Considering the importance of healthcare services costing in resource allocation, the aim of this study is to calculate the cost of rehabilitation services in an outpatient rehabilitation clinic in Tehran, Iran.The data for this study were categorised as financial data and information about the process of rehabilitation services. The first category was extracted from the financial documents and the second was obtained by observation of patient flow and interviews with the clinic staff in 2016. The cost of rehabilitation services has been estimated using the time-driven activity-based costing approach.The findings show that the cost of physical occupational therapy in the Asma rehabilitation center was $18.79 per unit of service. This amount for speech therapy services was $17.23 to $19.40, taking into account the difference in the quality of the service delivered. The cost of mental health occupational therapy service was between $19.46 and $23.57. Comparing the cost of these services with the government’s tariffs makes it clear that there is a huge gap.The limited number of patients referred to the center makes the cost of one unit of rehabilitation services much higher than the official tariffs. This is true for almost all similar institutions and makes the profitability of small rehabilitation institutions extremely unstable. Therefore, proper marketing for rehabilitation services by promoting patient referral links with larger healthcare centers and reallocation of resources to the formation of integrated rehabilitation complexes can play a significant role in their profitability. One of the main challenges in the sustainability of healthcare providers is the development of a cost information system that is suitable for pricing decisions, strategic management and resources allocation , 2. HealDifferent costing methods have been used for healthcare services, and over time, the accuracy of these methods have been enhanced. The traditional approaches, where there are no solid connections between the activities and the amount of resources used, can be only be employed in cases with limited activities and confined costs . Due to Due to the importance of human resources in providing healthcare, personnel costs have a significant share in the costs of these services . DiffereThe Ministry of Health and Medical Education (MOHME) is responsible for public health in Iran. Healthcare services are accessible through a number of different public and private providers. A significant portion of health services in Iran are financed by the Ministry of Welfare and Social Security through specialised insurer organisations including Social Security Organisation, the Army Medical Insurance Organisation and Health Insurance Organisation . The covDespite the challenge of chronic disease in the present century, the increase in the elderly population and the significant relationship between the need for rehabilitation services and catastrophic expenditures –24, the Rehabilitation service providers in Iran include public, private, and charitable organisations. Their tariffs are set annually by the MOHME. The Asma Rehabilitation Centre (ARC) is affiliated to the University of Social Welfare and Rehabilitation Sciences and is funded by the MOHME. Therefore, the ARC is an educational therapeutic center and provides internship opportunities for the students of rehabilitation sciences as well as providing rehabilitative services. The ARC has a range of specialised departments, including speech therapy, physical occupational therapy, mental health occupational therapy, psychology, audiology, neuro-feedback, hand occupational therapy, and physiotherapy.The present study used TDABC , 27 to cThere are two main approaches to healthcare costing; highly detailed bottom-up costing and top-down approach. If detailed information is available, the bottom-up approach is recommended . HoweverIdentifying the various departments: First, a map of the rehabilitation services process was drawn up by observing the process of activities in different departments and talking with the authorities in each department. Figure Estimating the total cost of each resource group: The cost groups were identified through interviews with the Finance and Property Departments. Cost groups include buildings, overheads, human resources, and equipment . Through the aggregation of these costs, the total cost of each section was determined. Then, direct and indirect costs were calculated for each department. Costs that could not be categorised in the above categories  were included as other overhead costs. The area (squared meter) occupied by each department was considered as basis of cost sharing for the building, and maintenance of other assets.Estimating the practical capacity of resource groups: At this stage, the practical capacity of each group was calculated. Staff hours are from 8:00 am to 2:30 pm from Saturday to Wednesday, and from 8:00 am to 1:00 pm on Thursdays. Therefore, the total working time is 1956.5 h per year . Subsequently, the practical capacity of each group was considered to be 85% of the theoretical capacity  equal to 99,781 min in a year. The practical capacity of medical and non-medical equipment and machinery was also considered in terms of the useful life of the equipment.Calculating the unit cost of each resource group: After calculating the total cost and practical capacity for each cost group, the cost of each unit was obtained by dividing these two variables into each other.Identifying the time equation: Once a patient has been referred to a unit, the time taken for them receive the service must be calculated. This time was measured by observation and timing for four patients, allowing time equations for rehabilitation services to be determined .Aggregating the costs and calculating the final price of each service: This step was acceded by multiplying the unit cost of each resource group in time equations.The data needed for this research can be divided into two categories: financial data and information about the process of providing the rehabilitation services. The first category includes personnel costs, depreciation cost for office equipment and medical equipment, rent, and overhead costs. These were collected from the financial documents of the ARC in 2016. The second part of data was gathered by observing the services processes and by conducting informal interviews with the staff of the ARC after obtaining the necessary permissions. In TDABC method, estimation of two parameters are necessary: cost of a unit of resources, and the time required for each activity , 29. MorThe number of rehabilitation sessions required by each patient varies according to the severity