 17)The subcategory Promoting setting goals refers to the act of phrasing one’s ambition when it comes to rehabilitation and returning to sports: the app helped with a selection of goals to choose from. With the benefit of hindsight, participants could go back to their previous goals and track their rehabilitation progression, regardless of how small the steps were. The app was Helping to accept feelings and sensations, and participants were supported in terms of self-compassion and body awareness. Participants described that during setbacks in their rehabilitation, the app helped them navigate feeling low; the app reinforced that it is okay to have such feelings. When being encouraged not to suppress feeling low, a sense of normalization could appear, and participants felt less lonely in their rehabilitation. Exercises with breathing and relaxation helped to access body awareness. Transferring knowledge to other times in life, illustrates that the participants learned of such aspects from the app, and implemented them in other life situations not related to sports.Setting goals has helped me to stay focused on what’s most important in my rehabilitation. (ID 10)The app made me aware of my knee and my fear. It helped me to understand more about my feelings and how to accept them. (ID 11)I think I would use the experience from the app if I would have a new injury. (ID 13)if and how the app corresponded to their individual needs and their progress in rehabilitation (or lack thereof).Psychological factors can hinder full recovery after ACLR. Individuals in rehabilitation and health professionals (such as physiotherapists) have expressed a need for psychological support during rehabilitation , 23, 24.Interacting with the app, and particularly the subcategory Choosing app content from own interest and need, illustrated how the participants chose and repeatedly employed cognitive-behavioral content from the app (using a task menu) during rehabilitation. In another intervention with cognitive-behavioral based physical therapy (CBPT) as psychological support, participants received seven sessions with controlled breathing, grounding, setting activity goals, monitoring self-talk, setting daily intentions, present-mindedness, managing setbacks, and guided imagery delivered using motivational interviewing by a physiotherapist. The intervention stopped eight weeks after ACLR and all participants completed all sessions [In our analysis, the category sessions . With a Drifting out of focus when other things call for attention and Fading out of use when no longer needing it). Adherence is a problem in other interventions with psychological support after ACLR [Participants shared how they forgot to use the app in their daily life and stopped using the app in a gradual manner . Physiotherapists report that they felt ill-equipped to deliver adequate support during ACL injury rehabilitation [Missing individual feedback).Participants experienced that the BANG app provided psychological support and it had become an educational resource through rehabilitation  could address this tailoring request. Another program with influences from psychology is the novel MOTor Imagery to Facilitate Sensorimotor Re-Learning (MOTIFS) model, which is delivered as individual sessions by a physiotherapist after traumatic knee injury [e injury . Particie injury . The choLearning more about injury and fear). For some participants, Finding it hard to use the app when not making any progression with rehabilitation, revealed that it was less motivating to use the app at certain times. Using a self-directed tool in collaboration with a physiotherapist at selected time-points throughout rehabilitation might avoid the less helpful experiences of using the app. Instead of an independent and self-directed app, there is potential for the BANG app content to become integrated and individualized with assistance from the physiotherapist. Previous research has shown that digital support can complement face-to-face sports injury rehabilitation [For almost 20 years, psychological factors have been a common topic in ACL injury research , 42–44. litation , 19.Enabling reflection) as they were undisturbed in a private space. This indicates that a digital self-directed approach may be a favorable option to face-to-face treatment for some participants at some points in rehabilitation. Requiring and requesting psychological support can carry stigma [Participants appreciated using the app for reflections  during rehabilitation following ACLR. The analysis of the interviews illustrates athletes’ awareness in interacting with, and the challenging and supportive experiences of using the app. The BANG app might provide support for returning to sport, primarily psychological support, as an adjunct to regular physiotherapy-guided rehabilitation. Combining the BANG app with usual care after ACLR might be an accommodating strategy in rehabilitation for people who wish to return to sport. Athletes’ experiences of the BANG app could be improved by healthcare professionals providing additional advice about when to use which content and why.Below is the link to the electronic supplementary material.Supplementary Material 1


Depression, generalized and cardiac anxiety, and posttraumatic stress disorder negatively affect disease severity, participation, and mortality in patients with cardiovascular disease. Psychological treatments within cardiac rehabilitation may improve the outcomes of these patients. We therefore developed a cognitive-behavioral rehabilitation program for patients with cardiovascular disease and mild or moderate mental illness or stress or exhaustion. In Germany, similar programs are well established in musculoskeletal rehabilitation and cancer rehabilitation. However, no randomized controlled trials have evaluated if such programs achieve better outcomes in patients with cardiovascular disease compared with standard cardiac rehabilitation.Our randomized controlled trial compares cognitive-behavioral cardiac rehabilitation with standard cardiac rehabilitation. The cognitive-behavioral program complements standard cardiac rehabilitation with additional psychological and exercise interventions. Both rehabilitation programs last for four weeks. We enroll 410 patients with cardiovascular disease and mild or moderate mental illness or stress or exhaustion aged 18 to 65 years. Half of the individuals are randomly assigned to cognitive-behavioral rehabilitation and the other half to standard cardiac rehabilitation. Our primary outcome is cardiac anxiety 12 months after the end of rehabilitation. Cardiac anxiety is assessed with the German 17-item version of the Cardiac Anxiety Questionnaire. Secondary outcomes cover outcomes assessed by clinical examinations and medical assessments and a range of patient-reported outcome measures.This randomized controlled trial is designed to determine the effectiveness of cognitive-behavioral rehabilitation at decreasing cardiac anxiety in patients with cardiovascular disease and mild or moderate mental illness or stress or exhaustion.German Clinical Trials Register .The online version contains supplementary material available at 10.1186/s12872-023-03272-1. Cardiovascular diseases are the most frequent cause of death and a major risk factor for health-related impairments of activities and participation . About oIn Germany, cognitive-behavioral rehabilitation was developed for patients coping with a somatic illness who are also affected by psychological stress or comorbid psychological disorders. Cognitive-behavioral rehabilitation is a multimodal intervention that includes additional psychological and exercise interventions as compared to standard rehabilitation programs . In patiIn a recent study, we tested the implementation of a cognitive-behavioral rehabilitation program for patients with cardiovascular disease . Study pOur randomized controlled trial tests whether participants in the cognitive-behavioral rehabilitation program have more favorable outcomes, in particular mental health outcomes, than participants in standard cardiac treatment. Our primary outcome is cardiac anxiety measured with the Cardiac Anxiety Questionnaire 12 months after the end of rehabilitation , 16.We are conducting a single-center, randomized controlled trial comparing two groups of cardiac rehabilitation patients. The intervention group receives a cognitive-behavioral rehabilitation program. The control group receives the standard cardiac rehabilitation treatment provided at the study center. Participants are randomly assigned to cognitive-behavioral rehabilitation or standard cardiac rehabilitation after giving informed consent.The study has been registered on the German Clinical Trials Register (DRKS00029295). The items from the World Health Organization Trial Registration Data Set are available as Additional file 1. This paper contains the original study protocol. Any substantial modifications to the study protocol will be submitted to the Ethics Committee of the University of Lübeck for approval prior to implementation. These amendments will be documented in detail in the German Clinical Trials Register and described transparently in trial reports.The trial is investigator initiated, and the University of Lübeck is the primary sponsor of the trial. An advisory board incorporating researchers, physicians, and employees of Federal German Pension Insurance and the German Pension Insurance North accompanies the conduct of the study and meets once a year.https://www.muehlenbergklinik-holsteinische-schweiz.de) is