Rehabilitation is considered a key health strategy in the 21st century. The aim of rehabilitation is to optimize the functioning of patients. The International Classification of Functioning, Disability and Health (ICF) is a framework for describing and organizing information on functioning and disability. Current international efforts to implement ICF in rehabilitation practise include the implementation of ICF Core Sets and operationalize ICF tools for clinics. The aim of the study is to create simple, intuitive descriptions and an initial reference guide for the assessment of the ICF Rehabilitation Set in Polish practice. The development of the Polish version of ICF Rehabilitation Set involved the following steps: (1) identification of ICF Rehabilitation Set categories; (2) development simple, intuitive descriptions; (3) the drafting of the rating reference guide by a multidisciplinary panel following the process employed to develop the Japanese version. The Polish version of ICF Rehabilitation Set, the simple, intuitive descriptions for 29 categories and the rating reference guides were successfully developed. The Polish version of ICF Rehabilitation Set proposed by us is a reference framework for the harmonization of existing information on the functioning and disability of people participating in the rehabilitation process. Rehabilitation is considered a key health strategy in the 21st century. Demographic and epidemiological trends indicate that human functioning is one of the most important indicators of population health, apart from morbidity and mortality. Thus, it suggests that the main focus of healthcare will be on the real health needs generated by long-term treatment of chronic diseases, including comprehensive enhancement and intensification of rehabilitation2.According to the World Health Organization (WHO) rehabilitation is defined as “a set of interventions designed to optimize functioning and reduce disability in individuals with health conditions in interaction with their environment”3. ICF and ICD provide health information in relation to diagnosis (ICD) as well as functioning and disability (ICF)4. In 2019, WHO launched an eleventh version of ICD (ICD-11), which was supplemented with a section presenting an assessment of functioning containing selected ICF categories5.The aim of rehabilitation is to optimize functioning of patients. WHO indicates International Classifications and Terminologies including the International Statistical Classification of Diseases and Related Health Problems (ICD) and the International Classification of Functioning, Disability and Health (ICF) as the global standards for collecting data regarding health, clinical documentation and statistical data aggregation6. The model contains 6 components, including: (1) body functions which make up physiological and psychological functions of the body; (2) body structures that are its anatomical parts; (3) activity as performing actions or tasks by the individual; (4) participation meaning involvement in life situations; (5) environmental factors that constitute physical, social or attitudinal elements of the environment in which people live; (6) personal factors constituting the life background and the life situation of an individual, comprising the features not related to a health condition7. This approach provides a multifaceted and multidimensional view of human functioning. It also creates a useful framework for understanding the interaction between individual components of the biopsychosocial model.The ICF is based on a holistic biopsychosocial disability model that allows for a comprehensive and wide approach to the problems of an individual related to functioning in his /her living environment9. In order to make the classification more suitable for everyday use, ICF Core Sets have been developed. The ICF Core Sets provide lists of core categories that are relevant to specific medical conditions and healthcare contexts10. The ICF Rehabilitation Set includes 30 ICF categories and is used in the context of describing functioning in various clinical populations among adults, at various stages of patient rehabilitation in inpatient and outpatient rehabilitation facilities, as well as in day care centres11. The rating system proposed in the ICF consists of so-called "qualifiers". They are used to code the severity of a functional problem. The qualifiers are as follows: 0 (0–4%), no problem; 1 (5–24%), mild problem; 2 (25–49%), moderate problem; 3 (50–95%), severe problem; 4 (96–100%), complete problem; 8, not specified; and 9, not applicable7. Environmental factors interact with all components of the biopsychosocial model, making a facilitating or hindering impact of physical and social world and attitudinal system. Qualifiers linked to the category of activity and participation, by determining the level of performance, make it possible to observe the extent to which environmental factors act as a barrier or a facilitator7.The ICF was developed and approved in 2001 and many initiatives have been implemented since thenThe lack of detailed guidelines presenting how to use the qualifiers can make it difficult to assess the patients consistently. In order to solve this problem, it is important to collect the already existing data on the assessment of functioning by means of the ICF and to develop explanations for codes and qualifiers adapted to the clinical conditions in a given country.12. The Global Disability Action Plan (2014–2021) of the WHO recommends the use of the ICF as a superior component of the diagnosis and assessment related with the rehabilitation process13. The ICF is accepted by all the WHO Member States, but its use in clinical practice is still limited14. One of the reasons is the limited number of clinical tools that use language familiar to healthcare professionals while still following the ICF concept. Therefore, in order to facilitate the implementation of the ICF in daily practice, it is necessary to develop a universal and health worker-friendly assessment tool. Cooperative and uniform implementation of the ICF to the Polish healthcare system, as well as other public services, must be coordinated. It is very important to introduce a universal language that would allow us to transfer the information about the state of human functioning in healthcare systems. Consequently, the aim of the study is to create an initial reference guide for the assessment of the ICF Rehabilitation Set in Polish practice.The ICF as a classification is not an assessment tool in itself, but it is a framework based on a biopsychosocial model that enables a comprehensive description of functioning and disability of the examined person in the context of his/her lifeThe development of the Polish version of ICF Rehabilitation Set involved the following steps: (1) verification of ICF categories; (2) development of simple, intuitive descriptions; (3) development of the first Polish version of the rating reference guide.The first stage in the verification of the ICF categories was to conduct a discussion panel consisting of rehabilitation experts with a minimum of 3 years of work experience in an inpatient rehabilitation department in a hospital or a rehabilitation centre, a day rehabilitation centre or an outpatient rehabilitation facility. The members of the discussion panel included: a medical rehabilitation physician, a primary care physician, physiotherapists, an occupational therapist, a psychologist, a speech therapist, a nurse, a social worker and members of the Polish Council for the ICF. Specialists were recruited from inpatient and outpatient rehabilitation centres in the country. Medical facilities conducting rehabilitation were drawn from the register of medical facilities. The inclusion criteria for these units was to have an expert in the field of rehabilitation with at least 3 years of experience at work with the patient. If the centre refused to participate in the study or did not have an expert with the above-mentioned experience, another centre was drawn from the pool.The task of specialists was to assess the ICF categories in terms of their importance for rehabilitation. The expert survey was carried out in the form of an online questionnaire. This expert survey, consisted of open questions and the answers to the questions regarding the most common problems, barriers and facilitations of people participating in the rehabilitation process, were identified, distinguished and linked to the ICF. Selected categories were counted only once per expert. It was assumed that the ICF categories, which were considered very important by at least 3 experts, were included in the list of categories qualified for the next stage of verification.The second step aims were to identify the most common problems experienced by patient with health condition that was documented in a clinical setting. The discussion panel was attended by 21 adults and older people. People participating in rehabilitation at the Donum Corde Rehabilitation Centre were included in the mentioned group. The group of 21 patients included 13 women and 8 men, their mean age was 45.8 ± 13.4 years. The reasons for participation in inpatient rehabilitation were: cardiological (n = 4), neurological (n = 8), pulmonary (n = 3) and orthopaedic diseases (n = 6).People participating in rehabilitation took part in interviews carried out by physiotherapists. The interviews aimed to identify the most common problems with functioning related with the current state of health, recorded in a clinical setting. During the interviews, the subjects determined aspects specific to the rehabilitation process and a given health condition that were important to provide a comprehensive description of their functioning. ICF categories that were considered a problem, barrier or facilitator were included in the candidate categories. Interview protocols were developed by the principal investigator, supplemented with relevant medical data contained in patients' medical records relevant to ICD-10. During the analysis process, the physiotherapist also discussed the patients' problems in an interdisciplinary team consisting of: a physician, physiotherapist, nurse, pharmacist, occupational therapist, speech therapist and psychologist to determine their relationship with ICF. Categories that were identified as a problem, barrier or facilitator by at least 75% of interviewees were included in the list of proposed ICF categories.15 and was also based on the experience of other researchers17.The methodology of the discussion panel was compliant with the WHO guidelines18. The ultimate list of ICF categories consisted of codes that were considered relevant by at least 75% of the participants of the discussion panel.The final verification of the ICF categories proposed for inclusion in the Polish version of ICF Rehabilitation Set was carried out by a research team consisting of 4 persons with the title of associate professor and PhD. All researchers had experience in the use of the ICF classification in scientific research and clinical practice and they dealt with treatment, rehabilitation, education or medical statistics. The team analysed the obtained proposed ICF categories from the stage of experts (in the field of rehabilitation) examination as well as patients participating in physical rehabilitation. Individual categories from the ICF Rehabilitation Set were considered confirmed if the same category emerged in the course of interviews with patients and a group of rehabilitation experts. In consequence, three groups of categories have been identified: (1) ICF categories identified as relevant by patients and experts, hence represented in the ICF Rehabilitation Set, (2) ICF categories that were represented in the ICF Rehabilitation Set but not identified as important by the discussion panel, and (3) ICF categories that were