The objective of this study is to introduce the application of augmented reality (AR) navigation system developed by the authors in recontouring surgery of craniofacial fibrous dysplasia. Five consecutive patients with craniofacial fibrous dysplasia were enrolled. Through three-dimensional (3D) simulation, a virtual plan was designed to reconstruct the normal anatomical contour of the deformed region. Surgical recontouring was achieved with the assistance of the AR navigation system. The accuracy of the surgical procedure was assessed by superimposing the post-operative 3D craniomaxillofacial model onto the virtual plan. The pre-operative preparation time and operation time were also counted. In all patients, AR navigation was performed successfully, with a mean ± SD of the errors of 1.442 ± 0.234 mm. The operative time of the patients ranged from 60 to 80 min. The pre-operative preparation time was 20 min for each patient. All the patients showed uneventful healing without any complications, in addition to satisfaction with the post-operative aesthetics. Using our AR navigation system in recontouring surgery can provide surgeons with a comprehensive and intuitive view of the recontouring border, as well as the depth, in real time. This method could improve the efficiency and safety of craniofacial fibrous dysplasia recontouring procedures. Although it is a benign bone lesion, fibrous dysplasia results in destructive functional and aesthetic outcomes when the craniofacial skeleton is involved3.Fibrous dysplasia, first described by Lichtenstein in 1938, is defined as an abnormal replacement of normal bone with fibro-osseous connective tissue4. Surgical recontouring, as a minimally invasive procedure, is the preferred treatment7. However, it is very challenging to successfully achieve the desired results intraoperatively depending on visual exposure and the surgeon's own assessment and experience. At present, although many authors have studied the use of three-dimensional (3D) printing technology, surgical simulation and navigation techniques to assist with the process of surgical recontouring and have achieved some promising clinical effects12, there are still some difficulties. For example, it is difficult to measure the volume of the lesions to be removed during the recontouring process in real time4. In addition, the feedback of important neighbouring structures  in the lesion area is usually achieved through repeated intraoperative visual evaluation, while lesions often obscure normal anatomy, making vision calculations unpredictable. Moreover, intraoperative instruments cannot be accurately controlled to operate within the lesions to be removed. Therefore, improper surgical recontouring is likely to lead to unsatisfactory clinical outcomes.Restoration of facial symmetry and normal function is considered a treatment principle for craniofacial fibrous dysplasia13 carried out the Le Fort I osteotomy under the guidance of a video see-through AR system. Gao et al.14 completed a study of mandibular angle split osteotomy assisted by an AR system. However, the application of AR navigation to correct craniofacial fibrous dysplasia has not yet been reported in the literature. In our work, we developed an AR navigation technique for recontouring craniofacial fibrous dysplasia to overcome the aforementioned limitations. The technique combines virtually planned and real images into one prospect, enabling the viewing of the imaging, surgical field, and additional information such as the location of the surgical instrument in real time and improving the visualization of every possible narrow space16.With the continuous development and innovation of digital medicine, augmented reality (AR) has been gradually applied in the medical field and has gained much attention in recent years. Badiali et al.In this clinical series, we describe the application of our AR navigation system in five consecutive patients diagnosed with craniofacial fibrous dysplasia undergoing surgical recontouring. Accordingly, the objective of the current study was to investigate the feasibility of using AR system to enhance the surgical outcome measured by increasing accuracy and decreasing or minimizing errors.Five patients (3 men and 2 women) with unilateral craniofacial fibrous dysplasia were admitted to the Department of Oral and Craniomaxillofacial Surgery at the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, from January 2020 to June 2020. The average age was 29 years old (range 18–54 years old). The patients’ characteristics and demographics are thoroughly described in Table All medical practices followed the Declaration of Helsinki on medical protocols. This study was approved by the Shanghai Ninth People’s Hospital Institutional Review Board . All methods were performed in accordance with the relevant guidelines and regulations of our hospital. Informed consent was obtained from all the participants. Informed consent has been obtained to publish the information and images in an online open-access publication.AR-assisted facial recontouring of patients was performed by applying the following workflow, which was implemented by surgeons and biomedical engineers, as shown in Fig. 17. Cranial anatomy was assessed in 3D views and multiple planes . The normal anatomical contour of the deformed region was mimicked from the normal contralateral side through the mirroring function based on the median sagittal plane as a reference to determine the amount of bone lesion to be removed.A computed tomography (CT)  scan was performed pre-operatively, and then the CT images were configured in Digital Imaging and Communications in Medicine (DICOM) format and imported into planning software  to generate a 3D craniomaxillofacial model. For a more accurate placement of the teeth, the dental stone model was scanned with a high-resolution laser surface scanner , yielding a digitalized dental model that can then be merged into the 3D skull model. At this point, we obtained a computerized merged model interpreting highly accurate dental and bony elementsThe setup of the AR system consisted of different essential parts for preparation of an integrated image: Head Mounted Display , workstation and optical tracking system . Figure A digital reference frame (DRF) firmly fixed to the patient's craniofacial skeleton can be tracked by the optical tracking system and the Head Mounted Display camera, and the surgical drill with another clamped DRF can be tracked by the optical tracking system. The workstation is used to process and update the data of the surgical drill and the patient's craniofacial skeleton’s 3D location and to transfer the data to the Head Mounted Display. The Head Mounted Display mainly played a role in visualizing the hologram, and the 3D virtual planning and the position of surgical drill are displayed through the Head Mounted Display for an intraoperative guidance.After the registration step, the surgeon performs the surgery wearing the Head Mounted Display. Following the exposure of the dysplastic zygomaticomaxillary bone, the dysplastic bone was shaved using the surgical drill. With the assistance of the AR navigation system, the surgeon can directly visualize the craniofacial skull and lesion images, surgical field and additional information such as the position of the surgical drill in real time through the head mounted display Fig. . More imWhen the drill is still at a large distance from the designed surface (> 1 mm), the model is rendered in green, and real-time data will be displayed;When the drill is about to reach the designed surface (≤ 1 mm), the model is rendered in red, and real-time data will be displayed.The different stages of intraoperative real-time guidance are as follows:CT scanning was performed 3 days following surgery. The accuracy of the surgical procedure was assessed by superimposing the post-operative 3D craniomaxillofacial model onto the pre-operative virtual plan. The detailed evaluation procedure is as follows:The pre-operative virtual plan and post-operative 3D craniomaxillofacial models are aligned in a common coordinate space. A program was designed using VTK  to align the models. By selecting the paired points on the unchanged region of the skull, the planned model’s coordinate space was transformed into that of the post-operative model via point cloud matching. Next, both models were imported into 3D analysis software  to analyse the superimposed discrepancy. Hundreds of pairs of points were generated uniformly in the surgical area, and the position of each point was obtained, as shown in Fig. Preoperative virtual planning and AR techniques were successfully achieved in all the patients included in the study. Consistency was obtained through superimposition of the post-operative 3D craniomaxillofacial model into the pre-operative virtual plan. The minimum error range of all patients in the anterior of the zygomatic maxillary complex was 0.0053–0.2901 mm. The maximum error range posterior to the zygomatic maxillary complex was 2.4081–3.0903 mm. These findings are consistent with the fact that it is more difficult for the surgeon to recontour posterior lesions of the zygomatic maxillary complex than anterior lesions. The mean ± SD of the error across all patients was 1.442 ± 0.234 mm, indicating that the application of an AR-based navigation technique-assisted craniofacial fibrous dysplasia recontouring system can accurately achieve pre-operative virtual planning and meet clinical requirements , with little variability. The preliminary results indicated that AR-assisted craniofacial fibrous dysplasia recontouring can accurately achieve pre-operative virtual planning and is feasible for clinical application. The minimum error range of all patients in the anterior of the zygomatic maxillary complex was 0.0053–0.2901 mm. The maximum error range posterior to the zygomatic maxillary complex was 2.4081–3.0903 mm. These findings can be explained by the fact that the surgical instrument located posterior to the zygomatic maxillary complex interferes with the corner of the mouth during the recontouring procedure, which makes it more difficult for the surgeon to operate posteriorly than anteriorly. In addition to the errors caused by the operation itself, the registration accuracy and tracking accuracy may be other important factors affecting the system accuracy. Therefore, the DRF fixed to the patient's forehead must be firm and reliable.This study introduces the application of an AR navigation system in recontouring craniofacial fibrous dysplasia surgery, which can enable surgeons to better control the operation process, effectively improve surgical efficiency and safety, reduce patient trauma and shorten the operative time. However, the current study still has some limitations. First, the current report was designed to introduce a new technique with the AR system developed by the authors; as such, the sample size and follow-up period should be increased to better analyse the outcomes, which is the issue of upcoming future research in our group. Second, prospective longitudinal clinical studies are needed to obtain more valid results. Third, considering the impact of AR application on surgeons, overly use of AR may cause visual shortness, dizziness, and mental fatigues.Using our AR navigation system in the contouring procedure for craniofacial fibrous dysplasia can provide surgeons with a comprehensive and intuitive view of the recontouring border and its neighbouring anatomical structures, as well as the depth for the recontouring procedure in real time. This method could improve the efficiency and safety of the procedure and shorten the operative time. In summary, we believe that AR navigation is an important potential technology for craniofacial fibrous dysplasia recontouring.