located in northern Germany. It currently has 321 patient treatment slots, 130 of which are for cardiac rehabilitation. Thirty of the 130 treatment slots for patients with cardiovascular diseases are intended for cognitive-behavioral cardiac rehabilitation.In Germany, rehabilitation due to cardiovascular diseases for people of working age is mainly provided by the pension insurance agencies and is intended to prevent health-related exit from work and disability pensions. Medical rehabilitation is possible both after acute events  and due to chronic diseases . There are 115 cardiac rehabilitation departments used by the pension insurance agencies , and in We are including patients aged 18 to 65 years who receive rehabilitation at our study center due to cardiovascular disease (ICD-10 I05 to I71 as well as I95 and I97) assigned by the Federal German Pension Insurance or the German Pension Insurance North and for whom mild or moderate mental illness or stress or exhaustion are recorded in the application documents for cardiac rehabilitation. For patients assigned by the Federal German Pension Insurance, mental illness or stress or exhaustion is determined by the socio-medical service of the Federal German Pension Insurance. For patients assigned by the German Pension Insurance North, this is determined by a cardiologist at the study center. The determination of mild or moderate mental illness or stress or exhaustion is performed in advance of the program with only the documents used to claim the rehabilitation.Patients with severe mental illness , severe heart failure (at least stage III according to the classification of the New York Heart Association), and significant limitations with the German language are excluded.The cognitive-behavioral rehabilitation program was developed for cardiac rehabilitation patients with mild or moderate mental illness or stress or exhaustion. The program complements standard cardiac rehabilitation with additional psychological and exercise interventions and follows the framework of cognitive-behavioral rehabilitation formulated by the German Pension Insurance .Cognitive-behavioral rehabilitation is conducted as a group program, i.e., 8–12 patients who begin the rehabilitation program at the same time complete five specific additional cognitive-behavioral components together. Table A detailed description of the psychological group can be found in Benninghoven et al. . This grControls receive the standard cardiac rehabilitation program in accordance with the recommendations for the rehabilitation of coronary heart disease . To ensuOur primary outcome is cardiac anxiety 12 months after the end of rehabilitation. Cardiac anxiety is assessed with the German 17-item version of the Cardiac Anxiety Questionnaire , 16. AllThe three subscales of the Cardiac Anxiety Questionnaire, i.e., fear, avoidance, and attention, are collected as secondary outcomes , 16. FurPhysical health: Functional capacity is assessed with eight items of the IRES-24 . The items assess difficulties in engaging in physical activities, e.g., sports that really make you sweat, such as jogging, skiing, and mountain hiking [n hiking . DifficuGeneral health: To describe general health, we use a visual analog scale with scores ranging from 0 to 100 points [0 points . Further0 points , 29. Usi0 points . The heaMotivation to change lifestyle is assessed using a 5-point scale ranging from 1 (“certainly”) to 5 (“certainly not”), and we count who is motivated to change lifestyle (certainly or rather yes) . ConfideSelf-rated work ability is measured using the Work Ability Score . The WorThe dose delivered describes the number of intended intervention units provided to participants . TreatmeThe dose received describes the elements of the intervention the participants actually received . We recoFor our health economic evaluation, we use the Questionnaire for Health-Related Resource Use in an Elderly Population . The queRecommendations for 12 common subsequent services are extracted from the standardized medical discharge report at the end of the rehabilitation .Diagnoses are also extracted from the standardized medical discharge report at the end of the rehabilitation . In the These include data on native language, partnership, number of children, level of school education and vocational qualification.Table Although adverse events causally related to cognitive-behavioral rehabilitation are not expected, we will assess disability pensions and premature termination of rehabilitation in both groups.Ancillary and post-trial care are not planned. Compensation for harms due to study participation is also not planned.Table As a minimum important difference for the 12-month follow-up, we defined 0.2 points of the total cardiac anxiety score . Alhttps://www.muehlenbergklinik-holsteinische-schweiz.de). Eligibility is determined prior to the start of rehabilitation either by the referring pension insurance agency or by the rehabilitation center if a mild or moderate mental illness or distress or exhaustion is described in the application documents. A physician from the study center therefore continuously screens the incoming application documents of patients who are assigned to the rehabilitation center by the pension insurance agencies. A member of the study team (psychologist) in the study center informs potential participants in writing about the possibility of participating in the study, specifies the possible start of rehabilitation, and suggests a telephone consultation to the participants. The relaying of this information by telephone is supported by a manual. After receiving verbal information during the telephone conversation, the potential participants sign the consent form and return it to the rehabilitation center within 48 h. The written information and the consent form are provided as Additional files 3 and 4. A website provides supplementary and ongoing information about the study (https://www.vor-kardio.de).Study participants are recruited at a German inpatient rehabilitation center  using Stata 16.0. Blocks of different size were combined randomly. Allocation concealment is ensured by sealed, opaque, and consecutively numbered envelopes. The envelopes are opened in the study center for randomization only after participants have given informed consent. The group assignment is documented in the study center in the study list through the identification number.Participants and providers are not blinded. The data analysis is also not blinded.Data are collected via questionnaires, clinical examinations, and the standardized rehabilitation discharge reports. Questionnaires are completed at four measurement points: at the beginning and end of rehabilitation, as well as 3 and 12 months after completion of rehabilitation. The assessments have already been tested during the implementation of the intervention  and are Data from questionnaires are entered by trained research assistants. We use forms created with Microsoft Access for data entry. When entering the data, the research assistants can only select from a set of valid values. The first 20 questionnaires of each of the four measurement points and subsequently every 20th questionnaire are re-entered by a second independent person. Incorrect completion of questionnaires  are logged. Data entries are made immediately when the questionnaires are available to the University of Lübeck. The presence of the questionnaires at the beginning and end of the rehabilitation is documented in the study list of the rehabilitation center. The same applies to the mailing of the 3-month and 12-month follow-up questionnaires. The questionnaires received after 3 and 12 months are documented at the University of Lübeck. The identification numbers of the incoming questionnaires are transmitted by the University of Lübeck to the rehabilitation center. If receipt is confirmed by the University of Lübeck, no reminder is sent. A comprehensive plausibility check is performed for all data prior to our analysis of the data.An external data monitoring committee is not implemented. Short-term and long-term effects are analyzed and published separately. No further interim analyses are planned. No criteria for early study termination have been established.The status of recruitment, the treatment fidelity, the response rates of the questionnaires, and the number of questionnaires entered are discussed in bi-weekly video conferences between the Universität zu Lübeck and the rehabilitation center.Participants are added to a password-protected study list (Microsoft Excel) with their name, address, and social security number upon consent to participate and are assigned an identification number. This list also documents the treatment group, consent, and the planned start of rehabilitation after randomization. This study list remains at the recruiting rehabilitation center. The rehabilitation center transmits a pseudonymized copy of this list exclusively with the pseudonym, treatment group, date of receipt of consent and planned start of rehabilitation electronically and encrypted to the University of Lübeck at the beginning of each week. The study list will be shredded by the rehabilitation center after all follow-up questionnaires have been sent out. As of that point, the data will be completely anonymized.The rehabilitation center documents in another list (Microsoft Excel), in which participants are recognized only by their identification number (pseudonymized list), their date of birth, gender, the actual start of rehabilitation and the end of rehabilitation , selected clinical data collected in the rehabilitation center , and the therapeutic services performed during rehabilitation.The participants’ questionnaires are provided with the identification number in the rehabilitation center and handed over to the study participants by a member of the research team for completion at the beginning and end of the rehabilitation. The participants deliver the questionnaires in an envelope at the reception of the rehabilitation center. Thereafter, these questionnaires are personally handed over to MB by DB every two weeks. The questionnaires of the follow-up assessments after 3 and 12 months, also tagged with the identification number, as well as the reminders, are sent from the rehabilitation center. A stamped return envelope addressed to the University of Lübeck is enclosed with this letter.https://www.synapse.org).Data, pseudonymized with the identification number, are recorded electronically at the University of Lübeck using Microsoft Access. All questionnaires received by the University of Lübeck are stored in locked cabinets at the University of Lübeck and will be shredded in accordance with data protection regulations upon completion of the study on 28.02.2025. Computers at the University of Lübeck that allow access to the data entered are located in locked office rooms and require user authentication in the form of passwords. Accesses can be tracked. Data is stored in folders that are only visible and accessible to the research team at the University of Lübeck. The network in which the computers are integrated is protected against external access and manipulation by a regularly updated firewall system. The University of Lübeck will completely delete the electronically stored questionnaire data 10 years after the end of the study (28.02.2035). To ensure a transparent research process, anonymized data will be permanently stored in a data repository after our primary publication is accepted  and long-term effects (12-month follow-up) using different models and publish the findings separately. We will use mixed models if more than one measurement point needs to be considered, i.e., end of rehabilitation and 3-month follow-up, and generalized linear models otherwise. If baseline scores are available, these will be included as a covariate when estimating the treatment effect. If outcomes are binary, a logit link will be used. High depression, high generalized anxiety, and high cardiac anxiety, as well as age, sex, and education, are tested as possible moderators of the treatment effect on our primary outcome cardiac anxiety assessed at the 12-month follow-up.For health economic evaluation, we will calculate an incremental cost-effectiveness ratio . AssessmMissing data will be imputed using chained equations . We willhttps://www.vor-kardio.de. This study protocol was prepared using the SPIRIT checklist  [The purpose of our randomized controlled trial is to test the effects of a cognitive-behavioral rehabilitation program for individuals with cardiovascular disease and mild or moderate mental illness or stress or exhaustion, in comparison with standard cardiac rehabilitation. Our study will provide high-quality evidence to inform pension insurance providers if cognitive-behavioral rehabilitation is likely to improve outcomes in similar patients and could be recommended for implementation across Germany. All results of our study will be published as articles in peer-reviewed journals and at conferences, regardless of the magnitude or direction of the effects. The authors of this protocol will write the final trial publications. We do not intend to use professional writers. We will provide updated information on the trial on our website  Trials) .Recruitment has started and is ongoing.Below is the link to the electronic supplementary material.Additional file 1: Items from the World Health Organization Trial Registration Data Set.Additional file 2: Description of the components of the cognitive-behavioral rehabilitation program according to the TIDieR checklist.Additional file 3: Patient information.Additional file 4: Consent to study participation.


Physical rehabilitation and physical activity are known non-pharmacological methods of treating multiple sclerosis. Both lead to an improvement in physical fitness in patients with movement deficits while improving cognitive function and coordination. These changes occur through the induction of brain plasticity. This review presents the basics of the induction of brain plasticity in response to physical rehabilitation. It also analyzes the latest literature evaluating the impact of traditional physical rehabilitation methods, as well as innovative virtual reality-based rehabilitation methods, on the induction of brain plasticity in patients with multiple sclerosis.  Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system of unexplained etiology. The disease most often affects young adults, leading to developmental issues, such as paresis of the limbs, ataxia, visual disturbances, and sphincter disturbances . In addiDespite many studies, the exact etiology of multiple sclerosis is still not fully known. It is believed that the development of the disease is caused by abnormal activation of the immune system induced by external factors in genetically predisposed individuals . Since dThanks to the progress of pharmacotherapy in recent years, a significant slowdown in the progression and consolidation of neurological deficits in patients with MS has been achieved. Despite this, physiotherapy is still essential. New and more effective forms of rehabilitation are constantly being developed. In recent years, many studies have been conducted on the impact of various rehabilitation methods on improving both the performance and quality of life of MS patients . It has Recent years have seen the development of telerehabilitation for remote exercises using immersive interfaces such as virtual reality (VR), augmented reality (AR), and mixed reality (MR) . The posThree-dimensional visualization technologies have gained particular popularity in recent years. Thanks to the development of digital techniques and information and communication technologies (ICT), the use of immersive interfaces is no longer just an element of entertainment but has become a new and exciting tool used in various fields of science, including medicine. Studies have examined the use of VR and AR in various conditions beginning with psychiatric disorders , neurological diseases , and in the treatment of pain, as well as in physical rehabilitation and cognitive disorders ,11,12,13Brain plasticity is a key property of the nervous system allowing the formation of changes in response to stimuli from the body and environmental stimuli. The capacity for neuroplasticity is retained throughout life and is responsible for the ability to self-repair, learn, and adapt to emerging variables. It is a complex phenomenon caused by changes occurring at various levels of the nervous system’s organization. In the induction of neuroplasticity, the critical role is played by the bi-directional change in synapse strength, defined as synaptic plasticity, and expressed by long-term synaptic potentiation and long-term synaptic weakness . Long-teMultiple sclerosis is a heterogeneous disease in which different LTP and synaptic plasticity disorders are also observed in relapsing and progressive forms. In experimental autoimmune encephalomyelitis (EAE) in mice, synaptic transmission disorders have been shown to be based on an increase in glutamatergic transmission and a decrease in GABAergic transmission. In the case of MS patients, an analogous imbalance is observed, leading to a disturbed synaptic inhibition and excitation balance. In primary progressive multiple sclerosis (PPMS) patients, LTP is over-activated, which leads to excitotoxicity  and lossNeuroplasticity is associated with changes in the levels of specific proteins called neuroplasticity markers in the blood and cerebrospinal fluid. A significant role in the induction of brain plasticity is played by neurotrophins: NGF, BDNF, NT-3, and NT 4/5. They are formed as precursor proteins that acquire biological activity in reaction with metalloproteinases . NeurotrAmong the neurotrophins, BDNF has the best-known mechanism of action and the most significant impact on the induction of brain plasticity. BDNF plays a fundamental role in the development and repair of the nervous system and its maintenance of function. In addition, it is the best-known neurotrophin acting through the mechanism of LTP induction. BDNF stimulates neuronal and axonal survival, plays a vital role in myelin repair, and stimulates neurogenesis in the dentate gyrus of the hippocampus . In the Research is still underway to understand the exact mechanism that induces brain plasticity in response to rehabilitation. One of these mechanisms is the occurrence of the so-called muscle–brain endocrine loop , which pThe first myokine that was identified was interleukin-6 (Il-6); it is released from muscles during their activity and influences other tissues’ metabolic processes . It was Another myokine that plays a role in exercise-induced brain plasticity is cathepsin B. It acts as a link between working muscles and the hippocampus. Cathepsin B stimulates the reconstruction of axons and increases the number of dendritic spines. It was found that the level of cathepsin B in the plasma increases during muscle activity. This is due to the activation of the AMP-activated protein kinase . CathepsOne of the myokines that induce brain