The augmented reality system was tested at ALTEC facilities in Turin, Italy, where 39 participants were performing an actual real astronaut's procedure, the installation of Temporary Stowage Rack (TSR) on a physical mock-up of an ISS module. User experience evaluation was assessed using comprehensive questionnaires, and interviews, gathering an in-depth feedback on their experience with a platform. This focused on technology acceptance, system usability, smart glasses user satisfaction, user interaction satisfaction, and interviews, gathering an in-depth feedback on their experience with a platform. The analysis of the questionnaires and interviews showed that the scores obtained for user experience, usability, user satisfaction, and technology acceptance were near the desired average. Specifically, The System Usability Scale (SUS) score was 68 indicating that the system usability is already nearly acceptable in the augmented reality platform.This paper introduces Augmented Reality (AR) system to support an astronaut's manual work, it has been developed in two phases. The first phase was developed in Europeans Space Agency's (ESA) project called “EdcAR—Augmented Reality for Assembly, Integration, Testing and Verification, and Operations” and the second phase was developed and evaluated within the Horizon 2020 project “WEKIT—Wearable Experience for Knowledge Intensive Training.” The main aim is to create an AR based technological platform for high knowledge manual work support, in the aerospace industry with reasonable user experience. The AR system was designed for the Microsoft HoloLens mixed reality platform, and it was implemented based on a modular architecture. The purpose of the evaluation of the AR system is to prove that reasonable user experience of augmented reality can reduce performance errors while executing a procedure, increase memorability, and improve cost, and time efficiency of the training. The main purpose of the first phase evaluation was to observe and get feedback from the AR system, from user experience point-of-view for the future development. The use case was a filter change in International Space Station (ISS)—Columbus mock-up in the ESA's European Astronaut Centre (EAC). The test group of 14 subjects it included an experienced astronaut, EAC trainers, other EAC personnel, and a student group. The second phase the experiment consisted of an  This paper introduces AR system to support an astronaut's manual work. The work presented in this paper falls under two individual projects, which have a direct continuum. The first phase was developed in Europeans Space Agency's project called “EdcAR—Augmented Reality for Assembly, Integration, Testing and Verification, and Operations” Helin,  and the One promising application for AR guidance and training is in maintenance and assembly; also termed AR instructions  is becoming a central goal and design strategy in the design of digital artifacts and services. The main challenges related to UX can be divided to (1) designing a user experience that is pleasurable, engaging and stimulating, and appropriate in the user's context, and (2) evaluating the UX and overall acceptability of the applications Olsson, . The ISOThe AR-system is based on the Microsoft HoloLens mixed reality platform and IEEE Draft Standard for an Augmented Reality Learning Experience Mode. The whole system is configured around the Activity and Workplace JSON files. The Workplace JSON describes workplace-related information such as points of interest, sensors, etc. It is parsed with the Workplace manager and information is transferred to the data layer. The Activity JSON describes all action steps and the content that should be active for each step. It is parsed with the Activity manager and information is transferred to the AR layer via local storage. The current version can annotate:UI in 3D space Figure Warning symbols Figure Location based warnings Figure Symbols Figure 3D models Figure 3D animations Figure Video annotations Figure Audio annotations Figure The user can interact with the AR player via a multi-modal user interface  Replacement procedure in International Space Station  supports troubleshooting activities by interacting with the crew over voice and video loops (or watching images of the working area taken by the AR glasses) and by showing directly on the hardware via AR, the parts and the operations to be performedThe crew follows the instructions provided by COL-CC through voice and video loops, and the AR systemOnce the problem is fixed, the crew can continue following the procedure instructions given by AR glassesThe installation of the TSR is a real procedure that the astronauts had to perform on the ISS in the ATV modules. All the components and mock-ups are available at ALTEC facility as well as the complete procedure The operator executes the procedure following the instructions displayed in the Microsoft HoloLens:Localization of the seat track studs, brackets and ball bearings location through basic augmentation Installation of the studs in the correct position following short video and audio that explains how to use themOrientation of the TSR in the correct position supported by imagesConnection of the straps to the interfaces following short video and audio that explains how to fix the strapsOnce the installation is complete, the operator tightens the straps to fix the TSR to the rackThe operator check that the installation has been completed successfully comparing the final result with a 3D model that is over imposed on the working area.The first evaluation (EdcAR) of the AR-system has been performed in the ISS-Columbus training mock-up located in the ESA's European Astronaut Centre in Cologne, Germany. The test group included 14 subjects, an experienced astronaut, EAC trainers, other EAC personnel, and a student group. During the test of the AR-system all test subjects were able to test the Microsoft HoloLens based system. Figure The second evaluation of AR-system was tested at ALTEC facility  performing a real astronaut procedure on a physical mock-up of an ISS module , the development team evaluated the user experience and usability by observing the user test. The observations were written down while the users were testing the system, based on the comments, and performances of the users. The idea of the evaluation was to allow the users comment freely their experience, without predetermined questions or framework, and thus let the users define what is significant for them. The approach was applicable also because of the small amount of test users. After the test, the development team summarized the written observations and drew the conclusions.For the second test , a set of questionnaires was used to collect feedback from the participants and to enable the evaluation process after the trial. Of the original set of seven separate questionnaires, the results from three UX-related are presented in this paper. The questionnaires include System Usability Scale (SUS), Smart Glasses User Satisfaction (SGUS) and User Interaction Satisfaction (QUIS).SUS is a tool for measuring both usability and learnability. The SUS scores calculated from individual questionnaires represent the system usability. SUS yields a single number representing a composite measure of the overall usability of the system being studied. Scores for individual items are not meaningful on their own. SUS scores have a range of 0 to 100 Brooke, , 2013. ASGUS questionnaire was created for the WEKIT trials. SGUS measures subjective satisfaction focusing especially on test participants' experiences on the features that support learning. SGUS is based on the evaluation criteria for web-based learning by Ssemugabi and de Villiers  and statQUIS measures subjective satisfaction with specific aspects of the interface, including usability and user experience  participants were interviewed during the space case trial. Two (2) of the interviewees were experts and four (4) were students. The participants to be interviewed were chosen randomly according to the availability of the participants and to avoid wasting their time . The interviews consisted of four main questions or themes: (1) Please use 2–3 words (adjectives) to describe your experience with AR glasses and the player during the execution of the task; (2) Please describe freely your experience on using the player during the execution of the task; (3) How well did you manage to complete the task with the player?; (4) Please tell the good and bad sides of AR glasses (HoloLens). Two to three positive and negative aspects.This study was carried out in accordance with the recommendations of Norwegian Centre for Research Data and VTT's Code of Conduct, which includes Ethical guidelines. All subjects gave written informed consent in accordance with the Declaration of Helsinki.Based on the observation and expert evaluation of the ISS-Columbus mockups use-case, the HoloLens based AR-system is usable for basic daily use as its usability has reached a reasonable level. The 3D space user interface and annotations were working properly without any delay and the image quality was high enough for see-through glasses. The main downside of the system is the narrow field of view, the user has to move his/her head around and search for the information in some cases. Test subjects also highlight the potential of applying AR technology to this field, but AR-systems UI should be update to support Operations Data File (ODF) format which is common in the space domain.The System Usability Scale (SUS) questionnaire showed that the SUS scores were very close to 70 , which is considered as the minimum acceptable score for system usability. Thus, the WEKIT applications were considered nearly acceptable in terms of system usability. There is a clear difference between student and expert groups in deviation, indicating that there was a greater variation in the experience of system usability in student group. The lowest SUS score in a student group was 23 and the highest was 95  questionnaire results, the participants experienced that the system and content helped them to accomplish the task quite well (GL7) and their attention was captivated in a positive way (GL6). The provided content was also contextually meaningful (GL2). However, using the AR glasses was experienced as less natural , and following and understanding the task phases  not very easy. The overall average  was satisfactory on a scale 1–7 (see Table Based on ingful GL. HoweverUser Interaction Satisfaction (QUIS) showed that the scores were close to the average score for most of the questions. Taking a closer look at scores above or below the average, it was possible to assess that the current (HoloLens) version of AR is not yet considered fully reliable by the users, where the characters on display were found a bit hard to read and the organization of the information on the display was a bit too confusing and inconsistent. These results were observed in particular in the answers of the students.The analysis of the Questionnaire for On the other hand, the experience with AR glasses for performing that kind of task was considered good for both the experts, and the students, that found it easier to learn how to operate on such a task being guided by AR instructions (see Table N = 6), participants' overall experience on the system, the experienced pros and cons of the Microsoft HoloLens and improvement suggestions for UX are presented in this section.As the results of the interviews  was 14 and in the second case (WEKIT) 39. According to the recent review of AR usability studies , both the pragmatic and emotional aspects of the user experience were considered fulfilling. It can be suggested that the AR-system is potentially a useful tool for supporting and facilitating the assembly and training procedure in the space field, even though the tool is still in prototyping phase and it will be updated after the next trials in WEKIT project.There are however many usability issues still to be resolved. For instance, the narrow field-of-view is one the most significant issues.On the other hand, the experienced problems in perceiving the work area and finding or noticing AR objects could be solved by enhancing visual guidance , providing clear orientation to the task and by providing instructive materials of the area From the technical point-of-view, the main indications to operate the AR-system inside the ISS-Columbus module on orbit were investigated and aspects related to the system integration were considered: (1) in order to operate the proper selection of the COTS items requesting only minor changes for operations in the ISS, (2) in order to highlight possible criticisms and critical technologies to be further investigated in the prototype design, assembly and testing phases, and (3) in order to address the design of the flight concept.The developed system has been already tested and evaluated in two other domains: bioimaging and aircraft maintenance. These tests were completed together with the EU-WEKIT project. Microsoft HoloLens AR-system worked properly and its usability has a reasonable level for performing the required task. Over 40 persons from another domain evaluated the AR-system. This shows that current system is also suitable for other domains.This research has been following VTT's Code of Conduct, which includes Ethical guidelines. VTT's ethical committee: Matti Karhunen (chair), Seppo Viinikainen, Reetta Grenman and Richard Fagerström.KH: AR system description, EdcAR case and evaluation, conclusion; TK: Evaluation methods and results, conclusion; CV: ALTEC case description; JK: AR system description; AV: Evaluation methods and results.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. The reviewer JV and handling Editor declared their shared affiliation.