considered important by the members of the discussion panel, but were not included in the ICF Rehabilitation SetTo develop simple, intuitive descriptions of the Polish ICF Rehabilitation Set based on the original ICF category descriptions included in the classification, a consensus conference with multidisciplinary rehabilitation experts was conducted.19, Italy15 and China20. The participants were asked to read and discuss the initial suggestions for descriptions. Then, each participant voted on whether the description was simple and intuitive enough to be used in everyday clinical practice. It was important to maintain the context of the original description of the ICF categories. During the first vote, consensus was achieved when the description of each ICF category reached 75% agreement in each group. The categories that did not reach consensus in the first vote were re-described, discussed and voted on again. A similar voting was conducted three times. The final versions of the ICF category descriptions were approved by the members of the Council for ICF Implementation in Poland and redirected for translation by a native English speaker.The consensus process included 3 groups of 7 experts with experience in clinical work with the patient. In each group, one moderator and an assistant for the ICF implementation team in Poland were appointed. The assistant was responsible for taking notes during the conference, but did not have the right to vote. Each group received initial suggestions for simple, intuitive descriptions of each ICF category. Descriptions were prepared on the basis of the results of previous conferences in JapanThe rating reference guide Polish version of ICF Rehabilitation Set contains a description how to assess the different ICF categories and how to transfer the assessment results to qualifiers.19 and the Italian Team15.Rehabilitation experts have identified the main aspects to consider when rating ICF Body Functions, Activities and Participation, and Environmental Factors. The members of the working group proposed descriptive scales that can be used in practice to rate the categories. The proposed rating system was prepared during a discussion panel based on the applied methods of clinician assessment, cognitive interviews and reviews of available and already verified tools for the assessment of ICF categories. The available guide is also based on the results of the definitions developed by the Japanese TeamThis study was approved by the Bioethics Committee of the University of Rzeszów (Resolution No. 33/05/2019). All participants provided written informed consent. All methods were performed in accordance with the relevant guidelines and regulations.A Polish version of ICF Rehabilitation Set to assess and verify functioning levels of patients was developed for 29 categories, including 8 categories  from Body Functions, 16  from Activities and Participation, and 5  from Environmental Factors. Table As a result of the process of defining ICF categories, 6 initial description proposals were accepted in the first stage, and 15 proposals in the second stage. In the final third voting, the description of the remaining 8 definitions was settled. All participants agreed to the final simple, intuitive descriptions of the ICF categories indicated at the verification stage.The Polish final version of the simple, intuitive description of the ICF categories was shown on Table 7. For “Body Functions”, a general qualifier that scores the extent of the problem or the extent of the impairment is shown on a scale from 0 to 4 .The assessment of all three components of the classification was proposed using the same scale described by WHOcapacity and performance. Rating Capacity refers to the patient's ability to complete a task or take an action. It should be measured in a unified, standardized environment, so without the use of facilitators and specific barriers. Rating Performance refers to the patient's ability to perform a task or take an action in one’s own current environment, thus taking into account the facilitators or barriers posed by the social conditions in which the patient lives. Both qualifiers are scored using the following scale: xxx.0 NO problem; xxx.1 MILD problem; xxx.2 MODERATE problem; xxx.3 SEVERE problem; xxx.4 COMPLETE problem; xxx.8 not specified and xxx.9 not applicable.For “Activities and Participation”, two qualifiers are presented: 7.The coding of "Environmental Factors" should be done by assessing the patient's perspective. A qualifier assessing environmental factors may indicate a barrier or facilitator. The rating scale is as follows: xxx.0 NO barrier; xxx.1 MILD barrier; xxx.2 MODERATE barrier; xxx.3 SEVERE barrier; xxx.4 COMPLETE barrier; xxx.+ 0 NO facilitator; xxx + 1 MILD facilitator; xxx.+ 2 MODERATE facilitator; xxx.+ 3 SUBSTANTIAL facilitator; xxx.+ 4 COMPLETE facilitator; xxx.8 barrier not specified; xxx.+ 8 unspecified facilitator and xxx.9 not applicableSensation of pain), B710 (Mobility of joint functions) and B730 (Muscle power functions) was linked to existing methods and scales commonly used in clinical practice, i.e. pain assessment on the Visual Analog Scale21, goniometric assessment of the range motion22 and assessment of muscle strength using the Lovett Scale23.For the analysis of limitations in the range of body functions related to B130, B134, B152, B445 and B620, an assessment by means of specially prepared questions and an assessment carried out by healthcare professionals were proposed. The description of the possible limitation makes it possible to qualify the patient's problem for the appropriate qualifier. The assessment of B280 (Transferring oneself), D510 (Washing oneself), D530 (Toileting), D540 (Dressing), D550 (Eating) was based on the Activity of Daily Living Scale24, taking into account the range of patient limitations on a scale from 0 to 4, whereas the assessment of one of the complex daily activities D650 (Doing housework) was presented using the Lawton Instrumental Activities of Daily Living Scale25. For the analysis of categories D415 (Maintaining a body position) and D410 (Changing basic body position), the Berg Balance Scale was used26. With reference to mobility assessment D450 , the result of the Short Physical Performance Battery Test was used on the ICF scale27.In case of activity and participation, D230, D240, D465, D570, D710, D850 and D920 were assessed by means of questions asked by healthcare professionals. The assessment of categories D420 ; immediate family (e310) or individual attitudes of healthcare professionals (e450). These categories are important due to the fact that they allow for the identification of facilitating factors or barriers related to the process of functional rehabilitation of patients19. Previous studies on the clinical implementation of the ICF Rehabilitation Set suggested that clinicians were unable to distinguish effectively the line between different qualifiers30. Moreover, in the study carried out by Senju et al., experts also indicated that it is difficult to distinguish the difference between mild and moderate ICF problems. The WHO states that a moderate problem ranges from 25 to 49% of problem percentages, whereas a mild problem is from 2 to 24%31. The existing explanation of the problem causes some difficulties in assessing patients in clinics32.The development of a reliable rating tool increases the usefulness of the ICF classification for clinical and statistical purposes. The results on the patient's functioning can be transmitted to other specialists, as well as compared showing the effectiveness of rehabilitation in different institutions or regions17. What is more, some studies regarding the implementation of ICF sets were also carried out, however, the assessment of qualifiers differed from one study to another. The Chinese project used an intuitive rating scale ranging from 0 to 1033. The Japanese researchers developed a rating reference guide for the ICF Rehabilitation Set. They developed a rating system with a description for each answer and translated the assessment into ICF qualifiers34. The weighted kappa coefficient in the field studies was 0.61. The inter-rater reliability test showed moderate to high inter-rater agreement34. In the Polish version of the ICF Rehabilitation Set, apart from specially developed questions for the assessment of individual categories, the most frequently used scales in clinical conditions in Poland were also proposed.Therefore, international efforts have been made to develop ICF-based clinical tools. The development of tools includes the development of ICF sets as well as simple intuitive descriptions of ICF categories included in the sets35. The ICF classification is closely related to the ICD-10. The ICD-10 is a health classification system and etiological framework, while the ICF is used to classify health-related functioning and disability. Thus, the ICD-10 provides information on the diagnosis of the disease, while the ICF offers an additional explanation of how the medical condition affects the functioning of people with various diseases36. Combining the ICD-10 reporting with the ICF can improve the quality of data regarding patients collected in the healthcare system.One of the most important issues in interdisciplinary communication in healthcare is the use of common terminology37. Therefore, the use of a unified rating system considering ICF qualifiers will be beneficial both in clinical and scientific discussion as well as in statistics39. The Polish version of ICF Rehabilitation Set proposed by us is a reference framework for the harmonization of existing information on the functioning and disability of people participating in the rehabilitation process.The World Health Organization and the World Confederation for Physical Therapy proposed the use of the ICF as a universal patient assessment in rehabilitation practice


Introduction: Epigenetic-targeted therapy has been increasingly applied in the treatment of cancers. Lysine (K)-specific demethylase 6B (KDM6B) is an epigenetic enzyme involved in the coordinated control between cellular intrinsic regulators and the tissue microenvironment whereas the pan-cancer analysis of KDM6B remains unavailable.Methods: The dual role of KDM6B in 33 cancers was investigated based on the GEO (Gene Expression Omnibus) and TCGA (The Cancer Genome Atlas) databases. TIMER2 and GEPIA2 were applied to investigate the KDM6B levels in different subtypes or stages of tumors. Besides, the Human Protein Atlas database allowed us to conduct a pan-cancer study of the KDM6B protein levels. GEPIA2 and Kaplan–Meier plotter were used for the prognosis analysis in different cancers. Characterization of genetic modifications of the KDM6B gene was analyzed by the cBioPortal. DNA methylation levels of different KDM6B probes in different TCGA tumors were analyzed by MEXPRESS. TIMER2 was applied to determine the association of the KDM6B expression and immune infiltration and DNA methyltransferases. Spearman correlation analysis was used to assess the association of the KDM6B expression with TMB (tumor mutation burden) and MSI (microsatellite instability). The KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis and GO (Gene ontology) enrichment analysis were used to further investigate the potential mechanism of KDM6B in tumor pathophysiology.Results: KDM6B was downregulated in 11 cancer types and upregulated across five types. In KIRC  and OV (ovarian serous cystadenocarcinoma), the KDM6B level was significantly associated with the pathological stage. A high level of KDM6B was related to poor OS  outcomes for THCA (thyroid carcinoma), while a low level was correlated with poor OS and DFS  prognosis of KIRC. The KDM6B expression level was associated with TMB, MSI, and immune cell infiltration, particularly cancer-associated fibroblasts, across