plasticity in response to training is the adipomyokine irisin. Irisin is known mainly for the “browning” effect of white adipose tissue, and its effect on the stimulation of brain plasticity is associated with an indirect effect on the expression of BDNF in the CNS. Irisin is formed as a result of the cleavage of the membrane protein fibronectin type III domain-containing protein 5 (FNDC5). Physical activity increases the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha , a transcriptional co-activator of mitochondrial biogenesis and a regulator of oxidative metabolism in brown adipose tissue and muscle . PGC-1 aIn addition to the involvement of muscle tissue in regulating brain plasticity, it is also worth considering adipose tissue and the adipokines it secretes. Adipokines are secreted by white adipose tissue and play a role in pro- and anti-inflammatory processes . An unheAmong the adipokines that play a role in the pathogenesis of MS, we ought to mention adiponectin. It may participate in both anti-inflammatory and pro-inflammatory processes, probably depending on its isoform. Adiponectin levels in the blood and cerebrospinal fluid of MS patients have been found to be higher than in healthy individuals and correlate with a more severe course and faster progression of the disease ,46. TherOxidative stress (OS) and nitroxidative stress (NOS) are considered to be significant factors in MS pathology. They occur as a result of the accumulation of reactive forms of oxygen and reactive forms of nitrogen, leading to mitochondrial dysfunction . MitochoBy appropriately increasing physical activity, mitochondrial metabolism is improved . HoweverIn the context of novel rehabilitation techniques using VR, there is a very limited base of work evaluating its effect on OS. A study evaluating chronic stroke patients who underwent training with VR found changes in OS t markers: there was a significant decrease in 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels and a significant increase in heme oxygenase-1 (HO-1) after the intervention. Moreover, changes in OS marker levels were associated with improvements in patients’ functional outcomes . SimilarA different theory explaining the development of brain plasticity in response to physical activity is epigenetic inheritance, the molecular basis of which involves changes and regulation of gene expression without intervening in DNA sequence changes . This leThe primary mechanism responsible for regulating gene transcription is histone modification. It occurs through the activity of histone acetyl-transferase (HAT) and histone deacetylase (HDAC). Gene transcription can be activated by HAT and inhibited by HDAC . PhysicaAnother mechanism affecting gene expression through epigenetic inheritance is DNA methylation. It occurs as a result of the addition of a methyl group by DNA methyltransferase to the 5′cytosine carbon of the genomic cytosine–phosphate–guanine (CpG) dinucleotides. It has been found that both incidental and regular exercise can affect DNA methylation. In a mouse model, BDNF levels were found to be elevated after aerobic training, accompanied by a decrease in the expression of the neurotrophic factor receptor p75 . ExercisWith the growing interest in using modern rehabilitation techniques using virtual reality, the question has arisen of whether and how these rehabilitation techniques affect the development of brain plasticity. The vast majority of studies evaluating the use of training with VR in patients with neurological conditions have found improvements in upper limb performance associated with increased strength and speed of movement ,62,63. TThe use of VR-based interventions leads to the activation of mirror neurons through multi-sensory feedback. As a result, areas of the sensorimotor cortex are activated, which translates into promoting and enhancing functional motor reorganization in the CNS .Initially, researchers were enthusiastic about mirror neurons, but a group of skeptics questioned the existence and role of MNS in humans . HoweverThere is a substantial database of publications on the use of different types of rehabilitation in treating multiple sclerosis. It has been shown that rehabilitation in MS patients can lead to improvements in physical performance, balance, and mobility. In addition, rehabilitation improves the quality of life and reduces symptoms of fatigue and depression. Studies of conventional rehabilitation frequently involve evaluations of various plasma biomarkers in addition to the standard assessment of physical status. There is also an interesting trend in assessing the effects of rehabilitation on cognitive function changes. Thanks to the growing interest in the concept of brain plasticity, in recent years, researchers have also evaluated the effect of rehabilitation on its induction. Unfortunately, for innovative rehabilitation methods, there are limited reports evaluating the levels of biomarkers of brain plasticity in blood or cerebrospinal fluid. Among the available types of conventional rehabilitation, we do not have a clear answer as to which training can produce the best results in improving health in patients with MS (PwMS). One possible problem with this evaluation concerns the use of different research protocols regarding the duration, intensity, and frequency of interventions. Determining changes in the levels of not only clinical but also biochemical biomarkers can more clearly answer the question of what type of training is more effective. Regarding the plasticity markers, levels of peripheral BDNF, in response to aerobic training and combined exercise, were found to be significantly increased after the intervention. In addition, no differences were found between aerobic and combined training or the duration of the intervention on serum BDNF levels .Moreover, chronic BDNF levels were also significantly elevated after rehabilitation. In contrast, exercise has not been shown to affect chronic NGF levels. However, a minor trend toward an increase in ciliary neurotrophic factor (CNTF) in response to exercise has been found . It is well-known that BDNF plays a crucial role in inducing rehabilitation-induced brain plasticity. To identify ways to explain variability in the development of plasticity changes in response to the same inducers during neurorehabilitation, studies have searched for differences in BDNF polymorphism between patients. Among patients with MS, a correlation between atrophy of the brain’s gray matter and the BDNF Val66Met polymorphism has already been found. It was found that Met carriers demonstrated less atrophy of the gray matter and a smaller volume of demyelinating lesions in the T2 sequence . Other sIn comparison to RRMS forms, there are fewer papers on the evaluation of progressive forms of MS in terms of plasticity markers in the blood. One by Briken et al. assessed the effect of training on a bicycle ergometer on serum levels of irisin, IL-6, and BDNF in patients with progressive forms of MS. The study reported a significant increase in BDNF measured immediately after training, which decreased substantially after 30 min, even below pre-training baseline levels. No significant changes in myokine and BDNF levels were observed after a nine-week exercise cycle . The lacUnfortunately, research confirms that MS patients are less physically active than healthy people. In addition, it was found that patients with PPSM, compared to RRMS patients, are less physically active . Most of the studies conducted to date confirm the beneficial effect of virtual reality training on improving upper limb dexterity and on improving strength and speed of movement in patients with neurological conditions ,62,63. Un = 8) and the control group (n = 8). Another study comparing traditional training with video-based exergaming (VBE) in PwMS showed that both groups had a significant improvement in the agility of the upper limbs. In addition, a significant improvement in cognitive functions, balance, and reduction of fatigue was found. On the other hand, in the VBE group, the BDI scores decreased significantly [In a study using VR and a special upper limb motion controller (Leap Motion Controller), on a group of 16 patients with MS, a significant improvement in strength measured with a dynamometer was found for the hand stronger at the baseline. Compared to the control group, PwMS receiving traditional motor rehabilitation showed improved limb manual dexterity . In a stficantly . Other ificantly . Improveficantly ,60,61 anficantly ,84. The ficantly . Despite the growing number of publications on rehabilitation using VR or AR in patients with neurological conditions, only a few researchers have addressed the effects on levels of plasticity biomarkers in blood or cerebrospinal fluid ,64. The It is worth noting that patient involvement plays a significant role in the rehabilitation process. Patients participating in traditional forms of rehabilitation frequently complain about their monotonous nature and, over time, become reluctant to participate in exercises. Moreover, it has been found that the level of patient involvement plays a crucial role in developing rehabilitation-induced neuroplasticity . When inIn addition to motor symptoms, PwMS struggle with many other symptoms, such as cognitive decline, fatigue, and depressive disorders . FatigueOne of the significant accompanying symptoms of MS is ataxia. Unfortunately, pharmacological treatment brings little effect in reducing the symptoms of ataxia, which is why forms of non-pharmacological treatment of these symptoms are sought. The studies conducted so far do not show unequivocal results in terms of reducing the symptoms of ataxia in patients with MS ,91,92. TAn essential role in the progression of disability in PwMS is played by cognitive impairment; it is found in more than 65% of patients and can occur at the early stages of the disease . As ther