Caries in the elderly has been associated with dependence, oral-health status and -care practices. This cross-sectional study aimed to investigate the association between root/coronal caries and individual factors among institutionalised elderly people in Bogotá, Colombia, using the International Caries Detection and Assessment System severity and activity criteria (ICDAS).A total of 226 institutionalised elderly were clinically examined for root and coronal caries in 40 institutions. Caries risk was assessed with Cariogram, and demographics, oral health knowledge and practices, oral health-related quality of life and denture-use habits using a questionnaire.Participants  presented a mean number of 10.8 ± 7.3 teeth and 19.4 ± 18.8 exposed root surfaces. Prevalence of Coronal-ICDAS caries experience (C-ICDAS DF) was of 100% and of 54.4% for C-ICDAS D; mean number of C-ICDAS DFS was 16.76 ± 27.36, with 50.9% of subjects having ≥ one active C-ICDAS DS. Prevalence of Root Caries Index was of 49.1% and of R-ICDAS DF of 46%; mean number of R-ICDAS DFS was 2.03 ± 2.78, with 40.3% of subjects having ≥ one active R-ICDAS DS. Most individuals had a systemic condition (94.2%) and required oral-hygiene assistance (58%). Logistic regression analyses showed significant associations (p < 0.05): for coronal active caries when having over six teeth (OR: 2.7), and for root caries, when having coronal caries (OR: 2.41), being a man (OR: 1.95), and having over 14 teeth (OR: 0.30). Those presenting with > eight exposed root surfaces were 4.04 more likely to have root caries and 2.4 times more likely to have active root caries.In the institutionalised elderly population in Bogotá significant associations were found, both for the presence as for the activity status of root and coronal caries, with individual clinical factors including coronal caries, exposed root surfaces and number of teeth.The online version contains supplementary material available at 10.1186/s12903-021-01520-4. There is a rapid increase in life expectancy throughout the world. The global population aged 60+ years was 962 million in 2017 and is expected to double by 2050, when it is projected to reach nearly 2.1 billion. The proportion of older adults in Colombia is expected to increase from 11.6 to 27.5% during the same period [Geriatric institutions are a more frequently residence option for the elderly and living in these institutions has been described as a factor associated with a high presence of caries , 4. HighRoot Caries Index (RCI) is one of the most frequently used epidemiological indices to measure the root caries experience at the tooth/surface level , 19. ThiFor coronal caries, the conventional DMF index has been the most widely used . This inThe International Caries Detection and Assessment System (ICDAS) is a detection and assessment system classifying stages of the caries process based on histological extent and activity (when included). Its caries criteria include besides extensive-stage caries lesions (cavitated deep dentine lesions), moderate- and initial-caries lesions . The Conventional DMF index considers as its D component the extensive  caries lesions. Thus ICDAS can be used both to compare with other studies using DMF and in addition can separately incorporate the initial caries lesions to have a wider and more current epidemiological caries profile description . ICDAS cThe identification of factors associated with root/coronal caries in institutionalised elderly using more sensitive caries criteria and considering the caries lesions’ progression status contributes to guide proposals focused on reducing the caries burden in this population. Thus, the aim of this cross-sectional study was to investigate the association between root/coronal caries and individual factors among institutionalised elderly people in Bogotá, Colombia, using the ICDAS severity and activity criteria.This cross-sectional study counted with IRB (012-2016) and followed the STROBE guidelines.Based on the number of registered geriatric institutions n = 152) , the est , the esThe inclusion criteria were adults aged 60+ years from institutions who did not present reported terminal illness or severe mental impairment in the medical records, nor edentulism, and who agreed to participate by providing a personal/legal representative signed informed consent forms. The exclusion criterion was mouth opening limitation at the time of the clinical examination.In November 2016, we received from the District Secretary of Social Integration the list of geriatric institutions in the city of Bogota, indicating 152 registered geriatric institutions . From NoThree examiners who had been previously clinically calibrated for the coronal ICDAS caries visual severity and activity criteria by an ICDAS core examiner (SM)  underwent a theoretical and preclinical training and a clinical calibration with natural teeth and elderly patients for root ICDAS caries visual severity and activity criteria by an expert consensus group , previously discussing and solving difficult cases with the ICDAS core group .epi-merged assessment of caries lesions (without air drying) included: C-Sound, C-Initial (C-ICDAS 1–2), C-Moderate (C-ICDAS 3–4) and C-Extensive (C-ICDAS 5–6); activity assessment based on: the severity of caries (non-cavitated vs. cavitated), the colour and opacity of the tooth, the tactile sensation and if located in a plaque stagnation area [After assisted tooth brushing, clinical examinations were conducted in the geriatric institution facilities during the morning hours, assessing the subjects in their beds, wheelchairs, or communal sitting areas, using headlamps, mouth mirrors, tweezers, ball-ended probes, and cotton rolls. The tooth surfaces were dried with cotton rolls for the examination. Data regarding clinical factors, including use of partial/total dentures; the number of teeth; number of exposed root surfaces, thick plaque, hyposalivation, fillings and caries lesions  were collected. The coronal (C) ICDASion area , 24. Theion area , 24, 25.For the coronal (C) conventional (Co) caries experience, the prevalence/mean number of decayed (D) and filled (F) surfaces (S) (C-Co DFS) was used, where D corresponded to the C-ICDAS-merged-Moderate-Extensive caries lesions; the C-ICDAS-Initial caries lesions were included in the corresponding C-ICDAS caries experience (C-ICDAS DFS).For the root (R) conventional (Co) caries experience , the RCI was used, which included for its calculation both D (cavitated caries lesions) and F. R-ICDAS-Initial caries lesions were included in the corresponding R-ICDAS caries experience (R-ICDAS DFS).An interview-led questionnaire on individual factors containing 18 items based on validated tools or international guidelines , 25–30 wThe individual caries risk was calculated using the Cariogram software , 29 usinThe categorical variables are expressed in terms of the distribution of absolute and relative frequencies and the continuous variables as measures of central tendency (mean and standard deviations). The subjects’ cut off points for both ICDAS coronal and ICDAS root caries were: those without presence and those with presence of ICDAS caries lesions, and those without presence and those with presence of ICDAS active caries lesions. The associations between the individual factors investigated and the outcome measures: presence of ≥ 1 root caries lesion; ≥ 1 active root caries lesion; ≥ 1 coronal caries lesion, and ≥ 1 active coronal caries lesion, were analyzed using the chi-square test and Fisher's exact test.In the absence of previously established intrinsic categories the variables "number of teeth in the mouth" and "number of exposed root surfaces" were analyzed using the approach to data analysis by tertiles exposure , 32.The first step of the analyses was crude logistic regression analysis. Subsequently, non-conditional multivariate logistic regression models were used to estimate the odds ratio and 95% confidence intervals (CI) between the individual variables investigated and the presence of coronal ICDAS/root ICDAS caries lesions, and active coronal ICDAS/active root ICDAS caries lesions, respectively. Furthermore, the relative effect of each individual factor on caries risk was adjusted for age, sex, systemic disease, daily sugar-free frequency intake, thick dental plaque, dental floss use, fluoridated toothpaste use, dental-pain/discomfort medication/food-type change during the last 3 months.Final multivariate models were created through stepwise elimination of variables of interest from bivariate analysis. Main independent variables and the covariates were incorporated in the multivariate logistic regression model, if they had a significant (p < 0.05) or marginal association p < 0.20) with the outcome measures, while variables considered in the literature or contextually as clinically and biologically relevant were retained. These included age to compare at a national level with corresponding ages 0 with th, as wellOut of 152 geriatric institutions in Bogotá, 52 were not reachable after sending post mail and at least three phone calls, and 60 did not agree to participate in the study. In total, 40 geriatric institutions located in five of the 20 municipalities in Bogotá accepted the invitation (26.3%). From a total of 468 elderly geriatric-institution residents, 242 were not included (51.7%): 15 did not agree to participate; 37 were not within the age range; 15 were terminally ill, and 174 were edentulous. The remaining 226 agreed to participate in the study providing signed consent forms and none were excluded at the clinical examination, for a total examined sample of n = 226. The mean age of the subjects was 80.1 ± 9.3 years; 63.7% were women.The subjects presented with a mean number of 10.8 ± 7.3 teeth  and a population distribution of 1–6 teeth (35.4%), 7–14 teeth (30.1%) and 15–28 teeth (96.3%). The mean number of exposed root surfaces was 19.4 ± 18.8  with a population distribution as follows: 0–8 exposed surfaces (36.7%), 9–21 exposed surfaces (29.3%) and 22–96 exposed surfaces (34.9%). The majority  had partial tooth loss (67.7% in both jaws and 31% only in the lower jaw); 30.5% (n = 69) of the subjects wore removable partial dentures . A total of 80 subjects (35.4%) were edentulous only in one jaw (28.8% upper jaw and 6.6% lower jaw), whereas 25.2% of them wore complete dentures (upper: 22.1% and lower: 3.1%).The prevalence of C-Co DF was 83.6% and that of C-Co D was 46.5%, which increased to 54.4% with the inclusion of Initial C-ICDAS D. The mean number of C-ICDAS-DFS was 16.76 ± 27.36 . Around half of the subjects (50.9%) showed at least one coronal ICDAS active caries lesion with a mean number of 4.7 ± 9.6 involved surfaces.Concerning root caries, the prevalence of RCI root caries was 49.1%, with a mean number of 1.09 ± 2.24 involved surfaces. The prevalence of R-ICDAS DF was 46%, and the mean number of R-ICDAS DFS was 2.03 ± 2.78 . Over a third of the subjects (40.3%) showed at least one active root ICDAS caries lesion and a mean number of 1.25 ± 2.44 involved surfaces.The repetition of clinical coronal and root ICDAS caries examinations reported perfect intra-examiners’ agreement of 78.5% for coronal ICDAS and of 91.0% for root ICDAS.The questionnaire showed that most of the participants had a systemic condition (94.2%), had last visited the dentist over a year ago (84.1%) and reported daily free sugar consumption above the recommended level (77.4%). Furthermore, 58% of the subjects required assistance to perform oral hygiene. Among those wearing denture (n = 69), 79.7% used it for < 2 years, 75.4% cleaned it at least once a day, with dentifrice/mouthrinse (97.1%), and 50.7% did not remove it before bedtime. Over a third of the population perceived the need for dental care (35.4%) and reported emergency as the reason for the last dental visit (38.1%). A small number reported the presence of painful sites in the mouth (5.3%) or were diagnosed with hyposalivation (3.1%).p < 0.05).Table p < 0.05) and between having over 8 exposed root surfaces  and root ICDAS active caries lesions.Table In Table Those who had coronal caries lesions and were men, were correspondingly 2.41, and 1.95 times more likely to have root ICDAS caries lesions. Furthermore, those who had over 21 exposed root surfaces were 3.51 times more likely to have root ICDAS caries lesions and 2.39 times more likely to have root ICDAS active caries lesions. Conversely, those who had over 14 teeth were 70% less likely to have root ICDAS caries lesions.In general, most caries occurs in adulthood  and the This cross-sectional study was carried out in 40 geriatric institutions in the city of Bogotá, Colombia.. [. [The institutionalised elderly population presented with a high coronal and root caries experience and high frequency of coronal/root ICDAS active caries lesions, which were associated with individual factors. The reported association between coronal caries and root caries , 13, 34 . [.   and at least one active caries lesion with the variables in this study. A 10 times higher prevalence of active caries lesions (40.3%) was observed in the current study when compared with that from Denmark (3.7%) [In the study conducted by Christensen et al. , they ask (3.7%) , which mk (3.7%) . Anotherk (3.7%) , 6, 40. k (3.7%)  includedk (3.7%) , 38.On average in our study we a higher mean number of remaining teeth (10.8) in comparison to that of 8.0 in the most recent Colombian National Oral Health Study . HoweverThe variable ‘Not cleaning the denture at least once a day’ was the only one that showed any association with root active caries lesions, possibly indicating better self-care. Unlike other studies, we did not find any association between caries and the dependence to deliver oral hygiene , 6, 42. It is important to note that the results of this study have to be interpreted in the light of institutionalised elderly population. The selected sample represents a higher-than-usual social-economic level within the institutionalised elderly population according to their location within the city in 5 out of 20 municipalities . About oAlthough logistic regressions can overestimate the effect size of statistical associations under a cross-sectional design and are considered a limitation of this study, we chose this type of statistical analysis following same design conducted by Christensen et al.  who alsoFinally, the classification of elderly into with and without caries groups could guide oral care activities. The coordinators and caregivers of geriatric institutions, with the support of health entities, could apply measures that help reduce the prevalence and development of new caries lesions in the affected group, while keeping the "no cavities" group healthy. Taking into account the challenges that the rapid increase in life expectancy with higher number of retained teeth pose, as well as the raise of other health and quality of life aspects, there is an increasing need of education within the dental profession in the comprehensive understanding of the pathology within other oral and general health aspects, as well as current individual- and tooth-surface level caries care guidelines , 37, 44.In the institutionalised elderly population in Bogotá significant associations were found, both for the presence as for the activity status of root and coronal caries, with individual clinical factors including coronal caries, exposed root surfaces and number of teeth.Additional file 1: Questionnaire on individual factors.