various cancer types with different correlations. Furthermore, the enrichment analysis revealed the relationship between H3K4 and H3K27 methylation and KDM6B function.Conclusion: Dysregulation of the DNA methyltransferase activity and methylation levels of H3K4 and H3K27 may involve in the dual role of KDM6B in tumorigenesis and development. Our study offered a relatively comprehensive understanding of KDM6B’s dual role in cancer development and response to immunotherapy. Epigenetic dysregulation leads to aberrant transcriptional programs that promote tumor occurrence and progression . Thus, evia demethylating H3K27me3 to prompt the expression of cyclin D1 cooperating with smad2/3  or Jumonji domain-containing protein-3 (JMJD3), belonging to histone H3 lysine 27 (H3K27) demethylases, regulates the gene expression by histone demethylation in response to an intracellular or extracellular stimulation under physiological or pathological conditions. KDM6B regulates a wide range of pathways involved in development, inflammation, and specifically cancers, such as WNT, NF-κB, and BMP . Over-ex smad2/3 . Howeverblastoma . NeverthConsidering the potential link between genes and cancers, it is necessary to explore the correlation between genes of interest and the occurrence and prognosis in pan cancers and the underlying molecular mechanisms. The publicly funded Cancer Genome Atlas (TCGA) project and the Gene Expression Omnibus (GEO) database contain functional genomic datasets for different types of tumors , which mhttp://timer.cistrome.org/)   was used to explore the KDM6B levels across 20 tumor types by entering “KDM6B” (We used the TIMER2.0 (Tumor Immune Estimation Resource 2nd Edition) website (me.org/)  to detecnalysis)  to gain Ex data” . FurtherEx data” . Also, t “KDM6B” .via the log-rank test. Moreover, we used the Kaplan–Meier plotter web server  to perform an analysis of OS, DMFS , FP (first progression), PFS , RFS , and PPS   and DFS  outcomes of high and low KDM6B expressions across all tumors in TCGA. As previously reported, cutoff-high (50%) and cutoff-low (50%) values were applied as thresholds to divide the samples into the high- and low-expression cohorts . We alsourvival) . As prevre drawn .https://www.cbioportal.org/)   value were calculated. We also analyzed the relationships between the expression of KDM6B and four DNA methyltransferases  with TIMER2.0  . After tess.be/)  to invesTIMER2.0 . Heatmap+ T cells, CD8+ T cells, Treg, follicular helper T cells, NK T cells, NK cells, neutrophils, common lymphoid progenitors, hematopoietic stem cells, common myeloid progenitors, endothelial cells, DCs (dendritic cells), granulocyte–monocyte progenitors, myeloid-derived suppressor cells, eosinophils, and cancer-associated fibroblasts. The purity-adjusted Spearman’s rank correlation test was used to obtain the p-values and sectional correlation values. Finally, the data across all the immune cells were shown in the form of heatmaps, and the data on cancer-associated fibroblasts were also visualized as scatterplots.We explored the association between the KDM6B expression and immune infiltration across all tumors in TCGA with TIMER2.0. CIBERSORT, CIBERSORT-ABS, TIMER, quanTIseq, MCP-counter, xCell, and EPIC algorithms  were appTMB represented Mut/Mb (the number of mutations per mega base) of DNA in tumor cells . MSI reshttps://string-db.org/)  web server (db.org/)  with thedb.org/) . Finallydb.org/) , we obta, and LIHC (liver hepatocellular carcinoma) (p < 0.01). On the contrary, the KDM6B expression level increased in the tumor tissues of CHOL (cholangiocarcinoma), HNSC (head and neck squamous cell carcinoma) (both p < 0.001), ESCA  and KIRC  (both p < 0.05). For DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), LAML (acute myeloid leukemia), OV (ovarian serous cystadenocarcinoma), and TGCT (testicular germ cell tumors), the KDM6B level decreased in the tumor tissues of DLBC, TGCT, and OV, while KDM6B expressed higher levels in the tumor tissues of LAML, all with p < 0.01 , as previously reported . In addition, a low KDM6B expression was correlated with the poor OS prognosis (p = 0.006) and DFS prognosis (p = 0.00029) for KIRC. Moreover, we used the Kaplan–Meier plotter tool to explore the survival data on other cancers (p = 0.0012) prognosis for breast cancer. However, a high KDM6B expression was correlated with a poor DMFS prognosis (p = 0.0075) for breast cancer (p = 5.3e-05) and FP (p = 8.3e-11) prognosis for lung cancer (p = 1.3e-10), FP (P = 1e-09) and PPS (p < 1.0e-16) prognosis for gastric cancer  and TGCT (p = 1.87e-14).We detected a genetic change of KDM6B in several tumor cases from TCGA projects with cBioPortal. The highest frequency of change in KDM6B (>8%) was found in melanoma patients with “mutation” as the main type . The “mup < 0.001 and R = 0.464), as shown in Next, as shown in via the tumor microenvironment. Analysis executed with all or most of the above-mentioned algorithms showed significantly positive correlations between immune infiltration and KDM6B expression in different cancers, for example, Tregs and COAD; endothelial cell and COAD, KIRC, PAAD, SKCM-metastasis, and STAD; neutrophils and PCGC, PRAD, and THCA; and follicular helper T cells and GBM and UCEC .To explore the potential mechanisms of KDM6B in tumor pathophysiology, we sought to highlight KDM6B-binding proteins and KDM6B expression-related genes in several pathway enrichment analyses. We gained top 50 experimentally determined KDM6B-binding proteins with STRING. The protein–protein interaction network of these proteins is shown in Furthermore, we executed GO and KEGG analyses with the KDM6B-related proteins. The top three significant terms in BPs , MFs (molecular functions), and CCs (cellular components) and KEGG pathways are shown in Above all, the results showed that the KDM6B expression had a different impact on the prognosis among various cancers. Genetic alteration of KDM6B also affected the prognosis of acute myeloid leukemia (LAML) and testicular germ cell tumors (TGCTs). The KDM6B expression level was associated with DNA methyltransferase activity, TMB, MSI, and immune cell infiltration, across various cancer types with different correlations, through which KDM6B may impact on the clinical outcomes and immune therapeutic effects. The top 50 proteins most correlated were recognized using STRING. GO and KEGG enrichment analyses showed that the most closely related pathway was histone methylation regulation.via certain pathways remains unclear. This study intended to explore the dual role of KDM6B widely. Therefore, with the TCGA and GEO databases, we thoroughly characterized the KDM6B expression in 33 types of tumors and examined the related molecular properties of the expression level, genetic alterations, or DNA methylation. We found that the expression of KDM6B differed in different cancer types and stages. With the KM survival analyses, we revealed that an irregular KDM6B expression may serve as a prognostic biomarker in several cancers, such as breast cancer, lung cancer, and gastric cancer. Moreover, we also predicted that the KDM6B expression level was correlated with the tumor immune microenvironment. GO and KEGG analytic approaches were performed to recognize the KDM6B‐related biological mechanisms.As a histone demethylase, KDM6B is responsible for the enzymatic removal of the repressive chromatin mark H3K27me3 which is involved in biological pathways such as differentiation, development, and apoptosis. These biological pathways tend to impact response reactions against intracellular or extracellular stimulations and suggest a functional link between KDM6B and clinical diseases, including inflammations, especially, cancers . HoweverTo improve the chances of cures for cancer patients, it is necessary to explore tumor-specific target molecules for a precise treatment through identifying the differentially expressed genes . TherefoAlthough immunotherapy has shown increasing the therapeutic impact in tumors, we barely obtained any reports focusing on the relationship between the KDM6B expression and tumor immunity through literature search. Therefore, we explored the potential association between KDM6B expression and immune infiltration levels of various immune cells with TIMER2. As a result, we detected a positive relation between the KDM6B expression and the immune infiltration level of Treg in COAD and LUSC; endothelial cell and COAD, KIRC, PAAD (pancreatic adenocarcinoma), SKCM (skin cutaneous melanoma)-metastasis, and STAD (stomach adenocarcinoma); neutrophil and PCGC (pheochromocytoma and paraganglioma), PRAD, and THCA; and follicular helper T cells and GBM (glioblastoma multiforme) and UCEC . Remarkably, for example, the immune infiltration of T-cell gamma delta was negatively correlated with the KDM6B expression in BRCA-LumA and CESC based on all or most algorithms. In addition, the results showed that the KDM6B expression correlates with the degree of the infiltration of tumor-associated fibroblasts in various cancers, including BRCA, HNSC (head and neck squamous cell carcinoma), HPV−, LIHC, LUAD (lung adenocarcinoma), OV, PAAD, SKCM, and SKCM−metastasis. Cancer-associated fibroblasts have been reported to have an important impact on tumor metastasis, progression, and prognosis . The evivia TMB, MSI, or MMR, offering a new predictor for the immunotherapy efficacy.Generally, higher TMB was related to a favorable overall survival and better response to immunotherapy . MSI staInduced by DNA methylases, characteristic hypermethylation in the promoter or enhancer of certain genes, especially cancer-associated genes, was a specific feature of cancer cells . Based ovia promoting the nuclear translocation of FOXO1 (via HOXC9 to remove the repressive chromatin marker H3K27me3 and induce neuronal differentiation, thus inhibiting cell proliferation and tumorigenicity (via the same way in certain cancers, such as neuroblastoma sphere-forming cells and squamous cell carcinoma cells. KDM6B upregulates the expression of specific genes, such as p53, p21, HOX, and ERβ so as to restrain cell growth and boost differentiation, senescence, and apoptosis through the histone demethylase activity in tumor cells (via binding to IL‐25R on tumor cells (via various mechanisms and its clinical significance remained to be further established.KDM6B plays oncogenic roles in several tumors. In prostate cancer, KDM6B demethylates H3K27me3 at cyclin D1 promoter and prompts the expression of cyclin D1 cooperating with smad2/3 . Upregulof FOXO1 . In squaof FOXO1 . In neurgenicity . To explgenicity . In addigenicity . H3K4 trgenicity . Hypermegenicity . A similgenicity . Furthergenicity . In epiggenicity . These por cells . The KEGor cells . KDM6B, or cells . Substanor cells . In pancor cells . Howeveror cells . In breaor cells . Thus, tin vitro or in vivo experiments should be performed to conduct a more solid conclusion.Some limitations to this study should be addressed. The survival analysis in several cancers was not assessed due to a lack of available data. In addition, the functions of KDM6B in pan-cancers were only tested with the bioinformatics analysis; therefore, In summary, this study assessed the dual role of KDM6B in tumor progression and clinical outcomes across all types and stages of cancers in TCGA for the first time. KDM6B expression was associated with TMB, MSI, and immune cell infiltration, particularly cancer-associated fibroblasts. Thus, KDM6B may affect the response to immunotherapy and clinical outcomes. Dysregulation of the DNA methyltransferase activity and the methylation level of H3K4 and H3K27 involve in the dual role of KDM6B in tumorigenesis and development. It is of great value to investigate the exact role of KDM6B in tumorigenesis and immune microenvironment to generate more precise preventive measures and immunotherapy.