Antimicrobial resistance (AR) poses a major global threat, with a reported 1.27 million deaths in 2019. This resulted in years of life lost as well as a large burden on the healthcare system. Transmission of resistant pathogens is not limited to one geographic area and can easily be influenced by international travel and trade of food and other products. Addressing the global threat of AR is recognized as a major action item within the U.S. National Action Plan for Combating Antibiotic Resistant Bacteria. In 2021, the Centers for Disease Control and Prevention (CDC) established the Global Antimicrobial Resistance Laboratory and Response Network . The network provides financial and technical support to partners, with a focus on detecting and monitoring existing threats, identifying risk factors, preventing and controlling spread, and mitigating the emergence of new threats.In just over 2 years, CDC has invested more than $40 million through the Global AR Lab & Response Network on projects with more than 20 organizations in nearly 50 countries. Using a multi-pathogen approach, the network focuses on healthcare-associated, sexually transmitted, fungal, enteric, and invasive bacterial and respiratory pathogens.CDC’s Global AR Lab & Response Network is strengthening capacity to detect and respond to AR threats identified in the CDC 2019 AR Threats Report. Accomplishments include implementing trainings for high quality laboratory methods; monitoring resistance in bacteria and fungi to inform on changes in resistance and improve interventions; training and implementing activities to prevent, detect, and respond to AR in healthcare settings; and enhancing data sharing nationally, regionally, and globally.Through this innovative approach with plans for expansion, the Network will continue to use a One-Health methodology to focus on enhancing global capacity to quickly detect AR threats and built upon existing laboratory and response efforts. Findings from these projects will also inform intervention strategies to address current and future AR threats.All Authors: No reported disclosures


Bioflavonoids, as naturally derived polyphenolic compounds, have been studied extensively due to their potential health-promoting attributes . Their aIntense physical activity can induce inflammation and oxidative stress in athletes, which may lead to muscle damage and prolonged recovery periods . BioflavBioflavonoids have been reported to improve vascular function by increasing nitric oxide (NO) production, a potent vasodilator, and reducing endothelial dysfunction . EnhanceCollagen, a primary structural protein in connective tissues, plays a vital role in maintaining the integrity and strength of tendons, ligaments, and cartilage . Some biThe immune system plays a crucial role in the body's response to injury and tissue repair. Intense exercise can temporarily suppress immune function, increasing the risk of infection and delaying the healing process , 13. BioPain is a common symptom associated with athletic injuries and can negatively impact the rehabilitation process . Some biEnergy metabolism is critical for athletes' performance and recovery. Bioflavonoids, such as quercetin , have beBioflavonoids often coexist with other nutrients and phytochemicals in plant-based foods, which may lead to synergistic effects that can enhance their overall impact on athletic rehabilitation , 27. ForWhile bioflavonoids are present in many plant-based foods, the specific types and concentrations of these compounds can vary greatly among different sources . MoreoveThe current understanding of bioflavonoids' nutritional mechanisms from a kinesiology perspective suggests that these compounds may have potential applications in supporting athletic rehabilitation , 36. TheAs the understanding of bioflavonoids' nutritional mechanisms and their potential role in athletic rehabilitation grows, it is crucial for practitioners working with athletes, such as sports dietitians, physiotherapists, and strength and conditioning coaches, to stay informed about the latest evidence and practical applications . This wiHowever, several knowledge gaps and limitations need to be addressed to fully understand and optimize the use of bioflavonoids in athletic rehabilitation. For instance, more research is needed to determine the optimal dosages, bioavailability, and efficacy of various bioflavonoids for different types of athletic injuries and rehabilitation needs . AdditioIn conclusion, an improved understanding of the nutritional mechanisms of bioflavonoids from a kinesiology perspective holds promise for their potential application in athletic rehabilitation. While evidence suggests that these compounds may have a variety of beneficial effects, further research is needed to elucidate their specific mechanisms, identify optimal sources and dosages, and evaluate their efficacy in well-designed clinical trials . While bQT wrote the first draft. BC was responsible for collecting data. CW was responsible for checking the format. TS was responsible for conceiving ideas. All authors contributed to the article and approved the submitted version.


For years it has been stated that the need for prevention and rehabilitation is not always identified early enough. Although many individuals have regular contact with a general practitioner (GP), this access path for applying for a prevention or rehabilitation service has not been fully exploited. The important role of GPs in supporting the intention to apply is highlighted in the research. This study aims to evaluate the effectiveness of the “check-up 45 + ” to support GPs both in identifying the need for prevention and rehabilitation services and in submitting applications.n = 1,654) aged from 45 to 59 years will be recruited by medical assistants. In addition to usual care, both study groups will receive a questionnaire covering socio-economic and occupational variables to be filled out immediately in the waiting room. The intervention group passes through the “check-up 45 + ”. This includes the completion of the “screening 45 + ” that aims to assess the need for prevention and rehabilitation services. Medical assistants will immediately evaluate this 2-page screening tool. If a need is identified and confirmed by the GP, information and application documents will be handed over. Moreover, the application process for rehabilitation services is simplified.The study is designed as a two-arm, pragmatic 1:1 randomised controlled study (RCT), which will be conducted in about 20 general practices in the German states of Berlin and Brandenburg. Patients .The online version contains supplementary material available at 10.1186/s12913-023-09392-w. Due to later retirement age and the increasing lack of qualified personnel, the importance of maintaining or restoring the work ability of older employees is increasing , 2. In oA central function of the German Pension Insurance is to promote and maintain the work ability of insured persons by financing rehabilitation measures in specialized rehabilitation facilities with a usual duration of three to four weeks. Furthermore, the German Pension Insurance is to provide prevention services for insured persons with initial health impairments. These prevention services include about three to five full-time days in specialized health care provision  and three to six months extra-occupational group services. Both prevention and rehabilitation services require an application by the insured person to the German Pension Insurance. The prevention application can be filled out online by the patient in a few minutes. The application for rehabilitation is more time-consuming and includes extensive application documents that must be filled out by the patient and physician. Within the study, the application procedure will be simplified by providing the necessary forms, reducing the number of forms required and replacing the doctor’s medical report with a short form.Flexirentengesetz) provides, according to § 14 (3) of the Social Security Code VI, that for insured persons aged 45 and over, the "introduction of voluntary, individual, work-related health care for insured persons […] is to be tested in model projects". The study presented here is based on this law and is one of about ten model projects in Germany. In addition, the German Federal law to strengthen the participation and self-determination of persons with disabilities (Bundesteilhabegesetz) provides that the need for rehabilitation of insured persons is to be recognized at an early stage by the rehabilitation providers using appropriate instruments. So far, there is no active screening to identify a possible need for prevention or rehabilitation. For years, it has been stated that prevention and rehabilitation needs have not always been identified in time and that rehabilitation services should be applied earlier to prevent or mitigate the progression of chronic diseases and disabilities. The application behaviour differs from the subjective and objective need for medical rehabilitation. It is assumed that the need is significantly higher than the number of applications .Recruitment has started and is currently ongoing.Additional file 1. Additional file 2.