ANOVA showed a high correlation (0.90) with a decrease in the number of crashes and time required. It was meaningful to users when performing rehabilitation training more than four times and significantly impacted the analysis. This study analyzed the upper limb and cognitive rehabilitation of able-boded people in three-dimensional space in a virtual environment; the performance difficulty could be controlled through variations in rehabilitation models.With aging, cerebrovascular diseases can occur more often. Stroke cases involve hemiplegia, which causes difficulties in performing activities of daily living. Existing rehabilitation treatments are based on the subjective evaluation of the therapist as the need for non-contact care arises; it is necessary to develop a system that can self-rehabilitate and offer objective analysis. Therefore, we developed rehabilitation tools that enable self-rehabilitation exercises in a virtual space based on haptics. Thirty adults without neurological damage were trained five times in a virtual environment, and the time, number of collisions, and coordinates were digitized and stored in real time. An analysis of variance (ANOVA) of the time and distance similarity changes revealed that as the number of rounds increased, no changes or increases occurred ( Paralysis is a state in which the nerves innervating the muscles lose their function, without changes in the form, resulting in limb numbness or the inability to move. Paralysis can be divided into sensory paralysis and motor paralysis, which occur when there is a disorder in any part of the exercise path from the motor center of the brain to the peripheral nerve and muscle fiber. Upper limb paralysis can result from different causes of brain damage, of which stroke is the most common . Fifty pSpecifically, haptic and virtual reality-based rehabilitation therapy maximizes the user’s sense of immersion within a low-cost and safe environment, allowing users to perform self-rehabilitation exercises ,31. In ahttp://newsite.fittofunctiontherapy.com (accessed on 1 April 2021)) form (Range of motion (ROM) is a method used by clinicians to check the health condition of the joint and improve the range of motion, affecting the qualitative characteristics of active motion . The dou1)) form  used in The coaching system, used video-based motion recognition and motion tracking, and provided subject feedback. Haptic is a coaching device used for virtual reality or remote rehabilitation and is detected by a sensor . In addix, y, and z coordinates were obtained in real time, and the user’s position was tracked within a three-dimensional (3D) virtual space using the OpenHaptics-based Chai3D API written in C++. A 3D model rendered in a virtual space creates a mathematical model that can be reproduced in space. A 3D object is represented by a 3D line through the rendering process and can have a texture and volume similar to those of a real object. The 3D model was created using the Auto Desk Inventor   haptic device  for translational motion was used. There are a maximum of 3DOF joint motions in the body ,44. The CA, USA) .Based on the algorithm presented in Three preprocessing steps were applied to identify changes in performance through the location coordination of the rehabilitation system, which was applied five times: (i) a principal component analysis (PCA); (ii) normalization; and (iii) warping.High-dimensional data processing makes it difficult to properly express the meaning of the data owing to the exponential increase in observation and memory usage, which is called the curse of dimensionality . A PCA bSubsequently, the category of the data was transformed by Equation (2) into  to minimize data duplication and reduce its impact on the relative size ,50.(2)NThe lengths of the normalized datasets were bound to vary from one person to another. A 1:1 convergence is difficult when vectors differ in length; therefore, the sequence of the two time series to be compared was intended to be matched. Here,  and (4) .(3)W=w1In this study, the Euclidean distance (ED) and Manhattan distance (MD) were used to analyze the similarity through the distance between two vectors expressed in n-dimensions to observe the difference from the first results of the same person after using the rehabilitation system five times. In addition, we analyzed the similarity with the dynamic time warping (DTW) method, which is a distance-based similarity measurement commonly used in a time series. It is also intended to analyze whether the two vectors have a linear relationship through cosine similarity (CS) and correlation coefficient (CC), which are methods of measuring the similarity based on the angles that they form. The similarities were analyzed using MATLAB software. We wanted to verify the hypothesis by setting it as null and void the idea that the change in performance will increase or be equal to the number of applications. The confidence interval of the ANOVA was set at 95% using Minitab software ; it was used to verify the hypothesis of the distance similarity analyzed earlier and the significant difference between the number of collisions and the time required in the experiments conducted by 30 subjects. Tukey’s post hoc test was used to analyze the correlation between the number of crashes and the time required to complete the course.This study was conducted after receiving approval from the Research Ethics Review Committee(1040875-202009-BM-068) and 10 min of prior explanation of experiments, consent descriptions, etc., to the participants. Experiments were performed five times every 30 s and took a total of 15 min. A total of 30 healthy people in their 20 s (22.6 ± 2.37 years) who were not diagnosed with neurological impairment participated in this study. Before starting the training program, the participants were verbally instructed to move from the entrance to the exit. The time taken to complete the course and crash records were not mentioned.The ED, a measure well suited to the meaning of the actual distance, is a measure of the shortest direct route election between two vectors using Equation (5) ,53.(5)EThe MD is also called a taxicab geometry and is the concept of substituting coordinates for cities. In other words, the idea is that, in real cities, when moving from the start point to the arrival point, the shortest distance must be recalculated based on the roads and any blocks because they are not accessible, similar to calculating ED . These eDTW is used to compare the similarities between two time-series patterns at different speeds . By matcThe CS is the similarity of two vectors, which can be obtained using the cosine angle between two vectors, with a value of 1 if the two vectors are in the same direction and milarity . The expThe CC, an indicator of the degree and direction of the relationship between variables, is expressed as a value between ned as − ,59. WhenDatasets preprocessed using PCA were preserved at an average rate of approximately 99.7%.x-axis travel path of the haptic probe. y-axis, and z-axis. x, y, and z-axes identified by the directions of the arrows and numbers indicated in x-axis of the probe in the first row, the y-axis of the probe in the second row, and the z-axis in the third row are identified. Movements were unstable until the third rehabilitation session, and the direction changed dramatically and tended to be overly skewed in the on-direction. However, the number of crashes in four and five rehabilitation sessions decreased with repeated learning.x-coordinates can show the characteristics of tremors in which the subject approaches or moves away from the subject in the model when applying the system. The y-coordinates are from the side of the moving position from start to end, and the z-coordinates can show the characteristics of the upward and downward tremors in the model.In the pre-processed dataset, the x-axis is greater than that of the y-axis, for which it is believed to be more difficult to recognize the depth of the space than the height. In addition, for an angle-based similarity, the average was high , and the correlation was strongly positive  [.: 0.98) , indicatp = 0.00), there was a large difference in the number of collisions (For the number of collisions showing significant differences (llisions . The resllisions .p = 0.32)  . However = 0.32) .p > 0.05). The x-axis requires spatial recognition horizontal to the ground, whereas the scatter size of the y-axis, which indicates the total travel path in space, and that of the z-axis, which requires vertical spatial recognition, shows that the perceived tremor values are concentrated and that the deviation of the target is insignificant.Motion paralysis occurs when there is a disorder in any part of the movement path from the motor center of the brain to the peripheral nerves and muscle fibers. Among them, upper limb paralysis is the most common disorder after a stroke, for which various treatment methods have been proposed, including rehabilitation exercise therapy through initial collaboration with rehabilitation medicine to prevent future effects and help relieve paralysis symptoms and restore motor function through repeated rehabilitation exercises . HoweverVirtual reality allows the input and output of information from various objects in a three-dimensional virtual space, and reverse-tactile information helps to realistically recognize various environments and increase user immersion. Moreover, virtual reality technology is in the spotlight for applications in rehabilitation because it can create individual environments and perceptual stimuli that motivate patients while preserving the potential to interact with real environments, including the patients . HoweverIn this study, a total of five rehabilitation evaluations were conducted using subjects in their 20 s (22.6 ± 2.37 years) with no neurological damage at intervals of 30 s. Subsequently, the hypothesis was verified after visualizing the change in the time and number of collisions and the distance similarity using the data stored in real time.x, y, and z were significantly reduced in the fifth round compared with the first round. At this point, the x-axis shows the movement of the haptic probe of the subject inside and outside of the target, the y-axis shows the path from beginning to end, and the z-axis shows the characteristics when the haptic probe moves up and down. The movement in and out of a space indicates that the depth of the space is level with the ground and differs from subject to subject based on use. Conversely, the z-axis shows the difference between vertical surfaces within a space. It can be interpreted that the abnormal values shown in y-axis with which the path of movement can be viewed