Traditionally, α1-AR antagonists were first used as anti-hypertensives, as α1-AR activation increases vasoconstriction, but they are not a first-line use at present. The current usage of α1-AR antagonists increases urinary flow in benign prostatic hyperplasia. α1-AR agonists are used in septic shock, but the increased blood pressure response limits use for other conditions. However, with the advent of genetic-based animal models of the subtypes, drug design of highly selective ligands, scientists have discovered potentially newer uses for both agonists and antagonists of the α1-AR. In this review, we highlight newer treatment potential for α1A-AR agonists  and non-selective α1-AR antagonists . While the studies reviewed here are still preclinical in cell lines and rodent disease models or have undergone initial clinical trials, potential therapeutics discussed here should not be used for non-approved conditions.α Upon further tissue characterization and molecular cloning, α1-ARs were further subdivided into the α1A-, α1B-AR, and α1D-AR subtypes based upon the subsequent cloning of the receptors [1C-AR is missing from the current α1-AR nomenclature due to misclassification and incomplete pharmacological characterization of the α1A-AR subtype [Receptors that are activated by the adrenaline-type catecholamines, epinephrine (Epi) and norepinephrine (NE), are called adrenergic receptors (ARs). They belong to the G-Protein Coupled Receptor (GPCR) superfamily, which are receptors that transduce their intracellular signals through G-proteins. According to their physiological effects on the body, they were initially assigned as classifications α and β . α-ARs weceptors ,4. The α subtype .1A-ARs can be pharmacologically distinguished in tissues and cell lines from the α1B-AR subtype based upon a 10–100-fold higher binding affinity for several ligands that are commercially available [1D-ARs share more pharmacological similar and genetic homology with the α1A- than the α1B-AR but buspirone analogs  have been developed that have at least a 10-fold higher binding affinity for the α1D-AR over the α1A-AR subtype [1B-AR subtype. α1B-AR does not have sufficiently selective ligands developed yet, but with the recent crystal structure of the α1B-AR bound with the antagonist cyclazosin [αvailable ,7  bind Epi and NE with comparative affinities, the three different families couple to different G-proteins and effector pathways that allow specificity in function. While all GPCRs can couple to multiple G-proteins, they strongly couple to only a few. α-ARs couple more efficiently to Gαs, which stimulates adenylate cyclase and increases cAMP levels. α2-ARs are transduced by Gαi, which inhibits the production of cAMP. α1-ARs couple to Gαq to activate phospholipase C that causes the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to release inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 binds to IP3 receptors located on the endoplasmic reticulum which causes the release of calcium. DAG activates protein kinase C (PKC), an enzymatic effector that can phosphorylate many proteins to amplify signals downstream in the signaling cascade. α1-ARs, as in all GPCRs, can signal directly or through cross-talk to couple to many other signaling pathways, both G-protein-dependent and independent, and through spatio-temporal as well as biased-agonistic mechanisms [While the nine subtypes  in the rat CA1 hippocampus [1A-AR systemically overexpressing transgenic mice increased synaptic plasticity, LTP, and performance in a battery of cognitive tests of spatial memory, while α1A-AR KO mice performed poorly [1B-AR KO mice had impaired spatial learning to novelty and exploration [1D-AR KO mice did not show deficits in spatial learning [1-AR subtypes are localized in the brain and expressed in overlapping domains, the α1A-AR subtype appears to have greater expression in cognitive areas such as the hippocampus and amygdala, as well as particular areas of the cortex and neurogenic regions involved in learning and memory [1A-AR selective agonist cirazoline increased cognition and BrdU incorporation in normal adult mice, while the α1A-AR overexpressing transgenic mice had increased BrdU incorporation in both the subventricular and subgranular neurogenic regions [αunctions . α1-AR apocampus , neocortpocampus , and maypocampus ,81,82,83d poorly . α1B-AR loration , and a dloration . While αlearning , they dilearning . While ad memory ,89. The  regions .1A-AR agonists to treat heart failure, cardiac ischemia, or Alzheimer’s disease, PAMs with sufficient signal bias would need to be developed that could regulate heart or brain function without effects on the vascular system to increase blood pressure. PAMs will increase a receptor activation and function but in such a way that it does not bind to the same site as the endogenous agonist , such as NE [1-AR agonists which limit their use in neurological conditions. The first PAM at the α1-ARs with high selectivity for the α1A-AR subtype has been developed [In order to develop suitable therapeutic αch as NE . Allostech as NE . Anothereveloped  that caneveloped ,95,96,971-AR antagonists affect the contraction of smooth muscle in several organ systems. α1-AR blockage results in the relaxation of smooth muscle in the prostate and ureter to increase urinary flow [1-AR antagonists are frequently used medications in the management of benign prostatic hyperplasia (BPH), kidney stones, and in therapy-resistant arterial hypertension, two conditions frequently found in older adults. As a powerful anti-hypertensive, α1-AR antagonists are not recommended as a first-line treatment [1-AR antagonists are effective in the relief of urinary symptoms and improve the quality of life in BPH, they appear less effective in preventing disease progression [1-AR blockers are also used to treat pheochromocytoma, a rare condition where a tumor forms on the adrenal gland or other paraganglia to cause excessive catecholamine release and severe hypertension. The tumor is excised immediately under the use of an α1-AR blocker to reduce hemodynamic instability, morbidity and mortality [1-AR antagonists will be discussed at the end of this article.As in the vascular system, αary flow ,99,100. reatment ,102 as tgression ,104. α1-ortality . GeneralCoronavirus disease 2019 (COVID-19) and the causative agent, severe acute respiratory syndrome coronavirus 2 (SARS), can elicit a vigorous systemic immune response  in the lungs as well as multiple organs, resulting in heart and kidney failure, liver damage, precipitating severe illness, and increased mortality . Recent 1-ARs have been shown to modulate the immune system [1-ARs have been identified on a wide variety of immune cells. Identification of immune cells using flow cytometry depends upon highly avid antibodies whose specificity are questioned for the current commercially available antibodies for the α1-ARs and many other GPCRs [1-AR subtypes [1B- and α1D-ARs [1-ARs but the subtypes are not clearly defined.Beyond their role in neurotransmission, cardiovascular, and the stress response, αe system ,109, inne system , and infe system ,112. α1-er GPCRs . Howeversubtypes . Monocyt α1D-ARs ,116. NK  α1D-ARs ,118,119, α1D-ARs ,121, als1-AR antagonists may reduce morbidity and mortality in patients at risk for hyperinflammation and cytokine storm that is often associated with COVID-19 and other conditions that result in severe respiratory tract conditions. Blockade of α1-AR function with prazosin prevents cytokine storm following pro-inflammatory conditions and increases survival in preclinical studies [n = 18,547) and three cohorts with pneumonia  found that patients exposed to α1-AR antagonists had a significantly lower risk (34%) for mechanical ventilation and death [https://clinicaltrials.gov/ct2/show/NCT04365257, accessed on 7 February 2023) but this trial is currently halted due to lack of recruitment. These results extend circumstantial findings that prazosin may be an early preemptive therapy in COVID-19 and may prevent the cytokine storm and severe complications due to hyperinflammation.Several studies indicate that α studies . A retrond death . Similarnd death  and anotnd death . These snd death  caused b1-AR blockers against COVID-19 were recently challenged in a study using meta-analysis of millions of patients prescribed α1-AR blockers , compared to alternative medications  or tadalafil (PDE5 inhibitor) to treat BPH. This study found no reduction in the risk of COVID-19 infection due to the sustained use of α1-AR blockers [1-AR blocker treatments for BPH as the study of Thomsen et al. [1-AR blockers to confer protective benefits against death and ICU admission due to COVID-19.The protective effects of αblockers . The negn et al.  also inc1-AR blocker effects on more severe outcomes . Therefore, one interpretation is that α1-AR blockers do confer protection, but the amount of pre-emptive protection is not that significant for use in the general population but only for a subset of severely ill patients once the cytokine storm has developed, and then used to reduce mortality. All of these studies have limitations in that they measured outcomes on men who are more likely to be prescribed α1-AR blockers due to BPH and may not reflect possible outcomes for women. Nevertheless, these results suggest the need for further clinical trials to include women and whether α1-AR blockers first ameliorates the severe symptoms of lower respiratory tract infection-associated hyperinflammation and the risk of death.The study of Nishimura et al.  suggeste1-AR blockers to reduce hyperinflammation. Prazosin prevents cerebral infarction by inhibition of the inflammatory cascade [1-ARs may use to combat hyperinflammation is through their association with chemokine receptors. Chemokines are a group within the cytokine family whose general function is to induce cell migration and are potential therapeutic targets in numerous inflammatory diseases, such as COVID-19. Several chemokine genes have been associated with disease severity and susceptibility to infection with COVID-19 [1B or α1D-AR subtypes and inhibited their function and were detectable in human monocytes [1A-AR in prostatic smooth muscle [1-ARs) reduced cytokine release and protected mice against COVID-19 lethal complications [1-AR, were observed in patients after SARS infection and suggested to cause impaired blood flow, the formation of microclots, and autoimmune dysfunction contributing to long-COVID symptoms [1-AR antagonists in blocking α1-AR mediated adverse effects in hyperinflammation.There is precedent in preclinical studies for the ability of α cascade . One mecCOVID-19 . At leasonocytes . The CXCh muscle . Many GPh muscle ,134,135.h muscle . In animications . Furthersymptoms ,138. The1-AR mediated through activation of phosphoglycerate