of the disability and the type of service required. Sometimes, due to the financial hardship, the patient does not attend all the sessions. Based on the time equations, the total personnel costs of each department were obtained per unit of service Table .Table 1PFor departments where services are provided by a workforce with varying degrees of expertise , rehabilitation activities were divided in terms of quality. High quality (HQ) services were defined as those delivered by rehabilitation specialists with a Ph.D. in a relevant field while medium quality (MQ) services were delivered by rehabilitation experts with a bachelor's or master’s degree in that field. By placing the cost per minute for the rehabilitation specialists (HQ) and rehabilitation (MQ) experts in to the time equations, the cost of HQ and MQ services can be calculated. In order to estimate the average rehabilitation session for each patient, we have considered a regular 10-session course of treatment for these services. Given that, the counseling and evaluation of the general practitioner are provided only twice, so this item is counted only at the beginning and the end of a regular 10-session rehabilitation.The non-personnel costs of each department are also admeasured according to the number of visits or the area occupied by the department. For this purpose, the cost of depreciation of equipment, rent of the building, repair and maintenance costs, and administrative costs were calculated for the different departments according to the contribution of each department to the activities of the institute.The cost of rehabilitation services, categorised by personnel and non-personnel costs, of each department is also presented in Table A closer look at the cost components shows that the shares of employee compensation and benefit in the total cost of speech therapy, physical occupational therapy, and mental health occupational therapy services were 4.20$, 3.15$ and 5.54$, respectively.The government’s tariffs for a session of speech therapy, physical occupational therapy, and mental health occupational therapy in 2017 are shown in Table The practical capacity of personnel was calculated throughout the year. By multiplying the practical capacity of an individual and the number of personnel in each department, the practical capacity of that department is specified.The comparison of unused capacity shows that the clinic’s cashier has the highest rate of idle time Table . Total cThe unused capacity of human resources in the ARC shows that a new combination of work reassignments and better management of human resources could lead to more efficient workforce utilisation and lower average cost of personnel. For example, if the clinic has plans to expand rehabilitation activities, given the unused capacity calculated, it only requires more general practitioners to be appointed. Therefore, the development of rehabilitation activities at the ARC not only does not have high costs, but also reduces per capita indirect costs and ultimately reduces the cost of the services.The literature lacks case studies of TDABC application in different rehabilitation services settings , which mThe highest non-personnel costs are due to "other overhead costs”; the most important of which is the building leasing cost. Therefore, avoiding the leasing costs  can play an important role in the profitability of the institution's services. The rent and cost of buildings depreciation in Iran is burdensome , 32, andHealthcare is one of the services that has a high diversity of human resources with cost variations of more than 10:1. In thisIn the present study, firstly, costs are calculated using a more accurate method based on the allocated time for each activity. Then, through mapping the process, all stages of the rehabilitation service have been identified for the ARC. This framework facilitates the identification of cost drivers of providing rehabilitation services at the ARC. The findings indicate a significant gap between the cost of services and government tariffs. One of the main reasons for this gap is the existence of unused human resource capacity in the ARC.Thereupon, if the ARC’s staff operate in a situation where their utilised capacity increases, fixed costs per unit of service will be reduced and result in lower prices.One consequence of the high cost of rehabilitation services at the ARC was the formation of the gap between the cost of service and government tariffs. As a result of this gap, the ARC activities have been financially detrimental. However, a more accurate costing framework may contribute to lower the costs and increase revenues for rehabilitation activities.This study attempts to apply a more accurate method for costing rehabilitation services, in addition to sensitisation about the loss of rehabilitation activities for small institutions. Long-term losses for rehabilitation clinics will limit the future of such services and will put the welfare community at a greater disadvantage. This study aimed to show that the government tariffs for rehabilitation services are significantly lower than the costs of providing these services. Second, most of rehabilitation services are not covered by Iranian social insurances, and tariffs are not affordable for families in need of rehabilitation services. In addition to inspire advocacy of social insurance provision for low-income families, this study sought to suggest an improved costing method for rehabilitation providers. Evidently, TDABC method may provide a more realistic cost structure information and other valuable information, such as a realistic profit/lost overview, unused human capacity and a basis for improved resources management for health care providers. All of this may enhance the availability and accessibility of rehabilitation services.The research has been accompanied by a few limitations. One of the limitations of the research was that not all rehabilitation activities cost has been calculated. This selection of activities was based on the number of patients referred to departments. Time and money constraints made it impossible for calculations to be made for all departments. In addition, it should be stated that all information was provided by the ARC and the accuracy of this information has not been independently verified. Another issue is the lack of links between the cost and value of rehabilitation services for patients. The actual value of the utility that rehabilitation services given to clients may be more or less than the estimated cost. Ultimately, these calculations were related to a small-scale rehabilitation center and the findings cannot be generalised to all active businesses in the rehabilitation services.