is ideal to move from left to right, but if reversed by a collision during rehabilitation, if the space of the curve is not understood and if the understanding of the time of termination is insufficient, a zigzag path will appear. Thus, as shown in x-axis. The interpretation of the x, y, and z-axes is also associated with hypothesis verification. As the number of uses increases, the cognitive skills of the user in the space are improved, particularly when rehabilitation tools are applied more than four times. Over time, however, the null hypothesis was not rejected, resulting in a significant or equal change in time. This means that there was time variability in each subject of the study. The results in As shown in x, y, and z-axes were not constant, the mean increased compared to the first and fourth rounds on the x-axis, meaning that there would be a significant change in the spatial recognition of the inside and outside when the rehabilitation was repeated four times. The y-axis decreased until four iterations and then increased after five iterations. In addition, given that the path was different from the first time when was repeated five times, the opposite situation on the ideal path was reduced by the fifth times, which means that the opposite situation (zigzag movement) on the ideal path was reduced, and the values were concentrated owing to the small spread. Finally, as the results of the z-axis indicate, the mean increased when compared with the first and third rounds and the first and fifth rounds, which means that there was a significant change in the spatial cognitive abilities above and below when the process was repeated three times and five times. In conclusion, the changes in the number of collisions, time, and distance similarity can produce meaningful results in at least four training sessions and can be analyzed.In addition, the number of collisions can be described as a result of a change in distance similarity depending on the number of rounds compared to the beginning, which is the result of a tremor. An example of the change in the distance of a vector shows that the electron has a large rate of change in its distance between each point, compared with two vectors having the same start and end points and two vectors having the same shaking condition. As a result of this study, the null hypothesis that there will be the same large difference between the two vectors was adopted in In this study, compared to subjective evaluations of therapeutic performance by rehabilitation specialists and rehabilitation therapists, experimental measurements of the resistance, track speed, and location error due to unnecessary forces allow for the digitalization of data for more objective performance and treatment evaluations. Existing rehabilitation methods have shown that physical therapy conducted based on a therapist’s subjective judgment can be analyzed and evaluated objectively through haptic-based rehabilitation receptors called muscle spindles and Golgi tendon organs . TherefoBased on the double-curved shoulder arc form, which is rehabilitation tool to check an active movement, we developed a tool to analyze self-rehabilitation exercise through haptic device and rehabilitation change processes. As a basic study analyzing the three-dimensional cognitive ability of the rehabilitation environment applied in a virtual world by selecting an able-bodied person as a target, it was possible to visually identify the changes in time, number of collisions, and distance similarity according to the rotation, and it was found that a meaningful analysis is possible only after at least four training sessions. The results of this study indicate that, the level of difficulty in terms of performance is thought to be controllable through a future transformation of the rehabilitation model, and the approach can be used in occupational therapy. In addition, as a basis for the Rood approach, it is possible to provide repetitive feedback and accumulate data accordingly, which can be utilized as big data. Furthermore, the system will be used in clinical and rehabilitation sites to manipulate the perceived use, perceived case of use variables in the technology acceptance model, and external variables to analyze the interaction with technology, user attitudes, and behavior.


The pathogenesis of allergic rhinitis is associated with genetic, environmental, and epigenetic factors. Genotyping of single nucleotide polymorphisms (SNPs) is an advanced technique in the field of molecular genetics that is closely correlated with genome-wide association studies (GWASs) in large population groups with allergic diseases. Many recent studies have paid attention to the role of epigenetics, including alteration of DNA methylation, histone acetylation, and miRNA levels in the pathogenesis of allergic rhinitis. In this review article, genetics and epigenetics of allergic rhinitis, including information regarding functions and significance of previously known and newly-discovered genes, are summarized. Directions for future genetic and epigenetic studies of allergic rhinitis are also proposed. The prevalence of allergic rhinitis (AR) has been on the increase worldwide. The frequency of hypersensitive reactions to multiple allergens has increased. Over 40% of populations in the United States and Europe suffer from allergies . AR contAR is characterized by sneezing, nasal itching, airflow obstruction, and watery rhinorrhea resulting from IgE-mediated responses to inhaled allergens and type 2 helper T (Th2) cell-induced mucosal inflammation . It is cThe pathogenesis of AR and other allergic diseases is complicated. Associations with genetic and environmental factors are known to be involved in these immunologic situations . EnvironSLFN12 gene when exposed to grass pollen [Many recent studies have paid attention to the importance of epigenetics . It is as pollen . HistoneWith the advance of biomedical techniques in the field of molecular genetic science, approaches to elucidating the genetics of allergic diseases have also evolved. In 2002, genome-wide genotyping was introduced, allowing genotyping of hundreds of thousands of single nucleotide polymorphisms (SNPs) and simple variants, such as insertions/deletions and repeats. The opportunity to genotype markers has increased discernment of the genetic structure of humans, revealing that SNPs are closely correlated with each other . This haSeven loci significantly associated with AR have been identified through genome-wide association studies (GWAS). Many other loci have also been identified through GWAS of related allergic diseases, such as asthma, atopic dermatitis, and allergic sensitization ,19,20,21AR is one of the most common atopic diseases worldwide. The heritability of AR has been estimated to be over 0.65, indicating a strong genetic component . Twin stORMDL3/GSDML, IKZF3, and ZPBP2, have been reported to be associated with childhood asthma [ORMDL3/GSDML variants are associated with a high risk of asthma in diverse ethnic groups [ORMDL3 in lymphoblastoid cell lines. Another large-scale GWAS for asthma has identified six asthma-susceptible loci, including IL1RL1/IL18R1, SMAD3, ORMDL3/GSDM, IL-33, IL2RB, and HLA-DQ [CDHR3 and ORMDL3 being the loci associated with induced eosinophilia and Th2 inflammatory or viral responses [Asthma concordance for identical twins is about 50% . The herd asthma . Subsequc groups ,33,34,35esponses ,36. ThymIL1R1, IL1RL1, IL13, SLC25A46, IL18R1, GSDMB, SMAD3, and HLA regions can influence allergic sensitization [SLC25A46 and TMEM232 encode transmembrane proteins involved in solute transport. SMAD3 is related to inflammation progression due to immune responses of T-helper 2 (Th2) cells and immune-related cytokines [C11orf30 is involved in increased total serum IgE levels, with higher susceptibility to poly-sensitization [D2HGDH and GAL3ST2 encode D-2 hydroxyglutarate dehydrogenase enzyme-related metabolism and tumor metastasis-related galactose-3-O-sulfotransferase 2 enzyme, respectively [For asthma, several asthma-associated loci, including tization . In bothytokines . C11orf3tization ,39. D2HGectively ,41.Fine-mapping of IgE-associated loci 1q23, 5q31, and 12q13 confirmed the associations of SNPs in 1q23, 5q31, and 12q13 with IgE regulation. For 1q23 and 5q31, the majority were associated with mild to moderately elevated IgE levels, while in the 12q13 locus, single-nucleotide polymorphisms (SNPs) were found to strongly elevate IgE levels. In 1q23, 5q31, and 12q13 loci, SNPs were found to correlate specifically with atopic asthma .Genetic association studies have identified about a total of 50 genes, many of which have been identified by linkage and gene mapping. Effect estimates are measured as odds ratios ranging from 0.5 to 1.5. These gene variants can predict less than 10% of the heritability of asthma .The concept of united airway disease appears to be due to the close relationship and overlapping genetic characteristics involving AR and asthma. The pathogenesis that involves respiratory epithelial cells and the inflammatory response of AR and asthma are similar . AlthougC11orf30/LRRC32 on chromosome 11 is a significant locus associated with both AR and asthma [Genes and cytokines including IL3, IL4, IL5, IL13, IL33, and GM-CSF are immune-related and involved in susceptibility to AR . C11orf3d asthma ,46. Expod asthma ,48.ZBTN10 and CLEC16A were significant [NFIA) gene in the 1p31 chromosomal region is significantly associated with combined asthma and AR phenotype [IL4-KIF3A in patients with AR and asthma [In a GWAS study, eleven significant variants associated with the risk of asthma with rhinitis were identified, of which nificant . A genethenotype ,50. The d asthma ,52.HLA-B and HLA-DQB1 were identified as the strongest associated HLA class genes [Most of the shared genes are associated with inflammatory processes. Related genes or cytokines, including IL4, IL4R, IL5, IL13, TSLP, IGHG4, and FCER2 are involved in Th2 cell immune response with specific functions. FCER2, expressed in macrophages, eosinophils, B cells, and platelets, regulates IgE responses and is involved in antigen presentation, differentiation of T and B cells, and cellular adherence ,54. It iss genes . In GWASss genes .SDAD1, CXCL10, CXCL9, RANTES, CXCL11, IL1R1, IL13, IL18, IL21/IL2, IL23R, IL12RB1, IL27, C11orf30, SMAD3, TLR1, GATA3, and HLA-DQ.In AR studies, many genes are involved in diverse immune-related diseases, including allergic and autoimmune disorders. These genes include BCAP on chromosome 10q24.1 and MRPL4 on chromosome 19p13.2 were commonly associated with AR and atopy in a GWAS of a Chinese cohort [HLA-DQ and NPSR1 loci, were also replicated. The 19p13.2 locus regulates allergic