kinase (PGK1)-mediated ATP production. Terazosin and its related “osins” are postulated to mediate protective mechanisms by binding adjacent to the ADP-ATP site of PKG1 and facilitating its activation. PGK1 is the first enzyme in glycolysis where ADP enters the cleft of the active site and is converted into ATP and shown to inhibit apoptosis [1-AR antagonists that contain quinazoline motifs, such as alfuzosin, prazosin, and doxazosin [1-AR blocker but with some selectivity for the α1A-AR [1-ARs may confer therapeutic levels of protection against inflammation and morbidity through non-α1-AR -mediated effects of increasing glucose metabolism by binding to the active site of PGK1.It is possible that the protective anti-inflammatory effects of prazosin, doxazosin and terazosin may be non-αpoptosis ,140. Teroxazosin  and COVIoxazosin ,143. Teroxazosin . The effe α1A-AR , does noe α1A-AR ,146. Fure α1A-AR . Therefo1-AR quinazolines  have also been shown in several studies to induce apoptosis in different cell lines and in vivo through non-α1-AR mechanisms [While the above protective effects of PGK1 appear to be metabolic, αchanisms ,149,150.chanisms . Therefo1-AR subtype, or disease-dependent. All of the pro-apoptotic effects of quinazolines are non-α1-AR mediated and mostly found in cancer cell lines, while metabolic effects are more systematic and may be α1-AR subtype dependent. The non-quinazoline tamsulosin does not exhibit cytotoxic or apoptotic activity in cancer cell lines [1-AR antagonist reduced inflammation and immune cell infiltration and improved insulin signaling in the adipose of fructose-fed rats [These two different but protective mechanisms (metabolic verses apoptotic) may be cell-type, αll lines . Prazosill lines . A non-qfed rats , as wellfed rats .1-AR subtype-dependent effects of antagonists, there is evidence that α1A-AR activation is protective, while chronic α1B-AR activation is damaging and neurodegenerative. Therefore, α1A-AR agonists would be protective and in systems where chronic α1B-AR activation is damaging, non-selective blockers may exert protective effects. Systemic overexpression of the α1A-AR in mice has anti-tumor effects [1B-AR subtype in mice was neurogenerative, induced autonomic dysfunction, heart failure, apoptosis [1A-AR selective antagonist [1B-AR blockage since tamsulosin (α1A-AR blockage) does not induce apoptosis nor binds with PGK1. The study of Koenecke (2021) [1B or α1D-mediated pro-inflammatory effects. There is an increased expression and cellular proliferation of the α1B-AR subtype in prostatic cancer cell lines that exhibit apoptosis with prazosin [1B-AR activation mediates unchecked cell cycle progression and induced foci formation [1-AR blockage might indicate that the α1B- or α1D-AR subtype is being blocked in the particular tissue or disease.Concerning α effects , precond effects , reverse effects ,65,66, a effects . In contpoptosis ,155,156,poptosis . Tamsulotagonist ,157. Thetagonist , while fe (2021)  found thprazosin . α1B-AR ormation , support1-AR quinazoline antagonists may also be useful in other neurodegenerative diseases.Neuroprotection, just like cardioprotection, may be mediated through increased metabolism . As the 1-AR blockers (non-quinazolines) are effective. α1A-AR stimulation has been suggested to mediate stress-induced memory formation and consolidation [1A-AR antagonistic effect.Posttraumatic stress disorder (PTSD) is associated with elevated noradrenergic activity ,169,170.1-AR quinazoline antagonists appear to improve symptoms in neurodegenerative diseases and PTSD, regardless of whether they are α1-AR or non-α1-AR mediated, antagonists that are selective for the α1A-AR subtype may potentiate neurodegeneration and dementia. This would be consistent with α1A-AR activation demonstrating increased cognitive performance and reversing Alzheimer’s disease as discussed in this review. Just as tamsulosin does not follow the protective properties of quinazoline antagonists as discussed in the above sections, tamsulosin, which is α1A-AR selective, increases the risk of dementia modestly and other adverse cognitive effects, in particular among patients over age 61 [1-AR blockers) for BPH as well as those taking no medications and followed them for 20 months after the first prescription was filled. However, two subsequent clinical studies contradict these results [While non-selective αr age 61 . This st results ,179. Whi results , and 76. results  used you results ,181 or g results , the amo results , while t results . Again, 1A-AR subtype [1A-AR but not the α1B-AR showed antidepressant behavior [1A-AR blockage with WB4101 induces learned despair in mice [1A-AR blockers may increase depressive and anxiety-based disorders as hypothesized.The amygdala can regulate psychological stressors and anxiety, besides regulating fear-conditioned memory and memory consolidation ,183, and subtype ,184. Trabehavior . α1A-AR  in mice  and tams in mice . While a in mice , BPH its in mice ,190 and  in mice . Further1-AR antagonists, or a combination, found a 22% increased risk of cardiac failure among the users of α1-AR blockers [1-AR blockers  were significantly associated with an 8% higher risk for heart failure compared with selective α1A-AR blockers (silodosin and tamsulosin). Silodosin is 500-fold more selective for the α1A-AR than α1B-AR [1A-AR is theorized to be cardioprotective and agonists protect against heart failure [1A-AR blockers then not associated with a higher risk of heart failure compared to non-selective blockers? There may be other non-α1-AR mediated effects associated with the increased risk of heart failure, such as increased apoptosis [1-AR quinazoline blockers. While α1-AR blockers are still a popular treatment for BPH, and particularly in younger men who may not display heart failure, it is advised that physicians assess the cardiovascular health of the patient before long-term use.The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) is a large, randomized double-blind study comparing four different classes of antihypertensive agents in patients older than 55 years . The useblockers . Non-seln α1B-AR , while tn α1B-AR . The α1A failure , but whypoptosis ,149,150,1-AR antagonists is intraoperative floppy iris syndrome (IFIS), that increases serious complications and is characterized by a poor pupillary response, iris billowing, and prolapse during cataract surgery [1-ARs, and particularly the α1A-AR subtype, regulates the dilator smooth muscle of the iris [1-AR antagonists and causing severe IFIS [1-AR antagonists can also cause it. A large meta-analysis of over 6000 cases using various α1-AR antagonists indicate that most α1-AR blockers associate with a higher risk of IFIS [1-AR blocker.Another adverse effect of the long-term use of α surgery . α1-ARs,the iris ,196, intthe iris ,198, andthe iris . Tamsuloere IFIS ,203,204, of IFIS . With th1-AR agonists to potentially treat heart failure, cardiac ischemia, Alzheimer’s disease, and other dementias are targeted to the α1A-AR subtype. However, all of these studies are preclinical in cell lines and mouse models or in initial clinical trials and it is not currently recommended to use these agents for non-approved use. Current development of positive allosteric modulators would be the choice as first-in-class therapeutics to avoid issues with increasing blood pressure to reduce other adverse side effects. The use of non-selective α1-AR antagonists of the quinazoline class to treat severe COVID-19/SARS, PTSD, and neurodegenerative disorders, such as Parkinson’s disease and ALS, have extensive evidence of efficacy in many clinical trials. However, the mechanism of action may be non-α1-AR mediated. Counterindications for α1-AR blockers are focused on those with established heart disease. Future clinical studies and larger, randomized, cross-over trials are required before drawing firmer conclusions about the counterindications of tamsulosin or other α1A-AR selective blockers.The use of α


Transcription factors play key roles in orchestrating a plethora of cellular mechanisms and controlling cellular homeostasis. Transcription factors share distinct DNA binding domains, which allows to group them into protein families. Among them, the Forkhead box O (FOXO) family contains transcription factors crucial for cellular homeostasis, longevity and response to stress. The dysregulation of FOXO signaling is linked to drug resistance in cancer therapy or cellular senescence, however, selective drugs targeting FOXOs are limited, thus knowledge about structure and dynamics of FOXO proteins is essential. Here, we provide an extensive study of structure and dynamics of all FOXO family members. We identify residues accounting for different dynamic and structural features. Furthermore, we show that the auto-inhibition of FOXO proteins by their C-terminal trans-activation domain is conserved throughout the family and that these interactions are not only possible intra-, but also inter-molecularly. This indicates a model in which FOXO transcription factors would modulate their activities by interacting mutually.  •FOXO forkhead domains exhibit different dynamics.•Helix 4 displays highest variations in dynamics among FOXO family members.