Tobacco smoking is a leading cause of preventable death worldwide. The perinatal period provides a unique opportunity for intervention, as many smokers quit smoking during pregnancy but relapse postpartum. Novel relapse prevention interventions that reduce the burden of treatment attendance in this population are needed. Attentional retraining (AR) has been shown to reduce attentional biases toward smoking-related stimuli, a cognitive process implicated in smoking, AR has not been applied to perinatal smokers, and the effect of AR on craving and smoking is not clear. The goal of this study was to evaluate the delivery of AR for smoking cues in perinatal smokers utilizing a mobile intervention.N = 17) for up to 2 weeks both before and after delivery.This pilot study utilized Ecological Momentary Assessment (EMA) methodology delivered on a mobile device to examine the relapse process and evaluate the utility of AR in former smokers attempting to remain abstinent postpartum. AR (or Control Training) was administered to abstinent smokers . There was no evidence that AR reduced craving. An exploratory analysis revealed that there was no evidence that AR reduced smoking during the study period.via a mobile device is feasible in perinatal smokers. Further research using larger samples is required to evaluate the utility of mobile AR in reducing craving and smoking.AR using EMA methodology  The heaOther than contingency management , effectiTo develop effective interventions to prevent postpartum smoking relapse, it is imperative to understand the factors and psychological processes that influence return to smoking following delivery. Negative affect, stress, and urges/cravings have been implicated in relapse , 22. TheAnother factor that influences smoking is “attentional bias” (AB) to smoking cues. AB is defined as the tendency to automatically attend to and maintain attention on smoking cues, and may be causally related to craving and use/relapse . EmpiricAR has not been evaluated in perinatal smokers or perinatal former smokers. In a perinatal population, it may be useful to administer AR on a mobile device, given the promise of these methods in this population . In this2.2.1.N = 17) were recruited from the obstetrical clinics at Yale New Haven Hospital. Participants self-identified their race/ethnicity as: 9 Black, non-Hispanic; 4 Black, Hispanic; 2 White, Hispanic; 1 White, non-Hispanic, 1 other (West Indian). Inclusion criteria were: 1) a history of smoking 5+ cigarettes/day and having achieved abstinence by 32 weeks’ gestation; 2) aged 18 to 40 years; 3) able to speak and write English; 4) Edinburgh Postnatal Depression Scale (EPDS) score < 10. Exclusion criteria included: 1) current substance use ; 2) current major depressive disorder, minor depression, or history of any of such in the last 6 months; 3) presence of an Axis I psychotic disorder; 4) plan to relocate out of the area; 5) imminent incarceration; 6) planned inpatient hospitalization during study period. All participants had to meet these eligibility requirements before they could be enrolled and randomized to either condition. Data collection took place between May 2014 and February 2015.Participants  and control with respect to age and severity of nicotine dependence. Participants and investigators were blinded as to study condition.2.3.Following randomization participants were instructed to carry a smartphone (LG Fathom) as they went about their daily lives for 2 weeks (Phase 1). Participants were locked out of all functions other than the program and told they were to complete four random assessments (RAs) per day. To increase adherence, participants could use the “delay” feature if they needed to delay the task by 5 min (up to four times per day). A “suspend” option could be used if a participant needed to prevent the phone from presenting assessments for a specific time period. Participants could also “make-up” a training/assessment if they missed an RA or experienced technical difficulties.via phone (Visit 2) and instructed that Phase 1 was completed and daily RAs were suspended. Approximately 4 days following delivery, participants began Phase 2, and were instructed via phone (Visit 3) to repeat the procedures from Phase 1. After 2 weeks they returned to the research clinic and completed Visit 4 assessments  levels were used to confirm reports of abstinence. Participants’ expired breath CO level was measured with a Vitalograph Breath CO device (CO level of <4 ppm was used to indicate abstinence from smoking). The NicAlert® assay was used for the urine cotinine analysis which gives an output on a “0″ to “6″ ordinal scale; <3 was used to indicate abstinence from smoking.2.4.2.EMA items, administered at RAs and make-up assessments, included the following: (1) overall mood and seven affect items  on a 7-point scale ; (2) four items adapted from the Parenting Stress Index ; (3) two items assessing recent smoking; (4) three items assessing general context; (5) two items assessing smoking context ; and (6) an item assessing craving for cigarettes a 7-point scale (as above) following exposure to a picture containing both smoking and non-smoking stimuli presented for 1 s, as described in Kerst & Waters. .2.5.At each assessment (RA or make-up), participants completed either a training task (AR or Control) (75% of RAs/make-ups), or a “standard” visual probe (VP) task (assessment of AB) (25% of RAs/make-ups).2.5.1.In a standard VP task, a pair of pictures  is briefly presented (for 500 ms) simultaneously side by side on a computer screen. When the pictures disappear, a probe stimulus  is presented in the location that had been occupied by one of the pictures (either on the left or the right), and participants are required to press a key as quickly as possible in response to the probe. AB for smoking-related cues is revealed by a faster response to a probe that replaces a smoking-related stimulus , since attention will have been allocated to the location where the smoking picture had been. Note that the standard VP task is an assessment of AB, and the assessment is not intended to change AB. The standard VP task was scored using typical procedures see .2.5.2.On 3 of the 4 RAs scheduled each day, participants were scheduled to complete a training task (AR or Control), 160 trials each. On 1 of the 4 RA scheduled each day, participants were scheduled to complete the standard VP task (for assessment of AB), 80 trials each. During the standard VP task, the dot is equally likely to replace the neutral or smoking picture. Fifteen picture sets consisting of 20 picture pairs  each were used for the tasks. Images were displayed for 500 ms. One picture set was administered on each study day (days 0–14 in pregnancy and days 0–14 postpartum). For the AR condition the VP task was modified so the dot always replaced the neutral picture. In the Control condition the dot was equally likely to replace the smoking stimuli and the neutral stimuli ensuring no correlation between the picture type and dot location, thus avoiding training of attention. This type of control condition also ensures equivalency between the AR and control conditions in terms of task duration, motor practice and stimuli presented .2.6.For both AB and craving, a linear mixed model (LMM) was used. Models included Group (AR vs. Control), Phase (Pre- vs. Postpartum), Day (within Phase) and, where appropriate, the Group x Day interaction. The primary analyses tested the main effect of Group and the Group x Day interaction. For Smoking, a binary outcome, a generalized linear mixed model was used (GLMM). Sample size considerations are reported in the 3.3.1.Seventeen subjects enrolled in the study, and all attended the final laboratory visit and reported completing at least some training (AR vs. Control). Fourteen subjects contributed EMA data. One subject returned the phone with the memory card removed (resulting in loss of EMA data), and EMA data from two other subjects could not be retrieved due to technical problems see . Partici3.2.p = 0.40), number of children (p = 0.70), prior smoking rate (p = 0.77), FTND (p = 0.38), or EPDS score at baseline (p = 0.62).The 14 participants who provided EMA data completed 575 trainings/assessments in total, with 290 from participants in the AR group and 285 from participants in the Control group. In the Control group, 164 of the trainings/assessments were RAs and 121 were make-up. In the AR group, 93 of the trainings/assessments were RAs and 197 were make-up. In total, there were 257 RAs and 318 make-up trainings/assessments see . On days3.3.n = 7) completed a mean of 28.29 (SD = 13.47) AR trainings, and Control participants (n = 7) completed a mean of 23.71 (SD = 12.63) Control trainings. The two groups did not differ in the number of trainings completed, t  completed a mean of 8.14 (SD = 4.26) VP assessments, and Control participants (n = 7) completed a mean of 8.00 (SD = 4.58) VP assessments. The two groups did not differ in the number of VP assessments completed, t , corresponding to an effect size r = 0.66 when using the formula used by Kashdan et al. (p = 0.69), meaning there was no evidence that AB changed across phases. The effect of AR on AB remained significant when controlling for recent smoking . To examine whether AB declined more over time in the AR group (vs Control) within Phases, a Group x Day interaction term was tested. Day within Phase, and the Group x Day interaction term, were included in a model that also included Group and Phase. When coefficients for Day were treated as fixed, the Group x Day interaction was significant , indicating that AB declined more over time in the AR group than Controls. When coefficients for Day were treated as random , the Group x Day interaction was not significant . As shown in n et al. . Phase w3.4.2.p = 0.26), meaning there was no evidence that craving changed across