Rehabilitation nutrition care process includes assessment and diagnostic reasoning, diagnosis, goal setting, intervention, and monitoring. The positive psychology perspective in the rehabilitation nutrition care process may be useful for providing higher quality rehabilitation nutrition. Positive psychology is defined as the scientific study of human thoughts, feelings, and behavior, with a focus on strengths and a better life. Psychological assessment and intervention are necessary in general and family medicine and in rehabilitation nutrition. The relationship between positive psychology and general and family medicine and rehabilitation has been reported.Positive psychology can be useful for maximizing function and quality of life (QOL). Rehabilitation nutrition elicits the highest body functions, activities, participation, and QOL by incorporating both rehabilitation and nutrition.The positive psychology perspective in the rehabilitation nutrition care process may be useful for providing higher quality rehabilitation nutrition Figure . The rehRehabilitation nutrition assessment and diagnostic reasoning include a comprehensive assessment by the International Classification of Functioning, Disability and Health (ICF), detailed nutritional assessment, and diagnostic reasoning of malnutrition, sarcopenia, and nutrient intake. Diagnostic reasoning of causes of anorexia, weight loss, and sarcopenia is important in rehabilitation nutrition.Rehabilitation nutrition diagnosis comprises three major categories: nutritional status, sarcopenia, and excess and/or insufficient nutrient intake. The domain of nutritional status includes undernutrition, overnutrition, at risk of undernutrition, at risk of overnutrition, lack of nutrients, and excess of nutrients. Positive diagnosis is important from the perspective of positive psychology. For example, high muscle mass, strong handgrip strength, and fast gait speed are positive diagnoses in patients with sarcopenia.Rehabilitation nutrition goal setting includes both rehabilitation and nutrition goals. The concept of SMART  goal setting should be used in rehabilitation nutrition.Rehabilitation nutrition intervention has two aspects, which are “nutrition care management in consideration of rehabilitation” and “rehabilitation in consideration of nutrition.” Psychological interventions, such as mindfulness and cognitive behavioral therapy, are important in rehabilitation nutrition. Moreover, interventions for environmental factors are also important in rehabilitation nutrition. For example, as presented at the 13th Annual Conference of the Japan Primary Care Association in 2022, hospital art classes may be useful for maximizing function and QOL in hospitalized patients.Rehabilitation nutrition monitoring includes monitoring goal achievement, general condition, nutritional status, nutritional intake, and physical and mental function such as depression, activities of daily living, and social participation. Moreover, well‐being and QOL including ikigai should be monitored from the perspective of positive psychology.Enhancing the well‐being of healthcare professionals themselves is also important. The number of healthcare professionals who suffer from depression, adjustment disorders, burnout, and other psychological disorders is not small in general and family medicine and rehabilitation nutrition. Any healthcare professional can develop a psychological disorder. Enhancing the well‐being of healthcare professionals themselves by mindfulness seems to be helpful in preventing psychological disorders.Further clinical research and clinical practice from the perspective of positive psychology are required in general and family medicine and rehabilitation nutrition.The author declares no conflict of interest for this article.


Previous research has revealed that skewed X chromosome inactivation (SXCI) and androgen receptor (AR) CAG polymorphisms are associated with increased risk of recurrent pregnancy loss (RPL); however, the results are conflicting, and the underlying mechanisms remain unclear. This study investigated the role of SXCI and AR CAG polymorphisms in patients with RPL and explored whether the underlying mechanisms were related to the ovarian reserve and preimplantation embryo aneuploidy.This was a prospective case-control study carried out in a tertiary hospital-based reproductive medicine center. An external validation RPL cohort was recruited during the study period. Data on baseline and cycle characteristics were collected. X-chromosome inactivation (XCI) was measured using a human AR assay. AR polymorphisms were assessed using quantitative fluorescent polymerase chain reactions and direct sequencing. Blastocysts of the patients with RPL were tested by single nucleotide polymorphism microarray based preimplantation genetic testing for aneuploidy.In total, 131 patients with idiopathic RPL and 126 controls were included for the case-control study. Patients with RPL exhibited a significantly more skewed XCI distribution pattern , as well as significantly shorter bi-allelic mean  and X-weighted bi-allelic mean  of AR CAG repeats. Multivariate logistic regression models indicated that CAG repeat < 20, SXCI, and duration of stimulation were independently associated with the risk of RPL. However, SXCI and AR CAG polymorphisms were not associated with ovarian reserve or preimplantation embryo aneuploidy in the RPL group, and the same results were attained in a separate validation cohort of 363 patients with RPL.SXCI and AR CAG polymorphisms