•The ability for forkhead - transactivation domain interaction is conserved among FOXOs. BesidesMany TFs harbor small discrete domains, that allow them to bind to DNA . Based oGiven the large number of TFs that share the same folds and target DNA sequences, it is puzzling how homologs of TFs can fulfill biologically very different functions. Here, we focus on the FOXO TFs, which are considered to be tumor suppressors due to their functions in cell cycle arrest , apoptosPKB/AKT  and CR3 . FOXO6 iewed in . Recentliewed in .Fig. 1SeOverall the sequences of FOXO proteins are more than 30% identical, with up to 90% identity within the DNA-binding FH domain B Jacobs. The FH FOXO function is often described as collective family function . This isSystematic KO studies in mice of FOXO isoforms revealed that FOXO1-KO is embryonal lethal, due to incomplete vascular development, whereas FOXO3- and FOXO4-KO mice were viable but grossly indistinguishable from their littermate controls. FOXO3-KO females additionally showed age-dependent infertility and had abnormal ovarian follicular development . The impOpposite effects in context of senescence have been observed for FOXO4 and FOXO1/3 . In oncoAt the atomic level the structure of the FOXO family members are highly similar when bound to DNA. However, the structures reveal different networks of hydrogen-bonds and water-mediated interactions in the major groove of the DNA . FOXO4 aHere, we aimed to reveal whether variations in the structures and dynamics of the FOXO FH domains can be detected and if these differences affect key FOXO interactions. To this end, we studied the structures and dynamics of the FH domains of all human FOXO members  in solution using NMR spectroscopy under identical experimental conditions, and extensive all-atom molecular dynamics (MD) simulations. We identified regions in FOXO FH domains that differ in terms of their dynamics, and we determined that these differences are due to slight variations in amino acid sequence. Furthermore, we show that the intra-molecular interactions between the FH and transactivation domains (TADs) recently discovered in FOXO3 and FOXO4  are also215N isotope labelling and recorded 1H, 15N heteronuclear single-quantum coherence (HSQC) NMR spectra  and FOXO6 (2.2 Å)  B. The dianalysis D. Next, analysis . In Fig.analysis C.Fig. 2FFH adopts a similar fold as all other FOXO family members. However, despite the high sequence identity, our MD simulations studies show that there are conformational differences within the FH domains of the FOXO proteins. The most variations are found in H4, the transition to H3 and in the loop regions connecting the β-strands S2 and S3.In summary, we found, as expected, that the FOXO62.115N longitudinal (R1) and transverse (R2) relaxation rates and the steady-state 15N,1H-Nuclear Overhauser Effect (NOE) for all FOXO FH domains. Heteronuclear 15N{1H} NOE values are indicators of the motions of amide bond vectors on the ps/ns timescale: values close to the theoretical limit of 0.83 at a static magnetic field strength of 600 MHz suggest rigidity whereas lower values indicate increased local flexibility. The regions that adopt stable secondary structure elements show 15N{1H} heteronuclear NOE values above 0.5 for all FOXO FH domains (lower flexibility), whereas the terminal ends show negative 15N{1H} heteronuclear NOEs, indicating high flexibility with rapid ps/ns motions . As observed with our prior NMR-based analysis, most of the secondary structure elements including H1, H2, H3, as well as S1, S2 and S3 remain stable in all isoforms . During To follow the molecular motions on a residue-specific level, we calculated the root-mean-square-fluctuation (RMSF) for each of the FH isoforms A. As exp1), (ii) the carboxyl group of Asp202 (CO) (d2), and (iii) the hydroxyl group of Ser206 (OH) (d3). These FOXO1 residues correspond to Gly198, Asp199, Ser203 and Lys207 in FOXO3, to Gly142, Asp143, Ser147 and Lys151 in FOXO4, and to Gly129, Asp130, Ser134 and Lys138 in FOXO6, respectively  and (i) the carboxyl group of Gly201 (CO)  can be explained by the formation of a stable hydrogen bond between the phenyl hydroxyl group (OH) of Tyr196 and the amino group of Lys200 (NZ) .The high structural stability shown by the first helical turn in H4 in all isoforms (Tyr196-Lys200 in FOXO16). We found out that in FOXO6 the amino group of Lys is closer to H2 compared to the other FOXO isoforms, which behave similarly throughout the MD simulations.Additionally, we measured the relative orientation of H4 towards H2 along the MD simulations  by measu15N}1H heteronuclear NOE values above 0.7 and R2 rates above 10 s−1 , Thr (FOXO4), Cys (FOXO3) or Tyr (FOXO6) A. In all3-5-W, we monitored the distance during the MD simulations between the heteroatom of the side chain of the terminal residue Xaa (Ser/Thr/Cys) and the center of the indole ring of the interacting Trp ting Trp A and B. le α see A in FOXO−1), whereas the other energies are quite similar. The interaction of Tyr with Trp has a medium value and is not a face-to-face π-π stacking interaction but an edge-to-face interaction. Thus, our MD simulations and quantum mechanical calculations have shown variable interaction strengths between the Xaa-Trp residues within the W-[2-5]-[S/T/C/Y] motifs of FOXO FH domains, with FOXO4 having the most favorable interaction (Thr-Trp).In addition, we also computed the interaction energy between the former two side chains C, using D. rerio and in C. elegans . Interestingly, we found a population in our MD simulations of FOXO3FH (and not in the other isoforms) where the N-terminal Trp157 interacts with Cys190 thereby displacing Trp186 which then interacts with Arg189. This suggests that there may be plasticity between the Xaa-Trp interactions within the repeated W-[2-5]-[S/T/C/Y] motifs in FOXO FH domains, and further investigations on the Xaa-Trp motifs are planned.Looking at the full-length protein sequences, the W-[2-5]-[S/T/C/Y]motif is repeated across the sequence of FOXOs, although to a varying degree between the four isoforms . Whereas elegans . We cann2.4Transcription is a highly important cellular process and thus, DNA binding of transcription factors is tightly controlled. It has been shown for a few TFs, including p53, CCAAT/Enhancer-binding Protein β, FOXO3 and FOXO4, that their disordered regions can interact with their folded DNA-binding domains and regulate DNA binding . In the 15N isotope-labeled FOXO FH domains with their recombinantly expressed CR3. The addition of FOXO1CR3 to 15N labeled FOXO1FH resulted in progressive chemical shift perturbations (CSPs), that are indicative of binding , and thus the smallest CSPs are observed in FOXO3, suggesting the weakest interaction.The CR3 of FOXO4 forms two transient α-helices upon binding to the FOXO4 FH domain and bends around H3 in a manner similar to DNA  Weigel . To idenCR3 to mediate FOXO4FH binding, is highly similar in all FOXOs  line broadening ii) strong CSPs or iii) weak CSPs of 1H,15N cross-peaks of the FH domain. 1H,15N cross-peaks of FOXO1FH, FOXO3FH and FOXO6FH in the presence of FOXO4CR3 and for FOXO3FH in the presence of FOXO1CR3. Strong CSPs were observed for 1H,15N cross-peaks of FOXO1FH in the presence of FOXO6CR3, of FOXO4FH and FOXO6FH in the presence of FOXO1CR3. Weak CSPs were observed 1H,15N cross-peaks of FOXO1FH, FOXO4FH and FOXO6FH in the presence of FOXO3CR3 and of FOXO3FH and FOXO4FH in the presence of FOXO6CR3 . This brought us to identify a common 3D-motif of sequence W-[2-5]-[S/T/C/Y] motif in all FOXO FH domains, where the former residue (Xaa) interacts via the side chain with the indole of a Trp residue. In the FH domains these interactions for Ser/Cys/Thr/Tyr with Trp are in the same range of energy but strongly vary in the relative orientation of the interacting side chains. In our quantum chemistry calculations , the dif1H,15N cross-peaks of FOXO1FH, FOXO4FH or FOXO6FH, respectively upon addition of their corresponding CR3, there are only weak chemical shift perturbations found in 1H,15N cross-peaks of FOXO3FH, indicating a weaker interaction in FOXO3. Thus, it is likely that the auto-inhibitory mechanism is conserved among the FOXO family and might be less effective in FOXO3.One way that FOXO4 is regulated is by the intramolecular interaction of the CR3 with its corresponding FH domain . This waLastly, we show that the FH – CR3 interaction can occur in an intermolecular manner involving different FOXOs. This might allow transcription regulation involving multiple FOXO proteins. We found that intermolecular interactions between FOXOs vary in strength, with the CR3 of FOXO4 showing the strongest binding to other FH domains, and FOXO3 showing the weakest interactions. For all FOXOs, residues in the N-terminal loop and H3 of the FH domain of FOXO proteins are affected most upon CR3 binding. This is intriguing since the N-terminal Trp residue shows different conformations in the MD simulations of the FOXO FH domains, which might account for the differential CR3 binding.The biological function of FOXO transcription factors is regulated by the binding of transcriptional co-activators such as β-catenin . β-catenFOXOs might form higher-order complexes, and these complexes might be important for transcription regulation. As an example, FOXO1 can dimerize upon binding to the palindromic DNA element DIV2 . The dimIn summary, we found distinct variations in the structure and dynamics of the FH domain of FOXO proteins. Especially the FH domain of FOXO3 exhibits relatively higher flexibility and more pronounced ps-ns dynamics, which may be due to Cys190 at the C-terminus of H2 and the conformational exchange between binding to the Trp187 and binding to Trp157 located N-terminal of H1. We describe intra- and intermolecular interactions between the CR3s and FH domains of FOXO proteins of different affinities. We present the first comparison study of the structure and dynamics of all FOXO proteins and their intramolecular interactions. Additionally, we show that FOXO proteins can interact with each other via FH – CR3 interactions at different strengths. Taken together, this study provides essential clues for better understanding of the functional difference of FOXO proteins in biomolecular pathways.44.1Q12778) spanned amino acids143 to 270 (FOXO1FH) and amino acids 587 to 636 (FOXO1CR3), for human FOXO3 (Uniprot ID O43524) from amino acid 141 to 260 (FOXO3FH) and from amino acid 593 to 646 (FOXO3CR3) and for human FOXO6 (Uniprot ID A8MYZ6) from amino acid 71 to 191 (FOXO6FH) and from amino acid 418 to 472 (FOXO6CR3). These constructs were generated by synthesis of the Escherichia.coli codon optimized DNA (Genscript) that were sub-cloned into pETM11-ZZ-His6 vector via NcoI/BamHI restriction. Expression constructs for FOXO4 (Uniprot ID P98177) were described previously  or 2 g of uniformly 13C-labeled glucose (Cambridge Isotope Laboratories) and 1 g of 15NH4Cl (Sigma). Each of the 1 L expression cultures were grown until an OD (600 nm) of 0.7 was measured, after which protein expression was induced by addition of 0.5 mM IPTG and allowed to continue for 16 h at 20 °C for FH constructs or 15 °C for CR3 constructs. After pelleting the cells via centrifugation at ca. 8000×g, the pellet was resuspended and lysed by sonication in either a denaturing lysis buffer  for CR3 constructs or in a non-denaturing lysis buffer phosphine) for FH constructs. His6-protein A-tagged recombinant proteins were then purified using Ni-NTA agarose (Qiagen) and the His6-protein A-tag was cleaved with His6-tagged TEV protease treatment. Untagged proteins were then isolated performing a second affinity purification using Ni-NTA beads. A final size exclusion chromatography purification step was performed in the buffer of interest on a gel filtration column . Protein concentrations were estimated based on their absorbance at 280 nm.For expression of recombinant unlabeled or 4.3NMR experiments were performed at 25 °C on 700 MHz spectrometer equipped with a TCI triple-resonance cryo-probe or on a Bruker 600 MHz Avance Neo NMR spectrometer equipped with a TXI room temperature probe.1H, 15N HSQC spectrum, the following three-dimensional spectra were acquired for NMR resonance assignment of FOXO6FH: HNCACB, CBCA(CO)NH, (H)CC(CO)NH-TOCSY and HCCH-TOCSY using 550 μM 13C15N labeled FOXO6FH. Previous assignments for FOXO1FH (BMRB 27894) and FOXO3FH (BMRB 15939) were confirmed using HNCACB and CBCA(CO)NH spectra using 500 μM 13C15N labeled FOXO1FH or using 300 μM 13C15N labeled FOXO3FH respectively. Resonance assignments have been deposited under BMRB accession number XXX (will be updated upon acceptance).Alongside the 2D CR3, FOXO3CR3 and FOXO6CR3, HN(CA)NNH, H(NCA)NNH, HNCACB and CBCA(CO)NH were recorded using 300–400 μM 13C15N labeled FOXO1CR3, FOXO3CR3 and FOXO6CR3 respectively. Resonance assignments have been deposited under BMRB accession number 51426. Spectra were processed using TOPSPIN 4.1 (Bruker) and analyzed using CcpNmr 2.5.For resonance assignment of FOXO14.4The CSPs was calculated according to the following equation (1):pNMR 2.5 .The correlation matrix of FOXO FH domains was calculated using the Pearson function following equation (2):RMSD values for the FOXO structures were obtained used the PyMol align method using only backbone atoms and zero cycles of outlier detection. Pairwise alignment and RMSD calculations were performed using the ColorByRMSD python module for PyMol using the align method and backbone atoms.4.515N labeled samples at 300 μM protein concentration in a buffer containing 50 mM NaH2PO4/Na2HPO4 (pH 6.5), 0.04% NaN3, 2 mM DTT and 10 %D2O. 15N R1 values were measured using the HSQCT1ETF3GPSI3D.2 Bruker pulse sequence , using delays 0.01, 0.2, 0.012, 0.131, 0.0151, 0.086, 0.0199, 0.0575, 0.0274, 0.0575 and 0.0392 s 15N{1H} heteronuclear NOE was measured using the HSQCNOEF3GPSI Bruker pulse sequence  using 3 s saturation period/interscan delay. For the NMR binding assays, all protein samples were equilibrated in the same buffer containing 50 mM NaH2PO4/Na2HPO4 (pH 6.5), 0.04% NaN3 and 5 mM DTT. 1H, 15N HSQC experiments were recorded according to the NMR section.NMR relaxation experiments for all FOXO FH domains were recorded using T1, T1ρ) or 1H pulse saturation (hetNOE) were encoded in the pseudo-third dimension. In the T1 and T1ρ experiments, 11 different 2D planes were recorded with maximum relaxation delays of 2 s and 0.2 s, respectively, and two duplicate measurements were collected to compute the error in measured intensities. The data were processed in NMRPipe :1/Ω), with ω1 equal to the spin-lock field strength (in rad/s) and Ω the 15N resonance offset. The fitted R1 and R1ρ rates were obtained via least-squares minimization using the standard Levenberg-Marquardt algorithm included in the lmfit Python package (https://lmfit.github.io/lmfit-py/). The errors in measured peak intensities were derived from the pooled standard deviation of the duplicated delay measurements, with the noise computed as: i refers to the peak associated with residue i, si,1 and si,2 refer to the signal intensity associated with the two duplicated time delays for residue i, and k refers to the total number of peaks in the analysis. Errors in the fitted relaxation rates were obtained via Monte Carlo analyses performed with an in-house Python script. In this analysis, 1000 synthetic datasets were generated for each residue in which each intensity value was randomly sampled from a normal distribution centered at the measured intensity with a width corresponding to two times the noise . The least-squares minimization procedure was performed on each of these 1000 synthetic datasets, and the error in the fitted rate constant was calculated by fitting the histogram of the best-fit values to a normal distribution: A the amplitude for normalization. The error was obtained from σ, which corresponds to the standard deviation of a normally distributed parameter and the reported errors in 4.6AlphaFold2 prediction were performed using the easy to use AlphaFold2  a protei4.7the FOXO4 FH-CR3 complex has been solved as described previously  and the theoretical model (version 1) for the FH domain of human FOXO6 was gotten from the AlphaFold2 Protein Structure Database . The soleviously . In the eviously ) and theeviously .Periodiceviously .−1 Å−2 were imposed to the rest of the atoms in each stage and the system was thermally equilibrated for 20 ps from 100 K to 298 K using the Langevin thermostat. The harmonic restraint was removed in 5 steps where the system equilibrated from a NVT to an NPT ensemble for 100 ps. Then, each of the systems was further simulated for 500 ns in three independent simulations  using the Langevin thermostat at 298 K and with a time step of 0.2 fs. MD simulation trajectory analysis was performed using the program cpptraj.The protonation state of each of the residues of the proteins were fixed at pH 6.5 by means of the server H++, version 3.2 . The pro3-5-W motif in the different isoforms was competed following the cluster method and using the counterpoise approach approach : first, approach  whereas approach  and a la6QVW, 2K86 and 1E17. Uniprot accession nos. Q12778, O43524, P98177 and A8MYZ6.Protein Data Bank accession nos. Emil Spreitzer: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. T. Reid Alderson: Formal analysis, Conceptualization, Writing – review & editing, Supervision. Benjamin Bourgeois: Conceptualization, Writing – review & editing, Supervision. Loretta Eggenreich: Conceptualization, Investigation, Writing – review & editing, Visualization. Hermann Habacher: Investigation, Writing – review & editing, Visualization. Greta Brahmersdorfer: Conceptualization, Investigation, Writing – review & editing. Iva Pritišanac: Writing – review & editing, Visualization. Pedro A. Sánchez-Murcia: Conceptualization, Methodology, Formal analysis, Investigation, Writing – review & editing, Visualization, Resources, Data curation. Tobias Madl: Conceptualization, Methodology, Writing – review & editing, Resources, Supervision, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Castration-resistant prostate cancer (CRPC) is an advanced form of prostate cancer with a low survival rate, as the CRPC patients only survive for 9–13 months on average. In this narrative review, we first outline the most common androgen receptor (AR) receptor-related mechanisms, highlighting the important role of ARs in the development of CRPC. We also discuss the key importance of non-coding RNAs (ncRNAs) in this setting, including long ncRNAs and microRNAs. Overall, studies of the molecular biological mechanisms governing the CRPC will facilitate the development of appropriate targeted therapeutics, improving treatment options for the CRPC patients.Despite aggressive treatment and androgen-deprivation therapy, most prostate cancer patients ultimately develop castration-resistant prostate cancer (CRPC), which is associated with high mortality rates. However, the mechanisms governing the development of CRPC are poorly understood, and androgen receptor (AR) signaling has been shown to be important in CRPC through AR gene mutations, gene overexpression, co-regulatory factors, AR shear variants, and androgen resynthesis. A growing number of non-AR pathways have also been shown to influence the CRPC progression, including the Wnt and Hh pathways. Moreover, non-coding RNAs have been identified as important regulators of the CRPC pathogenesis. The present review provides an overview of the relevant literature pertaining to the mechanisms governing the molecular acquisition of castration resistance in prostate cancer, providing a foundation for future, targeted therapeutic efforts. Prostate cancer is the second leading cause of cancer-related death among men worldwide . In ChinThe androgen receptor (AR) is a 110 kDa 919 amino acid nuclear receptor (NR) family member encoded on the X chromosome (q11-q12) A. The ARSeveral different mechanisms link AR to the development of CRPC, including AR upregulation, de novo androgen synthesis, co-regulatory factor activity, AR gene mutations, and altered splicing .The AR signaling reactivation most commonly occurs at the level of gene amplification or protein upregulation, with up to 80% of the CRPC patients harboring high AR gene copy numbers, among whom 20–30% of patients exhibit high gene amplification levels ,13,14. SThe persistent de novo production of androgens within the CRPC tumors can additionally contribute to enhanced AR activation and the progression of hormone-refractory prostate tumors . Even foHSD3B2, AKR1C3, CYP17A1, and CYP11A1) are present [When multiple steroids  was additionally identified as a critical source of intratumoral androgen production ,37,38, aOver 180 such AR co-regulatory proteins have been identified to date, with both co-repressors and co-activators functioning in a synergistic manner to regulate AR transcription . These pAEEB1 gene encoding the regulatory protein βArr1 was found to be upregulated in the CRPC tumor tissues, with the βArr1 deletion resulting in impaired PCA tumor growth, invasion, and metastatic progression in vitro and in murine model systems [The interaction of specific co-activating proteins with AR, including JMJD2C, LSD1, 37CBP/P300, p160/SRC, and SUV39H2, can drive enhanced AR activation and the concomitant upregulation of the AR-dependent genes to augment tumor growth. Consistently, the inhibition of these activators has been linked to reduced AR expression and PCA tumor growth in ex vivo analyses ,52,53,54 systems . The AR  systems ,57. Tan  systems . As such systems .Between 10 and 30% of the CRPC patients harbor point mutations that alter the conformation of the AR protein ,59. The The AR splice variants (AR-Vs) were initially identified over two decades ago, and over 22 of such variants have been identified to date  ,75,76,77The AR signaling can drive the onset of CRPC under the conditions of persistent ADT treatment. The AR-Vs are thus commonly expressed in the CRPC tumors as an alternative to AR-FL signaling, potentially functioning in a manner distinct from other AR isoforms ,86,87,88The ARV-567es is a common AR-V that lacks LBD exons 5–7, while retaining the hinge region encoded by exon 4 that is involved in the receptor nuclear localization ,94,95,96The GR and AR are highly homologous steroid receptors, particularly within the DBD domain. As it harbors the same chromatin binding site, the GR can regulate the expression of a large number of the AR-specific genes ,101,102.DNA damage repair is one of the most fundamental defense mechanisms that governs cell survival ,110. TheThe Hedgehog (Hh) pathway has increasingly been reported to be derepressed in PCA cells under therapeutic treatment, contributing to the progression of CRPC ,125. WitA majority of the CRPC tumors exhibit the activation of the PI3K-AKT-mTOR pathway ,142,143,Under the ADT treatment or in the absence of androgens, reductions in the oncogenic PTEN expression contributed to PI3K activation and downstream AKT/mTOR activation that is evident in 63% of the CRPC tumors ,151,152.The altered Wnt pathway activity has been tied to malignant growth and progression in many tumor types ,162,163.The non-classical Wnt pathway was also recently identified as an important regulator of the CRPC tumor cell growth . For exaThe non-coding RNAs (ncRNAs) are a large class of RNAs that lack protein-coding capabilities, and are subdivided into microRNAs (miRNAs), small nuclear RNAs (snoRNAs), PIWI-interacting RNAs (piRNAs), and long ncRNAs (lncRNAs). These ncRNAs can play diverse roles as the regulators of processes such as differentiation, metabolic activity, and apoptosis ,175,176.As the first ncRNA subtype to have been identified as a driver of CRPC, the miRNAs are short 20–24 nucleotide RNAs that can bind to complementary 3′-untranslated region (UTR) sequences of target mRNAs, thereby suppressing their expression or promoting their degradation ,182. OveThrough a qPCR analysis, Das et al. determined that the overexpression of miR-4719 and miR-6756-5p was associated with significant reductions in IL-24 expression within cells, which is noteworthy, given that IL-24 can promote tumor cell death via multiple mechanisms . The CRPAt over 200 nucleotides in length, the lncRNAs represent a distinct ncRNA class that nonetheless serve as important regulators of a range of physiological and pathological processes ,194,195.The interactions between lncRNAs and miRNAs have also recently been shown to be mechanistically important in CRPC ,201,202.Prostate cancer remains among the leading causes of cancer-associated death in males, yet the mechanisms underlying the development of CRPC remain incompletely understood. While AR signaling clearly remains important in this pathological context, a growing body of evidence supports the importance of other mechanisms and pathways independent of the AR pathway as drivers of castration-resistant disease . This pa


Statistical Topology emerged as topological aspects continue to gain importance in many areas of physics. It is most desirable to study topological invariants and their statistics in schematic models that facilitate the identification of universalities. Here, the statistics of winding numbers and of winding number densities are addressed. An introduction is given for readers with little background knowledge. Results that my collaborators and I obtained in two recent works on proper random matrix models for the chiral unitary and symplectic cases are reviewed, avoiding a technically detailed discussion. There is a special focus on the mapping of topological problems to spectral ones as well as on the first glimpse of universality. Statistical Topology aims at combining, in a generalizing form, topological questions appearing in physics with the powerful concepts of Random Matrix Theory (RMT) which is capable of describing spectral statistics in a huge number of systems, stemming from different areas of physics and beyond. The focus in this work is exclusively on winding numbers and associated statistical quantities studied in the framework of a random matrix model; other topological invariants, such as the Chern numbers, which are also of considerable interest, are not discussed. The long-term aim is to study the emergence of universalities whose identification and usage is always, in all branches of statistical physics, the most rewarding enterprise. I have two goals. First, I want to present an introduction to Statistical Topology, restricted to statistical problems which are related to winding numbers, for readers without a pertinent background. Neither physics expert jargon, nor heavy mathematics and mathematical physics terminology are used. Second, I want to review and summarize results that my collaborators and I obtained in two recent studies ,2. We caThe paper is organized as follows: In After briefly revisiting the occurrence of winding numbers in complex analysis in W.The winding number is a topological concept encountered in complex analysis. Before discussing applications in physics, we briefly sketch the mathematical background. The winding number n on the chain. Moreover, t is the hopping strength. The Hamiltonian may be reformulated in terms of Majorana fermions whose number is twice that of the electrons. Remarkably, depending on the parameters, there are two possibilities, as schematically depicted in To illustrate the occurrence of topological invariants in physics, we look at the Kitaev chain ,4 as a pk and the three parameters t. Importantly, the first component is zero, In Fourier space, the Kitaev chain corresponds to the Bloch–Bogolyubov–de Gennes Hamiltonian matrix ymmetry,{H(k),C}=k on the curve, t determine its shape. In x axis, the band gap disappears, marking the phase transition point.To see how topology enters, we notice that the vector t from probability distributions. Hence, the contour can be different for a particular choice, i.e., it becomes random, and the winding number W will be random as well. In general, the dynamics of a system under consideration, described by the Hamiltonian, and the distributions of its parameters will determine the distribution When studying such topological invariants in statistical physics, the closed contour might be a random quantity, for example, generated by a proper ensemble of Hamiltonians. In the case of the Kitaev chain, this ensemble may be realized by choosing the parameters ry (RMT) ,7 is knory (RMT) ,9. The cIn Quantum Chromodynamics, the chiral symmetry of the Dirac operator is broken spontaneously as well as explicitly by the quark masses. The chiral condensate is the order parameter of the phase transition that occurs at a high temperature and that restores chiral symmetry, which is related to the confinement–deconfinement transition. To investigate statistical properties of lattice gauge calculations, chiral RMT ,15,16,17W indicates the topologically non-trivial situation with In the terminology of condensed matter physics, the winding number comes in as characterization of translationally invariant one-dimensional chiral systems that are gapped at the centre of the spectrum. The winding number is the integer topological index with respect to the bundle of negative-energy bands. A non-zero winding number boundary ,27,28,29boundary ,31 reveaAnother intriguing direction might be the application of Statistical Topology to classical wave phenomena such as microwaves or acoustics and, furthermore, to photonics where topological issues are already in focus .After introducing chiral random matrix ensembles with a parameter dependence in H, as most of the present application of winding numbers seem to stem from Condensed Matter Physics. The matrices H are complex Hermitean or quaternion real, i.e., self-adjoint, with even dimension K have no further symmetries. We draw the matrices H from the chiral Gaussian Unitary, respectively, Symplectic Ensembles . To study questions of topology, we give these random matrices a parametric dependence p lies on the unit circle. The winding number corresponding to these Hamiltonians is then [p, see We derived results ,2 for th is then ,3410)WWH, as moTo produce explicit results, we choose a particular realization of the parameter dependence. With two smooth and k different points k-point correlators of winding number densitiesk, we calculated the generatorsk and l being different has technical advantages. We notice that k and l are the numbers of determinants in denominator and numerator, respectively. The k-fold derivativeConsidering  in Ref. , see SecneratorsZk|lneratorsZk|lensitiesCktructure ,9.Furthermore, we also computed the distribution of winding numbers ase AIII , see SecY.At first sight, the computation of the correlators  and the Equation  encodes  one hasK(p)=a(p)N complex eigenvalues Y and readsY has a complex conjugate Y and The task to be solved is the derivation of the probability density for the random matrices pherical ,36 ensemherical [G˜(β)(Y)=The correlators for the unitary case are addressed in Y, one hask-fold products of k winding number densities W must have a symmetric distribution with vanishing first moment. The integral of In Ref. , we calcEquation  in the uquations  and 20)Ckp,…,pk as W between N. Here, m eigenvalues are inside the unit circle and the remaining ones outside which may be written asIn Ref. , we alsoe choice . Using Cunctions . The com choice (P(W)=rW+NThe quest for universality is twofold, first, there is the question of whether the same statistical effects, distributions or scalings, etc, can be identified in empirical or experimental data of different physical systems. Second, there is the theoretical and mathematical side concerned with often schematic models and their ability to describe or even predict the results from data analysis. In the case of spectral correlations, universal statistics is found on the local scale of the mean level spacing, i.e., universalities are revealed after a rescaling of the energies, referred to as unfolding. The unfolded correlators of, on the one hand, RMT for infinite level number and of, on the other hand, numerous physical systems of very different nature with large number of levels coincide, see the discussion in Refs. ,7. The tIn the case of statistical topology, universality is of equally high importance, but it appears to be considerably more complicated. Already, on the theoretical and mathematical side, there are several natural questions to be posed: First, is there an unfolding scale comparable to the local mean level spacing and how is it related to the scale of the level velocity as in the parametric correlations ,9,37? SeN, the unfolded two-point function approaches the limit (ψ1,N. More precisely, its second moment behaves like N.In Ref. , we alsoWe computed the generators , respect in Ref. ,39. It iEquation . In the Statistical Topology is an emerging branch in statistical physics, with connections to various branches of mathematics. It is triggered by the identification of topological questions in many areas of physics, ranging from quantum mechanics and quantum field theory over semiclassics to QCD and Condendsed Matter Physics. First, I tried to give an introduction to winding number statistics for newcomers who do not have any background, avoiding usage of expert jargon and of burying the key ideas under the adavanced terminology developed in mathematics and mathematical physics. Second, I reviewed results that my collaborators and I obtained in two recent works. We studied winding numbers and associated statistical quantities in a random matrix model. There are, of course, also other topological invariants of considerable interest in physics, most notably, the Chern numbers.I presented our first, probably awkward, steps towards looking at universal behavior. In my opinion, the most fascinating challenge for the future is the further study of universality in statistical topology, more precisely, of both of its aspects, the experimental–empirical as well as the theoretical–mathematical one.