phases. We examined whether Craving declined more over time in the AR group (vs Control) within Phases by testing a Group x Day interaction term. Day within Phase, and the Group x Day interaction term, were included in a model that also included Group and Phase. The Group x Day interaction was not significant when coefficients for Day were treated as fixed , or random .There was a non-significant main effect of AR on the EMA measure of craving . Across 3.4.3.p = 0.65), meaning there was no evidence that levels of smoking changed across phases. Regarding assessment of relapse, defined as any self-reported smoking during the study period, 3 participants (21.4% of 14 participants) reported no smoking during the entire study period, with abstinence confirmed with biochemical assessments, and 11 participants (78.6% of 14 participants) reported relapse. Two abstinent participants were in the AR group (28.6% of AR group) and 1 abstinent participant was in the Control group (14.3% of Control group).There was no evidence for a significant main effect of AR on smoking . Phase w3.5.F  = 5.92, p = 0.04, indicating that Craving declined over time. This model included Group and Day (Day was a level 1 numeric variable and coefficients for Day were treated as random). Additionally, when the Group by Day interaction was added to the model it was not significant, F  = 0.21, p = 0.66. This means that the declines in Craving over time were not significantly different between the AR and Control groups.Exploratory analyses revealed that there was no evidence that the effect of Group was different in the two phases see . In a su4.The main results of this pilot study of AR were as follows. First, there was evidence that AR reduced AB to smoking cues in perinatal women. Second, women in the AR group did not report significantly less craving than women in the Control group. Third, women in the AR group did not report a significantly lower rate of smoking than women in the Control group. Additional separate analyses by phase demonstrated that there was no evidence the effect of AR on study outcomes was different in the two phases (pre vs. postpartum).Compared to Control training, there was evidence that AR reduced AB, as assessed by the VP task. AB was reduced about 50 ms in the AR compared to the Control group. This finding is consistent with other data suggesting that AR administered by the modified VP task can reduce AB as assessed by VP task . This sn = 14) were included in analyses . However, the effect of Group was not significant in analyses restricted to individuals who were abstinent at baseline and who provided EMA data (n = 13). Therefore, more research is required to examine if the effect of AR in abstinent perinatal smokers is robust. Second, it was interesting that participants in the Control group did not exhibit significant AB. This is in contrast to data from participants who received Control training in previous studies (n = 7) was smaller than sample sizes in other studies.Although a significant effect of AR was observed, the following caveats should be noted. First, as noted earlier, the effect of Group was significant when all participants who provided EMA data , this suggests a similar decline in craving in the two groups. Other researchers have reported that both AR and Control training can yield positive outcomes , 41. ForThere was no significant effect of AR on smoking assessed on the smartphone, or on a biological measure of smoking assessed at the lab. Wiers and colleagues have argued that the effects of AR on drinking outcomes are more robust in clinical populations, who are generally strongly motivated to maintain abstinence, than in student samples or samples recruited online . AlthougThis study had a number of limitations. First, the sample size was small, which reduced power of analyses. The analytic sample size was further reduced by loss of data due to technical limitations. Therefore the findings, particularly the null effects of AR on craving and smoking, should be interpreted with caution pending further research with larger sample sizes. Nonetheless the data and findings may be useful for researchers for estimation of effect sizes and/or for use in meta-analyses. Second, due to participants’ extensive use of make-up assessments (rather than RAs) data from the study is likely less “random” than data from a study solely using random assessments. Use of make-up assessments reduces the generalizability of study findings and can potentially lead to bias in parameter estimates. Third, there were limitations regarding the assessment of AB. There was no baseline assessment of AB, meaning that it was not possible to determine whether the two groups differed at baseline. The study did not assess whether the effect of AR on attention generalized to different stimuli type  or to performance on a different attention bias task. Fourth, the use of a single item for craving of unknown reliability could be considered a limitation. Finally, while the main focus of the study was to examine AR in abstinent perinatal former smokers, there was evidence that one participant had smoked prior to randomization. Results should be interpreted in light of the fact that both abstinent and the non-abstinent individual were included in the intent-to-treat sample.The study also had strengths. First, and most importantly, this was the first study to develop and administer an AR intervention for perinatal former smokers, a group at high risk of relapse. Second, another strength was the recruitment of an underserved minority population who are at risk of relapse and lifelong smoking.Results from this study provide evidence that perinatal women can tolerate several days of training and that AR reduces AB in the field. Future research can build off the results of this study. It is possible that the effect of AR on outcomes is diluted by the presence of assessments administered in the field. Assessments were similar to Control trainings, and so future studies might manipulate the proportion of assessments to AR trainings in order to examine whether changes in proportion influence the effect of AR. As noted in the introduction, AR can be easily modified and has been modified for various health conditions and behaviors, such as healthier eating  and anxiety  , 46. FutThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by Yale University Institutional Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.AF and AW contributed to the study’s conception, design, and data acquisition. AF, AW, and RG contributed to the analysis and interpretation of the data and the first draft of the manuscript. AW and RG performed the statistical analysis. CM wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.


Treatment-induced AR alterations, including AR alternative splice variants (AR-Vs), have been extensively linked to harboring roles in primary and acquired resistance to conventional and next-generation hormonal therapies in prostate cancer and therefore have gained momentum. Our aim was to uniformly determine recurrent AR-Vs in metastatic castration-resistant prostate cancer (mCRPC) using whole transcriptome sequencing in order to assess which AR-Vs might hold potential diagnostic or prognostic relevance in future research. This study reports that in addition to the promising AR-V7 as a biomarker, AR45 and AR-V3 were also seen as recurrent AR-Vs and that the presence of any AR-V could be associated with higher AR expression. With future research, these AR-Vs may therefore harbor similar or complementary roles to AR-V7 as predictive and prognostic biomarkers in mCRPC or as proxies for abundant AR expression. Therapeutic interventions for advanced prostate cancer (PCa) are based on the inhibition of the androgen receptor (AR) and downstream of the signaling pathways. Resistance to androgen deprivation therapy is inevitable, and molecular mechanisms driving castration-resistant prostate cancer (CRPC) primarily involve alterations in AR expression and activity.AR is a nuclear receptor transcription factor that is directly activated by androgen hormones. AR is not only considered to be a ligand-activated transcription factor, but is also able to rapidly engage and activate many signaling effectors in the cytoplasm of target cells, leading to cell cycle progression, invasion, cytoskeleton changes, and so on ,2. The gThe LBD plays a key role in the molecular mechanism of AR activity, and persistent AR activity has been extensively explored as a mechanism linked to AR splice variants (AR-Vs). Several of these AR-Vs lack the LBD, thus promoting constitutive gene transcription in the absence of androgen ligands . To thisTherefore, our aim was to uniformly determine recurrent AR-Vs in a large cohort of mCRPC patients, using whole transcriptome sequencing in order to re-evaluate the landscape of recurrent AR-Vs in mCRPC patients in order to prioritize subsequent research. Our unique cohort of WTS analyses of tissue biopsies allows us to provide a complete landscape of AR splicing.We utilized the whole-transcriptome sequencing (WTS) data derived from needle biopsies of 278 distinct patients with metastatic castration-resistant prostate cancer (mCRPC) from the CPCT-02 study to inventory the expression of recurrent AR splice variants AR-Vs; a.n = 278) with available clinical records (n = 213) are reported in n = 48), enzalutamide (n = 18), Radium-233 (n = 3), abiraterone (n = 2), carboplatin (n = 1), and estramustine (n = 1).Baseline characteristics of the included patients from the CPCT-02 study (n = 49) and normal-adjacent prostate tissue constituting mostly normal-adjacent tissue .In addition, we utilized a publicly-available WTS dataset comprising 89 biopsies from non-matched hormone-sensitive prostate cancer (hsPC) patients prior to ADT, comprising both prostate tumor tissue  cryptic exons from the literature  to