are related to RPL; however, these two factors do not lead to RPL by affecting the ovarian reserve or increasing embryo aneuploidy. The roles of SXCI and AR CAG in RPL may involve other mechanisms that require further investigation.https://www.clinicaltrials.gov .NCT02504281,  The diagnostic criteria for recurrent pregnancy loss (RPL) vary between three or more consecutive pregnancy losses , 2 and t12 In heterozygous females with SXCI, the X-linked transcriptional and allelic dosages of silenced genes are unbalanced and may be functionally homozygous ), which is the gold standard for XCI analysis . PolymorDNA from a healthy man was used as a control in each run of the assay because the male X chromosome is always unmethylated. Samples homozygous for the AR (CAG)n gene locus were excluded from the XCI analysis due to the inability to distinguish between the two alleles. The degree of XCI was calculated for the heterozygous samples according to a previously published protocol . Both 85The number of CAG repeats was calculated relative to a series of standards obtained using direct sequencing. The AR (CAG)n repeat polymorphism profiles were analyzed in three modes  as folloAn antagonist protocol for controlled ovarian hyperstimulation was used for each participant. Treatment with recombinant human FSH  was initiated on the 2nd or 3rd day of the menstrual cycle with a starting dose of 150 − 300 IU/day adjusted for age, BMI, AFC, FSH, and AMH levels. Gonadotropin-releasing hormone antagonist  was administered at a dose of 0.25 mg/day when the dominant follicle reached 14 mm in size or the serum E2 level reached 350 pg/mL. This treatment continued until a leading follicle reached 18 mm or two follicles reached 16 mm in size. Subsequently, 5,000 − 10,000 IU of human chorionic gonadotropin  was administered as a trigger and oocytes were retrieved 36 h later. ICSI and blastocyst culture were performed for all participants in accordance with IVF laboratory guidelines, and single nucleotide polymorphism microarray based PGT-A was administered to the patients with RPL as per the manufacturer’s instructions . Mosaicism calls were made when 20–80% of the biopsied cells were aneuploid.For the case–control study, the median CAG repeat number of the control group  was usedValues are presented as average ± standard deviation for continuous data and were compared using Student’s t-test or Mann − Whitney U-test. The chi-square and Fisher’s exact tests were applied as appropriate for categorical variables. Departures from Hardy-Weinberg equilibrium (HWE) were tested to determine whether the frequencies of AR CAG repeat polymorphisms were consistent with the genetic balance. The adjusted ORs and 95% confidence intervals (95% CIs) of SXCI and AR CAG polymorphisms associated with RPL risk were examined by multivariate logistic regression analyses. The relationships of SXCI and AR CAG polymorphisms with ovarian reserve or blastocyst euploidy were evaluated using logistic regressions (Backward LR) that were adjusted for age, AFC, AMH, and other possible confounders. Data were analyzed using the Statistical Package for the Social Sciences (SPSS) software . The statistical significance level for all tests was set at P < 0.05.The case–control study included 131 patients with RPL and 126 controls . The distribution of XCI demonstrated a significantly more skewed pattern in patients with RPL than in controls . The multivariate logistic regression analysis indicated CAG < 20 was independently associated with DOR in the control group, while SXCI and AR CAG polymorphisms were not associated with DOR in the RPL group  . HoweverTo the best of our knowledge, this is the first study to investigate the correlation between SXCI and CAG polymorphisms with embryo euploid status at the preimplantation blastocyst stage. Previously, only three studies have investigated the association between SXCI and embryo aneuploidy, and all have examined the products of conception. Sangha et al. demonstrated that RPL patients with SXCI > 90% had more miscarriages secondary to fetal aneuploidy than those without extreme SXCI . Beever The limitations of the present study need to be addressed. First, the sample size of the case-control study is limited considering the overall size of the Chinese population and the prevalence of RPL. Despite that we enrolled a separate RPL cohort with statistically sufficient sample size to validate the results and that we adjusted many potential confounders by logistic regressions, the potential confounding factors cannot be entirely ruled out. Second, this study was carried out in the Chinese Han population, while the race and ethnicity differences may have an effect on the genetic polymorphisms, such that further research are needed to estimate the role of SXCI and AR CAG repeat polymorphisms in RPL risk within other populations.According to the above results, we concluded that mechanisms other than ovarian reserve or embryo aneuploidy may account for this association of SXCI and AR CAG polymorphisms with RPL. Chromosome X is also enriched for immune-related genes, and skewed XCI patterns could cause the breakdown of thymic tolerance induction processes, conferring an increased predisposition to develop autoimmunity , which mIn summary, SXCI and AR CAG polymorphisms are related to RPL; however, these two factors do not cause RPL by affecting the ovarian reserve and increasing embryo aneuploidy. The roles of SXCI and AR CAG in RPL may involve other mechanisms that require further investigation.


The cosmetic appearance of the AR-assisted implants was rated as very good (median 25.00 out of 30 points) and significantly improved in comparison with the conventionally shaped implants (median 14.75 out of 30 points). Our experiments showed outstanding results regarding the possibilities of AR-assisted procedures for single-step reconstruction of craniofacial defects. Although patient-specific implants still represent the gold standard in esthetic aspects, AR-assisted procedures hold high potential for an immediately and widely available, cost-effective alternative providing excellent cosmetic outcomes.Defects of the cranial vault often require cosmetic reconstruction with patient-specific implants, particularly in cases of craniofacial involvement. However, fabrication takes time and is expensive; therefore, efforts must be made to develop more rapidly available and more cost-effective alternatives. The current study investigated the feasibility of an augmented reality (AR)–assisted single-step procedure for repairing bony defects involving the facial skeleton and the skull base. In an experimental setting, nine neurosurgeons fabricated AR-assisted and conventionally shaped (“freehand”) implants from polymethylmethacrylate (PMMA) on a skull model with a craniofacial bony defect. Deviations of the surface profile in comparison with the original model were quantified by means of volumetry, and the cosmetic results were evaluated using a multicomponent scoring system, each by two blinded neurosurgeons. Handling the AR equipment proved to be quite comfortable. The median volume deviating from the surface profile of the original model was low in the AR-assisted implants (6.40 cmThe online version contains supplementary material available at 10.1007/s10143-022-01784-6. Cosmetic reconstructions are usually required after surgical resection of bony lesions of the skull, particularly in frontal localization or involvement of the facial skeleton and the skull base , 27. WhiThe implementation of augmented reality (AR) promises new possibilities in this area. With regard to applications in cranial neurosurgery, various AR-based simulations for cerebrovascular, brain tumor, and skull base procedures have been described so far , 31, 39,The present study is the first to investigate the feasibility of AR-assisted reconstruction of bony defects involving the facial skeleton and the skull base for potential application in affected patients.A computed tomography (CT) scan  of an x-ray dense skull model was performed , serving as ground truth. The three-dimensional (3D) reconstructed scan was transferred to the AR device . Thereafter, a defect involving the frontal skull and orbital rim was inserted into the model, with localization and extension corresponding to common pathologies of the anterior skull base involving the orbital rim . Subsequently, nine participants (three experienced neurosurgeons and six less experienced younger neurosurgery residents) each produced two AR-assisted and two conventionally shaped (“freehand”) implants from PMMA  for defect repair. AR assistance was provided using Magic Leap 1 combined with Brainlab® Mixed Reality Viewer  by manually positioning the holographic projection of the original skull model (ground truth) over the real model with the inserted defect. To simulate the real intraoperative situation, the clamp-fixated skull model was covered after AR alignment (except for the defect to be repaired), as shown in Fig. E1) and the lacking (error volume E2) volume of the implants in comparison with the surface profile of the original model were measured, as illustrated in Fig. E1 and E2 .Volumetric deviations of the surface profiles of the implants in comparison with the original skull model were each quantified by two independent, blinded investigators using the SmartBrush device on Brainlab Origin Server 3.2 , and the corresponding mean values were used for further analysis. Both the overlapping . The mean values were used for further analysis. Points between 0 and 10 were awarded for the overall cosmetic appearance and for two specific regions of interest (ROIs) .Methods of descriptive statistics were used. For numerical data, median values and the interquartile range (IQR) were calculated. Interrater reliability in volumetric analysis and cosmetic scoring was validated using a Bland–Altman plot. Statistical differences were evaluated using a Mann–Whitney test as an unpaired nonparametric test . The level of significance was set to The AR hardware and software were easy to use for both experienced and less experienced neurosurgeons in the operating theater. The manual registration process  took 5–10 min, depending on the individual experience. The well-fitting AR glasses allowed free head movement and the associated equipment was small and could be easily worn on the body. The battery life was adequate, and individual adjustment of the light made it possible to vary the translucency of the image in the glasses to see more of the real image or the AR projection as required. The surface profile of the holographic object was well visualized, even in a complex region. By altering the distance of the glasses to the object, the image was also displayed in slices, like scrolling through a CT scan, which was additionally helpful. After the registration process, the time needed for implant fabrication (preparation of the situs with cottonoids and PMMA fitting) was measured. This procedure took 10–20 min, depending on the experience of the neurosurgeon, with no relevant difference between the AR-assisted and the conventionally shaped implants. The AR-assisted fabrication process is demonstrated in Video Etotal AR) was low and significantly reduced in comparison with that of the conventionally shaped implants , at 6.40 cm3 (IQR 2.24) versus (vs.) 13.48 cm3 (IQR 5.26), p < 0.0001  showed no significant difference between the AR-assisted and the conventionally shaped implants (3.78 cm3 (IQR 2.39) vs. 3.66 cm3 (IQR 6.63), p = 0.9626). The median error volume lacking in comparison with the original skull model (E2) showed a significant difference between the AR-assisted and the conventionally shaped implants (2.25 cm3 (IQR 2.73) vs. 8.84 cm3 (IQR 9.32), p = 0.0004). Detailed volumetric values are listed in Table Compared with the surface profile of the original skull model, the median total error volume of the AR-assisted implants  and that of the conventionally shaped implants was 14.75 (IQR 9.5), Patient-specific CAD/CAM implants are increasingly used because of their better cosmetic results in comparison with, for instance, prefabricated titanium implants or freehand-shaped PMMA implants, particularly in patients with benign lesions who undertake normal life activities and do not require immediate surgery , 3, 14. In the presence of a relatively intact bone flap that needs to be replaced, such as in resection of intraosseous meningiomas or calvarial metatases, an efficient and cost-effective method is described that has no special technical requirements. With this simple procedure, a PMMA implant is fabricated based on the bone flap to be replaced and its negative form, which appears to be an excellent method for selected cases, but seems less suitable for complex defects with craniofacial involvement or in cases where skull continuity is lost .Recent publications report a highly interesting, cost-effective alternative for covering cranial defects based on 3D-printed molds, which were used to intraoperatively fabricate PMMA implants , 30, 34.In this context, AR holds high capability for future concepts. In spinal neurosurgery, AR-assisted procedures have been applied especially for screw placement in combination with navigation or robot assistance within the last decade , 23, 35.Handling of the AR equipment was quite intuitive and after registration both hands were free to work on the implants. The AR image allowed adaptation of the implant to the holographic projection of the original skull model, which was accurately visualized even in complex regions. The evaluation of our participants reflects that the AR equipment being worn on the body was not experienced as disturbing and the entire AR handling was found to be easy to deal with, regardless of the individual expertise. The use of AR was definitely considered to be a benefit for implant fabrication. Participants mostly had the subjective impression of achieving good cosmetic results when using AR, and this usually correlated with satisfaction with results after finishing the implants. General satisfaction with cosmetic results of AR-assisted implants was high. During the fabrication of the conventionally shaped implants, subjective impressions varied about whether good results were achieved, and participants later were rather dissatisfied with the results. The manual registration of the real object and the holographic projection, which had to be readjusted for each run, was seen as problematic because it was time-consuming and a potential source of error. There is a need for optimization regarding the lack of an automatic registration mechanism. Nevertheless, additional manual adjustment should still be possible, since the automatic registration mechanisms may not yet guarantee the high accuracy that is required for skull base applications in particular .Patient-specific CAD/CAM implants represent the gold standard for skull reconstruction. With these, only minimal deviations from the original surface profile and an excellent esthetic appearance can be assumed. Reports on less expensive implants manufactured with 3D-printed molds also describe very good cosmetic results, but an objective quantitative analysis, as performed in the current work by means of volumetry, cannot be found in any of these articles , 22, 30.With regard to cost calculation in a Western country, a price of at least 4000–5000 USD can be assumed for a patient-specific CAD/CAM implant made of PMMA, given the size and complexity of our study implant. Comparative data can be found in a recent Canadian publication on cost-effectiveness in cranioplasty . Here, 1Etotal = 6.40 cm3), whereas this was twice as much for the conventionally shaped implants . The cosmetic appearance of the AR-assisted implants was confirmed to be very good by two blinded raters. Thus, with a maximum score of 30 to be achieved, the median score of the AR-assisted implants was high at 25.00.Our presented method therefore offers the potential of a cost-effective and widely available single-step procedure for the treatment of complex, cosmetically challenging skull defects, even in the context of immediate reconstruction in malignant lesions or after trauma. Despite the limited number of implants, the statistical results seem to be promising. Compared with the ground truth, the AR-assisted implants deviated only slightly in volume Below is the link to the electronic supplementary material.


Low frequency magnetic fields are often present in our everyday life due to the multitude of electronic devices. High magnetic fields can occur in the workplace from a wide variety of machines and systems, which must be measured and evaluated from the point of view of occupational safety. To facilitate the understanding of magnetic fields by supervisors and employees in the workplace, an augmented reality (AR) application was developed to visualize the measured flux densities and the resulting safety distances. The application was deployed on two smartphones, allowing for the simultaneous viewing of the same scene without the use of additional markers. Whether the application creates a better understanding of the exposure situation was evaluated with the help of an online survey. In this survey, participants received either a classic measurement report or a report enhanced by augmented images. The evaluation shows that it subjectively felt less difficult for participants with the augmented report to answer questions about the exposure situation. Furthermore, they also objectively performed better in answering the questions than did the group with the classic report. Therefore, this work shows that AR enhanced images can improve the understanding of an exposure situation, and it describes how such images and videos can be created. Low frequency magnetic fields are one of the many physical agents that can potentially be hazardous in a work environment . In contImages or sketches of the exposure situation might help employees to better understand the extent of the magnetic fields present and the safety distances which must be observed. However, creating such imagery can be a difficult task, since three-dimensional information about the exposure situation has to be visualized. Here, we present an augmented reality solution which embeds measurement and safety evaluation data directly into live imagery of the workplace being reviewed. Images or videos visualizing the exposure situation and the necessary safety distances can easily be created with this system. An evaluation study was performed investigating the influence of such imagery on the objective understanding of the exposure situation and on the subjective perception of the hazard communication. Evaluating information visualization is a challenging task in itself, and therefore, the study design took previous research into account . This woThe aim of the system is to visualize data specifically for an OSH evaluation and not to visualize magnetic fields in general. Therefore, it is not necessary to visualize the field at any given point in time and space in its vectorial form . For anIn general, AR technology becomes increasingly more feasible for practical use cases in industry settings and is estimated to continue growing to a ~USD 100 billion market by 2028 . Its abiThe aim of this work is to present the developed AR solution for visualizing magnetic fields in an OSH context and to show that media created with this system can improve the understanding regarding an exposure situation. Please note that the magnetic field measurement and assessment process can be very complex in itself. It is not the focus of this work and therefore, it is not described in detail, but is rather treated as a black box. The interested reader can find further information in .This chapter is subdivided into two parts. The first part describes the setup of the AR measurement system, and the second part provides a description of the evaluation study.2 area. The EFA 300 can measure magnetic fields for frequencies between 5 Hz and 32 kHz and with amplitudes between 100 nT and 32 mT. The typical uncertainty is specified as ±3%. In this study, peak values of the magnetic field were measured in broadband mode. A Samsung S20+ 5G smartphone was used for running the AR application. Bidirectional serial communication with an optical cable between the smartphone and the EFA 300 was established following the EFA 300 remote control protocol. A custom designed 3D printed casing combined the EFA 300 with the attached probe, the smartphone, and the necessary adaptors for the remote control connection in such a way that the probe was not visible in the field of view of the smartphone’s RGB and depth cameras. The whole system is called AURA—an abbreviation for augmented reality measurement system.The main components of the AR system for visualizing magnetic field measurements and safety evaluation data are shown in The software development was conducted with Unity . Google’In an initial “scanning” phase after starting the system, sensory data  are gathered, while moving the AURA system around an object of interest until the ARCore system reports “good” localization accuracy. This is an ARCore internal accuracy estimate, and we chose to initially gather data and wait for “good” accuracy to be able to provide stable AR objects after the initial scanning phase. The achieved accuracy depends on many factors . In general, we achieved reasonably stable AR objects with our system see  allowingThe safety evaluation method can be chosen from a dropdown list containing different regulations, e.g., for workers with or without a pacemaker . DependiIn practice, many people are present during a magnetic field safety evaluation at a workplace. To allow them to explore the AR scene independently of the person with the AURA measurement system, the Cloud Anchors functionality of ARCore is used, which allows for viewing the same scene from a different perspective with another device . A CloudThe viewer application allows the user to change the evaluation method, which influences the coloring of the measurement spheres. Furthermore, the user can switch between a gradient and a binary (green/red) coloring scheme, indicating if a limit value is exceeded.A video showcasing the AURA system and its capabilities can be found on the project web page  or in thThe driving force behind developing the AURA system was to improve the understanding of magnetic field exposure situations by visualizing the measurements and the safety evaluation. From our practical experience in OSH, we know that people have difficulties understanding safety evaluation reports. The AURA system allows people to explore the exposure situation in AR. However, in practice, it is much more likely that people will read a report instead of being able to explore the AR scene with a smartphone at the workplace. Therefore, images  augmented with the additional virtual information coming from the AURA system will probably be the main media which people will encounter.To test if such augmented media could improve the understanding of a safety evaluation report, an online survey was conducted. A total of 2 groups of 15 participants each received a short report about the safety evaluation of magnetic fields from a resistance welding machine. Participants were recruited among colleagues and acquaintances. The study was anonymous, and participants acknowledged a data privacy statement. Participation was voluntary and was not compensated.The “Classic Report” group received a report which was similar to what is currently provided from OSH experts when evaluating a workplace (including result tables and images). The “Augmented Report” group received the same report, along with three additional images created with the AURA system, visualizing measurements and hazard zones . Note that the survey was conducted in German.All answers in the “AR” group were correct. Four of fifteen participants in the “CR” group answered incorrectly. Six participants of the “AR” group reported that they extracted the information from the AURA images.p = 0.009) between the groups, showing that the “AR” group rated the task as less difficult.Additionally, the participants were asked to rate the perceived difficulty of the question on a 5-point Likert scale. The results are shown as a box plot. There was a significant difference  between the groups, but a strong trend towards the “AR” group rating the task as less difficult. The trend is supported by a median of 4 for the “AR” group and of 2 for the “CR” group.Again, participants were asked to rate the perceived difficulty of the question on a 5-point Likert scale. There was no significant difference (Three statements about the picture were given, and the correct statements were to be selected. Selecting all three answers together equaled a correct response. At this stage, participants could not refer to the report anymore, but had to solve the task from memory with the knowledge they had acquired so far.Only ten of all the thirty participants answered correctly. Seven were from the “AR” group and three from the “CR” group, which is in line with the other results. Interestingly, three of the seven people who indicated that they have experience with magnetic fields in an OSH context did not answer correctly.In the last part of the survey, participants from both groups were shown an image  and a viThe presented images and videos showcase that the AURA system can be used to easily create visualizations of measured flux densities and relevant safety information for workplace exposure scenarios. Almost all of the utilized software assets are freely available, and a high-end smartphone is sufficient for the intended task of achieving stability of the AR objects. Therefore, the AURA system represents a prototypical example, which can be improved and built upon without the need for proprietary knowledge.The survey results show that AR enhanced images improve the understanding of the exposure situation, since AR participants made fewer mistakes when answering safety relevant questions about the exposure assessment report. Furthermore, there were significant differences in the perceived difficulty of the questions between groups, showing that participants provided with AR enhanced images felt that the questions were easier. These are very important and helpful results, since