reactions by affecting soluble intercellular adhesion molecule 1 [TNF-α gene are also known as a high risk factor of AR [C11orf30 and LRRC32 have genome-wide significance. C11orf30 encodes a protein with epithelial barrier function [LRRC32 encodes a surface receptor of T cell for latent TGF-β and plays an important role in immune tolerance by regulatory T-cell function [FERD3L on chromosome 7p21.1 is significant in a genome-wide meta-analysis for all ethnic populations [e cohort . Linkagelecule 1 . SNPs inor of AR . A largefunction . LRRC32 function . These tfunction ,46. A loIL7R at 5p13.2 that is previously associated with eczema [SH2B3 on chromosomal 12q24.12 is related to blood eosinophil count [CEBPA have been previously linked to eosinophilic esophagitis [CEBPG plays a role in an enhancer binding protein for IGH, IL4, IL6, and IL8 [Recently the largest GWAS has found 41 significant risk loci of AR, including 20 novel loci . Most ofh eczema  and invoh eczema . SH2B3 oil count  and inflil count . A locusphagitis  and assophagitis , respect and IL8 .CXCR5 encodes a B-cell chemokine receptor and follicular T-cells. It functions in the migration of B-cells and helps interactions between B- and T-cells within lymph nodes [FCER1G at chromosomal 1q23.3 encodes γ chain of IgE receptor, which is an essential component of allergic responses. NFKB1 at 4q24 encodes a subunit of the NFκB complex by functioning in the activation of inflammatory pathways [BACH2 at 6q15 plays an immune-modulating role in the production of memory B- and T- cells induced by antigen [LTK and TYRO3 at 15q15.1 can regulate Th2 immunity and TLR signaling, respectively. SPPL3 and OASL at 12q24.31 are required for mature NK cell and IFN-αsignaling, respectively. RORA at 15q22.2 plays a role in the development of Th2 innate lymphoid cell and inflammation process. TNFSF11 at 13q14.11 functions in T-cell activation by dendritic cells. VPRBP at 3p21.2 has roles in T-cell proliferation after antigen recognition. It is involved in V(D)J recombination during B-cell development [Most of the additional novel loci have known immune-related characteristics. A locus at 11q23 near ph nodes . FCER1G pathways . BACH2 a antigen ,76. LTK elopment ,79,80,81Studies conducted on patients with AR and immune cells isolated from patients with AR have shown that histone deacetylase (HDAC) is increased in immune cells and that inhibition of HDAC can help improve the condition of AR. A study of patients with AR has shown that HDAC1 is upregulated in the nasal epithelial cells compared to that in healthy controls . InterleStudies have suggested that changes in DNA methylation might differentiate allergic patients from the healthy people. Patterns of DNA methylation are correlated with patterns and numbers of CD4+ T cells in AR. A study has revealed that allergic children have changes of DNA methylation at CpG sites in blood mononuclear cells and airway epithelial cells . It has MicroRNAs (miRNAs) are small (20–25 nucleotides) non-protein coding RNA molecules that regulate gene expression by cleaving and silencing the target transcript and inhibiting the post-transcriptional process. With the premise that regulation of miRNA-mediated gene expression is one of the epigenetic mechanisms , there hMiR-29a is highly expressed in the nasal mucosa of AR patients. It can suppress nasal epithelial cell apoptosis and enhance the development of AR by down-regulating FOS expression . CirculaOther studies have also shown that highly expressed miR-221 and miR-142-3p on nasal mucosa can be biomarkers for AR by promoting mast cell degranulation and reinforcing the degranulation of mast cells, respectively ,104,105.CDK2AP1 and C12orf65. Many genes including CDK2AP1, ABCB9, ARL6IP4, C12orf65, SBNO1, SNRNP35, MPHOSPH9, OGFOD2, PITPNM2, RILPL2, and SETD8 function with DDX55 as enhancer-promoter in various immune cells of blood and lung tissues. However, none of these genes has any obvious AR-related features. Many other related genes, such as NEGR1 at 1p31.1, RERE at 1p36.32, FOSL2 at 2p23.2, and JAZF1 at 7p15.1, are similar in blood and lung tissues [Despite recent advances in GWAS, which may provide more information regarding disease etiology, certain limitations remain in that individual effect and possible interactions have not been resolved. Pinpointing causal SNPs can be challenging due to linkage imbalance, which can span considerably more than one gene. In addition to genetic factors, many other factors can determine treatment responses. Difference in treatment responses between individuals may contribute to the clinical course of AR and treatment responses. They might be caused by diverse factors, such as tissue-specific epigenetic influences and environmental factors, such as smoking and allergen exposure, that can potentially interact with genetic factors. This provides a basis to investigate how eosinophil targeting antibody to novel gene- based cytokines might be correlated with modifications to DNA methylation in eosinophils and pre-treatment epigenetic profiles. Epigenetic profiling could also yield future potential biomarkers that could aid in the classification and selection of patients who would most benefit from potential biological therapies and for whom little improvement could be otherwise expected. Although many target genes involved in AR have been identified recently, how medical science can use this information for the development of novel drugs or repurposing existing drugs remains unclear. Recent findings of genetic variants using genomics-based drug repositioning may provide promising results for AR patients. However, a number of novel AR loci have unknown functions in the pathogenesis of the disorder. Associated loci at 12q24.31 are between  tissues . Future Currently main pharmacotherapy for AR includes oral/nasal anti-histamines, intranasal corticosteroids, leukotriene antagonists, anti-cholinergics, anti-IgE monoclonal antibodies, and mast cell stabilizers. These medications mainly aim at reducing symptoms related to AR. Limitations of these currently used pharmacotherapies are that the lasting time of effects is short and symptoms begin to appear when stopping medications . Among cEpigenetic studies began with the goal of controlling the long term production of cytokines and T cell proliferation . It has SMAD3 and TGFβ2 [Epigenetic controls function as resistance to self-antigens, regulating immune responses. Epigenetic dysregulations can lead to immune disorders . An epignd TGFβ2 . These fAlthough still in its infancy, allergic disease-associated SNPs are linked to potential drug targets. Main SNPs were associated with several gene transcripts. It may be due to functional regulation of several genes by one SNP or disequilibrium at a high linkage level. Coding mutations can alter amino acid sequences, resulting in changes in protein function. These mutations result in protein truncation or production of different amino acids by mRNA splicing . ChangesWith the rapid development of genetics, we can present new drug targets and potential drug repositioning based on genomics for the therapy of AR. Drug repositioning means identifying drugs that are available in the market or in development, and repurposing them for specific uses. This method increases the efficiency of drugs and reduces time and cost due to the established safety framework .HMGCR gene that encodes the HMG-CoA reductase protein is a target for statins used to lower blood cholesterol levels. This finding is significant in that gene-targeting drugs discovered by genetic studies are more than twice as successful as drugs without genetic basis in reaching the market [GWAS can identify disease associated genes, providing a powerful technique for validating existing targets and uncovering new drug targets. Genes that encode proteins targeted by drugs already on the market have been identified by a number of genetic studies. For instance, a GWAS for dyslipidemia has revealed that the e market . Althouge market . IndividPharmacogenetics is a promising topic in the field of allergy genetics. Among them, individualized medicine is sought-after the most to identify medications that should be used for specific patients. In patients with severe allergic disease who do not respond to standard therapy, the lack of appropriate management is the main cause of treatment failure. The aim of genetic researches for allergic disease is to achieve improved therapeutic outcomes and minimize side effects by tailoring individualized therapy to specific genotypes. Using genetics, it will be possible to identify and early discriminate patients who are difficult to treat disease, allowing them to proceed with novel personalized treatments. Personalized treatment options in which specific, targeted genetic modifications are implemented are likely to play important roles in monitoring or predicting outcomes for personalized therapy .In this study, we reviewed the current status of genetic and epigenetic studies. Through expression quantitative trait locus (eQTL) mapping and coding sequence variation, we provided the latest genetic updates concerning GWAS information and AR, with an emphasis on genetic functions. Although research involving AR and asthma has made great progress to date, there remain many unexplored areas of allergic diseases among GWAS studies . Many stGenetic associations do not function equally to biological functions. Consequently, research is necessary to apply initial findings to biological insights that will further accelerate development of clinical diagnostic techniques and therapeutic applications. The final purpose of our research regarding genes implicated in AR and large scale GWAS studies on AR is to use the information of novel identified variants for diagnosis and treatment in the clinic. In the near future, using individual whole-genome sequencing to identify individual genetically at-risk patients and administer individualized medicine seems to be feasible. Future research might also include detailed genotyping of diverse ethnic populations, improving our understanding of environmental and epigenetic factors, and applying new tools using genome sequencing, epigenetics in specific tissues, and a systemic biologic approach. Systemic biology includes the aggregation of big data for integrated profiles of disease, providing better understanding of pathogenesis and disease outcome . It invoMany current GWAS studies tend to be limited to specific ethnic groups, especially Europeans. Thus, whether GWAS results can be applied more broadly to various ethnic groups remains unclear. Designing genetic studies that include racially and ethnically diverse participants with allergic diseases is needed in the future .