also n = 10; Using the supporting spliced reads for each splice junction distinctive to each AR-V and comparing this against the support for the canonical splice junction between the first two canonical exons of AR, we calculated the sample-wise relative expression of each AR-V within our cohort b. From ln = 184; 66%), AR-V3 , AR-V7 , AR-V9 , AR23 , and AR-V1 . AR45 was observed in the majority of the samples.When evaluating for minimal expression (relative expression of ≥1% compared to AR), we noted that certain AR-Vs could be more prevalently observed than others: AR45  and no support for distinct splice junctions for any AR-V, incl. AR-Vs which were also not observed in our mCRPC cohort. Therefore, the absence of AR-Vs in treatment-naïve (hormone-sensitive PCa) and NAP samples points toward the emergence of AR-Vs due to treatment or disease progression rather than occurring within early-stage malignant or normal-adjacent tissue.p < 0.001).We evaluated whether harboring expression (≥1%) of any AR-Vs was associated with more abundant expression of AR, in general. We observed statistically significant differences in which samples that harbored an AR-V indeed displayed more abundant expression of AR . From these univariate analyses, the total number of treatment lines (AR45 and AR-V9) and treatment with enzalutamide (AR-V1 and AR-V7), cabazitaxel (AR45), and Radium-223 (AR23 and AR-V3) had significant interactions with the relative expression of specific AR-Vs.To perform exploratory analysis into possible trends with relative AR-V expression (tested per AR-V) versus clinical characteristics, we first performed univariate regression of age at biopsy, given treatment line at any moment prior to biopsy , and the total number of treatment lines to determine potentially significant univariate features (p < 0.05) with AR-V specific relative expression between cabazitaxel treatment and AR45 (p = 0.0265), enzalutamide treatment and AR-V7 (p = 0.0276), and Radium-233 with AR-V3 (p = 0.0499). These effects were therefore seen as significant whilst correcting for the total number of treatment lines within the model.Next, we performed multivariate regression analysis using these four features within the same model and retained significant trends (n = 48) or enzalutamide (n = 18) next to ADT. Between these two patient groups, we observed univariate significance (p < 0.05) of age at biopsy with relative expression of AR-V3 and AR-V7, enzalutamide treatment with AR-V7 and AR-V9, and docetaxel with AR-V7. Next, we performed multivariate regression analysis utilizing all three features and observed the retainment of enzalutamide treatment with both the relative expression of AR-V9 (p = 0.0104) and AR-V7 (p = 0.0157), as shown in p = 0.0381; As these prior treatments constituted a heterogenous mixture of treatments, we performed a similar analysis within the substantial subset of patients who received only a single line of treatment of either docetaxel , followed by AR-V3 and AR-V7. However, we also observed expression of AR-Vs below this threshold, meaning that our currently selected threshold may be under-representing for the actual occurrence of an AR-V, yet this allowed us to better capture highly-expressed AR-Vs from lowly-expressed signals. AR45, as named after its molecular mass of 45 kDa, has previously been described as an enigmatic factor in various mammalian tissue, which uniquely retains the LBD and either represses or co-activates AR activity depending on certain co-factors and active pathways ,15,16,17With our current cohort, we cannot robustly discern the potential roles of AR-Vs within this disease context due to the large heterogeneity of treatment lines and included patients. In addition, we also lacked detailed clinical characteristics such as the Gleason score and histological features. Therefore, characterizing the exact role of AR-Vs with dual roles such as AR45, known as both a repressor or pro-oncogenic factor of AR activity , would nOf these recurrent AR-Vs, AR-V7 has shown much potential as a predictive and prognostic biomarker in mCRPC ,19. FromWe also report that the majority of mCRPC patients expressed one or more AR-Vs in a non-mutually exclusive (co-expressed) manner . FurtherIn conclusion, this uniform inventory of AR-Vs in mCRPC shows that next to AR-V7, AR45, AR-V3, and AR-V9 are also recurrently observed and that future research might further establish their biological roles within mCRPC. Additional studies could furthermore establish the potential of these AR-Vs as predictive and prognostic biomarkers in mCRPC or as a proxy for abundant AR expression due to various somatic aberrations.n = 49) and normal-adjacent prostate tissue . The primary prostate cancer patients included prior to ADT are considered hormone-sensitive prostate cancer patients, whilst the normal-adjacent prostate (NAP) samples can be considered prostate cancer stroma. We used both groups of patients as our reference dataset to compare AR-Vs in distinct prostate cancer states.As part of the CPCT-02 Dutch nationwide multicenter trial (NCT01855477), patients with local prostate or metastatic prostate malignancies were eligible for inclusion and subsequent sampling for whole-transcriptome sequencing if the following criteria were met: (1) age ≥ 18 years; (2) indication for a new line of systemic treatment with registered anti-cancer agents; and (3) safe biopsy according to the intervening physician. All patients have given explicit informed consent for sequencing, data sharing for cancer research purposes, the collection of matched peripheral blood samples (reference DNA), and image-guided percutaneous biopsy from a malignant lesion. For the current study, patients were included who underwent biopsy between 18 February 2015 and 6 June 2020, resulting in a cohort of 278 distinct mCRPC samples. As a reference dataset, we interrogated the whole-transcriptome sequencing data from patients with primary prostate cancer prior to androgen deprivation therapy (ADT) from the NGS-ProToCol cohort (EGAD00001004215) [n = 278 biopsies from 278 distinct patients; one from each patient) was performed according to the manufacturer protocols using a minimum of 100 ng total RNA input. Total RNA was extracted using the QIAsymphony RNA kit  according to manufacturer instructions. Paired-end sequencing of (m)RNA was performed on either the Illumina NextSeq 550 platform  and NovaSeq 6000 platform , depending on the time of sequencing.Whole-transcriptome sequencing of the CPCT-02 cohort  from the CPCT-02 samples were pre-processed using fastp  The whole-transcriptome sequencing data of NGS-ProToCol was processed as previously described . In brieAll primary-aligned, properly paired, and non-duplicated spliced alignments overlapping with a predefined genomic locus representing the AR locus  were retrieved (with strandness) from the Binary Alignment Map (BAM) files using standardized BioConductor approaches in R . These sTo determine the sample-wise and relative expression of any given AR-V, we used the number of splice junction reads supporting the canonical exons 1 and 2 of AR as a reference to determine the following ratio:Equation (1):ESJE21 is the number of primary-aligned, properly paired, non-duplicated spliced alignments (reads) supporting the splice junction between Exon 1 and Exon 2 and xSJ being the similar splice junction support for the AR-V-specific splice junction x, as shown within Here, n = 213).To determine possible correlations or interactions between the relative expression of AR-Vs with clinical characteristics such as age at biopsy, the total number of treatment lines, and specific treatments, we performed univariate and multivariate regressions on all complete patient cases  to determine the univariate significance of these metrics in regards to AR-V expression, in which each AR-V was tested separately. For example, we used a univariate regression model with age at biopsy versus relative expression of AR45. Variables with p < 0.05 were considered to be significant contributors to relative AR-V expression.We first performed univariate regression of age at biopsy, the total number of treatment lines, and major treatment lines (treatments decoded as 1 for received or 0 if not received) on all evaluable patients (n = 48) and enzalutamide (n = 18) prior to biopsy.Next, we subsetted our cohort for patient groups with only a single line of treatment prior to biopsy. In order to robustly determine potential interactions, we only retained patient groups with a min. of 10 patients per group, which contained patients treated with docetaxel (n = 66), we performed a similar univariate analysis in which age at biopsy, enzalutamide treatment, and docetaxel treatment (treatments decoded as 1 for received or 0 if not received) were compared to relative AR-V expression, again per AR-V. Variables with p < 0.05 were considered to be significant contributors to relative AR-V expression. In addition, a multivariate analysis per AR-V with all statistically significant features from the univariate analysis was combined into a multivariate analysis , and similarly, a threshold of p < 0.05 was used to determine statistical significance to relative AR-V expression.Using these patients  under the accession code DR-071. The raw sequencing data of the NGS-ProToCol was previously deposited on the European Genome-phenome Archive (EGA) under accession number EGAS00001002816. The remaining data are available within this article, The WTS and corresponding clinical data used in this study were made available by the Hartwig Medical Foundation  after signing a license agreement stating data cannot be made publicly available via third-party organizations. Therefore, the data are available under restricted access and can be requested by contacting the Hartwig Medical Foundation  have been deposited on Zenodo/GitHub under the GPL-3.0: