When stratified by sex, the AR association with OS was only significant for both sexes. The multivariate Cox models with justifications of sex, age of diagnosis, stage of disease, and Breslow depth of the tumor confirmed the AR-OS association in all patients. However, the significance of AR was lost when ulceration was included in the model. When stratified by sex, the multivariate Cox models indicated significant role of AR in OS of female patients but not in males. AR-associated genes were identified and enrichment analysis revealed shared and distinct gene network in male and female patients. Furthermore, AR was found significantly associated with OS in RAS mutant subtypes of melanoma but not in BRAF, NF1, or triple-wild type subtypes of melanoma. Our study may provide insight into the well-known female survival advantage in melanoma patients.Androgen receptor (AR) is expressed in numerous tissues and serves important biologic functions in skin, prostate, immune, cardiovascular, and neural systems, alongside sexual development. Several studies have associated AR expression and patient survival in various cancers, yet there are limited studies examining the relationship between AR expression and cutaneous melanoma. This study used genomics and proteomics data from The Cancer Proteome Atlas (TCPA) and The Cancer Genome Atlas (TCGA), with 470 cutaneous melanoma patient data points. Cox regression analyses evaluated the association between AR protein level with overall survival and revealed that a higher level of AR protein was positively associated with a better overall survival (OS) ( Melanoma incidence continues to increase worldwide; in the US, it has increased by 320% since 1975 . While mAR is located on the X chromosome [As a male sex hormone receptor, the gene romosome . Two andromosome ,9,10. ARromosome ,12,13.Sex differences in cancer incidence have also been documented in several cancers, . For insCurrent literature has explored possible pathways into AR’s effects, both harmful and protective, on cutaneous melanoma development. One proposed mechanism explains how AR and the protein Early Growth Response 1 (EGR1) increase melanoma proliferation through coordinated transcriptional regulation of several growth-regulatory genes, including the repression of EGR1-mediated transcriptional activation of p21Waf1/Cip1, a known tumor suppressor gene . AnotherAR gene and AR protein expression in human cutaneous melanoma, and their association with OS in patients. Genomic network was further explored in an attempt to understand the sex-differentiated roles of AR in patient overall survival.The conflicting findings of AR’s protective or harmful role in melanoma progression shows the complexity of several convergent mechanisms likely implicated in melanoma’s AR dependency. In this paper, we use The Cancer Genome Atlas (TCGA) and The Cancer Proteome Atlas (TCPA) to evaluate the relationship between http://gdac.broadinstitute.org/, accessed on 14 June 2022), and proteomics data from The Cancer Proteome Atlas (TCPA) . The RNA-Seq data were retrieved as RSEM (RNA-Seq by Expectation-Maximization) and Z scores [The data source used for all analyses (TCGA-SKCM) were obtained from The Cancer Genome Atlas (TCGA), with mRNA sequencing (RNA-Seq) data downloaded from Broad Firehose GDAC . The regression model was further stratified by sex or adjusted to age, tumor stage (early and late), Breslow depth, and ulceration status of the tumors. Sex was also used as an adjusting co-variable in the overall model. The overall survival was defined as the period from date of diagnosis until death from any cause. Significance levels are set at 0.05 (two-sided) for all analysis.All statistics were analyzed using Stata 17. Linear regression was used to examine the association of mRNA and protein levels in tumor samples. AR levels (mRNA or protein) were compared between sex by Student https://www.cbioportal.org/, accessed on 14 June 2022) using sex-stratified patient information. The AR-co-expressed genes were processed using adjusted q values of 0.05, followed by cutoff value of Spearman’s coefficient of 0.3 [https://biit.cs.ut.ee/gprofiler/, accessed on 14 June 2022).AR-co-expressed genes (based on RNA-Seq) were extracted from the cBioportal website . However, when the protein levels were used for sex comparison, tumors from females (N = 144) showed a significant lower level of AR protein than those from males (N = 208) (p = 0.0099) (The TCGA SKCM dataset was downloaded from the Broad Institute Firehose website. The baseline characterizations of patients are listed in  0.0099) .p < 0.0001 for all samples together, p = 0.003 for females and p < 0.0001 for males)  .p = 0.0025), for male patients (p = 0.046), or for female patients (p = 0.0107) (p = 0.012) or for all patients (p = 0.003), and the significance level was reduced to 0.047  in male patients   for the entire cohort, while “AR-low” group of patients have tumor AR levels lower than the median AR. Our initial Kaplan–Meier survival analysis suggested that higher AR levels were associated with better OS , and thi 0.0107) a–c. Cox patients . Cox pro = 0.39) .p = 0.29) (p = 0.001), and the significance remained after adjusting to age of diagnosis, stage of disease, and sex .In the multivariate analysis, sex was used as a co-variable, and the AR association with overall survival was additionally adjusted by age of diagnosis, stage of disease  , Model 4p = 0.23 in Student t-test).These results may suggest an impact of AR on the ulceration status. However, tumors with or without ulceration did not show significant difference in the AR protein levels (p = 0.008)  and included this variable in our multivariate COX analysis. The AR association with overall survival remained significant when the result was adjusted to Breslow depth, along with other factors  , Model 5t-test was used to evaluate the AR protein levels, and no difference in means was found (p = 0.89 for men and 0.13 for women).Since testosterone levels are known to change with men’s age, we also examined whether AR levels in tumors were different in older versus younger patients (≤50 vs. >50 years). A Student In order to understand how AR expression levels are associated with patient overall survival in women but not in men, the TGCA SKCM mRNA data were used to extract the AR co-expressed genes using the online tool from the cBioportal website. The entire genome was included and the co-expressed genes were identified using a cutoff q value of q < 0.05. 6413 genes from men and 3384 genes from women were retained for further comparison. When the Spearman’s co-efficient for AR-association was set at ρ > 0.34, then 75 genes in women and 202 genes in men were retained for further comparison. Among these genes, 44 were unique for women, 171 were unique for men , and 31 https://biit.cs.ut.ee/gprofiler/gost, accessed on 14 June 2022). Genes were ordered All significant enrichments for females and partial of that for males are listed in The 44 and 171 genes identified in the female and male tumors, respectively, are subjected to enrichment analysis using an integrated web-based tool termed g:profiler  (p = 0.047). When only sex is adjusted, the significant also remained (p = 0.022), but it was reduced to borderline (p = 0.057) when both sex and age are included.The TCGA melanoma team classified this cohort of patients into four distinct subtypes with distinct somatic mutations in the tumors . We obta= 0.013) . The sigp = 0.10, not shown in results). Nevertheless, our finding is significant, as this is one of the first studies to show an association of tumor AR level with overall survival in melanoma patients.The findings of this study suggest that a higher level of tumor AR protein is positively associated with a better overall survival in cutaneous melanoma patients, which remains true after adjusting to age of diagnosis, stage of disease, sex of patients, and Breslow depth of the tumors. However, when patients are stratified by sex, the significant association was found only in female patients, but not in male patients, even though sex itself is not significantly associated with overall survival in this dataset. Additionally, when ulceration status is included in the model, the significance of AR association with OS was lost, suggesting that ulceration is still the most effective prognostic factor for melanoma OS. A statistical test of an interaction of AR with ulceration status revealed only borderline significance (A previous report suggested an opposite role of AR in melanoma patient survival, i.e., higher AR was associated with worse survival . That reFor melanoma, similar to many other cancer types, females in general show a survival advantage even after adjusting to many other prognostic factors. The underlying mechanism may be multi-fold, and we have been interested in the roles of sex hormones in such situations. Sex hormones and their receptors play critical roles in many pathophysiological conditions and impact many oncogenic pathways and cellular functions. AR was recently studied in melanoma cells, with a function of promoting proliferation, tumorigenesis, metastasis, and drug resistance ,3,4, whiThe possible explanation may be directly linked to the androgen levels as the majority function of AR is linked to locally available testosterone and dihydrotestosterone. Therefore, in most cases, we must study the function of AR/T or AR/DHT together. It is particularly important to study the sex-specific impact, as men and women are distinctly different in the circulating T or DHT levels. Our study also showed a distinct gene network in tumors from male and female patients, further strengthening the importance of sex-specific investigation. Another possible reason is related to how to interpret the data. In one study, loss of AR led to more DNA damage , suggestIt is also noticeable that AR plays distinct functions in the male and female tumors, with shared functions in both sexes. The enriched functions are much broader in male tumors, indicating male-biased significance of AR. Since AR is involved in many more gene networks in males, the ability of these functions to maintain a relative cellular balance may be strengthened, which may help to explain why AR in men did not show a significant association with overall survival.The weakness of this study is that we used only the TCGA data, with no replicating dataset. Therefore, this study requires further validation in a different patient cohort. As noted in one of our previous study , the patThe overall conclusion of this study is that tumor AR protein levels are associated with better OS in female patients, and not in male patients. We have also identified shared and distinct AR-associated gene networks in male and female tumors, which suggests AR exhibits common function in all tumors, and also exhibits distinct function in tumors from male and female patients. This study is the first to include data from a large database source with over 350 data points from melanoma patients’ tumors. Most of previous studies of AR in melanoma suggested that AR promoted tumor proliferation, metastasis, and drug resistance. Our study suggests that the role of AR should be considered in sex-specific manner, and in females, AR could be protective. Further investigation on these shared and distinct functions of AR in melanoma patients will help us to develop precise treatment strategies.


Androgen deprivation therapies (ADTs) are important treatments which inhibit androgen-induced prostate cancer (PCa) progression by either preventing androgen biosynthesis (e.g. abiraterone) or by antagonizing androgen receptor (AR) function . A major limitation of current ADTs is they often remain effective for limited durations after which patients commonly progress to a lethal and incurable form of PCa, called castration-resistant prostate cancer (CRPC) where the AR continues to orchestrate pro-oncogenic signalling. Indeed, the increasing numbers of ADT-related treatment-emergent neuroendocrine-like prostate cancers (NePC), which lack AR and are thus insensitive to ADT, represents a major therapeutic challenge. There is therefore an urgent need to better understand the mechanisms of AR action in hormone dependent disease and the progression to CRPC, to enable the development of new approaches to prevent, reverse or delay ADT-resistance. Interestingly the AR regulates distinct transcriptional networks in hormone dependent and CRPC, and this appears to be related to the aberrant function of key AR-epigenetic coregulator enzymes including the lysine demethylase 1 (LSD1/KDM1A). In this review we summarize the current best status of anti-androgen clinical trials, the potential for novel combination therapies and we explore recent advances in the development of novel epigenetic targeted therapies that may be relevant to prevent or reverse disease progression in patients with advanced CRPC. Acinar  cancers –9. Howev cancers –12. NePC cancers . This reAR gene is located on chromosome X and spans ~186,587 base pairs (bp). The protein coding region is ~2757 nucleotides long and the protein is comprised of ~920 amino acids and possess polymorphic glutamine and glycine tracts  (The androgen receptor (AR) is the primary mediator of androgen action. The main physiological androgens, testosterone and it more potent derivative, dihydrotestosterone (5-α-DHT), have long been recognized to play an important role in the initiation and progression of PCa . The tra (FQNLF) . This mo (FQNLF) . The tra (FQNLF)  and epig (FQNLF) Figure 3 (FQNLF) –30.It has long been recognized that PCa initiation and progression is androgen-dependent and for this reason depriving PCa cells of androgen and androgen receptor function through inhibition of androgen biosynthesis or AR function will act to suppress tumorigenesis. Luteinizing hormone releasing hormone (LHRH) ) agonists are androgen synthesis blockers which suppress androgen production by acting on the hypothalamus–pituitary–gonadal axis, whereby sustained stimulation of the LHRH receptor results in the downregulation of testosterone after an initial surge . LHRH anAR gene mutations, drug antagonist-to-agonist switching, ligand promiscuity, bypassing the AR signaling pathway, AR splice variants, AR genomic structural rearrangements (AR-GSRs), and alterations in transcription factors and AR co-regulators  . Previougulators , 47, 48.gulators .Several phase I and II studies demonstrated reductions in PSA (with varying success across cohorts) with abiraterone acetate treatment, often in conjunction with prednisone and subsequently is a standard of care treatment for metastatic castration resistant prostate cancer (mCRPC) . These results also confirm that CRPC commonly remains hormone driven , 51. AddFollowing Enzalutamide, another second-generation anti-androgen, Apalutamide, was developed. The addition of Apalutamide to conventional ADT was found to significantly improve patient outcome in non-metastatic CRPC  . In addiAnother promising drug is darolutamide (ODM-201) which is a structurally distinct androgen receptor antagonist, which holds a significant advantage over other anti-androgens, as it shows negligible brain penetrance, does not increase serum testosterone levels in mice, reduces the risk of seizures and was found to inhibit AR variants . A recenTherapies such as Abiraterone and androgen receptor antagonists  have been used to treat CRPC but are not curative . EffortsDespite the challenges of resistance, there are benefits of hormonal therapies and there are many on-going clinical trials to identify optimal combinations of hormonal therapies with chemotherapy, radiotherapy, and immunotherapy.Chemotherapy, commonly docetaxel or cabazitaxel, is often given only in late stages of prostate cancer disease to prolong the life of patients. Recently it has been shown Cabazitaxel plus prednisone use as a third line treatment after disease progression with docetaxel and abiraterone or enzalutamide, is significantly more effective than the hormonal therapy (NCT02485691) .Radiotherapy is both given after neoadjuvant treatment to treat localized prostate cancers and at late-stage prostate cancer to target metastatic sites. The Phase III RADICALS trial (NCT00541047) found that precautionary radiotherapy after surgery, resulted in no significant difference in recurrence after 5 years compared with observation . This prAnother potential new therapeutic approach, recently reviewed by Loizzo et al., is targeting autophagy to sensitize prostate cancer to other drugs . AutophaTheranostics (or Theragnostics) is a novel therapeutic strategy in the move towards precision medicine to co-deliver therapeutic and imaging functions within a single agent. This includes delivering cytotoxic levels of radiation which are targeted at a cellular level to specific biomarkers. Prostate-specific membrane antigen (PSMA) presents a potential target for this approach, [reviewed by ]. It hasThe identification of clinically relevant biomarkers and/or genotypes is critical for effective personalized treatment approaches to enable and inform prioritizing patients for immuno-and targeted therapies. While there is great interest in the development of immunotherapies for cancer, PCa is considered to have an immunosuppressive microenvironment, with few infiltrative lymphocytes. Recently the use of high dose-rate brachytherapy was able to alter this with increase in immune infiltrates . There aRecent advances in biomarker discovery have enabled the more precise use of targeted therapies in PCa. A new class of anticancer drugs called PARP inhibitors (PARPi) have been developed and trialled successfully on several types of cancers including PCa. Poly (ADP-ribose) polymerases 1 and 2 (PARP1 and 2) play a crucial role in DNA damage repair (DDR) by sensing DNA damage, binding to the damaged DNA and recruiting DNA repair proteins to the damage site, through a process called PARylation . PARP inA clinical trial in 2015 NCT01682772) tested the efficacy of olaparib, a PARP inhibitor, in treating mCRPC and found that treatment with olaparib prolonged radiographic progression–free survival (rPFS) and reduced circulating tumor-cell counts. Importantly, a better response was observed in patients with tumors that had aberrations in DNA repair genes . An alte772 testesynthetic lethality; a term used to describe cell death following the genetic or pharmacological inhibition of a cellular pathway in the context of a distinct loss of function mutation in a separate pathway  (via the homologous recombination repair (HRR) pathway. Where homologous repair of DSBs cannot be carried out, cells are forced to use more error-prone non-homologous end joining (NHEJ) which results in chromosomal instability and increased sensitivity to PARPi  . BRCA 1 to PARPi . BRCA geto PARPi . Additioto PARPi , 86 and to PARPi . Overallto PARPi , 89.via cell cycle checkpoint control) was found to inhibit HRR in the ATM knockout cells and although cytotoxic effect was modest in the context of ATM knockout alone, addition of PARP inhibition significantly increased cell death. The efficacy of inhibiting both PARP and ATR was then confirmed in a xenographic model of a patient derived mCRPC cell line with loss of ATM  aimed to compare olaparib against enzalutamide or abiraterone for the treatment of mCRPC with HRR gene alterations . It founs of ATM . This sts of ATM .So far, olaparib has been approved by the FDA for use in mCRPC with identified mutation in HRR and rucaparib has been approved for mCRPC with BRCA mutation. Ongoing trials of PARP inhibition in prostate cancer and completed trials awaiting results are included in In recent years the focus on understanding cancer progression has led to several multicenter studies being able to stratify prostate cancer. This stratification has also highlighted the crucial role of epigenetic factors in cancer progression –100. HerAs noted earlier, the AR depends on multiple enzymatic distinct epigenetic coregulators, many of which are independently implicated in PCa. For this reason the focus is now shifting to the chromatin modifying coregulator proteins such as lysine demethylases (KDMs) that are key cofactors for cancer drivers such as AR , 101–104SPOP gene in PCa often impart resistance to BET inhibitors  subfamily. It binds acetylated histone lysine residues and has been shown to be important for recruiting RNA polymerase II to facilitate transcription. In CRPC, BRD4 interacts with AR and inhibition of BRD4 prevents AR from binding to its target genes, disrupting AR transcriptional activity . Thus, hibitors . Howeverhibitors . BRD4 wohibitors . BRD4 alhibitors . Recentlhibitors .EZH2 (enhancer of zeste homolog 2) is a methyltransferase that typically functions as part of the polycomb repressor complex 2 (PRC2). However, recently it was shown that it also exhibits a separate activation function when it binds to the promoter region of AR and plays a role in AR signaling. It also acts as a co-activator of AR in CRPC [reviewed by Jones et al. ]. RecentThese studies provide hope for the development of combined epigenetic therapies in the future that effectively target two or more epigenetic factors significant in PCa.In conclusion, therapeutic strategies targeting the AR function remain the cornerstone for treating men with advanced PCa. However, the emergence of castrate resistant PCa and indeed treatment emergent neuroendocrine-like PCa, highlights the need for the development of newer more targeted pharmacological approaches to suppress pro-proliferative androgen signaling. With the emerging role of DNA repair and epigenetic factors in mCRPC and NEPC, identifying and repurposing drugs for these new factors in PCa could lead to better precision treatment options for individuals with advanced PCa.The development of epigenetic targeting therapies represents a novel approach to suppress androgen signaling and could be an additional line of therapy after resistance has occurred to other approaches. Indeed, a precision medicine approach could also be applied to identify and treat individuals that would respond to immunotherapies, including methods of enhancing immune response and immune infiltration for effective immunotherapy. The numerous ongoing clinical trials will continue to identify the most effective combinations of hormonal therapies with chemotherapy, radiotherapy, immunotherapy and other novel therapies to improve both survival and quality of life of prostate cancer patients.AH, AN, and NM contributed to conception and design of the study. AH, VM, JL-R, DV, NM, and JJ review preparation. AH, VM, DV, NM, CR, AN, SB, and JJ wrote first draft. AH, DV, CW, CE, MH, MT, MA, JB, SM, AN, SB, and JJ wrote sections of the review. JP, LG, ER, BR, FK, LM, JM, JB, SM, CA, VJ, CR, RF, AN, SB and NM critical revision of review. All authors contributed to the article and approved the submitted version.We gratefully acknowledge the financial support of the University of Nottingham, the BBSRC; Doctoral training program  and  and Prostate Cancer UK RIA15-ST2-005 (NPM) and Prostate Cancer Foundation & John Black Charitable Foundation (20CHAL06: NPM).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Teachers’ interpersonal communication skills and strategies have been widely considered effective pedagogical tools in academia. Despite the growing research in this area, unraveling the power of English as a foreign language (EFL) teachers’ self-disclosure in shaping classroom climate and immediacy has been relatively left intact. To fill the gap and provide new insights into this strand of research, the present article was an effort to present a theoretical analysis of the interplay of self-disclosure, classroom climate, and immediacy. In so doing, the definitions, conceptualizations, dimensions, underlying theories, and empirical evidence in support of the interaction among these three constructs were presented. Moreover, practical implications for EFL teachers, teacher trainers, and L2 scholars were provided to raise their awareness of interpersonal communication skills and their outcomes in academia. Finally, the study provided some suggestions for further research in this line of inquiry. Teaching is by nature a communicative act and the way the teacher communicates with his/her pupils and maintains that interrelationship influences various aspects of education . An inteIn second/foreign language education, teacher self-disclosure  refers to a teacher’s statements about self that may or may not be pertinent to course content, but reveal information about him/her that students are unlikely to obtain from other sources . The conThe concept of self-disclosure was originally studied in humanistic psychology, interpersonal relationships, and communication studies to refer to the act of revealing and sharing personal information with others . The terAs teaching is a communicative process in which the teacher and students constantly negotiate meanings and send messages, the concept of self-disclosure is of paramount significance in this job . The quaamount, duration, depth, positivity, negativity, relevance, and appropriateness , haptics (touch), vocalics , kinesics (body movement/orientation), oculesics (eye contact), classroom environment , and chronemics (time).As research signifies, there are two types of classroom immediacy, namely verbal and non-verbal immediacy. Verbal immediacy concerns verbal messages that demonstrate empathy, reward, kindness, willingness, inclusiveness, praise, openness, and humor in communication . On the Concerning theoretical underpinnings, three theories underpin classroom immediacy in education. They include The second related theory is the hierarchy of needs theory proposed by Undoubtedly, all these human needs are vital in one’s life and education, yet the psychological needs (belongingness and intimacy in communities) are more powerful in educational contexts in that they can pay off the other deficiencies. Because of this, good teachers usually form strong emotional bonds with their students by using different immediacy clues and behaviors to manifest their concern for students’ psychological needs . The thiDifferent empirical studies have been conducted on the variables reviewed in this article. Concerning self-disclosure, a growing body of research indicates that it increases EFL students’ classroom engagement, participation, attention, motivation, achievement, willingness to communicate, interpersonal skills, passion, social competence, speaking skills, writing skills, and decreases their anxiety . MoreoveMoreover, immediacy as one of the most outstanding consequences of TSD and positive classroom climate has been found to improve students’ motivation, attention, involvement, WTC, interpersonal communication skills, psychological empowerment, clarity, and credibility . AlthougIn this review article, it was pinpointed that TSD is a facilitator and predictor of EFL classroom climate and immediacy, which in turn, generate several positive academic outcomes of both teachers and students. It was also claimed that when EFL teachers provide more self-disclosure to their students, a strong sense of rapport would be created in the class that installs a positive classroom climate for education that is laden with immediacy cues. Based on these, it is contended that the present theoretical review would have valuable contributions to the field by acting as a response to the necessity and significance of including awareness-raising of interpersonal communication skills and their outcomes in academia. It can also be beneficial for EFL teachers in that they can understand the power of interpersonal communication strategies like self-disclosure in shaping the overall classroom climate and a sense of proximity in the classroom. They can also use verbal and non-verbal immediacy cues to reinforce classroom rapport and academic performance of the students. Furthermore, this study can be helpful for teacher trainers by showing them the criticality of interpersonal communication skills in many aspects of L2 education. Hence, they can offer training programs like workshops and seminars on different communication skills and practical ways of applying them in the classroom. Finally, L2 researchers may find this review fruitful in that they can carry out further studies on this line of research and bridge the existing gaps. More particularly, they can conduct mixed-methods, qualitative, and longitudinal studies on TSD, classroom climate, and immediacy to provide richer data as opposed to one-shot, correlational studies common in this area. Likewise, given the culture and context specificity of these three constructs, they are recommended to run cross-cultural studies to unpack the role of cultural factors in the interaction of the reviewed constructs. Additionally, future researchers are suggested to focus on the situated nature of the interplay among TSD, classroom climate, and immediacy considering the role of more variables, socio-economic factors, and power-related factors embedded in the context. Finally, the relationship between TSD and other interpersonal communication skills such as clarity, confirmation, credibility, stroke, care, etc. is also an interesting topic for future research.The studies involving human participants were reviewed and approved by the Xinxiang Medical University Academic Ethics Committee. The patients/participants provided their written informed consent to participate in this study.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Androgen receptor splice variant 7 (AR-V7) has always been considered a key driver for triggering enzalutamide resistance of castration-resistant prostate cancer (CRPC). In recent years, both the homeostasis of AR-V7 protein and AR-V7’s relationship with LncRNAs have gained great attention with in-depth studies. Starting from protein stability and LncRNA, the paper discusses and summarizes the mechanisms and drugs that affect the CRPC patients’ sensitivity to enzalutamide by regulating the protein or transcriptional stability of AR-V7, hoping to provide therapeutic ideas for subsequent research to break through the CRPC therapeutic bottleneck.Prostate cancer (PCa) has the second highest incidence of malignancies occurring in men worldwide. The first-line therapy of PCa is androgen deprivation therapy (ADT). Nonetheless, most patients progress to castration-resistant prostate cancer (CRPC) after being treated by ADT. As a second-generation androgen receptor (AR) antagonist, enzalutamide (ENZ) is the current mainstay of new endocrine therapies for CRPC in clinical use. However, almost all patients develop resistance during AR antagonist therapy due to various mechanisms. At present, ENZ resistance (ENZR) has become challenging in the clinical treatment of CRPC. AR splice variant 7 (AR-V7) refers to a ligand-independent and constitutively active variant of the AR and is considered a key driver of ENZR in CRPC. In this review, we summarize the mechanisms and biological behaviors of AR-V7 in ENZR of CRPC to contribute novel insights for CRPC therapy. As the most common urological malignant tumor, prostate cancer (PCa) occurs in the prostate epithelium, and is the fifth leading cause of death from cancer in men. New cancer cases among men worldwide exceeded 10 million in 2020, 1.4 million of which were newly diagnosed with PCa and accounted for 14.1% of all incident cases in men. And the number of deaths from PCa was 375,304, making up 6.8% of all cancer deaths in men [The introduction of second-generation ARSI has greatly impacted the treatment of CRPC . EnzalutENZ mainly acts through the blockade of the AR signaling pathway, including blocking the binding of androgens to AR, impeding the AR nuclear translocation, and inhibiting the AR transcriptional activity for the treatment of CRPC ,12,13. SAR-related signaling pathways have always been seen as one of the considerable mechanisms responsible for ENZ resistance (ENZR) in CRPC, such as the aberrant amplification and/or overexpression of AR ,18, AR mThe full-length AR (AR-FL) is mapped to chromosome Xq11-Xq12 . For theAR-Vs were first discovered over 20 years ago and reported by Tepper et al. . At presAR-V7 is a mimic of AR, or AR-V7 has unique activity or whether AR-V7 contains a unique binding site is the focus of researchers. A recent study has found that AR-V7 induces a slightly higher proportion of total regulatory genes in the cell model than AR-FL, and its differential binding with chromatin is stronger than AR-FL . Some stAR-V7 is derived from AR-FL and is almost always co-expressed with AR-FL in the clinical environment. Therefore, the topic of whether the role of AR-V7 in PCa depends on AR-FL has been widely debated. Some researchers have concluded that AR-V7 works with AR-FL or requires AR-FL to perform activities, because AR-V transcriptional activity requires AR-V homologous dimerization . AR-V7 cp < 0.001)Sharp et al. assessed the expression of AR-V7 protein in 358 primary prostate specimens and 293 metastatic biopsy specimens . The resp = 0.004). Moreover, it was also found that the AR-V7 positive patients had a shorter PSA progression-free survival , shorter clinical or radiographic progression-free survival  and shorter overall survival , indicating insufficient therapeutic efficacy and prognosis as a consequence of the decreased sensitivity of AR-V7 positive patients to ENZ. Similarly, research detected in various ways the AR-V7 in the CTCs of blood samples belonging to high-risk CRPC patients being treated by ENZ and came to the same conclusion: the AR-V7 expression is independently related to shorter clinical progression-free survival (PFS) and poorer overall survival (OS) [The generation of AR-V7 is one of the causes of the poor prognosis and low overall survival rate of CRPC patients after treatment. Compared with AR-V7 negative patients, the prognosis of PCa patients treated with ENZ became worse if they were also AR-V7 positive . The resval (OS) . MeanwhiA large number of preclinical research results show that AR-V7 can be used as a biological marker of ENZR in CRPC ,65. BesiAR-V7 has long been a crucial research focus in exploring the mechanism of ENZR. In cell-level experiments, it has been found that the ENZ therapy of CRPC enhances the level of AR-V7 and aids the expression of its target genes in PCa cells, and that the knockdown of AR-V7 can restore the sensitivity of cancer cells to ENZ, suggesting that AR-V7 is one of the salient factors leading to drug resistance . SimilarHeat shock proteins (HSP) refer to a group of proteins generated by cells under the induction of stressors, especially environmental high temperature . They arAR/AR-V7 is one of the client proteins of HSP . In the HSP70 has been identified as a biomarker for the survival and prognosis of PCa owing to the relationships between its overexpression and carcinogenesis as well as drug resistance in PCa . HSP70 iMoon et al. screened and identified the drug bruceantin (BCT) from a small antimalarial drug library, by directly binding to HSP90 which can not only disrupt the interaction between HSP90 and AR-FL/AR-V7 but also inhibit the chaperone function of HSP90, causing the degradation of AR-FL/AR-V7 through the ubiquitin-proteasome system . These rIn recent years, the application of natural products and their analogues in tumors has received much attention. AR-V7 is a substrate for the proteasome, as Liu et al. demonstrated . They exA novel protac small molecule named MTX-23 was designed and developed by Lee et al. , which wOne approach to treating cancer is to screen and create small molecule compounds from databases. Nevertheless, continuous discovery of natural products and their derivatives has demonstrated an extraordinary anti-cancer effect as well as their abilities to reverse tumor resistance, suggesting the crucial clinical value of natural products and their derivatives . MoreoveLncRNA has been widely researched in the past decade, which is defined as an RNA transcript longer than 200 nucleotides without protein coding . EarlierRecent research has displayed close relationships between LncRNAs and the occurrence and development of malignancies ,117. LncWang et al. revealed the relevance between LncRNAs and the expression of AR-V7 in CRPC. Specifically, LncRNA-Malat1 is essential for the generation of AR-V7 . They foThe LncRNA was related to the generation of AR-V7. Additionally, the most recent research uncovered that LncRNA might also change the expression level and/or function of AR-V7 through a series of mechanisms, affecting the drug sensitivity of ENZ in CRPC. Zhang et al. found that LncRNA-PCBP1-AS1 can accelerate the progression of ENZR in CRPC by binding to NTD to improve the stability of the USP14-AR-V7 complex. AR-V7 and USP22 were found in this experiment to be greatly reduced by PCBP1-AS1 depletion . TherefoNTD is one of the necessary structures for AR transcription. Hirayama et al.  found thGenerally speaking, LncRNAs and AR-V7 are closely related to each other, playing an important role through various mechanisms in ENZR in CRPC . In addiRecent studies have reported that many molecules regulate the expression of AR-V7 in different ways . AKR1C3,The kinesin family encompasses kinesin family member 15 (KIF15) which is essential for the development of bipolar spindles . KIF15 iHow AR-V7 mediates genome function in CRPC remains largely unknown. homeobox B13 (HOXB13), a member of the homeobox proteins family, is a key regulator of the epithelial differentiation in the prostate gland . Zhong eTherefore, if key regulatory molecules such as AKR1C3, KIF15, DBC1 and HOXB13 can be targeted, thereby reducing the stability of AR-V7, promoting the protein ubiquitin-proteasome of AR-V7 may be able to reverse ENZR of CRPC.ENZ, a second-generation ARSI, has given hope to CRPC patients and increased the overall survival rate. Nevertheless, ENZR has become challenging in the clinical treatment of CRPC. Scientific research has also focused on the mechanism of ENZR in CRPC. Numerous studies have thus far reported that the occurrence of ENZR is mediated by different mechanisms, which are AR-related mechanisms, glucocorticoid receptor-related processes, neuroendocrine transformation, related pathway activation, etc. There is no doubt about the role of AR in mediating ENZR in CRPC. As the most important AR-Vs, AR-V7 have been shown to play an important role in the development of drug resistance in CRPC and in predicting treatment response.In addition to expanding our understanding of the AR signal axis in the past, the research into AR-V7 in the mechanism of ENZR in CRPC also offers the most recent therapeutic approaches to treating CRPC. One of the recent research hotspots and foci are the relationships between LncRNA with proteostasis and AR-V7. In order to treat ENZR in CRPC, continuous investigation is explored on strategies targeting the stability of the AR-V7 protein or the AR-V7 transcription levels. For example, the conventional drug niclosamide has been applied in a new way. Some newly developed small-molecule compounds such as ARVib-7 and MTX-23, as well as natural products such as nobiletin and Hsp90, have also shown efficient therapeutic effects.Although the data of preclinical studies are encouraging, the specific mechanism of AR-V7 activation is not clear at present, and most drugs are limited to the results of preclinical studies now. No further clinical trials have been carried out. In addition, there are still problems of limited efficacy or unacceptable toxicity in clinical trials in clinical development. Therefore, there is still a long way to go to develop drugs that inhibit the expression of AR-V7 or promote the degradation of AR-V7 and finally put them into clinical treatment for patients with CRPC.


To describe cross‐sectional and longitudinal variation in neurorehabilitation content provided to young people after severe paediatric acquired brain injury (pABI) and to relate this to observed functional recovery.This was an observational study in a cohort of admissions to a residential neurorehabilitation centre. Recovery was described using the Pediatric Evaluation of Disability – Computer Adaptive Testing instrument. Rehabilitation content was measured using the recently described Paediatric Rehabilitation Ingredients Measure (PRISM) and examined using multidimensional scaling.The PRISM reveals wide variation in rehabilitation content between and during admissions primarily reflecting proportions of child active practice, child emotional support, and other management of body structure and function. Rehabilitation content is predicted by pre‐admission recovery, suggesting therapist decisions in designing rehabilitation programmes are shaped by their initial expectations of recovery. However, significant correlations persist between plausibly‐related aspects of delivered therapy and observed post‐admission recovery after adjusting for such effects.The PRISM approach to the analysis of rehabilitation content shows promise in that it demonstrates significant correlations between plausibly‐related aspects of delivered therapy and observed recovery that have been hard to identify with other approaches. However, rigorous, causal analysis will be required to truly understand the contributions of rehabilitation to recovery after pABI.Rehabilitation content varies widely between, and during, admissions for neurorehabilitation after paediatric acquire brain injury.Strong correlations are seen between plausibly‐related aspects of rehabilitation content and observed recovery, though careful interpretation is necessary. Rehabilitation content varies widely between, and during, admissions for neurorehabilitation after paediatric acquire brain injury.Strong correlations are seen between plausibly‐related aspects of rehabilitation content and observed recovery, though careful interpretation is necessary.We examine the content of neurorehabilitation provided to young people recovering after severe paediatric acquired brain injury (pABI). Content varies widely between and during admissions and is predicted by pre‐admission recovery suggesting rehabilitation programmes are shaped by therapists' expectations of recovery. However significant correlations persist between plausibly‐related aspects of delivered therapy and observed post‐admission recovery after adjusting for such effects. Rigorous causal analysis will be required to truly understand the contributions of rehabilitation to recovery after pABI 1057–1058 of this issue.This original article is commented on by Whyte on pages  MDSMultidimensional scalingpABIPaediatric acquired brain injuryPEDI‐CATPediatric Evaluation of Disability – Computer Adaptive TestingPRISMPaediatric Rehabilitation Ingredients MeasurePaediatric acquired brain injury (pABI) represents an increasing health challenge as developments in intensive care medicine have reduced the rate of mortality from critical illnesses.Achieving a better understanding of the role of rehabilitation in improving recovery after pABI presents three distinct challenges.This paper focuses on the second and third of these challenges. We have recently described a Paediatric Rehabilitation Ingredients Measure (PRISM) as a practical means of describing rehabilitation content.This paper aimed to describe variations in rehabilitation content during and between different individuals’ admissions, and to examine relationships between recovery to date, rehabilitation treatment content, and ongoing recovery. Compared to our previous work,The children in this study received residential neurorehabilitation at The Children’s Trust, Tadworth, Surrey between June 2019 and October 2020.https://www.youtube.com/watch?v=QW6ZwY1gacs and https://www.youtube.com/watch?v=‐GSjSiM_apE). It is used to describe the areas of focus of the rehabilitation multidisciplinary team, expressed as proportions among a range of required items selected from a pre‐defined ‘menu’ of 11 available options . It uses the analytical hierarchy processThe development of PRISM is described elsewherePRISM scores were estimated at monthly multidisciplinary team meetings using a bespoke online analytical hierarchy process calculator. PRISM scores come with an internal consistency ratio: a ratio < 10% is deemed acceptable and all PRISM estimations met this criterion.The functional outcome assessment tool used in these analyses was the Pediatric Evaluation of Disability Inventory – Computer Adaptive Testing (PEDI‐CAT),PEDI‐CAT scores were obtained at admission and discharge. PEDI‐CAT velocities (rates of change) were used rather than absolute score changes between admission and discharge. This was to avoid the potentially confounding effect of length of stay that varies between children and is itself a proxy for injury severity.The PEDI‐CAT ability estimate algorithm generates a (mis)fit score for each domain that if large (>2.0) suggests an unexpected pattern of scores across items and that the overall scaled score should be interpreted with caution; there were no instances of this in the data set.www.hra‐decisiontools.org.uk), therefore a research ethics review was not sought. The study was endorsed by The Children’s Trust research committee (TCT049 May 2017).As an observational study that used routinely collected clinical data, with no consequences for or effects on patient care, this study was deemed a service evaluation by standard criteria  using cmdscale . Multiple approaches to the visualization of PRISM data were used: hierarchical clustering was examined using Appendix . MDS is n = 15), anoxia (n = 9), and other (predominantly various types of stroke and tumour) (n = 37). There were no statistically significant differences in sex, age at injury, interval from injury to admission, or injury mechanism between the 61 children with PRISM scores and the 33 without. PEDI‐CAT data were available for 51 out of 61 children.PRISM data were available for 61 children , of a total of 94 admissions during this period. Median age at injury  was 11 years 2 months ; interval from injury to admission 128 days . Injury mechanism was trauma (x = 0 or y = 0 axes) were common, implying that many possible ingredient pairs were treated as mutually exclusive. If any one ingredient was scored, none of the others were. Modest but highly significant negative correlations were evident between the child, active practice, and each of family practice, child, other management, family adaptations, and environmental adaptation  at approximately monthly intervals. Figure x and y coordinates of the points on this scatterplot are, respectively, the Dimension 1 and 2 scores in Figure z scores around a mean of zero). Figure x) value correlates very strongly with active practice of skills by the child  and Dimension 2 (y) correlates positively with other management of child body structure and function  and Family Learning , and strongly negatively correlates with emotional support of the child .MDS converted PRISM observations to points on a scatterplot Figure . The x aConventional principal components’ analysis yields very similar results with a first component loading on Active Practice accounting for 31% of variance and a second loading negatively on Child Emotional Support and positively on Other Management and Family Learning accounting for an additional 18%. A similar structure also emerged from cluster analysis Appendix .y = 0 line, reflecting the compositional nature of PRISM data.Figure r > 0.98; p < 0.001 in all cases), implying comparable recovery rates across these domains before admission. Figure After deleting observations falling outside the 45‐day limits, pre‐ and post‐admission PEDI‐CAT velocities were available for 28 children . Figure p < 0.1 level) between median Dimension 1 (Active Practice) and pre‐admission Mobility and Social/Cognitive velocities; and between median Dimension 1 and post‐admission Daily Activity velocity. Univariate correlations between post‐admission Daily Activity and Responsibility velocities and median Dimension 2 scores reached the p < 0.05 threshold. More robust multivariate linear regressions were performed using the formal directed acyclic graph approach used in previous workR2 = 0.31, p < 0.05) of median Dimension 1 on post‐admission PEDI‐CAT Daily Activity subdomain velocity persists after adjustment for possible pre‐admission velocity effects on rehabilitation content. The Dimension 2 rehabilitation content score was negatively correlated with pre‐admission PEDI‐CAT subdomain velocities and positively correlated with post‐admission Daily Activity and Responsibility velocities.Figure To our knowledge, this is the first detailed quantitative examination of cross‐sectional and longitudinal variation in rehabilitation content after acquired brain injury, certainly in children. Three approaches – MDS, cluster analysis, and principal components analysis – yield very similar findings. Child active practice followed (with opposite signs) by child emotional support and other management of body structure and function emerge as the PRISM ingredients accounting for most of the variation in rehabilitation treatment content between and through admissions. It should be emphasized that PRISM’s conceptualization of ‘active practice’ encompasses all forms of ‘learning by doing’ irrespective of ‘target’. Depending on the clinical context, this could include practicing speaking or activities of daily living as well as many cognitive rehabilitation techniques and not just the active practice of gross and fine motor skills.x < 0 in Figure We focussed on the MDS approach here because of its explicitly spatial interpretation and a wish to explore trajectories in ‘rehabilitation content space’. The dominance of active practice is perhaps not surprising, but the opposing ‘polarities’ of Child Emotional Support and Other Management on Dimension 2 is somewhat unexpected. Further qualitative examination of what clinical populations these rehabilitation patterns are associated with will be informative. Large absolute (positive or negative) scores on Dimension 2 are associated with less active practice  in order to examine possible rehabilitation dose–response effects. The challenge of robust, causal inference here is underlined by the most important and novel observation of this study, namely the extent to which rehabilitation content can change during an admission. Figure p < 0.05) for median Dimension 2 on post‐admission Daily Activity and Responsibility velocities persist after adjustment for this.More generally, this finding underlines the importance and magnitude of the third challenge identified in the introduction: whether therapists’ decisions to alter rehabilitation content are determinants of, or are occurring in response to, observed recovery. The findings of Table Figure We have set this work in the context of the overarching goal of being able to identify rehabilitation dose–response effects with confidence (with the ultimate aim of improving rehabilitation outcomes through comparative effectiveness research). Previous efforts have largely been in adults, in stroke,R2 values are generally smaller than the previous study,The hoped‐for benefit of this approach to rehabilitation‐content parsing is the ability to examine rehabilitation dose–response effects. In a recently published pilot study that focussed specifically on rates of gross motor recovery,Despite these complications, however, the ready demonstration of statistically significant correlations in small samples here and in our previous workAppendix S1: Supporting material.Click here for additional data file.


C. elegans, odorant response abnormal 4 has been implicated in the delivery of olfactory GPCRs to the cilia of chemosensory neurons. However, the function and regulation of its human homolog, C1orf27, in GPCR transport or in general membrane trafficking remain unknown. Here, we demonstrate that siRNA-mediated knockdown of C1orf27 markedly impedes the ER-to-Golgi export kinetics of newly synthesized α2A-adrenergic receptor (α2A-AR), a prototypic GPCR, with the half-time being prolonged by more than 65%, in mammalian cells in retention using the selective hooks assays. Using modified bioluminescence resonance energy transfer assays and ELISAs, we also show that C1orf27 knockdown significantly inhibits the surface transport of α2A-AR. Similarly, C1orf27 knockout by CRISPR-Cas9 markedly suppresses the ER–Golgi-surface transport of α2A-AR. In addition, we demonstrate that C1orf27 depletion attenuates the export of β2-AR and dopamine D2 receptor but not of epidermal growth factor receptor. We further show that C1orf27 physically associates with α2A-AR, specifically via its third intracellular loop and C terminus. Taken together, these data demonstrate an important role of C1orf27 in the trafficking of nascent GPCRs from the ER to the cell surface through the Golgi and provide novel insights into the regulation of the biosynthesis and anterograde transport of the GPCR family members.The molecular mechanisms underlying the anterograde surface transport of G protein–coupled receptors (GPCRs) after their synthesis in the endoplasmic reticulum (ER) are not well defined. In  G protein–coupled receptors (GPCRs) represent the largest superfamily of cell surface signaling proteins that regulate a wide variety of cell functions under physiological and pathological conditions , 2. The 2-Adrenergic receptors (α2-ARs) are prototypic members of the GPCR family that have three subtypes, α2A-AR, α2B-AR, and α2C-AR, all of which play important roles in regulating the sympathetic nervous system, both peripherally and centrally. All α2-AR subtypes couple to the Gi/Go family of G proteins and regulate the activation of adenylyl cyclases, Ca2+ channels, and mitogen-activated protein kinases (2-ARs is that they have quite large third intracellular loop (ICL3) and relatively short C terminus (CT), both are important intracellular domains, mediating receptor interaction with G proteins, arrestins, protein kinases, and other molecules involved in regulation of signal initiation and termination, phosphorylation, and trafficking  cells and Chinese hamster ovary cells  when expressed in heterologous systems.Odorant response abnormal 4 (ODR4) gene was identified in chemotaxis-defective screens in  elegans  and its  elegans . The fun neurons , 25 and ry cells . ORs areC. elegans ODR4 and human C1orf27 share only 22% identity and 44% similarity. Recent studies suggest that C1orf27 is involved in regulation of protein UFMylation/deUFMylation via interaction with ubiquitin-fold modifier 1-specific protease 2, a deUFMylation enzyme  and a streptavidin-binding peptide (SBP) at its N terminus (NT) and the ER retention signal KDEL fused to streptavidin (Str-KDEL) is used as a hook to prevent the export of nascent cargo from the ER via streptavidin–SBP interaction. The ER export of cargo molecules is synchronized after incubation with biotin that binds streptavidin and thus disrupts SBP–streptavidin interaction. We first generated the RUSH construct Str-KDEL_SBP-EGFP-α2A-AR and tested the RUSH system in the transport of α2A-AR from the ER through the Golgi apparatus to the cell surface in live HeLa cells. α2A-AR was almost exclusively accumulated in the ER in the absence of biotin  and incubation with biotin induced α2A-AR export from the ER to the Golgi and the cell surface. After addition of biotin for about 10 min, the cargo α2A-AR was clearly concentrated at the Golgi, and the strongest Golgi expression was observed at 25 to 40 min (A and B). After 45 min of biotin induction, α2A-AR expression at the Golgi began to decline, indicative of transport to the cell surface (A and B). Although α2A-AR surface expression was visible after 1 h of biotin induction in some cells (C), robust receptor expression at the surface was not always easily detected, likely due to the mobility of live cells and low abundance of the receptor in cells chosen to be studied.To investigate the possible function of C1orf27 in the anterograde export of GPCRs in mammalian cells, we first used the retention using the selective hooks (RUSH) assays  to charaf biotin A, 0 min o 40 min , A and B surface , A and Bme cells C, robust2A-AR in HEK293 and HeLa cells after the cells were transfected with the RUSH plasmid and fixed at different time of biotin induction. Similar to the results observed in live cells, α2A-AR transport to the Golgi was obvious at 15 min after biotin induction and reached the maximum at 30 to 45 min (D). α2A-AR transport to the Golgi after release from the ER was confirmed by co-localization with the Golgi markers pmTurquoise2-Golgi and β1,4-galactosyltransferase 1  . In addition, the ER–Golgi transport of α2A-AR over time in HEK293 and HeLa cells was very much the same (D and E).We then compared the transport kinetics of αo 45 min D. α2A-AR1  . α2A-AR nduction , D and Ethe same , D and E2A-AR by quantifying its expression at the Golgi over time. Transfection of siRNA targeting C1orf27 almost abolished the expression of C1orf27 (A). In live cell RUSH assays, similar to the results observed in cells without transfection, α2A-AR was strongly expressed at the Golgi after 15 min of biotin addition, and the majority of α2A-AR was transported to the Golgi at 30 min after biotin treatment in control siRNA-transfected cells (B), suggesting that control siRNA did not affect the transport kinetics of α2A-AR. In marked contrast, α2A-AR was not clearly transported to the Golgi until biotin incubation for 30 min, and the strongest Golgi expression was observed at about 50 min in C1orf27 siRNA-treated cells (B). Quantitative data showed that the time course curves of α2A-AR expression at the Golgi were shifted to the right in C1orf27 siRNA-transfected cells as compared with control cells (C) and that the half time (t1/2) values were much higher in two C1orf27 siRNA-transfected cells (22.4 ± 1.6 and 25.7 ± 1.2 min) than that in control siRNA-transfected cells (13.4 ± 0.9 min) (D).We next measured the effect of siRNA-mediated knockdown of C1orf27 on the ER–Golgi transport of α C1orf27 A. In lived cells B, suggesed cells B. Quantiol cells C and tha0.9 min) D.Figure 2A-AR expression at the Golgi was significantly less in C1orf27 knockdown cells as compared with control cells at 15 and 30 min in both HEK293 and HeLa cell types (A and B). Together, these data strongly demonstrate that siRNA-mediated depletion of C1orf27 remarkably slows down α2A-AR transport from the ER to the Golgi.In fixed cell RUSH assays, αll types , A and B2A-AR, we modified bioluminescence resonance energy transfer (BRET) assays  marker Venus-kRas and control siRNA or individual siRNA targeting C1orf27. We first measured the time course of α2A-AR surface expression and found that similar to the results quantified by imaging (E), the maximal receptor expression was observed after 2 h of biotin induction (B). SBP-α2A-AR-Rluc8 was able to activate extracellular signal–regulated kinase 1 and 2 (ERK1/2) in response to stimulation with UK14304, an α2-AR agonist, suggesting that the receptor was functional (C and D). The cell surface expression of α2A-AR was significantly attenuated in C1orf27 siRNA-transfected cells as compared with that in control siRNA-transfected cells after 2 h of biotin induction (E).In order to quantify the effect of C1orf27 depletion on the surface transport of α) assays , 31 to m imaging E, the manduction B. SBP-α2nctional , C and Dnduction E.Figure 2A-AR at steady state. In this experiment, HEK293 cells were transfected with HA-α2A-AR together with control or C1orf27 siRNA, and the cell surface expression of α2A-AR was measured in enzyme-linked immunosorbent assays (ELISA). The surface expression of α2A-AR was strongly reduced by more than 35% in cells transfected with C1orf27 siRNA as compared with cells transfected with control siRNA (F). These data demonstrate that in addition to the ER–Golgi transport, C1orf27 also regulates the surface expression of α2A-AR at steady state.We next measured the effect of C1orf27 depletion on the surface expression of transiently expressed αol siRNA F. These 2A-AR, we measured the activation of ERK1/2. Consistent with the reduction in surface expression of α2A-AR, ERK1/2 activation after UK14304 stimulation was markedly reduced in C1orf27 knockdown cells as compared with control cells (G and H). In contrast, ERK1/2 activation by epidermal growth factor (EGF) was not affected by C1orf27 knockdown  by the CRISPR-Cas9 genome editing technology via transient transfection of KO plasmids. Because the control and KO plasmids carried GFP, the transfected cells were defined by the GFP signal. As α2A-AR functions mainly in neurons, human-derived neuroblastoma SHSY5Y cells were also used in these experiments. In the first experiment, the control or KO plasmids were transfected together with the RUSH plasmid Str-KDEL_SBP-mCherry-α2A-AR. In the absence of biotin, α2A-AR was expressed in the ER in cells transfected with control or C1orf27 KO plasmids. After induction with biotin for 15 or 30 min, α2A-AR expression at the Golgi was obvious in control cells expressing GFP or cells without transfection (indicated by arrows), whereas the receptor largely remained in the ER at 15 min and only partially exported to the Golgi at 30 min in C1orf27 KO cells (A–C).To further confirm the role of C1orf27 in αKO cells , A–C.Fig2A-AR in cells stably expressing HA-α2A-AR. After transfection with control or C1orf27 KO plasmids, the cell surface expression of α2A-AR was revealed by imaging after staining with HA antibodies in nonpermeabilized cells. As expected, α2A-AR was robustly expressed at the surface in cells transfected with control plasmids expressing GFP alone or without transfection, whereas the receptor was almost undetectable in cells expressing C1orf27 KO plasmids (D).In the second experiment, we measured the effect of C1orf27 KO on the surface expression of αplasmids D.2A-AR after transient expression of α2A-AR-RFP. α2A-AR clearly expressed at the cell surface in cells transfected with control plasmids or without transfection (indicated by arrows), whereas the receptor was largely arrested in the perinuclear region, unable to transport to the surface, in cells transfected with C1orf27 KO plasmids defined by the GFP signal (E). These data further demonstrate a crucial role of C1orf27 in the ER–Golgi-surface traffic of α2A-AR.In the third experiment, we measured the effect of C1orf27 KO on the subcellular localization of αP signal E. These 2-AR, dopamine D2 receptor (D2R), and epidermal growth factor receptor (EGFR) in RUSH assays. Similar to α2A-AR, the ER-to-Golgi transport of both β2-AR and D2R at 15 and 30 min after addition of biotin was much less in C1orf27-depleted cells than in control cells . In contrast, C1orf27 knockdown had no effect on the transport of EGFR (C and D).We next investigated if C1orf27 could regulate the export trafficking of other GPCRs and non-GPCR PM proteins. For this purpose, we measured the effect of siRNA-mediated knockdown of C1orf27 on the ER-to-Golgi trafficking of β of EGFR , C and D2A-AR, β2-AR, D2R, and EGFR at steady state after transient transfection in BRET assays. The surface expression of all three GPCRs, but not EGFR, was moderately but significantly attenuated by C1orf27 depletion (E). These data suggest that C1orf27 is likely a specific regulator for the transport of GPCRs.We then measured the effect of C1orf27 siRNA on the surface expression of αepletion E. These 2A-AR transport, we determined if C1orf27 could interact with the receptor. In co-immunoprecipitation assays, C1orf27 tagged with GFP and α2A-AR tagged with hemagglutinin (HA) were co-expressed in HEK293 cells and were found to form a complex following immunoprecipitation with HA antibodies (A).To study the mechanisms underlying the function of C1orf27 in regulating αtibodies A.Figure 2A-AR in glutathione S-transferase (GST) fusion protein pulldown assays using the ICL3 and the CT of α2A-AR (B) which are known to mediate receptor interaction with a number of regulatory proteins involved in signaling and trafficking (C). The progressive deletion strategy revealed that the shortest fragment-bound C1orf27 was S296-L332 in the ICL3, which further split abolished the interaction (D and E).We next sought to identify the binding domains of αf α2A-AR B which afficking , 21, 22.fficking C. The preraction , D and EB) and C1orf27 is an acidic protein with a calculated pI of 5.6, we determined the effect of increasing salt concentrations on their interactions. Incubation with NaCl inhibited the fragment S296-L332 interaction with C1orf27, and inhibition was in a dose-dependent fashion (F and G). Similarly, the CT interaction with C1orf27 was also inhibited by increasing concentrations of NaCl (H and I). These data suggest that the interactions between C1orf27 and α2A-AR are most likely ionic.As the fragment S296-L332 and the CT possess 10 and nine positively charged residues, respectively B and C1o fashion , F and G of NaCl , H and IC. elegans, was first demonstrated to assist the localization of some ORs to olfactory cilia almost a quarter of century ago , and dynabeads protein G were from Thermo Fisher Scientific. UK14304 was obtained from Sigma-Aldrich. MagneGST glutathione particles were from Promega. All other materials were obtained as described elsewhere , 61.2A-AR tagged with GFP at its CT or HA at its NT was generated as described previously . The plasmids encoding Venus-tagged C terminal 25 amino acid residues of kRas4B, β2-AR-Rluc8, and D2R-Rluc8 were kindly provided by Nevin A. Lambert (Augusta University) as described  without changing the encoded amino acid sequence and then amplified by PCR using the primers containing Fsel and XbaI restriction sites . The PCR product and the plasmid Str-KDEL_SBP-EGFP-Ecadherin (Addgene #65286) (2A-AR by using Str-KDEL_SBP-mCherry-Ecadherin (Addgene #65287)  and then amplified by PCR using the primers containing Sdal and Xbal restriction sites  and subcloned into Str-KDEL_SBP-EGFP-Ecadherin after release of EGFP-Ecadherin by Sdal and Xbal. C1orf27 tagged with GFP at its NT in the pEGFP-C1 vector was generated by PCR using the primers . To generate the plasmid Str-KDEL_SBP-β2-AR-EGFP, β2-AR-EGFP (2A-AR were generated in the pGEX-4T-1 vector as described previously (αeviously . α2A-AR escribed . To gene #65286)  were dig #65287) . To gene-AR-EGFP  was ampleviously . A similHEK293 and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). SHSY5Y cells were cultured in F12/Minimum essential medium (V/V =1:1) with 10% FBS. Transient transfection of cells were carried out by using Lipofectamine 2000.2A-AR were generated as described for HA-α2B-AR (2A-AR in the pcDNA3.1(+) vector for 24 h using FuGENE HD transfection reagent according to the manufacturer’s protocol. The cells were split into four 100-mm dishes and selected with G418 at a concentration of 600 μg/ml for 2 weeks. Stable transfectants were isolated and grown in DMEM containing 300 μg/ml of G418. The cells stably expressing HA-α2A-AR were confirmed by immunoblotting using HA antibodies and radioligand binding using [3H]-RX821002.HEK293 cell lines stably expressing HA-αA-α2B-AR . Briefly2A-AR per well for 6 h and split into 12-well plates for 20 to 24 h. Depletion of C1orf27 was confirmed by immunoblotting.Two Stealth RNAi duplexes (siRNA) targeting human C1orf27 (targeting sequences are GGGUGCUGUGAAAUGCAGAGCUUAU and CCACAGCAGUAAACCCAAAGUUAAA), as well as negative control med GC duplex, were purchased from Thermo Fisher Scientific, and siRNA-mediated depletion was carried out as described previously . Briefly2A-AR, cells were cultured on 12-well dishes and transfected with C1orf27 KO plasmids plus α2A-AR-RFP (0.5 μg each) for 30 h before imaging. As control and C1orf27 KO plasmids carry GFP, transfected cells were defined by the GFP signal.The CRISPR-Cas9 C1orf27 KO plasmids targeting human C1orf27, as well as control plasmids, were purchased from Santa Cruz Biotechnology, and the experiments were essentially carried out as described previously . The C1o2A-AR transport in live cells, images were captured using LAS X software at an interval of 1 min with a 63x objective on a Leica Stellaris five confocal microscope equipped with an Okolab UNO stage top incubator. The cells with low receptor expression without aggregation were chosen to be studied. Receptor expression at the Golgi and the whole cell were quantified by measuring the fluorescence intensities. The Golgi area of individual cells was defined by the region with highly concentrated receptors after biotin induction. To measure the effect of C1orf27 depletion on the subcellular localization and ER export to the Golgi and the cell surface of α2A-AR in fixed cells, the cells were fixed with 4% paraformaldehyde for 15 min. To measure the surface expression of HA-α2A-AR in stable cell lines, the cells were fixed and blocked with 0.24% normal donkey serum for 1 h. The cells were then stained with HA antibodies overnight and Alexa Fluor 594-conjugated secondary antibodies for 1 h.To measure the effect of C1orf27 depletion on α2A-AR with or without cotransfection with siRNA. After washing twice with Dulbecco′s phosphate buffered saline and addition of 1 ml of DMEM (no phenol red) containing 10% FBS, 1 ml of biotin (80 μM), dissolved in no phenol red DMEM with 10% FBS plus cycloheximide  was added to induce receptor export. For fixed cell RUSH assays, cells were seeded on 12-well plates with coverslips overnight and transfected with 500 ng of Str-KDEL_SBP-EGFP-α2A-AR together with siRNA or Str-KDEL_SBP-mCherry-α2A-AR plus CRISPR-Cas9 KO plasmids for 20 h. The cells were incubated with biotin plus CHX for different time periods as indicated in each figure and then fixed. The data were expressed as the ratio of Golgi expression to the total expression. In some experiments (C), the Golgi/total ratio at each time point was subtracted from the ratio at time 0 and then normalized to the highest ratio which was defined as 100%.RUSH assays were essentially carried out as described . For liveriments C, the Go2A-AR-Rluc8 and 750 ng of Venus-kRas or pcDNA3.1 together with control siRNA or individual siRNA targeting C1orf27 with Lipofectamine 2000. The cells were transferred to 6-well plates and cultured for additional 20 to 24 h. The cells transfected with RUSH plasmids were incubated with biotin at 40 μM for 2 h. The cells were harvested and split onto black 96-well plates. After addition of coelenterazine h (5 mM), luminescence was immediately measured using a Mithras LB940 photon-counting plate reader (Berthold Technologies GmbH). The BRET signals were calculated by dividing the emission intensity at 520 to 545 nm by the emission intensity at 475 to 495 nm.The live cell–based BRET assays were used to measure surface receptor expression in HEK293 cells as described previously , 31. Bri2A-AR together with control or C1orf27 siRNA and split into 24-well plates. After 24 h, the cells were fixed with 4% paraformaldehyde for 20 min, washed three times with cold Tris-buffer saline, blocked with 1% bovine serum albumin for 1 h, and incubated with horse radish peroxidase–conjugated HA antibodies at a dilution of 1:5000 for 1 h. After washing for three times, the cells were incubated with 200 μl of 1-Step Ultra TMB-ELISA substrate solution for 15 min. 100 μl solution from each well was transferred into 96-well plates, and 100 μl of H2SO4 (1 M) was added to stop the reaction. The absorbance at 450 nm was measured in a SpetraMax M2 microplate reader (Molecular Device).HEK293 cells cultured on 6-well plates were transfected with 1 μg of HA-α2A-AR. After 24 h, the cells were starved for 12 h before stimulation with UK14304 at 1 μM for 5 min. Stimulation was terminated by addition of 100 μl SDS gel loading buffer. ERK1/2 activation was determined by measuring their phosphorylation by immunoblotting as described previously (Cells were cultured on 12-well plates and transfected with αeviously .2A-AR together with GFP-C1orf27 constructs (10 μg each) for 24 h. The cells were harvested and lysed with 500 μl of lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1% protease inhibitors for 1 h. After centrifugation, the supernatants were incubated with 2 μg of HA antibodies  overnight at 4 °C, followed by incubation with 30 μl of protein G dynabeads for 1 h at 4 °C. The beads were collected and washed three times with lysis buffer. Immunoprecipitated proteins were solubilized with SDS gel loading buffer and detected by immunoblotting.Co-immunoprecipitation assays were carried out as described previously . BrieflyGST fusion protein pulldown assays were carried out using the MagneGST pull-down system (Promega) as described essentially , 34. Brip < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.Statistical differences were analyzed by using one way ANOVA. All the data were expressed as mean ± SE. Significance levels are ∗guwu@augusta.edu)All data presented are available upon request from Guangyu Wu (This article contains The authors declare that they have no conflict of interest with the contents of this article.


Androgen Receptor (AR) gene, resulting in expanded glutamine tract in the AR protein. Polyglutamine (polyQ) expansion renders the mutant AR protein toxic, resulting in the formation of mutant protein aggregates and cell death. This classifies SBMA as one of the nine known polyQ diseases. Like other polyQ disorders, the expansion of the polyQ tract in the AR protein is the main genetic cause of the disease; however, multiple other mechanisms besides the polyQ tract expansion also contribute to the SBMA disease pathophysiology. Posttranslational modifications (PTMs), including phosphorylation, acetylation, methylation, ubiquitination, and SUMOylation are a category of mechanisms by which the functionality of AR has been found to be significantly modulated and can alter the neurotoxicity of SBMA. This review summarizes the different PTMs and their effects in regulating the AR function and discusses their pathogenic or protective roles in context of SBMA. This review also includes the therapeutic approaches that target the PTMs of AR in an effort to reduce the mutant AR-mediated toxicity in SBMA.Spinal and Bulbar Muscular Atrophy (SBMA) is an X-linked adult-onset progressive neuromuscular disease that affects the spinal and bulbar motor neurons and skeletal muscles. SBMA is caused by expansion of polymorphic CAG trinucleotide repeats in the  These forms of modifications are required to regulate proteome homeostasis, biological pathways, or other cellular functions , allow the genome to drastically increase its ability to produce proteins that can meet the demand of life. Both these mechanisms allow the cell to produce multiple different functional proteins from a single gene. Alternative splicing accomplishes this through modification of pre-mRNA constructs, producing differently spliced mRNAs that are then translated to different mature proteins. In contrast, PTMs are chemical modifications that are added to the mature proteins to change or modify their function. PTMs involve addition of acetyl, methyl, phosphate groups, or ubiquitin molecules unctions . Dysreguunctions , includiunctions . PolyQ dunctions . This grunctions .Androgen Receptor (AR) gene  gene   gene . The loche males . The heas of age  with a ss of age . The diss of age .in vivo and in vitro models have been developed that recapitulate many aspects of SBMA phenotypes  models have also been developed and used to study the SBMA pathology  in humans (The n humans . Its seqn humans . AR is an humans . Structun humans . The amin humans . The DNAn humans . A flexin humans . The nucn humans . The nucn humans .In the absence of ligands such as testosterone or dihydrotestosterone (DHT), AR exists as a complex with the heat-shock proteins (HSPs), such as HSP90 and HSP70, within the cytoplasm. However, when the ligand is present, AR protein binds the ligand, resulting in a conformational change in the AR structure that promotes dissociation from HSPs and allows it to translocate into the nucleus . Within Posttranslational modifications are biochemical modifications that add or remove specific chemical groups, such as acetyl, methyl, phosphate, or ubiquitin, to specific proteins that change the properties of those target proteins. PTMs help expand the potential of a protein to carry out cellular functions beyond its functional capacity . Aberranin vivo and in vitro models - and carboxyl (C)-terminal interdomain (N/C) interaction is known as one of the potential mechanisms that contributes to SBMA pathogenesis . The potin vitro  (Drosophila homolog of NLK (nmo in flies) reversed the severe retinal degeneration phenotypes in the trAR112Q SBMA fly model (A subsequent study found that AR S81 phosphorylation contributes to SBMA phenotypes . Nemo-lirylation . To checrAR112Q) . Loss ofly model . The trAly model . Taken tly model .in vitro, in a manner that potentially involves inhibition of CDK2  was found to suppress the polyQ-AR induced neurodegeneration phenotypes in the fly eye  in the wild-type AR, resulted in delayed nuclear localization , a SUMO-1 activating enzyme, was found to worsen the polyQ-AR mediated degeneration in SBMA fly model  bind to the target proteins and play crucial roles in multiple cellular functions, including neuronal development and differentiation , DNA damly model ; howeveractivity , indicat(in vivo . In this(in vivo . In thes(in vivo . Taken tCurrently, there are no known cures for SBMA. Available treatment strategies provide only limited improvement, underscoring the need for development of more effective therapeutic options for the patients. There have been several approaches used to modify SBMA pathogenesis, including lowering the levels of androgens such as DHT , modulatin vitro and in vivo (in vitro models of SBMA, treatment of forskolin reduced phosphorylation of polyQ-AR at S96 and decreased polyQ-AR aggregation (The role of phosphorylation as PTM in modulating the polyQ-AR mediated toxicity has shown great potential. A study reported that CDK2 phosphorylates polyQ-AR at S96 site . This CD in vivo . Similar in vivo . In in vregation . Taken tin vitro studies, IGF-1 was found to enhance clearance of AR aggregates through the ubiquitin-proteasome system in a mechanism mediated by phosphorylation of AR by Akt (in vivo (Previous studies have clearly shown the toxic effect of polyQ-AR in skeletal muscles in SBMA . Thus, tR by Akt . Overexp(in vivo . Similar(in vivo . Taking (in vivo .in vivo and in vitro models. An in-depth understanding of the exact roles of PTMs on AR in SBMA context may help in developing more efficient and reliable therapeutics for the SBMA patients in the future.The results obtained from the clinical trials further raised questions on whether targeting the PTMs can be utilized in potential therapeutics to treat SBMA. The safety and efficiency of therapeutics utilizing this mechanism and their performance in the clinical trials are still major concerns. Thus, it is important that the therapeutic candidates are extensively tested and validated for their therapeutic potential in in vivo and in vitro models that accurately model all aspects of the disease may also aid in more effective translation of findings to therapeutic options for the patients. Gaining detailed insights in these aspects may help discern the gaps in terms of regulatory mechanisms that are involved in SBMA pathogenesis and may aid in the detection of novel biomarkers, drug candidates, or effective and reliable therapeutics to treat SBMA.Since the genetic underpinnings of SBMA were identified over 30 years ago, our understanding of the molecular and cellular mechanisms underlying SBMA pathogenesis has advanced substantially. Multiple mechanisms have been implicated in SBMA pathogenesis, including structural alterations in AR, transcriptional dysregulation, altered protein-protein interactions, and formation of toxic polyQ-AR fragments and AR aggregation. PTMs of AR have been of particular research interest, as dysfunction of PTM regulation is implicated in AR dysfunction and subsequent SBMA pathology, and manipulation of specific AR PTM sites have shown potential in modulating SBMA pathogenesis as effective therapeutic strategies. Although our understanding of diverse PTM sites of AR, as well as the functional impact of manipulating these sites, has advanced substantially, the translation of these findings to viable therapeutic options for SBMA patients has achieved limited success only. Absence of effective methods to detect the novel and precise PTM sites may serve as a major limitation factor in this aspect. The advancements in genomic and proteomic techniques may help in identification and use of more accurate methods that may facilitate detection of novel and accurate PTM sites that are difficult to access and have yet remained to be discovered. Besides the PTMs discussed in this review, the roles of additional PTMs in AR biology and SBMA disease are the potential areas that may provide more insights in disease pathogenesis and thus needs to be explored. It is also imperative to understand the effect of interactions of PTMs at protein level in regulating AR function to understand the complexity of the disease better. Furthermore, the development of NG and JL developed the concept. NG, LN, and VO were involved in writing, reviewing, and editing the manuscript. FH and KL were involved in preparation of figures, tables, and editing the manuscript. JL was involved in reviewing, editing, and providing comments on the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Prior research shows that in a particular science domain, students’ identity depends on their self-efficacy, perceived recognition by others, and their interest in that domain. In this study, we investigated how the end of the semester physics self-efficacy and perceived recognition by others for bioscience majors enrolled in the second semester of a traditionally taught mandatory physics course sequence predict their overall science identity aligned with their disciplinary major. We find that bioscience majors’ physics self-efficacy and perceived recognition not only predict their physics identity but also their overall science identity. These relations between physics self-efficacy and perceived recognition and the overall science identity of bioscience majors suggest interdisciplinary connections that may provide additional pathways for boosting students’ science identity, e.g., by enhancing their self-efficacy and perceived recognition in their other mandatory courses such as physics. We also find that on average, women majoring in bioscience had lower physics self-efficacy, perceived recognition, physics identity, and overall science identity than men even though women were not underrepresented in the physics course. One possible reason is that the societal stereotypes and biases pertaining to who can excel in physics can impact women who are constantly exposed to them throughout their life. Students’ science, technology, engineering, and mathematics (STEM) related motivational beliefs, e.g., self-efficacy and identity, have implications for their short and long-term outcomes in those disciplines regardless of their performance. Identity frameworks in STEM disciplines have been used to explore undergraduate students’ participation in college classes and career outcomes –8. For eScience identity in this context refers to identifying with science or a student’s view about whether they see themselves as a “science person” , 15. TheSelf-efficacy (closely related to competency belief) is defined as a person’s motivational belief that they can succeed in a particular activity or course , 22. It Another motivational belief hypothesized to influence identity in a particular discipline, interest, may also affect students’ perseverance, persistence, and achievement –36. One The third factor influencing identity, perceived recognition by others, has been shown to be an important factor in motivation . In one All three of these factors  play an important role in identity formation. However, identity can be context dependent  and thusHere we use the framework in which self-efficacy, interest, and perceived recognition play a central role in identity formation. RQ1 Are there gender differences in students’ motivational beliefs ?RQ2 To what extent can the components of student physics identity  predict students’ overall science identity?RQ3 What is the covariance between the physics identity and overall science identity?The study was conducted at a large, public research university. We analyzed the results from responses to motivational beliefs survey administered to 873 students who took the survey at the end of the semester in an introductory algebra-based physics 2 course over two years. This research was carried out at the University of Pittsburgh in accordance with the principles outlined in the institutional review board (IRB) ethical policy, which approved the study. All interviewed participants provided oral consent for this research, which was documented orally and which was approved by the IRB. This class is taken by bioscience majors for whom both physics courses in this sequence are mandatory and is usually taken in their junior or senior year of undergraduate studies. Both physics courses in this sequence were traditionally taught with lectures as the primary mode of instruction with a few clicker questions per week. The university provided demographic information such as age, gender, and ethnic/racial information using an honest broker process by which the research team received the information without knowledge of the identities of the participants. The gender data provided by the university includes only binary options of “male” and “female” for students. We recognize that gender is a socio-cultural and nonbinary construct; however, we are limited to the binary data provided by the university in this study. Based on the university data, 38% of the participants identified as male and 62% as female students.physics self-efficacy questions measured students’ confidence in their ability to solve and understand physics problems [interest in physics questions measured students’ enthusiasm and curiosity to learn physics and ideas related to physics [perceived recognition questions measured the extent to which the students thought that other people see them as a physics person [science identity question evaluated whether the students see themselves as a science person. The physics identity questions evaluated whether the students see themselves as a physics person [The motivational belief survey instrument used in this study is a validated instrument. This study focused on students’ physics self-efficacy, interest, perceived recognition, and identity as well as their overall science identity for the students enrolled in the physics 2 course using the validated survey. The problems –47. The  physics . The ints person . The scis person . The phys person , 48, 49.s person .The survey was adapted from previous research , 51 and r values signify the strength of the relationship between variables. The inter-correlations vary in the strength of their correlation, but none of the correlations are so high that the constructs cannot be separately examined. The highest intercorrelation was the value between Physics Identity and Perceived Recognition (0.82). Perceived recognition questions ask about external identity , whereas the physics identity question asks about internal identity  so past research has found a high correlation between these constructs [The pair-wise Pearson correlations are given in nstructs , 20, 54.nstructs . Furthernstructs .t-tests and for the effect size using Cohen’s d [d is d = (mμ−fμ)/pooledσ, where mμ is the average score of male students, fμ is the average score of female students, and pooledσ is the pooled standard deviation for all students. To quantify the statistical significance and relative strength of our framework’s links  and outcomes  for statistical significance using ohen’s d . Cohen’sinks see , we usedThe model estimates were performed using moderation analysis to check whether any of the relations between variables show differences across gender by using “lavaan” to conduct multi-group SEM  InitiallRQ1, women had statistically significantly lower mean values than men for all constructs in our model, including science identity  to see if the relationships between the motivational beliefs were different across gender. There were no group differences at the level of weak and strong measurement invariance including no difference at the level of regression coefficients. Therefore, we proceeded to gender mediation analysis to understand how gender mediates physics identity at the end of the yearlong introductory physics sequence.RQ2, we find that students’ science identity is predicted by physics self-efficacy and perceived recognition with student self-efficacy in physics having the largest direct effect on science identity (0.20). For bioscience majors, this physics 2 course is the second of the two physics courses they take, usually in their junior or senior year. In addition, gender is directly connected to perceived recognition (P.R.), self-efficacy, and interest. The relations between gender and both science identity and physics identity are mediated by the intervening variables and gender does not directly predict either science identity or physics identity. Thus, women have a lower physics and science identity because they have lower self-efficacy, perceived recognition, and interest. In response to RQ3, we investigated the covariance (dashed lines) between science and physics identity and find that science identity and physics identity covary with one another (0.21). Thus, while students’ overall science identity and physics identity are not the same construct, they could influence one another in meaningful ways.The results of the SEM are presented visually in In physics courses that bioscience majors are required to take for their major, women have lower mean values in their motivational beliefs than men . It is iIn our model, we found that some of the motivational beliefs that predict physics identity also predict science identity at the end of the course. The identity model in We also find covariance (0.21) between the overall science identity and physics identity which may be useful for devising strategies for boosting students’ science identity and career aspirations. Since physics 2 is usually taken in the students’ junior or senior year, it is one of the last non-major STEM courses they take before they graduate. Therefore, physics instructors and TAs may have the opportunity to improve students’ science identity by recognizing and affirming their students positively for making progress. Furthermore, at the end of a traditionally taught two-semester mandatory physics course sequence, we find that on average, women majoring in bioscience had lower physics self-efficacy, perceived recognition, physics identity, and overall science identity aligned with their disciplinary major than men even though women were not underrepresented in the physics course. One possible reason is that the societal stereotypes and biases pertaining to who can excel in physics can impact women who are exposed to these stereotypes and biases from an early age through differential upbringing, media coverage, K-12 education, and the culture of college physics classes. Moreover, although both men and women had an average perceived recognition below the positive lower threshold (score of 3 corresponding to “agree”), women’s averages were lower than men’s. The fact that women feel less recognized by their instructors and TAs than men could influence women’s self-efficacy, interest, and identity in physics in particular and science in general.Physics instructors may unwittingly reinforce gender stereotypes about physics and communicate lower expectations for female students in physics classes. By not letting men dominate the conversations in class and explicitly praising effort and affirming women when they do well/achieve in the class, the gender gap in students’ perceived recognition could be decreased. This is especially important when implementing active learning pedagogy in the classroom. If not implemented using teaching strategies that are equitable and inclusive, the gender gap in the courses may increase . In prio