Self-focused augmented reality (AR) technologies are growing in popularity and present an opportunity to address health communication and behavior change challenges.We aimed to examine the impact of self-focused AR and vicarious reinforcement on psychological predictors of behavior change during the COVID-19 pandemic. In addition, our study included measures of fear and message minimization to assess potential adverse reactions to the design interventions.A between-subjects web-based experiment was conducted to compare the health perceptions of participants in self-focused AR and vicarious reinforcement design conditions to those in a control condition. Participants were randomly assigned to the control group or to an intervention condition .P=.03) and susceptibility (P=.01) when compared to the control. A significant indirect effect of self-focused AR and vicarious reinforcement on intention was found with perceived threat severity as a mediator . Self-focused AR and vicarious reinforcement did not result in higher levels of fear (P=.32) or message minimization (P=.42) when compared to the control.A total of 335 participants were included in the analysis. We found that participants who experienced self-focused AR and vicarious reinforcement scored higher in perceived threat severity (Augmenting one’s reflection with vicarious reinforcement may be an effective strategy for health communication designers. While our study’s results did not show adverse effects in regard to fear and message minimization, utilization of self-focused AR as a health communication strategy should be done with care due to the possible adverse effects of heightened levels of fear. With self-focused augmented reality (AR) usage increasing in recent years , the utiIn response to the recent increase in self-focused AR usage ,6-8, we Research in psychology suggests that heightening self-focused attention (manipulated by using a mirror or video camera) has implications for perception, affective experiences , and behavior . ObjectiResearch prototypes have explored self-focus  and selfWe present findings from an online experiment on the impact of combining self-focused AR with vicarious reinforcement, visualizing the cause and effect of risk-mitigating behavior layered onto one’s reflection. Our study took place during the COVID-19 pandemic, focusing on hand hygiene behavior as an effective measure against pathogen transmission ,17. We dVarious health behavior change models ,19 highlResearch suggests that self-focused attention can result in action consistency with behavioral standards . ObjectiResearch suggests that fear may play a large role in health behavior, especially during public health emergencies such as the current pandemic. Harper et al  found COIt is important to consider the potential negative impact of heightening fear and risk perceptions. While Harper et al  found inOur study investigated the impact of vicarious reinforcement outcome expectancy when combined with self-focused AR. Research suggests that outcome expectancy mediates the impact of self-focused attention on behavior. For someone who has been made aware of a discrepancy between “actual state” and “desired state,” if they don’t believe a suggested behavior change will result in the “desire state,” they are more likely to change the “desired state” . When thWhile studies combining self-focused attention and vicarious reinforcement have yet to see much direct utilization in human-computer interaction research, a few studies on health smart mirrors ,12,13, sWhile applications of smart mirrors for health care are limited, exploratory research prototypes have shown their potential to detect emotional states, monitor physiological parameters, and encourage behavior change. The Wize Mirror  encouragA recent study by Jung et al  used a pOne notable finding from Jung et al  was thatYee and Bailenson  found thOur study expands on existing research by investigating how health behavior self-focused AR may impact specific predictors of behavioral intentions and what negative implications may exist in regard to fear control responses.In our research, we examined the impact of self-focused AR and vicarious reinforcement on perception and emotion as it relates to hand washing health beliefs and behavioral intentions. Below, we present our hypotheses:H1: The combination of self-focused AR and vicarious reinforcement will result in higher levels of perceived positive outcome expectancy, perceived threat severity and susceptibility, fear, and message minimization when compared to a control.H2: Using avatar representations in self-focused AR with vicarious reinforcement will result in higher levels of threat severity, susceptibility, fear, and message minimization compared to a control.To study the effects of self-focused AR on behavioral intention and perception, we conducted an online experiment. Participants interacted  with a wWe conducted a between-subjects experiment where users interacted with a web application and then responded to an online questionnaire. The study followed a posttest-only control group design to avoid a testing threat to internal validity. The design of the web application differed depending on the intervention condition each participant was randomly allocated to. All five conditions displayed the same information about COVID-19, including how it is spread and preventative measures as described by the US Centers for Disease Control and Prevention (CDC)  and the In the control condition , we dispIn the reinforcement condition , the hanThe self-focus condition  utilizedThe self-focus AR × reinforcement condition  visualizIn the avatar condition , particiParticipants were recruited via Prolific (a crowdsourcing platform ) and werThe experiment took place between August 6-21, 2020. In all five conditions, after receiving consent, we described the experiment as a study on health information presentation and provided instructions to review the information given carefully. In the three self-focused-AR–based conditions, we displayed information on how to set up the web camera for the study.All conditions provided information about COVID-19. Details focused on how the virus is spread and preventative measures as described by the CDC . ParticiThe questionnaire included questions to check whether the participant is paying attention. Three multiple-choice questions asked the user about the information displayed in the study . To validate that self-focused AR interventions were delivered correctly using the camera, participants in these conditions were informed, prior to the study, that screenshots would be collected randomly throughout the animation. The screenshots were reviewed to ensure that participant’s reflections were displayed to them and that those in the self-focus AR × reinforcement condition had their hands within the view as instructed. Only those who followed the instructions, verified by screenshots, were included in the final data set.We collected measures of self-reported health beliefs, behavioral intention, and self-reported perceptions of COVID-19 , along wAlthough adapted from previous research, cross loading was a concern due to the rewording of items and the difference in factors present compared to the adapted questionnaires. For example, we added items to measure opinions about perceived threat severity and susceptibility of family and friends. To examine the validity and reliability of our measures, we conducted exploratory and confirmatory factor analysis using a split-sample approach, with one half to develop a model and the other half to validate. Factors loaded as expected .Data on demographics and the COVID-19 risk of a severe illness of loved ones were collected. Participants were asked to report their age, gender, and the state in which they currently reside. In addition, they were asked to report if they have a family member or friend who is at high risk of severe illness if they are infected with COVID-19. It was noted that one is considered high risk if they are ≥65 years and/or have underlying medical conditions.P values ranged from 6.35e-08 (efficacy) to 1.927e-25 (intention). Therefore, hypothesis testing was conducted using the nonparametric Wilcoxon-Mann-Whitney test, comparing perceived threat severity, susceptibility, outcome expectancy, fear, and message minimization scores between intervention conditions. If condition pairs had the same distribution shape, medians were compared. If the shapes were different, the mean ranks were compared. Additionally, mediation models for dependent variables and design conditions with significant findings were tested. A bootstrapping method using PROCESS macro models 4 and 6 [P<.05. Statistical analysis was performed using Python (Python Software Foundation) [Analysis of our data using histograms and the Shapiro-Wilk test showed that the data were not normally distributed. Shapiro-Wilk  4 and 6 , 5000 bondation) , and thendation)  and SciPndation)  was resondation)  was implProcedure section) and were included in the analysis. Of the 335 participants, 77 were randomly assigned to the control condition, 61 to the self-focused AR condition, 70 to the reinforcement condition, 63 to the self-focus AR × reinforcement condition, and 64 to the avatar condition.A total of 502 individuals participated in the study. Of this, 335 participants met the attention and screenshot verification checks .Although our study focused on predictors of behavioral intentions, we began with results pertaining to intention  to proviP=.43), message minimization scores of those in the self-focus AR × reinforcement condition were not significantly different. This was also the case for outcome expectancy (P=.41) and fear (P=.23)  . HoweverU=1983, P=.03). Regarding perceived threat susceptibility, median scores for the self-focus AR × reinforcement and control groups were 6.00 and 5.33 . Our results partially supported H1 regarding perceived threat severity and susceptibility; however, we did not find support for outcome expectancy, fear, and message minimization.For perceived threat severity, median scores for the self-focus AR × reinforcement and control groups were 6.25 and 6.00, respectively ; the twoand 5.33 ; the twoP=.48), threat severity (P=.39), susceptibility (P=.40), fear (P=.10), and message minimization (P=.47). Conversely, when compared to the control, those in the self-focused AR condition (without vicarious reinforcement) did not have a significant difference in medians for outcome expectancy (P=.26), threat severity (P=.21), susceptibility (P=.45), and message minimization (P=.39). Median scores for the self-focused AR and control groups were 5.0 and 5.3 .Additional analysis revealed that the use of self-focused AR and vicarious reinforcement individually did not impact measured predictors of intention, except for in the case of fear. When compared with participants in the control condition, those in the reinforcement condition (without self-focused AR) did not have a significant difference in medians for outcome expectancy . An additional model tested whether the self-focus AR × reinforcement condition affects message minimization through fear, threat severity, or threat severity. A significant negative indirect effect of the self-focus AR × reinforcement condition on message minimization was found with severity as the mediator .P=.42), severity (P=.49), susceptibility (P=.15), fear (P=.23), or message minimization (P=.17).Hypothesis 2 proposed that combining vicarious reinforcement and self-focused AR while using an avatar would result in higher levels of positive outcome expectancies, fear, perceived threat severity, perceived threat susceptibility, and message minimization when compared to a control. Compared to the control, those in the avatar condition did not have significantly different levels of outcome expectancy .P2I hadn’t thought about separately lathering and washing my thumbs.P3Comments on personal concerns about risk indicate that future work measuring these variables may consider the time spent around others vs alone and personal risk:I answered questions knowing that my husband’s and my job allow us to work from home, which decreases our risk significantly, and that most of my family lives in a rural area, also less susceptible to infection.P4I know that I take it way more seriously due to the cancer treatment drug I take than most of my friends and peers because if I get it, I am not strong enough to fight it off. I think that factors in way more than friends and family risk, at least for me personally.P5Lastly, a few participants in the self-focus AR × reinforcement group expressed confusion about the design. P7, for example, expressed difficulty in positioning themselves on the screen.It was an interesting survey, but the instruction given for the hand part was kind of hard to complete because the outline of the hands and the picture did not match. However, I tried my best to make it work.P7Found the movements in the video hard to follow along with, but I tried my best!P8The handwashing directions confused me. At first, I didn’t understand that I wasn’t supposed to mimic the exact instructions.P9The responses of P8 and P9 suggest that participants may have practiced along with the video animation. Practicing was not a requirement of participation but appeared to be a trend among those in this condition.This study explored the impact of self-focus and vicarious reinforcement design interventions on psychological predictors of behavior change during the COVID-19 pandemic. Our results showed that combining self-focused AR with vicarious reinforcement increases perceived threat severity and threat susceptibility and could potentially impact behavioral intentions.Our results did not show any direct effects on behavioral intention . Given tOur findings also revealed interesting relationships between the self-focus AR × reinforcement condition, perceived threat severity, threat susceptibility, fear, and intention. Mediation models showed the self-focus AR × reinforcement condition to positively affect intention and fear through increased perceived threat susceptibility and threat severity. We found self-focus AR × reinforcement to increase perceived threat severity through increased perceived susceptibility. Lastly, we found self-focus AR × reinforcement to indirectly affect intention with fear as the mediator. These results suggest that design strategies that layer a health threat directly on an individual’s reflection may increase one’s perceived threat susceptibility, threat, severity, fear, and indirectly behavioral intention. While such strategies might help meet behavior change design objectives, it is essential to note the potential consequences of designs that increase fear, especially in the context of a public health emergency.Based on the EPPM, Li  tested aSelf-focused AR and vicarious reinforcement embedded as features independently (versus combined) did not show a significant result on any of the tested predictors of behavior change except fear. We provide a few possible explanations for these results. First, regarding mirror self-focus, threat severity, and susceptibility, there may have been too large of a time gap between when participants reviewed the health information provided and when they looked at their self-reflection. In the conditions combining the features, there was content on the screen, reminding participants of the threat at hand. Future work may account for this difference by providing text-based information over one’s reflection. Second, regarding vicarious reinforcement and outcome expectancy, It is important to note that this research took place during a long-term global public health emergency with restrictions on lifestyles that can take a while to get adjusted to. Health perceptions related to current circumstances are subject to change throughout the lifecycle of a pandemic. Our findings warrant replication studies that consider changes in severity, government mandates, social perceptions, and the availability and range of tools for risk mitigation .The data used in this study are self-reported and susceptible to response biases, specifically social desirability bias. Due to the severity of the pandemic, government mandates, news coverage, and social discussions may have increased the pressure to respond in ways that align with social norms. Future work should aim to use methods to decrease the impact of this limitation.It is important to note that focusing on individual constructs may create an ineffective design system if the construct only works in combination with other constructs . This shWhile our study indicates that combining self-focused AR with vicarious reinforcement may affect health behavior change by influencing threat severity and susceptibility, we lack a data-driven explanation of why. Future work may benefit from the inclusion of quantitative measures for self-focused attention to compare with severity and susceptibility scores.Future work may lend itself to developing experimental methods to explore the extent to which self-monitoring, reflective thinking, self-evaluation, and emotion management naturally occur (or do not occur) when using self-focused AR. Conducting these experiments will provide deeper insights into how self-focused AR impacts the psychological mechanism related to behavior change and possibly inspire experiments on how the combination of self-focused attention and other design features could enhance this effect.According to user feedback, future work should also consider accounting for time spent around others vs alone and personal health when measuring perceived threat severity. Responses also indicate that when measuring perceptions about new messaging, participants should be instructed to respond based on their preferred news source to limit confusion related to the different opinions they hold for individual new sources.Two comments from the user feedback indicated that participants might have been actually practicing handwashing movements while viewing the animation. During the screenshot verification process, it was noted whether a participant was observed practicing along with the handwashing video. Practicing was not a requirement of participation and was not mentioned in any instructions provided to them. A total of 32 out of 63 participants were observed practicing along with the video in the self-focus AR × reinforcement condition. The self-focused AR and avatar conditions had 7 and 6 individuals observed practicing, respectively. These results cannot be used to make any claims due to the study’s technical setup. Those in the self-focus AR × reinforcement group were instructed to have their hands in view of the camera. Those in other conditions may have practiced off-camera. However, as practicing may affect behavior, this is another potential area for future research.As self-focused AR technologies grow in popularity, it is important to understand how such experiences could impact perceptions, emotions, and behavioral intentions. Previous research ,12-14,28We found that displaying germs disappearing directly from the user’s self-reflection during a handwashing animation will result in higher scores for perceived threat severity and susceptibility when compared to the control or conditions that implemented self-reflection and a display of germs disappearing separately. Increased perceived severity and susceptibility were found to increase behavioral intention. These findings indicate that combining self-focused AR with vicarious reinforcement may be an effective strategy for health communication designers. However, we also voice concern about the possible adverse effects of heightening levels of fear as a design strategy. While our study did not show concerning results, prior research indicates that heightening fear as a health communication strategy can lead to defensive reactions (versus changing behavior) ,25 and c


Androgen Receptor (AR) alterations  are master players in metastatic castration resistant prostate cancer (CRPC) progression and central therapeutic targets for patient management. Here, we have developed two multiplexed droplet digital PCR (ddPCR) assays to detect AR copy number (CN) and the key point mutation T877A. Overcoming challenges of determining gene amplification from liquid biopsies, these assays cross-validate each other to produce reliable AR amplification and mutation data from plasma cell free DNA (cfDNA) of advanced prostate cancer (PC) patients. Analyzing a mixed PC patient cohort consisting of CRPC and hormone sensitive prostate cancer (HSPC) patients showed that 19% (9/47) patients had AR CN amplification. As expected, only CRPC patients were positive for AR amplification, while interestingly the T877A mutation was identified in two patients still considered HSPC at the time. The ddPCR based analysis of AR alterations in cfDNA is highly economic, feasible, and informative to provide biomarker detection that may help to decide on the best follow-up therapy for CRPC patients. Prostate cancer (PC) remains one of the leading causes of cancer related deaths in the western world. Molecular profiles indicate that increased androgen receptor (AR) activity is one of the main drivers during PC development. Therefore, PC responds generally well to androgen deprivation therapy (ADT). However, eventually PC develops ADT resistance mechanisms, commonly via AR mutations, amplification, and splice variants . There iAR amplification correlates with castration resistant prostate cancer (CRPC) and is found in approximately 20–60% of PC recurring during ADT while very rare in primary PC (<1%) ,4,5,6,7.AR mutations, especially if affecting exon 8 of the AR gene and the ligand binding domain (LBD) of the AR protein, can modulate ligand and cofactor affinity, and result in gain-of-function through increased sensitivity to other steroid ligands   or the cIt is generally difficult to obtain advanced PC tissue biopsies for biomarker evaluation, in part due to anatomical locations of metastases such as in the bone. Liquid biopsy is emerging as a surrogate non-invasive and economic resource for biomarker detection with high potential, as repeated liquid biopsies are highly feasible and allow longitudinal monitoring of biomarker changes during disease progression. There is also evidence that liquid biopsy may overcome the bias of inter- and intra- tumor heterogeneity and sample accessibility, especially in the advanced PC setting . In addiHere, we developed multiplexed ddPCR assays to analyze AR copy number (CN) and the most common AR T877A mutation using cell free DNA (cfDNA) isolated from plasma of advanced PC patients and detected both markers in our proof of concept patient cohort.TM  gradient centrifugation as described before [Prostate cancer cell line LNCaP , VCaP (high AR CN) , and mamd before .g, 10 min at room temperature within 4 h and supernatants were transferred and spun at 16,000× g, 10 min at 4 °C. Plasma were aliquoted and stored at −80 °C till further use.PC patients were recruited from Sydney St. George and Liverpool Hospitals. Patients and healthy controls gave written consent under the ethic HREC/13/LPOOL/158 approved via the South Western Sydney Local Health District Ethics Committee. Peripheral blood was drawn into 9 mL BD Vacutainer EDTA tubes  and processed with a double spin protocol as described before . BrieflyGenomic DNA (gDNA) from PBMC and cell lines were extracted with Bioline genomic DNA extraction kit according to manufacturer’s instructions . gDNA concentration and quality were determined with NanoDrop 2000 . Plasma cfDNA extraction was performed with QIAamp circulating nucleic acid kit plus QIAvac 24 Plus vacuum manifold  as described previously ,26.Droplet digital PCR (ddPCR) was performed with a Bio-Rad QX200 droplet digital PCR system (droplet generator and C1000 Touch thermocycler and droplet reader). All assays were optimized firstly by determining the best annealing temperature using a temperature gradient (52–60 °C) revealing 57 °C as the best annealing temperature for multiplexed reactions. Primers and probes for AR and appropriate reference genes  were desData and figures were analyzed and generated using GraphPad Prism 8 . Correlation analysis of 2 methods were performed with Pearson r and different groups  were compared with a Mann–Whitney test.The AR-Amp-1/T877A assay multiplexes amplification and detection of the AR sequences encoding the T877A mutation by using a T877A specific probe (FAM conjugated) and the corresponding wild-type probe (HEX conjugated). Another set of primers and a probe (HEX conjugated) to detect the reference gene RPP30 located on human chromosome 10 is included to normalize for AR amplification status. For assay optimization, gDNA from cell lines, LNCaP (AR point mutation T877A+) as well as from VCaP and MFM-233 cells (AR amplification positive), were used. Annealing temperature 57 °C with “standard” 500 nM primers and 250 nM probes resulted in the best separation of the four main event populations: negative , T877A (MT), wild-type (WT), and RPP30 . The ideGiven the challenge to reliably detect AR amplification status with high sensitivity and specificity from plasma derived cfDNA, we decided to improve assay accuracy by developing a second multiplexed validation assay (AR-Amp-2) based on DNA amplification with two sets of AR specific primers and probes (both FAM conjugated) targeting two independent AR amplicons: AR exon 1 (AR-X1) and exon 2 (AR-X2). The three AR-loci used in our two assays are distinct and separated from each other by approximately 75,000 base pairs, to ensure coverage of the AR gene. AR-Amp-2 includes primers and probes (both HEX conjugated) independently targeting two reference genes: X-chromosomal zinc finger MYM-type protein 3 (MYM) and TATA-binding protein (TBP) located on chromosome 6.To develop this assay, individual probe concentrations were first titrated to identify the corresponding fluorescent amplitude. Increasing population density and separation between fluorescent and background events was expectedly observed with higher probe and primer concentrations . Primersp < 0.01) and are used to determine a threshold for defining AR amplification. Conservatively, AR amplification threshold was set as 2 to ensure high specificity of higher AR CN calls. In our patient cohort (n = 47), nine patients showed cfDNA based evidence of increased AR CN. Importantly, AR amplification was consistently detected with both methods, ranging from 3.93 to 48.13 fold of amplification detected using the AR-Amp-1/T877A assay (mean ± SEM: 3.74 ± 1.32) and 2.91 to 35.59 using the AR-Amp-2 assay (mean ± SEM: 2.98 ± 0.92), while the remaining patients show normal AR CN similar to healthy controls 0.82–1.92  (p < 0.0005), which further validated our threshold setting and suggests reliable detection of AR CN in PC patients. Also in this cohort, none of the HSPC patients (n = 16) show AR amplification and 29% (9/31) of CRPC patients have detectable AR amplification. It is noteworthy that both methods show very strong correlation  and thereby present a critical way of internal cross-validation of cfDNA derived AR CN data  . CN of A CN data . The AR- CN data .The AR-Amp-2 assay, by including an X-chromosomal (Chr X) reference gene, not only provides the information of AR CN but indicates potentially larger ChrX amplifications . Chr X copy number can be calculated via the ratio of two times MYM vs. TBP (Chr X CN = 2 × MYM/TBP). In one patient, we detected increased copy numbers of both MYM and AR indicating major Chr X amplification (CN of Chr X: 4.30), also indicating possibly heterogeneous Chr X amplifications considering that cfDNA is a pool DNA released by cancer and normal cells .We further examined whether AR CN status correlates with previous treatments in 30 CRPC patients (one patient had missing treatment information). In the patients with previous chemotherapy, 42% (5/12) were AR amplified compared to 22% (4/18) of patients with no previous chemotherapy. For patients that have received second generation ADT  at some stage during prior therapy, 29% (4/14) were AR amplified compared to 31% (5/16) of patients that never received second generation ADT treatment. In the patients with both chemotherapy and second generation ADT, 50% (2/4) had increased AR CN compared to 27% (7/26) patients who never received chemotherapy or second generation ADT. While these data may show interesting trends, there was no signification correlation between AR CN status and any of the treatment regimens in our small cohort by Fisher’s exact test see .AR amplification is rarely detected in HSPC (around 1%); however, the incidence is increased to about 30–50% in CRPC with CN gains as high as 56 copies . ImportaDetection of point mutational biomarkers from cfDNA with ddPCR is common practice and becoming part of clinical management, for example, testing EGFR mutations in non-small cell lung cancer . The oveIn addition, 10–40% of PC patients have been reported to have entire chromosome X amplification and additional AR copies have been reported even in treatment naive patients due to polysomy of chromosome X . Increasn = 19, 53%) than on abiraterone  and other agents  [We applied our assays to a cohort of 47 PC patients to test translatability and found AR amplification in nine patients, with sometimes very high amplification of the AR gene. The highest AR CN detected by the AR-Amp-1/T877A assay was 48. According to our corresponding AR-Amp-2 assay data, this amplification can at least in part be explained by larger scale X-chromosomal amplifications as the distant X-chromosomal reference gene MYM was also clearly increased over the TBP reference gene in the patient. AR CN gain detection with our assays was restricted to CRPC patients . These data agree with another study which examined the prognostic relevance of plasma cfDNA based detection of AR amplification in pre-chemotherapy metastatic CRPC and found evidence for AR CN gain in 27% of patients , while Ap < 0.0005). Thus, we conclude that our assays, especially if used in combination, show high sensitivity and specificity.DdPCR is one of the most sensitive and economic ways to detect rare gene point mutations, amplification, and expression levels. Compared to FISH or Whole genome sequencing (WGS), ddPCR is more time- and cost-economic with superior sensitivity despite the superiority of FISH in discriminating cell-cell heterogeneity and WGS in wider coverage . Given iThe limitations of this study include testing the assays on a relatively small number of patients which were heterogeneous in the disease stage and treatment schedules. Therefore, correlation between AR CN status and disease progression still needs to be evaluated in a larger more homogeneous patient cohort over time.We have established two multiplexed ddPCR based methods to detect AR CN changes from cfDNA. A simple combination of both assays produces a high security to call AR amplification from cfDNA samples that can be obtained longitudinally during PC patient management. Reliable and sensitive detection of AR amplification and T877A status may have application for patient monitoring and to define eligibility to certain clinical trials. Larger studies are warranted to define correlation of AR CN status with treatment types and disease outcomes.


Blumeria graminis f. sp. tritici (Bgt), has limited wheat yields in many major wheat-production areas across the world. Introducing resistance genes from wild relatives into cultivated wheat can enrich the genetic resources for disease resistance breeding. The powdery mildew resistance gene Pm60 was first identified in diploid wild wheat Triticum urartu (T. urartu). In this study, we used durum as a ‘bridge’ approach to transfer Pm60 and Pm60b into hexaploid common wheat. Synthetic hexaploid wheat , developed by crossing T. urartu (AuAu) with durum (AABB), was used for crossing and backcrossing with common wheat. The Pm60 alleles were tracked by molecular markers and the resistance to powdery mildew. From BC1F1 backcross populations, eight recombinant types were identified based on five Pm60-flanking markers, which indicated different sizes of the introgressed chromosome segments from T. urartu. Moreover, we have selected two resistance-harboring introgression lines with high self-fertility, which could be easily used in wheat breeding system. Our results showed that the durum was an excellent ‘bridge’ for introducing the target gene from diploid T. urartu into the hexaploid cultivated wheat. Moreover, these introgression lines could be deployed in wheat resistance breeding programs, together with the assistance of the molecular markers for Pm60 alleles.Powdery mildew, caused by the fungus  Blumeria graminis f. sp. tritici (Bgt) is the disease-causing fungus of wheat powdery mildew, which is one of the most destructive diseases in many major wheat production areas. The disease could be well controlled by genetic resistance in the host; however, throughout history, the pathogen has overcome widely deployed host resistance genes rather rapidly [Pm) have been identified in wheat [PmG16, Pm41, and MlNFS10 are derived from Triticum turgidum L. var. dicoccoides [Pm13 and Pm66 from Aegilops longissima [Pm12 and Pm53 from Ae. speltoides [Pm21 from Dasypyrum villosum [Pm60 from T. urartu [Pm8 from Secale cereale [ rapidly . Until nin wheat . A signioccoides ,4,5, Pm1ngissima , Pm12 aneltoides ,9, Pm21 . urartu , and Pm8T. urartu , is the A-genome donor of tetraploid wheat T. turgidum subsp. durum  and hexaploid common wheat T. aestivum  [T. urartu has extensive genetic diversity for many traits, including disease resistance and phenological and morphological characteristics [T. urartu are extremely diverse, with adaptive traits that have accumulated over evolutionary time, which allow them to grow under a wide range of environmental conditions [T. urartu materials have good resistance to wheat diseases, including powdery mildew, stripe rust, and stem rust [T. urartu a genetic resource for wheat improvement to enrich the gene pool of wheat [T. urartu can be transferred to tetraploid and hexaploid cultivated wheat through direct hybridization and gene introgression [The wild diploid wheat,  AABBDD) . T. urarnditions . Some T.tem rust ,17,18, wof wheat . Becausegression ,21.T. urartu or other wild wheat relatives with hexaploid wheat [Sr60, Yr15, Yr36, and PmG16 [T. urartu is the donor of the A genome of T. durum, and durum shares the same A and B genomes with T. aestivum. Therefore, durum wheat might be suitable also as a ‘bridge’ between T. urartu and common wheat for the introduction of exogenous genes.Wide crossing has been an important approach for crop improvement, in which segments containing favorable genes from wild relatives can be introduced into existing crop species through chromosomal recombination . Howeverid wheat . In someid wheat . There and PmG16 ,25; revind PmG16 ). T. uraPm60, has been cloned from T. urartu [Pm60 encodes a protein with a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain. Three functional resistance alleles were identified on the Pm60 locus , distinguishable by an 80 amino acid deletion (Pm60a) or insertion (Pm60b) in the LRR region as compared with Pm60 [To date, only one powdery mildew resistance gene, . urartu . Pm60 enith Pm60 .Pm60 and Pm60b from T. urartu into common wheat using the durum wheat line Mo75 as a ‘bridge’. During the introduction, Pm60 and Pm60b alleles were selected in the progenies via functional molecular markers, and verified according to the resistance phenotype to powdery mildew. Based on graphical genotype analysis, eight different recombinant introgression types were identified that carry different lengths of chromosome segments derived from T. urartu with one of the Pm60 alternative alleles. At the same time, we selected two resistant lines with high self-fertility. These resistant introgression lines provide excellent materials for future wheat powdery mildew resistance breeding.In this study, we introgressed T. urartu accessions CITR17664 , PI428215  and PI428315  carrying the powdery mildew resistance genes Pm60 and Pm60b were used as the Pm60 donor lines [Bgt isolate E09, was used as a bridge for the introgression of Pm60 alleles into common wheat . These plants were removed from the soil at the five- or six-leaf stage for artificial chromosome doubling (AABBAuAu). After the soil was gently washed off the roots, the plants were submerged in a mixture of 2.5mM colchicine  and 2% dimethyl sulfoxide (DMSO) and kept in darkness for 24 h at 7 °C–10 °C in the growth chamber [uAu. The succeeded synthetic hexaploid wheat (SHW) plants could be self-fertile, or were otherwise sterile. The SHW plants were used as the parent to cross with the susceptible common wheat line Xuezao, and the hybrid progeny were evaluated for powdery mildew resistance in the greenhouse. To introduce the powdery mildew resistance genes into more common wheat backgrounds, the resistant hybrid plants were used to cross and backcross with several susceptible wheat lines Fielder, Xuezao, and Shiluan 02–1 in the next crossing process.To introduce the  devised . The sus chamber . After tBgt isolate E09, a prevailing pathotype in the Beijing area, is virulent to the resistance genes Pm1a, Pm3a, Pm3c, Pm5a, Pm7, Pm8, Pm17, and Pm19 [Bgt isolate E09 was maintained on the susceptible wheat line Xuezao by transferring spores to new plants each week. Inoculations were performed by dusting or brushing conidia from neighboring sporulating susceptible seedlings onto the two-week-old wheat seedlings. Approximately 10–14 days after inoculation, when susceptible control plants were fully infected, the infection types (IT) of the tested plants were scored using the 0–4 scale, where 0 indicated immune with no visible symptoms, 0; for necrotic flecks we noted a hypersensitive response (HR), 1 for necrosis with little sporulation, 2 for necrosis with moderate sporulation, 3 for moderate-to-high sporulation without necrosis, and 4 for full sporulation without necrosis [The and Pm19 , and wasnecrosis . ITs 0–2Total genomic DNA was extracted from young seedling leaves by means of the cetyltrimethylammonium bromide (CTAB) method . DNA samPm60 alleles were tested via amplification with M-Pm60 primers [Pm60 used in the experiments were all from Zou et al. [2O. PCR amplification was performed using the marker M-Pm60 with a protocol of 5 min at 94 °C; 35 s at 94 °C, 35 s at 55 °C, 1 min 40 s at 72 °C, and termination after 10 min of extension at 72 °C for a total of 35 cycles. For the flanking markers, the primer extension time was 30 s at 72 °C and the other PCR conditions were the same as those described above. PCR products were separated in 1% agarose gels or via 10% non-denaturing polyacrylamide gel electrophoresis (acrylamide: bisacrylamide = 39:1), and gels were visualized with silver nitrate staining [The  primers . The flau et al.   and Chinese Spring (RefSeq 1.1) from the Triticeae Multi-omics Center database (http://202.194.139.32/getfasta/index.html) (accessed on 3 March 2021).Analysis of the introgression fragments flanking T. urartu accessions CITR 17664 contained Pm60, PI 428215, and PI 428315 contained Pm60b. To introduce Pm60 and Pm60b into common wheat, the susceptible durum wheat line Mo75 was used as a ‘bridge’. Firstly, we crossed those three T. urartu accessions  with the female parent Mo75 (AABB), and obtained 3–10 F1 seeds for each cross. The hybrid plants were treated with colchicine for artificial chromosome doubling. Finally, we got 1–3 plants with high self-fertility from each cross, suggesting that the chromosomes in these plants were successfully doubled. Compared with the durum wheat line Mo75, synthetic hexaploid wheat  showed some new morphological traits, such as spike brittleness, larger seeds, and longer spikes crossed with several susceptible wheat lines, such as Xuezao, Fielder, and Shiluan 02–1. We obtained six BC1F1 populations using different male parents  met the 1:1 segregation ratio, except for Z2 and Z14  based on the size of the amplification product [M-Pm60 confirmed the presence of either the resistance gene Pm60 (PCR product size: 1551 bp) or the Pm60b allele (PCR product: 1791 bp) in all the powdery-mildew-resistant plants, which were absent in all of the susceptible plants  using the Pm60 flanking markers on the long arm of chromosome 7A  from T. urartu  showing high self-fertility with 83.3% and 84.6% fertility rates, respectively  of T. urartu natural populations and approximately 25.2% (58 out of 230) wild emmer wheat accessions [Pm60 locus is one of the important Bgt resistance loci in the natural populations of T. urartu and wild emmer wheat. The different Pm60 alleles might be resistant to different Bgt isolates. TdPm60 has been transformed from wild emmer wheat into common wheat [Pm60 and Pm60b genes had never been used in common wheat before, in this study, we introduced the Pm60 and Pm60b genes into common wheat and found that their resistances to Bgt E09 were maintained in the hexaploid background  and T. urartu (AuAu). Since SHW lines directly combined the whole genome of diploid wild relatives, they showed many undesirable traits, such as late maturity, taller plants, and difficulty in threshing  and created highly resistant and fertile derivative lines (Synthetic hexaploid wheats (SHWs) have been successfully used to combine the AB genome of tetraploid wheat and the D genome of DD wheat . Diseaseand Sr33 ,40. In ohreshing . Therefove lines . Howeverve lines , which c