However, the progression-free survival rate for the CRPC patients on antiandrogen therapies is only 8–19 months. Hence, there is a need to understand the mechanisms underlying CRPC progression and eventual treatment resistance. Here, we have leveraged next-generation sequencing and newly developed analytical methodologies to evaluate the role of AR signaling in regulating the transcriptome of prostate cancer cells. The genomic and pharmacologic stimulation and inhibition of AR activity demonstrates that AR regulates alternative splicing within cancer-relevant genes. Furthermore, by integrating transcriptomic data from in vitro experiments and in prostate cancer patients, we found that a significant number of AR-regulated splicing events are associated with tumor progression. For example, we found evidence for an inadvertent AR-antagonist-mediated switch in IDH1 and PL2G2A isoform expression, which is associated with a decrease in overall survival of patients. Mechanistically, we discovered that the epithelial-specific splicing regulators (ESRP1 and ESRP2), flank many AR-regulated alternatively spliced exons. And, using 2D invasion assays, we show that the inhibition of ESRPs can suppress AR-antagonist-driven tumor invasion. Our work provides evidence for a new mechanism by which AR alters the transcriptome of prostate cancer cells by modulating alternative splicing. As such, our work has important implications for CRPC progression and development of resistance to treatment with bicalutamide and enzalutamide.Androgen receptor (AR), is a transcription factor and a member of a hormone receptor superfamily. AR plays a vital role in the progression of prostate cancer and is a crucial target for therapeutic interventions. While the majority of advanced-stage prostate cancer patients will initially respond to the androgen deprivation, the disease often progresses to castrate-resistant prostate cancer (CRPC). Interestingly, CRPC tumors continue to depend on hyperactive AR signaling and will respond to potent second-line antiandrogen therapies, including bicalutamide (CASODEX Androgen receptor (AR) is a member of the superfamily of hormonal nuclear receptors . In the ®) and enzalutamide (XTANDI®) [Interestingly, studies employing xenograft prostate tumor models have shown that castrate-resistant prostate cancer (CRPC) tumors that emerge after androgen-ablation therapy, continue to express AR and AR-regulated genes . Recent XTANDI®) –28. AlthXTANDI®) . In addiThe recent advent of high-throughput RNA sequencing and splicing microarrays has unveiled new layers of regulation of gene expression and highlighted the extreme complexity and versatility of the transcriptome. The majority of human genes encode multiple transcripts through the use of alternative promoters, alternative splicing (ASE), and alternative polyadenylation . ASE is Herein, we hypothesized that modulation of AR signaling either during prostate cancer progression or in response to treatment with AR antagonists might dysregulate the transcriptome of prostate cancer cells by modulating ASE. We employed a multitude of genomic approaches including Affymetrix splicing array, whole transcriptome RNA-seq analysis, and RT-PCR to show that AR signaling regulates the transcriptome of prostate cancer cells by modulating ASE of a wide array of genes involved in regulating protein function. Furthermore, leveraging publicly available transcriptome data of primary-site samples from patients with prostate cancer at various stages of progression, we found a subset of AR-driven splicing events that are associated with progression of prostate cancer. Mechanistically, we found that epithelial splicing regulator proteins (ESRP1 and ESRP2) are the splicing factor through which AR may regulate splicing of pre-mRNA in prostate cancer cells. Interestingly, the inhibition of ESRPs suppressed AR-antagonist-mediated increase in the invasion of prostate cancer cells. Collectively, we provide the evidence for a novel and critical mechanism of prostate cancer progression that is regulated by AR and that the treatment with AR antagonist may inadvertently promote invasion by dysregulating splicing of critical genes. Our analytical approach is described in Supplementary Fig. To study the effect of pharmacological manipulation of AR signaling in prostate cancer cells, we performed expression profiling of LNCaP cells that were cultured for 72 h in charcoal-stripped fetal bovine serum and stimulated with 10-nM AR agonist, dihydrotestosterone (DHT), or 10 µM of the AR-antagonist casodex for 24 h. The array consisted of >6 million probes, of which 70% covered exons for coding transcripts while the remaining 30% covered exon–exon splice junctions and noncoding transcripts; hence, allowing us to monitor transcriptional changes at the level of the exon. Besides, the well-characterized gene expression changes  was observed when we compared the transcriptome of LNCaP treated with enzalutamide with that of DHT Fig. . Furtherp ≤ 0.05) altered splicing in response to casodex treatment of LNCaP cells. In addition, the rMATS analysis showed that enzalutamide significantly (p ≤ 0.05) induced splicing in 19 out of 100 prostate-cancer-relevant genes. The heatmap comparing expression of differentially expressed HTA probes spanning exon–exon junction of a prostate cancer genes and PSI of the prostate-cancer-relevant genes across samples is shown in Fig. AKT2, AKT3, GSK3β, PIK3R, PIK3CD, RB1, EGFR, PDGF, MAPK3, KRAS, IDH1, and MTOR. IDH1 was one of the RT-PCR-validated gene and is a key metabolic gene regulating TET2-mediated epigenetic re-programing in prostate tumor cells [p = 1.84e − 43) in the TCGA-prostate adenocarcinoma tissue (n = 495) in comparison to the GTEX-normal prostate tissue (n = 100) and is also associated with decreased overall patient survival  overrepresentation analysis. We used all the genes that have an annotation as reference in our analysis as recommended by clusterProfiler. The alternatively spliced genes were enriched in pathways related to modulation of gene expression including nucleic acid, protein transport, mRNA splicing, and proteosomal degradation whereas the differentially expressed genes were enriched in pathway related to EMT including those involved in GTPase activity, cell–cell interaction/junctions, and cytoskeleton organization Fig. . We nextAAK1, SYNE4, and MAN1A1, which were predicted to undergo ASE in response to casodex treatment. We found a robust fivefold decrease in the expression of AR in 22RV1 cells transfected with siRNA in comparison to a scrambled siRNA , SYNE4 (SI: −1.36), and MAN1A1 (SI: −3.22) in the same direction as that of casodex  RNA-seq data to further study the effects of genomic inhibition of AR on ASE in MDA-PCa-2b cells, a model for advanced prostate cancer (advPC) bone metastasis cells that express PSA, AR, and are androgen sensitive. The rMATS analysis revealed that siRNA knockdown of AR in MDA-PCa-2b cells induced 3841 ASE events after a stringent filtration of FDR ≤ 0.05 and delta PSI of ≥10%. Also, similar to our observations with enzalutamide-treated LNCaP cells, we found that the MEE formed the largest fraction of ASE events induced by the siRNA knockdown in MDA-PCa-2b and were followed by CE and IR events Fig. . In addi AR Fig. . Similar AR Fig. . In addi AR Fig. . Overallp value = 2.96e − 08) were deregulated by treatment with casodex in comparison to DHT  are key proteins that bind to mRNA or noncoding RNAs and play a wide variety of roles in posttranscriptional processing including regulation of ASE. Therefore, we hypothesized that RBPs, which are differentially expressed in response to modulation of AR-axis, might provide mechanistic insight into the observed regulation of ASE. To test our hypothesis, we curated a list of 112 RBPs from the published literature  with a kESRPs are reported to regulate splicing in a position-dependent manner. Binding of ESRPs in the downstream intron promotes exon inclusion, while binding within or upstream from an exon promotes exon skipping . AccordiESRP1/2 is involved in EMT and invasion of cancer cells , 44. HowOur analysis in prostate cancer cell lines shows that modulation of AR signaling dysregulates splicing of functionally relevant genes. Since dysregulation of AR signaling is the hallmark of prostate cancer progression, we hypothesized that ASE of functionally relevant genes would be associated with the progression of disease. To test our hypothesis, we employed rMATS splicing analysis using GSE80609 . This daTRIM37, PTGR1, CREM, HMOX2, UPF3A, TNRC6B, PLA2G2A, and ESYT2. The direction of PSI for these events varied during disease progression, suggesting a differential role for these genes in disease , BPH in comparison to L.PC (unpaired samples) and in response to the pharmacological or genomic inhibition of AR signaling in LNCaP and MDA-PCa-2b cell lines, respectively  and list of splicing events induced by either pharmacological and genomic inhibition of AR in LNCaP and MDA-PCa-2b cells  in comparison to healthy prostate tissue . In contrast, the percentage of the ENST00000400520.7 isoform that does not contain exon-2 was significantly lower in patients with prostate cancer in comparison to healthy prostate  had significantly (p = 0.021) shorter overall survival compared to those difference of <4.08 (n = 123)  Fig. . In concIn this study, we leveraged a combination of next-generation sequencing and molecular methodologies to provide critical pieces of evidence for a direct role of AR and its clinical inhibitors in regulating the transcriptome of prostate cancer cells by modulating global alternative splicing. A key finding from our study is that AR-driven alternatively spliced genes had a distinct physiological role from those that were transcriptionally regulated by AR in prostate cancer cells. Furthermore, we found that antiandrogens currently used in clinics may inadvertently contribute to the disease’s progression by dysregulating splicing. Given that prostate cancer patients on antiandrogens will only experience 8–19 months of progression-free survival , our stuASE is known to play a significant role in maintaining cellular physiology. Therefore, identifying the transcriptional splicing patterns may have the potential for early diagnosis, prognosis, and identification of therapeutic targets in tumor biology . The nucWe leveraged HTA-2.0, a newer generation of microarray that interrogates junctions between exons in the transcriptome as well as the exon themselves and RNA-Seq analysis to test whether modulating AR signaling would alter ASE prostate cancer cells. This approach leds to an unexpected finding that treatment with DHT or casodex induces a large number of ASE in prostate cancer cells. We confirmed our observations by conducting a thorough validation of predicted splice events using real-time PCR in three different prostate cancer cell lines, including androgen-sensitive LNCaP, castrate-resistant 22RV1, and metastatic PC3 cell lines. We also leveraged in-house and publicly available RNA-Seq data to validate whether inhibiting AR signaling with enzalutamide would also induce ASE. Indeed, our analysis found that enzalutamide in comparison to DMSO control induced ASE in LNCaP cells. Similar to casodex experiment, the highest number of ASE was observed when a comparison was made between enzalutamide- and DHT-treated LNCaP cells. However, a comparison between DHT- and enzalutamide-treated LNCaP cells involves comparing experiments from different groups; therefore, we cannot rule out the possibility of a batch effect in this comparison. Given the limitations of across study comparison, we also performed a within study comparison between LNCaP cell treated enzalutamide and DMSO. Supporting our hypothesis that treatment with AR signaling modulates alternative splicing, we found that enzalutamide induced >150 ASE in comparison to DMSO treated LNCaP cells. Further experiments would be needed to validate observed changes between DHT- and enzalutamide-treated prostate cancer cells. In conclusion, we leveraged in vitro experiments and publicly available genomic data to support our findings that AR signaling may alter the transcriptome of prostate cancer cells by modulating global ASE.AR molecules in the cytoplasm dimerize and translocate to the nucleus in response to androgens. Dimerized molecule of AR binds to ARE and regulates target gene expression. Therefore, a majority of published reports have primarily focused on genes transcriptionally regulated by AR to understand the physiological significance of AR signaling. Consequently, we tested whether the AR-signaling-regulated ASE has a different physiological role from differentially expressed genes. Consistent with earlier findings, we found that inhibiting AR signaling leds to expression changes of genes involved in EMT , 52. HowInterestingly, we observed that inhibiting AR signaling using casodex and enzalutamide significantly increased IR compared to DHT treatment. IR is known to generate abnormal transcripts that are translated into immunogenic peptides, loaded on MHC-1, and presented to the immune system . TherefoThe results from our work suggest that AR agonists and antagonists may dysregulate ASE within the pre-mRNA of genes that regulate tumor biology. Because pharmacological modulators may have nonspecific activity, it is possible that the observed ASE observed in response to treatment with AR modulators is not mediated through AR. Therefore, using RT-PCR we demonstrated that genomic inhibition and pharmacological inhibition of AR may induce similar splicing pattern, within a subset of genes in prostate cancer cells. In addition, we also found a significant overlap as well as unique ASE events induced in different prostate cancer cells treated by pharmacological inhibitor or the genomic inhibition of AR. For our global splicing analysis, we leveraged multiple prostate cancer cell lines; therefore, some of the common or unique ASE events identified could be because of cell line differences and not an effect of treatment. Therefore, the global comparison of ASE between the pharmacological inhibitor and genomic inhibition of AR signaling in prostate cancer cell lines needs further validation. Also, we may not be able to rule out the possibility of nonspecific pathways being engaged by pharmacological inhibitors of AR to dysregulate splicing. However, based on analytical models that predict splicing in response to genomic and pharmacological inhibition and RT-PCR validation provides sufficient evidence to conclude that ASE events induced in response to treatment with AR inhibitor are in parts driven by direct modulation of AR expression.The regulation of ASE is primarily mediated by RBPs that interact with sequences flanking exon and introns as splicing enhancers or silencers, depending on the regulator and binding context. Because AR is a transcription factor, we argued that AR might regulate ASE by transcriptionally regulating the expression of RBPs. We tested 112 well-characterized RBPs and found that expression of ESRP1/2 in prostate cancer cells decreased in response to treatment by pharmacological inhibitors of AR. Interestingly, expression of proteins predicted to bind with ESRP1/2 was also deregulated by inhibition of AR signaling. We further confirmed that ESRP1/2 binding sites were enriched downstream from AR-enhanced CEs and upstream from AR-excluded CEs in response to pharmacological inhibition of AR in LNCaP cells. In support of our findings, Munkley et al. also reported that AR controls expression of ESRPs to regulate ASE in prostate cancer cells. However, contrary to the model proposed, we show that the AR-ESRP axis may be only of the mechanisms by which AR may regulate splicing events. To that extent, we found that the SART3, a RNA binding protein and a binding partner of AR, was predicted to be enriched downstream of AR-enhanced exons and underrepresented in the same region of AR-excluded exons in prostate cancer cells. The prediction of AR-SART3 axis being involved in regulating subset of ASE in prostate cancer cells suggests that AR may employ multiple mechanisms to modulate global splicing and they need to be carefully investigated. We will need to perform ChIP-Seq to directly validate whether androgens or antiandrogens dysregulate ESRP and SART3 binding around AR-axis driven CEs. Also, it would be interesting to study whether AR and ESRP or SART3 bind within the same region and are part of a protein complex regulating splicing.Because ESRP plays a critical role in EMT and tumor invasion in several tumor types , we hypoOur work using prostate cancer cell lines showed that AR signaling dysregulates the splicing of functionally relevant genes. Because AR signaling is a hallmark of prostate cancer progression, we also investigated whether AR-regulated ASE is associated with the progression of disease. Our analysis showed that significant splicing events are occurring during different stages of prostate cancer progression. Contrary to an earlier study , which hFurthermore, the transcriptomic analysis had only identified 15 genes that were differentially expressed between advPC and CRPC, thus making these two stages of prostate cancer challenging to differentiate. By focusing on ASE, we have now identified an additional 13 genes that are differentially spliced between advPC and CRPC. Thus, a combined gene expression and splicing panel could potentially have a higher diagnostic value to discriminate patients in an advanced stage from the one with the castrate-resistant disease.PLA2G2A gene in prostate cancer cells. The translational significance of the inclusion of exon-2 was validated in the TCGA-PRAD, where we revealed that the PLA2G2A isoform that includes exon-2 is a prognostic factor for outcomes, providing strong evidence for developing a therapeutic strategy that can mitigate the inadvertent pro-tumorigenic effects of inhibiting AR signaling.Lastly, we investigated whether the treatment of prostate cancer cells with pharmacological inhibitor enzalutamide may lead to an inadvertent switch in the splicing of a pre-mRNA and promote tumor progression. Our analysis found that dysregulation of AR signaling in prostate cancer driven by enzalutamide treatment increases the inclusion of exon-2 of the In conclusion, this study highlights the so-far undescribed role of AR in modulating gene expression via ASE in prostate cancer. This discovery also opens a new therapeutic path and supports the rationale for using ESRP modulators in combination with AR antagonists for the treatment of advanced-stage prostate cancer to counteract the AR-antagonist-driven invasive phenotype.5α-DHT, casodex, and Enzalutamide were obtained from Sigma and were resuspended in DMSO (Sigma). Primers were designed manually and were purchased from Integrated DNA Technologies. Anti-ESRP1 (#21045-111-AP) and anti-ESRP2 (#23317-1-AP) were obtained from the Proteintech. E-Cadherin (4A2) (#14472) and Vimentin (D21He) (#5741) were obtained from the CellSignaling, while anti-aTubulin antibody (#A01410) was obtained from Genscript. All other reagents if not specified were obtained from Thermo fisher Scientific.The human cell lines LNCaP (androgen-sensitive human prostate adenocarcinoma cells) and 22RV1  were obtained from ATCC and were cultured in RPMI-1640 medium. PC3  was maintained in F-12K medium. All culture medium was supplemented with 10% HyClone Defined Fetal Bovine Serum  and 1% Pen/Strep (Invitrogen) unless specified. Cell cultures were tested every 6 months for cross-contamination using human STR profiling cell authentication service provided by ATCC. Mycoplasma contamination was tested using MycoAlert Mycoplasma Detection Kit (Lonza).Cells were dissolved in RIPA buffer (sigma). Protein concentration was measured by BCA protein assay reagent kit , as described previously. Proteins were fractionated on 10% SDS-PAGE, and transferred by electrophoresis to nitrocellulose transfer membrane (GE). Membranes were incubated with primary antibodies for overnight. Horseradish peroxidase-conjugated antibody anti-mouse IgG and anti-rabbit IgG (Dako) were used to detect immunoreactive bands and binding was revealed using enhanced chemiluminescence (Pierce). The blots were then stripped and used for further blotting for control antibody. Unless otherwise specified, displayed western blots are representative images of at least two independent experiments.22RV1 cells were transfected with a mixture of four different siRNA targeting AR (siRNA #L-003400-00-0010) and non-targeting control pool (#D-001810-10-05). PC3, LNCaP, and 22RV1 cells were transfected with a mixture of four different siRNA targeting ESRP1/ESRP2 kinases and negative control . Cancer cells were seeded in a six-well plate. 200-nM siRNA/well were used for transfection using 5 µl/well of Hi-Perfect (Qiagen) following manufacturer’s recommendation.Reduced growth factors Matrigel (BD Biosciences) was polymerized in 8-mm pore cell inserts (Sarstedt) prior to the addition of cells. LNCaP and 22RV1 (5 × 106 cells) were seeded into the insert containing Matrigel in serum-free media. Twenty percent FBS medium was used as an attractant in the bottom chamber, and cells were allowed to migrate for additional 24 h. The inserts were removed, and migrated cells were fixed with 4% paraformaldehyde and stained with crystal violet. The inserts were then imaged, and migrated cells were counted, hence, providing a quantitative value for migrated cells across the membrane.LNCaP cells were cultured for 3 days in the RPMI-1640 with 10% charcoal-stripped fetal bovine serum and treated with either 10-nM DHT, 10-µM casodex, or DMSO for 24 h. Total RNA was extracted using the RNeasy mini kit (Qiagen) and quantified using Nanodrop ND-100 Spectrophotometer (Thermo Fisher Scientific). The quality of RNA was assessed using Agilent 2100 Bioanalyzer (Agilent). Biotinylated cDNAs were prepared from a minimal 100 ng of total RNA using Life Technologies WT-plus RNA Amplification system (Ambion). Following the amplification, cDNA was fragmented, hybridized on Affymetrix GeneChip Human Transcriptome Array 2.0 (HTA-2.0) chips, and nonspecific bindings were washed as per manufacturer’s recommendations. The fluorescence intensity of the arrays was scanned using the Affymetrix Scanner and the raw data. The raw data were analyzed using the Transcriptome Console Software (TAC 2.0) that allows for the identification of differentially expressed genes and leverages information from the junction and exons probes to detect ASEs and possible transcript isoforms that may exist in samples.p value < 0.05 in at least 50% of the samples were considered as statistically significant. Principal component analysis was used to calculate variance and confirm if the variance is similar between the groups being compared. Genes were considered to be differentially expressed when fold change (FC), log ≤ −2.0 or ≥+2.0 and FDR corrected p value ≤ 0.05. The splicing level analysis as also carried out using TAC 2.0 software, which determines the SI of a gene. The SI corresponds to a comparison of gene-normalized exon-intensity values between the two analyzed experimental conditions. Additional criteria used besides SI: FDR corrected p value ≤0.05, a gene is expressed in both conditions tested, a probeset ration (PSR)/junction must be expressed in at least one condition, a gene must contain at least one PSR value, and a gene cannot be differentially expressed.For microarray data analysis, two parallel analysis  were performed using HTA-2.0. Data were normalized using quantile normalization and the background was detected using the built-in detection above background algorithm (DABG). Only the probesets that were characterized by a DABG ® Green PCR Master Mix on the Applied Biosystems 7500 Detection System. Spicing-specific primers included two primer pairs, one for monitoring the expression of constitutive exons within all the isoforms of a gene and another for measuring changes in the alternatively spliced region. Furthermore, specificity and efficiency for primers were analyzed by running RT-PCR with series of cDNA dilutions, and specific amplification for every assay was confirmed by melt curve analysis. The amplified transcripts were quantified using the comparative ΔΔCt method. GAPDH and HPRT were used as the internal control. SI was calculated for (A) by normalizing FC to the average FC of (C) for each splicing event. All assays were run in triplicates and were repeated three times. A raw Ct of 35 is used as the limit of detection: Ct values are set at 35 for any replicates with Ct values not determined or >35.A total validation of 15 splicing events was performed on three prostate cancer cell lines including LNCaP, 22RV1, and PC3 at various disease state . Briefly, the prostate cancer cells were cultured for 3 days in 10% CSFBS and were treated with either DMSO, 10-nM DHT, or 10-µM casodex for 24 h. Total RNA was extracted, and cDNA was prepared using SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific). cDNA samples were amplified using SYBRhttps://bioconductor.org/packages/release/bioc/html/DESeq2.html) method as described earlier [http://rnaseq-mats.sourceforge.net) to identify differentially ASE between the two sample groups. Briefly, rMATS uses a modified version of the generalized linear mixed model to detect differentially ASE from RNA-Seq data with replicates, while controlling for changes in expression at gene level. In addition, it also accounts for exon-specific sequencing coverage in individual samples as well as variation in exon splicing levels among replicates. rMATS was run using the default parameters and then significant splicing events were filtered using a stringent cutoff of FDR ≤ 0.05 and delta PSI ≥ 10%. The Maser (v1.6.0) https://www.bioconductor.org/packages/release/bioc/html/maser.html was used for extracting and visualizing splice events.High-quality RNA samples were extracted, and illumine library was constructed as described earlier. Libraries were pooled and diluted for sequencing with a 1% PhiX spike-in according to Illumina protocol. The pool of library was loaded onto the HiSeq was performed using a 300-cycle high output v2 kit. Reads were obtained from sequencer or were downloaded from GEO. Adapter sequences and invalid reads containing poly-N and low-quality were removed using the FastX tool kit (v 0.0.14). The quality of reads was then confirmed using fastqc tool kit (v 0.11.5). All downstream analysis used the cleaned reads. The clean reads were mapped to the ENSEMBL built GRC38 using the STAR aligner (v2.5.3a) using ENCODE option as described in the STAR manual. Principal component analysis was performed to calculate variance within each group, and differential expression of genes was obtained using the DESeq2 (v1.28.0) ( earlier . Subsequhttp://rmaps.cecsresearch.org) analysis to determine the binding patterns of splicing factors and RNA binding proteins within significantly detected exon skipping ASE between two treatment groups. We collected well-characterized 115 known binding sites of RNA binding proteins. For each motif, the analysis scanned for motif occurrences separately in exons or their 250-bp upstream or downstream intronic sequences. Furthermore, for the intronic sequences our analysis excluded the 20-bp sequences within the 3′ splice site and the 6-bp sequences within the 5′ splice site. In addition, by default the alternative exons without splicing changes as defined by rMATS FDR > 50%, maxPSI > 15%, and minPSI < 85% were treated as control exons. For each motif tested, the analysis counted the number of occurrences and motif score in the upstream exon, upstream flanking intron, target exon, downstream flanking intron, and downstream exon separately. The p value for motif enrichment was calculated using the Wilcoxon’s rank sum test for each sliding window between upregulated versus control or downregulated versus control exons.We employed rMAPS2 (ReactomePA (v1.32.0) and the clusterProfiler (v3.16.0) were used to generate lists of the GO terms enriched in the DEGs and ASE. The integration of protein features to splicing events was carried out using maser package. Briefly, maser enables systematic mapping of protein annotation from UniprotKB to splicing events and determine whether the splicing is affection regions of interest containing known domains or motifs, mutations, posttranslational modification, and other described protein structural features.Supplementary MaterialData Set 1Data Set 2Data Set 3Data Set 4Data Set 5


COMT heterozygous-HEA-haplotype showed higher PLS1 FFA-MRI scores, which were positively associated with the FRA in an old/new face recognition task, than those with the COMT homozygous HEA haplotype and HEA non-carriers, suggesting that individuals with an appropriate (intermediate) level of dopamine activity in the FFA might have better FRA. In summary, our study provides empirical evidence for the genetic and neural basis for the heritability of face recognition and informs the formation of neural module functional specificity.Previous studies have shown that face-specific recognition ability (FRA) is heritable; however, the neural basis of this heritability is unclear. Candidate gene studies have suggested that the catechol-O-methyltransferase (COMT) rs4680 polymorphism is related to face perception. Here, using a partial least squares (PLS) method, we examined the multivariate association between 12 genotypes of 4 COMT polymorphisms (rs6269-rs4633-rs4818-rs4680) and multimodal MRI phenotypes in the human fusiform face area (FFA), which selectively responds to face stimuli, in 338 Han Chinese adults . The MRI phenotypes included gray matter volume (GMV), resting-state fractional amplitude of low-frequency fluctuations (fALFF), and face-selective blood-oxygen-level-dependent (BOLD) responses (FS). We found that the first COMT-variant component (PLS1) was positively associated with the FS but negatively associated with the fALFF in the FFA. Moreover, participants with the  Previous behavioral genetic studies have shown that face recognition ability is heritable6; however, the neural basis for this heritability is unclear. One candidate cortical region underlying inter-individual variability in face recognition is the human fusiform face area (FFA) especially in the right hemisphere, which shows more significant responses to visual face stimuli than to other non-face stimuli7 and represents a critical hub in the face network10. More importantly, features of the FFA are associated with face-specific recognition ability (FRA)3. However, information about the effects of genetic variability on FFA structure and function and their relationship with face recognition ability remain limited.Face recognition is a highly developed skill in humans that shows considerable variability between individuals11 and functional magnetic resonance imaging (fMRI) responses12 in the occipitotemporal cortex encompassing the FFA in monozygotic twins than dizygotic twins. Additionally, neural responses to facial expressions are modulated by common genetic variance in a set of regions constituting the face network that include the FFA13. These studies suggest that the FFA may be modulated by genetic variations. However, previous studies have not specifically localized the FFA, and it is unclear whether the observed genetic effects are derived from changes in the FFA or other regions. Notably, a previous genome-wide association neuroimaging study explored specific genes contributing to the FFA but failed to identify any significant genome-level correlations between single nucleotide polymorphisms (SNPs) and FFA-responses to facial expressions14. One possible reason for this negative result is insufficient statistic power due to a relatively small number of participants (N < 300).Previous twin studies have demonstrated the higher intra-pair similarity of cortical structure19. The COMT gene codes for the COMT enzyme, a protein that degrades catecholamine neurotransmitters and modulates dopamine metabolisms20. Among the COMT polymorphisms, Val158Met (rs4680) has been most reported to play a role in face processing24. For example, individuals with the Met/Met (rs4680) genotype are more sensitive to configural changes in faces than those with other genotypes15. Healthy homozygous subjects with the Met allele of rs4680 are better at recognizing facial emotions16 and show a stronger bias to perceive neutral faces as expressions of anger21 than those with other genotypes. Previous studies also investigated the impact of the COMT gene on the brain activity during face emotional processing25 and demonstrated that the COMT Met allele seems to contribute to neural substrates of negativity biases24, and the COMT Val allele is associated with increased amygdala activity in response to fearful/angry facial expressions25. It should be noted that several meta-analyses suggest that the effects of COMT Val158Met on cognitive abilities are either very small or nil due to insufficient statistical power, unknown moderators including genetic variations at different loci, publication bias, or effect heterogeneity28. Therefore, individual studies should pay more attention to the methodological rigor and scientific nature. Accumulating studies have revealed that there are multi-to-multi relationships between genotypes and brain phenotypes linked to learning and cognitive ability31. Previous studies have indicated that four COMT polymorphisms  have a strong linkage disequilibrium and always function as haplotype blocks to affect dopamine (DA) degrading enzyme activity33. However, little is known about whether certain patterns of the 4 COMT polymorphisms exert significant effects on the FFA and therefore affect the face recognition ability.Candidate gene studies have demonstrated the association of the catechol-O-methyltransferase (COMT) gene, located on chromosome 22q11.2, with face recognition37; accordingly, this study used multimodal MRI measures for the FFA , including GMV, fractional amplitude of low-frequency fluctuations (fALFF) from resting-state fMRI, and BOLD responses specific to faces  from task-state fMRI. Second, the partial least squares (PLS) approach was applied to examine relationships between sets of multivariate measures. This approach has demonstrated utility in studies with relative small samples and problems with multicollinearity40. Third, bilateral FFAs are involved in face processing simultaneously8, notwithstanding some hemispheric lateralization in their size and functional recruitments42. We conducted analyses for the right FFA (rFFA) and the left FFA (lFFA) separately to validate the genetic variants that modulate the FFA. Using these three strategies, we identified profiles of 4 COMT polymorphisms that would be robustly associated with profiles of structural or functional FFA, in a population of Han Chinese individuals.In this study, we employed a combined genetic and neuroimaging approach and incorporated three strategies to improve statistical power without dramatically increasing the number of participants. First, previous studies focused on a single measure of the FFA  or blood oxygen level-dependent [BOLD] responses); notably, multiple modals and joint modeling can improve sensitivity for identifying disease-associated biological pathways in common complex disordersCOMT genotypes contributed to the 3-modal right FFA-MRI phenotype in 338 participants. The PLSCA yielded 3 sets of paired latent variables (LVs) capturing the rFFA-SNP association, ordered by the size of the explained variance . Each component (LV pair) was a linear combination of the weighted COMT SNP scores that most strongly correlated with weighted MRI scores and was orthogonal to each other. The omnibus p value of the model was 0.004 and only the top PLS component (PLS1) was significant . Thus, the PLS1 represented a significant association  between a specifically patterned COMT genotypic profile and a specifically patterned rFFA phenotype , rs4818 AA genotype , rs4818 AG genotype , rs4633 TT genotype , and rs6269 AG genotype  reliably contributed to the PLS1 COMT-SNP component  and fALFF  reliably contributed to the PLS1 rFFA-MRI component  was conducted to examine whether the 12 p = 0.002, 95%CI = ), negatively correlated with fALFF , and not correlated with GMV of the rFFA   of the 12 COMT genotypes  modulates the 3 raw MRI measures of the rFFA, we correlated the PLS1 COMT scores with FS, GMV, and fALFF of the rFFA. The results showed that the PLS1 COMT profile was positively correlated with FS  Fig. . Thus, tp = 0.821). Moreover, the intra-class correlations (ICC) for the two half GMV, fALFF, FS, PLS1-SNP scores, and PLS1-rFFA scores were 0.985 , 0.995 , 0.989 , 0.999 , and 0.994 , respectively. Thus, both the rFFA measures and the association pattern between the rFFA phenotypes and the COMT polymorphisms exhibit high reliability. Second, to test whether the threshold for identifying the rFFA affects the main results, we conducted control analyses with 4 different thresholds  and found that the association between the 12 COMT genotypes and the rFFA-MRI phenotypes was significant in all of the 4 threshold conditions . Thus, the lFFA exhibits a similar association pattern with the COMT polymorphisms as the rFFA does.To valid the results and improve reliability, we conducted the following control analyses. First, we randomly split the voxels within the rFFA into two halves and entered each half of the rFFA measures along with COMT polymorphisms into a PLSCA. We found that both of the rFFA-COMT associations were significant  a high enzymatic activity (HEA) haplotype (rs6269/G-rs4633/C-rs4818/G-rs4680/G) with a frequency of 0.328, (2) an intermediate enzymatic activity (MEA) haplotype (rs6269/A-rs4633/T-rs4818/C-rs4680/A) with a frequency of 0.239, and (3) a low enzymatic activity (LEA) haplotype (rs6269/A-rs4633/C-rs4818/C-rs4680/G) with the highest frequency of 0.389  = 0.003, p = 0.958]. Tukey’s HSD post hoc tests showed that COMT heterozygous-HEA carriers had significant larger PLS1 rFFA-MRI scores than COMT homozygous HEA carriers  and non-HEA carriers . Thus, participants with the COMT heterozygous-HEA-haplotype (intermediate DA degrading enzymatic activity therefore an intermediate level of DA) showed increased FS and decreased rFFA-fALFF (positive FFA scores of PLS1), whereas participants with the COMT homozygous HEA haplotype (high DA degrading enzymatic activity therefore a low level of DA) and HEA non-carriers (low DA degrading enzymatic activity therefore a high level of DA) showed decreased FS and increased rFFA-fALFF (negative FFA scores of PLS1).Because the four COMT SNPs has been previously found to be in strong linkage disequilibrium and define three common haplotypes with functional consequences on dopamine (DA) degrading enzyme functionlotypes:  a high elotypes:  a high ep = 0.029; left Panel of Fig. p = 0.003, Cohen’d = 0.4, 95%CI = ), suggesting that the COMT-modulated rFFA-MRI profile was more relevant to the face recognition ability than to the flower recognition ability. Further, the association between the FRA  and the PLS1 rFFA-MRI scores was significant  when sex, age, and Raven scores were treated as nuisance covariates  , suggesting that the COMT-modulated bilateral FFA-MRI profile might have similar association patterns with the FRA. To make our results comparable to those in previous studies using the signal detection theory, we calculated the sensitivity index (d’) for face recognition and flower recognition. No significant correlations were found between face sensitivity (d’) and the PLS1 FFA-MRI score  and flower recognition accuracy score with the PLS1 rFFA-MRI scores and the PLS1 COMT scores, respectively. The results showed that face recognition accuracy was positively correlated with the PLS1 rFFA-MRI scores  face recognition and new (relatively unfamiliar) face recognition. To control the effect of response bias on the accuracy48, we calculated the response bias [c = −Z(hit rate) + Z]49 and regressed the bias out of the hit rate and correct rejection rate to obtain the old-face recognition score and new-face recognition score. A significant association was found between the new-face recognition score and the bilateral PLS1 FFA-MRI scores , suggesting that the COMT-modulated FFA-MRI profile was more sensitive to the new-face recognition ability than to the old-face recognition ability.Finally, because the FFA has been demonstrated to be involved in the process of recognizing familiar and unfamiliar facesCOMT polymorphisms. Participants with the COMT heterozygous-HEA-haplotype showed higher PLS1 FFA-MRI scores, which represented an increased FS and a decreased fALFF and were associated with a better face-specific recognition ability, than those with the COMT homozygous HEA haplotype and HEA non-carrier, suggesting that participants with an appropriate (intermediate) level of dopamine in the bilateral FFA may have better face-specific recognition ability. Our study provides empirical evidence of genetic modulation of a functionally-specialized cortical region in humans.In the present study, we explored the neural basis of the heritability of face recognition by investigating associations between 12 COMT genotypes and 3-modal FFA-MRI phenotypes. The results revealed that a profile of face-selective responses and resting-state neural fluctuations of the bilateral FFA were modulated by a profile of 3, and this association is domain-specific3. In this study, we revealed that a profile (linear combination) of higher face selectivity BOLD responses, as well as lower resting-state activities in the bilateral FFA, was associated with a higher face recognition  ability, and the association could not be accounted for by FFA responses to objects such as flowers, suggesting that the face-specific recognition ability might be modulated by a certain covariation pattern of resting-state and task-state activity in the FFA, which was modulated by a certain profile of the COMT polymorphisms.Previous studies have found that participants with higher face selectivity in the FFA have higher face recognition abilityCOMT Val/Met (rs4680) polymorphism has been suggested to play a role in face perception22. Our study extends previous findings by identifying a neural site where COMT affects face recognition. Specifically, we investigated a broader modulation pattern of COMT genetic effects on the FFA by including multiple SNPs. We found that individuals with the heterozygous HEA haplotype exhibited higher face-selective responses but smaller fALFF. In contrast, individuals with the homozygous HEA haplotype or HEA non-carriers had lower face-selective responses and larger fALFF. Because COMT encodes catechol-O-methyltransferase, a protein that degrades catecholamine neurotransmitters and modulates dopamine metabolism20, neural activity (including the face-recognition-task-state and resting-state activities) in the FFA is likely modulated by changes in dopamine. Moreover, we found that an intermediate level of dopamine in the FFA was associated with higher face-selective responses (in the task state) and smaller fALFF (in the resting state), and the profile of higher face-selective responses and lower fALFF in the FFA was associated with better face-specific recognition ability. Thus, an intermediate level of dopamine in the FFA is likely beneficial for behavioral performance in face recognition. That is to say, participants with an appropriate level of dopamine  in the FFA possess a functional characteristic profile of higher face-selective responses and lower intrinsic FFA activities, therefore, they might have better face-specific recognition ability.The COMT rs4680 and other dopamine-function related generic variants imply that dopamine-related neurons may affect the baseline brain resting state activity by synchronizing the low-frequency oscillation50. Moreover, dopamine could stabilize  neural signaling in brain networks responsible for behavior-stimuli oriented attention52. Thus, an appropriate level of dopamine may improve the functional segregation-integration of a functional-specialized region by maintaining excitation-inhibition stability of neural signal transduction to ensure that individuals with the HEA heterozygous haplotype have a relatively higher rFFA activation in face recognition tasks (which has been found to be related to a better face-specific recognition ability3), as well as a relatively lower rFFA activity during resting state . This is consistent with previous studies demonstrating associations between dopamine hyper- or hypo-function in cortical or subcortical regions and cognitive dysfunctions56. Therefore, it would be interesting to examine the level of dopamine in the FFA in individuals with developmental prosopagnosia59, who show specific deficits in face recognition with unclear genetic basis60 and those with mental disorders who might have disturbances in COMT modulation of face processing23.Previous studies have rarely reported the direct impact of COMT polymorphisms on resting-state FFA activity in healthy people. Some studies focusing on the interaction effect between the 47, there seems to be no difference in the increase of neural response activity of the FFA when recognizing familiar and unfamiliar faces47. Compared with recognizing unfamiliar faces, recognizing familiar faces involves more activity in the anterior and mid-fusiform gyrus and extended face areas45. In our study, the stimuli were not familiar to the subjects, and the social and emotional information involved in the face stimuli was relatively sparse; therefore, the recognition difference between the old face and the new face might be mainly reflected in the familiarity and unfamiliarity of the facial features. We found that the COMT-polymorphisms modulated PLS1 rFFA-MRI profile was sensitive to the recognition of new faces, but not to that of the old faces. The findings may be related to the fact that the novel signals can trigger the dopaminergic metabolic system55 in the task-related brain areas and enhance perception62.Our study found that the 4 COMT-polymorphisms-modulated PLS1 rFFA-MRI profile mainly regulated the recognition ability of new faces. Although previous studies have revealed that it takes more time and is more likely to make mistakes to recognize faces of different identities than faces of same identities63. The posterior superior temporal sulcus (pSTS) is responsible for dynamic features64 and sensitive to emotional information67 from faces, and the prefrontal-amygdala circuit is involved in social and emotional regulation in face processing23. In fact, face perception as a whole is a network involving multiple brain regions9. Studies have found that the COMT Met allele is related to the modulation of neural substrates of negative face emotional bias23. Therefore, in future studies, it would be interesting to evaluate the role of multiple COMT polymorphisms in the core face network and its relation with face-specific recognition ability and face-emotion recognition.The FFA is sensitive to the static or invariant properties of facesThis study has some limitations. First, although we have adopted multiple strategies to improve the statistical power, the findings still need to be validated using data from other cohorts of participants. Second, measurements for the face recognition ability and the general mental ability are limited to the face memory test and the Raven test, respectively. More multivariate measurements could be introduced in the future to improve the reliability of the individual face recognition ability and general mental ability, and further elaborate the specificity of the effect of COMT on the face recognition ability. Third, all participants had the same ethnicity ; therefore, how the ethnicity of the individual participants affected the results needs to be examined using data from multiple ethnicities in the future. Fourth, the Caucasian children’ faces were used as stimuli to define the FFA in Han Chinese participants. As demonstrated in previous studies, there is an other-race effect in face recognition; therefore, how our findings are affected by stimulus attributes still needs to be investigated in further studies.COMT gene affected both resting- and task-state neural activity in the FFA through rs6269-rs4633-rs4818-rs4680 haplotypes, and the COMT-polymorphisms-modulated FFA was associated with face-specific recognition ability. The present findings provide a basis for the behavioral observation of face-specific recognition heritability and the approach used in this study might be s a feasible method for exploring and validating the neural basis of heritable behavioral performance. In addition, this study may inform the genetic etiology of developmental prosopagnosia and other mental disorders showing abnormities in face processing.In conclusion, our study demonstrated that the Three hundred and sixty-two Han Chinese college students  were recruited from Beijing Normal University (BNU) in Beijing, China through campus advertisement. Ethnicity was determined by the ethnic information displayed on the ID card of the participants. All participants had normal or corrected-to-normal vision, and none of them reported a history of neurological or psychiatric disorders. All the experimental protocol and procedures were approved by the Committee for Protecting Human and Animal Subjects of the Faculty of Psychology at Beijing Normal University. All experiments of the study were conducted in accordance with the relevant guidelines and regulations of Beijing Normal University’s Institutional Review Board (Human Subjects Division). All participants gave written informed consent prior to their participation in this study.3). Resting-state scanning lasted for 8 min and consisted of 240 contiguous volumes. Participants were instructed to relax and remain still with their eyes closed during resting state scanning9. A GRE-EPI sequence was also used to acquire task-state fMRI . A high-resolution structural T1-weighted image was acquired with a 3D magnetization-prepared rapid acquisition gradient echo (MP-RAGE) sequence . Earplugs were used to attenuate scanner noise, and a foam pillow and extendable padded head clamps were used to restrain head motion3. None of the participants were excluded due to excessive head motion (>2 mm in translation or 2° in rotation) during MRI scanning.Functional and structural MRI was performed at the BNU Imaging Center for Brain Research on a Siemens 3 T scanner  with a 12-channel phased-array head coil. Resting-state fMRI was acquired using a T2*-weighted gradient-echo echo-planar-imaging (GRE-EPI) sequence  isotropic Gaussian kernel. Finally, the modulated images were masked using absolute masking with a threshold of 0.2 to exclude noisy voxels. Masked-modulated GM images were used for further statistical analyses69.Voxel-based morphometry (VBM) was performed using the SPM8 . First, unified segmentation in SPM8 was used to segment T1-weighted anatomical images into gray matter (GM), white matter (WM), and cerebral spinal fluid (CSF). Second, the Diffeomorphic Anatomical Registration through Exponential Lie algebra (DARTEL) registration method63. Movie clips of close-up faces of 7 Caucasian children (filmed when children were dancing or playing), objects (moving toys), scenes , and scrambled objects (constructed by scrambling each frame of the object movie clips) were included to examine category selectivity in the ventral visual cortex63. Participants were instructed to passively view movie clips during scanning. Because Adults’ FFA have been effectively identified by children’ faces in previous studies67 and the FFA is sensitive to the invariant properties of faces, but not to the dynamic or emotional information of faces70, we expected that the dynamic children’ faces would accurately and unbiasedly localize the FFA in adults.A dynamic face localizer was used to define the face-selective area and derive FSIn total, participants completed three 198-s runs. Each run consisted of 2 block sets intermixed with 3 18-s rest blocks at the beginning, middle, and end of the run. Each block set contained four 18-s (including six 3-s movie clips) blocks corresponding to 4 stimulus categories. The order of category blocks in each run was palindromic and randomized across runs Fig. .Figure 4www.fmrib.ox.ac.uk/fsl). Preprocessing included head-motion correction, brain extraction, spatial smoothing , grand-mean intensity normalization, and high-pass temporal filtering (120-s cutoff). A first-level analysis was performed separately on each run for each participant using FMRIB’s Improved Linear Model with a local autocorrelation correction. A boxcar kernel was convolved with a gamma hemodynamic response function and its temporal derivative was used to model BOLD signal. Six parameters from motion-correction were also included in the model as regressors of no interest to account for the effect of head movement. A second-level fixed-effect analysis was conducted to combine all runs. Specifically, the parameter  image from the first-level analysis was first aligned to the individual’s structural images using FMRIB’s linear image registration tool with 6 degrees of freedom and then warped to the MNI152 template using FMRIB’s nonlinear image registration tool with default parameters. The spatially normalized parameter images (resampled to 2-mm isotropic voxels) were then summarized across all runs using a fixed-effect model69. In this study, statistical images from the contrast between faces and objects were used to define the right fusiform face area and derive its face selectivity (FS), which was calculated as the average z-score (faces vs. objects) across all voxels in the defined face-selective region.Functional images were analyzed with the FMRI Expert Analysis Tool version 5.98, part of FMRIB’s Software Library , which is defined as the fractional sum of the amplitudes within the low frequency range divided by the sum of amplitude across the entire frequency range (0–0.25 Hz)3. Specifically, all contiguous voxels that surpassed the threshold within the posterior fusiform area  were defined as the FFA  was used to define the bilateral FFA based on the random-effect group-level Z-statistic image for faces vs. objects72. All the 4 COMT SNPs met the following criteria: genotyping call rate >0.95, minor allele frequency (MAF) > 0.05, and Hardy–Weinberg equilibrium (HWE) P > 0.05. Participants were excluded from analyses if they had a missing genotype rate of >25% . Twenty-four participants were excluded, and the remaining 338 participants  were included in the data analyses. Allele frequencies in this sample were very similar to those in the Han Chinese sample from the HapMap dataset (HapMap Data Release 27 Phase II + III).Genomic DNA was extracted from peripheral blood samples of each subject using QuickGene-Mini80 equipment and the QuickGene DNA whole blood kit S (Fujifilm). Sixty-four cognition-related candidate SNPs 48 was also calculated for face recognition and flower recognition.We used an old/new cognitive memory task to measure the face-specific recognition ability (FRA). There was a face block  and a flower block  in this task . PLS is particularly suitable for identifying associations between 2 sets of variables, especially when variables of each set are highly interdependent or multi-collinear74. The PLS approach maximizes the covariance between a set of variables (a linear combination of X measures) and another set of variables (a linear combination of Y measures). More specifically, the cross-correlation matrix between measures of SNPs and measures of brain-MRI was first computed. Then, a set of paired latent variables (LVs), which were uncorrelated  with each other and ordered by the explained covariance of brain and SNP measures, were generated via singular value decomposition. LVs are linearly weighted by the raw variables, and these weights or saliences measure the contribution of each raw variable to the LV73. Finally, the significance of the variance of singular values was tested by comparison with the distribution of variance arising from random permutation tests (1000 repetitions), and a bootstrap procedure (500 times) was used to test the reliability  of each variable in the significant PLS component73.The PLS correspondence analysis (PLSCA) was conducted using the R Package ExPosition  as categorical variables, and each variable was weighted according to the information it provided. The weight of a variable was defined as the inverse of its relative frequency because a rare variable provides more information than does a frequent variable75. This fully categorical coding scheme allows us to detects linear and nonlinear relationships within  and between datasets37. Thus, treating each genotype as a level of a categorical variable is suitable for genetic association studies, especially when the inheritance pattern, direction, and/or size of the effect are unknown76. Accordingly, a matrix of measures of 12 COMT genotypes estimated in the 338 participants and a matrix of measures of 3-modal FFA-MRI phenotype estimated in the 338 participants were entered into the PLS analysis.The SNPs-FFA multivariate association analysis was used to examine whether specific patterns of COMT polymorphisms contributed to the multimodal FFA-MRI phenotypes. First, GM, fALFF, and FS within the right FFA and the left FFA ROI were extracted for each participant. Then, each SNP was coded in accordance with genotypic (codominant) models77 was conducted to investigate how SNPs in strong linkage disequilibrium work as blocks to modulate FFA phenotypes. Haplotype blocks of SNPs affecting the FFA phenotype were assessed and illustrated using Haploview software version 4.2 (https://sourceforge.net/projects/haploview/) with the solid spine of the linkage disequilibrium method and parameter 0.80. Associations of haplotypes with the FFA were examined using R software version 3.5.2 with generalized linear models.A haplotype block analysisSupplementary Information


Moreover, we discuss the role of the individual adrenoceptors as targets for the treatment of retinal diseases.The retina is a part of the central nervous system, a thin multilayer with neuronal lamination, responsible for detecting, preprocessing, and sending visual information to the brain. Many retinal diseases are characterized by hemodynamic perturbations and neurodegeneration leading to vision loss and reduced quality of life. Since catecholamines and respective bindings sites have been characterized in the retina, we systematically reviewed the literature with regard to retinal expression, distribution and function of alpha Many ocular diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, retinal vascular occlusion and glaucoma frequently cause severe visual impairment substantially diminishing the quality of life ,2,3,4,5.Since the retina is the tissue with the highest metabolic demand in the body, it is not surprising that retinal hemodynamic perturbations play a critical role in the pathogenesis of numerous ocular diseases ,16,17,18Receptor families comprising multiple subtypes with organ-specific patterns of neurovascular expression and function appear to be attractive candidates for the development of targeted therapies against diseases associated with abnormal tissue perfusion. One such intriguing receptor family is the adrenoceptor (AR) family.1 (α1)-, alpha2 (α2)-, and the beta (β)-AR subfamily . Me. Me1 , carbon dioxide (CO2), nitric oxide (NO), and hydrogen sulfide (H2S) prazosin. The authors reported that α1-adrenergic binding sites were only enriched in the outer plexiform layer noradrenaline and [3H]clonidine [2-AR binding sites were identified in the inner plexiform layer of the rat retina by [3H]para-aminoclonidine ([3H]PAC) -RX 781,094 and [3H]-rauwolscine PAC) . Most of[3H]PAC) . Furtherwolscine . Matsuo iography .2-ARs, α2A, α2B, α2C, have been identified by molecular and pharmacological characterization techniques dihydroalprenolol in nearly all rat retinal layers , but with the lowest concentration in the outer nuclear layer [In 1986, Zarbin et al. localized β-AR binding sites by [ar layer . In the ar layer .1-AR, β2-AR and β3-AR, have been identified [4-AR was hypothesized by Granneman as a novel state of the β1-AR [4-AR was considered by Madamanchi as a low-affinity state of the β1-AR, which still needs pharmacological and genetic characterization [1-AR and β2-AR binding sites were found in bovine retinal vessels and in the neural retina [3-ARs has also been verified in rat retinal blood vessels [3-ARs were localized in the inner capillary plexus of the mouse mid-peripheral retina, whereas β1-ARs and β2-ARs were localized in rat cultured Müller cells [3-ARs has been shown for the first time on cultured human retinal endothelial cells in 2003 [3-ARs promoted migration and proliferation of endothelial cells [Three distinct β-AR subtypes, βentified . In 2001he β1-AR . Six yearization . Among βl retina . The pre vessels . By immuer cells . Howeverer cells . Express in 2003 . In thatal cells . Based oStress conditions, such as hypoxia, can cause catecholaminergic overstimulation in the cardiovascular system, which in turn may activate β-ARs . A studyIntriguingly, a novel β-AR agonist, compound 49b, was reported to decrease VEGF levels in the diabetic rat retina . The effβ-AR activation was also shown to increase human and mouse pericyte survival under diabetic conditions . ConversStimulation of β-AR by agonists can activate members of the G protein-coupled receptor (GPCR) kinase (GRK) family, which is the potential mechanism that leads to β-AR desensitization . A first1- and β2-ARs reduced retinal VEGF receptor-2 expression and abolished the development of vascular abnormalities in the superficial plexus of the retina [1- and β2-AR blockade by propranolol was shown to reduce the expression of VEGF and to ameliorate retinal dysfunction [2-AR blocker, decreased retinal levels of proangiogenic factors and reduced pathogenic neovascularization in a mouse OIR model, suggesting that β2-AR blockade may be effective in the blockade of retinal angiogenesis [Hypoxia was reported to trigger the release of catecholamines, which have been shown to contribute to the increase in retinal VEGF expression, causing pathologic neovascularization ,184,185.e retina . In anotfunction . Other sogenesis .2-AR desensitization that downregulates the expression of β2-ARs in the retina, which in turn exerts a downregulation effect on VEGF expression in OIR [2-AR KO mice exhibited certain features similar to diabetic retinopathy, resulting in retinal cell death [2-AR KO mice has shown a functional link between β-ARs and insulin receptor signaling pathways in the retina [2-ARs can maintain insulin receptor signaling in retinal Müller cells, which potentially supports neuroprotective effects promoted by β-AR stimulation in diabetic retinopathy models [1-AR agonist, attenuated iNOS expression in human retinal endothelial cells grown in high glucose medium [1-, β2-, and β3-ARs can cause dilation of retinal arterioles in rats, thus, increasing retinal blood flow [2-AR stimulation is mediated via a Gi protein by activation of large-conductance Ca2+-activated K+ channels [As we mentioned before, there are inconsistent results regarding the role of β-AR activation or blockade on VEGF expression and pathogenic vessel formation. While most studies reported on inhibitory effects of pharmacological β-AR blockade on VEGF formation some other studies observed blockade of VEGF formation by β-AR agonist exposure ,185,186.n in OIR . Jiang ell death . A studye retina . Furthery models . Xamotere medium . Studiesood flow ,208. Morchannels .3-ARs were shown to be involved in the neovascularization processes of various retinal vascular diseases [3-ARs were upregulated in response to hypoxia in an OIR mouse model with dense β3-ARs immunoreactivity in engorged retinal tufts, suggesting that activation of β3-ARs is likely to constitute an important part in pathologic angiogenesis [3-AR antagonists, L-748,337 and SR59230A, downregulated retinal VEGF release in hypoxia via modulation of the nitric NO signaling pathway [3-AR agonist, CL316243, was shown to reduce retinal damage following intravitreal injection of N-methyl-D-aspartate (NMDA) in rats [3-ARs, which differ from β2-ARs with regard to a lack of GRK, are resistant to agonist-induced desensitization [3-AR appears to be an attractive therapeutic target for the treatment of ischemic retinal diseases.βdiseases . For exaogenesis . It has  pathway . Moreove in rats . Furthertization ,212. Tak2-AR on agonist-mediated vascular desensitization, suggesting that the arginine at position 16 (Arg16) polymorphism (the substitution of glycine for arginine) of the β2-AR is associated with enhanced agonist-mediated desensitization in the vasculature [Notably, β-AR genes have genetic polymorphisms caused by mutations in the gene promoter, leading to changes in the expression of receptors and the regulation of signal transduction ,213,214.culature . However1-ARs play a potential physiological and/or pathophysiological role in the regulation of retinal vascular tone, retinal α1-adrenergic vasoconstriction is largely masked by endothelial mechanisms under physiological conditions and becomes more relevant when the endothelium is damaged. Therefore, α1-AR signaling pathways may represent therapeutic targets primarily in the context of retinal pathologies associated with impaired endothelial function. However, there are inconsistent findings regarding the subtype mediating adrenergic vasoconstriction in retinal blood vessels. While Mori et al. found that α1A- and α1D-ARs mediated vasoconstriction in vivo in the rat retina, Gericke et al. reported that the α1B-AR-mediated adrenergic vasoconstriction in the isolated mouse retina [1-AR subtype mediates vascular responses in the human retina. However, the lack of highly specific pharmacological ligands and antibodies for individual α1-AR subtypes hampered the progress in this research area so far. From a clinical point of view, subtype-selective agonists and antagonists would constitute a therapeutic approach to specifically influence retinal perfusion.Although αe retina ,84. It r1-ARs in general and of the three α1-AR subtypes in particular within the architecture of retinal vessels is still elusive but may be indicative of their vascular function(s) and relevant for their pharmacological accessibility. The endothelial mechanisms involved in masking retinal α1-adrenergic vasoconstriction have not yet been identified. In retinal diseases associated with endothelial dysfunction, the actual pathogenic contribution of α1-AR-mediated vasoconstriction remains an open question.It should be emphasized, however, that many issues remain poorly understood and need further investigation. The location of α2-AR agonist, brimonidine, in the retina [2-ARs [Various animal experiments and cell culture studies revealed neuroprotective effects of the selective αe retina ,150,216.a [2-ARs . In conta [2-ARs . In a pia [2-ARs . However1- and β2-AR subtypes, is the only β-AR antagonist that has been tested in clinical trials, so far [3-ARs appear to be involved in pathogenic vessel formation in the ischemic retina. Hence, future studies are needed to explore β3-AR ligands in human ischemic retinal diseases.Propranolol, which has a high affinity to β, so far . Propran, so far . However, so far . Based o1-, α2- and β-ARs in the retina of various species, including humans. α1-ARs function as stimulatory receptors involved mainly in the contraction of vascular smooth muscle, resulting in vasoconstriction. In contrast, α2-ARs are primarily expressed in neurons and glia. Its pharmacological activation was shown to lower IOP and to induce neuroprotective effects in the retina. β-ARs are expressed in blood vessels, neurons and glial cells, where they were shown to contribute to the regulation of vascular diameter and to responses to hypoxia. The results of numerous in vitro and in vivo studies suggest that these retinal receptors are functionally active and, hence, may constitute potential targets for the treatment of several retinal diseases.In conclusion, the studies reviewed in this article provide evidence for the presence of α