they show that it is worthwhile to create AR enhanced content. The results also indicate that the created content does not necessarily have to be presented in an interactive AR format to be helpful, which otherwise could be a technical challenge for some users. Instead, they show that classical media, such as images or videos, can benefit from AR enhancements.It might seem surprising that the survey results revealed some wrong answers, even for “simple” tasks like extracting a number from a table. However, this is congruent with our experiences from OSH consultations. People often do not have a background in magnetic field health effects. This unfamiliarity with the topic, in combination with the safety evaluation aspect, tends to result in difficulties in understanding the provided exposure assessment reports. The results support these anecdotal experiences by quantitatively showing the difficulties participants had with answering some of the questions.Being able to correctly answer questions such as, “At which distance is the high action level exceeded?” is very important. This knowledge is necessary to implement effective safety measures, like setting appropriate safety distances. The results show that AR enhanced images can help to reduce the number of mistakes; therefore, they can help to increase safety.Another aspect we observe when showing AR enhanced imagery in an OSH setting is that people tend to take the hazard more seriously when they “see” it. Since magnetic fields cannot be seen, the potential threat is often neglected. However, this is not as easy to do when the hazard zone is visualized directly at the workplace.The survey included an open feedback text field, and participants reported that they felt safer and more in control after seeing the AR enhanced imagery because they felt that they could better understand the necessary safety distances. Furthermore, they reported that seeing people directly in relation to the hazard zone as in  represenFuture improvements to the AURA system could include playing a warning sound whenever the chosen limit value is exceeded by the currently measured value. However, movement of the probe within a magnetic field can induce voltages in the probe’s coils which in turn can lead to measurement artifacts. Therefore, measurements should only be taken when the probe is relatively steady in space. This would have to be considered when implementing a live measurement warning feature.Electromagnetic compatibility of the smartphones when exposed to high magnetic fields is an important topic. High intensity fields could cause the devices to malfunction, or could damage them permanently. We could not find any data on frequency dependent magnetic field damage levels for smartphones. For the AURA system, it is important that the smartphones work at magnetic field levels which are in the range of the allowed limit values. Higher levels are not necessary since employees are not allowed to enter such areas anyway. Currently, we have used the AURA system at several workplaces, with different types of magnetic fields, and we have not yet encountered any electromagnetic compatibility issues .In general, it seems that AR enhanced imagery could also be helpful for agents other than magnetic fields. It could be helpful in the context of radiofrequency electromagnetic fields, e.g., visualizing hazard zones for wireless communication base stations. In that case, a concept very similar to the one presented here could be used by replacing the low frequency magnetic field measurement device with an appropriate high frequency measurement device.Another application could be in the field of acoustics for noise measurements, e.g., as presented in . In thatBroadly speaking, AR visualization solutions can be helpful whenever a measured quantity has a room scale spatial dependency. Therefore, the concept lends itself very well to visualize all kinds of hazards in an OSH setting at a workplace.In summary, we presented a novel system for visualizing magnetic flux density measurements for occupational safety in the workplace via augmented reality. Images and videos created with the system were used in an evaluation study to test whether such AR enhanced media can improve the understanding of the exposure situation. The results of an online survey showed that participants with access to such media made fewer mistakes answering safety relevant questions about the exposure. Furthermore, almost all participants perceived the AR enhanced content as very helpful. Therefore, we conclude that occupational safety and health workplace evaluations can benefit from AR enhanced media, and that such media can be created with reasonable effort given current technology.


Arabidopsis. In rice, several genes have been reported in conferring chilling tolerance, however, the chilling signaling in rice remains largely unknown. Here, we report the chilling-induced OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 6 (OsSAPK6)-IDEAL PLANT ARCHITECTURE 1 (IPA1)-OsCBF3 signal pathway in rice. Under chilling stress, OsSAPK6 could phosphorylate IPA1 and increase its stability. In turn, IPA1 could directly bind to the GTAC motif on the OsCBF3 promoter to elevate its expression. Genetic evidence showed that OsSAPK6, IPA1 and OsCBF3 were all positive regulators of rice chilling tolerance. The function of OsSAPK6 in chilling tolerance depended on IPA1, and overexpression of OsCBF3 could rescue the chilling-sensitive phenotype of ipa1 loss-of-function mutant. Moreover, the natural gain-of-function allele ipa1-2D could simultaneously enhance seedling chilling tolerance and increase grain yield. Taken together, our results revealed a chilling-induced OsSAPK6-IPA1-OsCBF signal cascade in rice, which shed new lights on chilling stress-tolerant rice breeding.Chilling is a major abiotic stress harming rice development and productivity. The C-REPEAT BINDING FACTOR (CBF)-dependent transcriptional regulatory pathway plays a central role in cold stress and acclimation in  Rice, a staple food feeding half of the world population, is a chilling sensitive plant that shows slow seedling growth, yellowing, stunting, withering, and ultimately death under low temperature stress4. Therefore, it is crucially needed to develop genetic tools for breeding cold stress-tolerant rice varieties, which can prevent low temperature damage and minimalize the constraint of rice cultivation area6.Cold is a major abiotic stress that adversely affects plant growth and crop productionArabidopsis, and the C-REPEAT BINDING FACTOR/DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 1 (CBF/DREB1)-dependent transcriptional regulatory pathway plays a central role in cold stress and acclimation7. The expressions of three CBF genes are rapidly induced under cold stress, and CBF proteins could directly bind to the CRT/DRE cis elements in the promoters of COLD REGULATED (COR) genes, leading to enhanced cold tolerance in Arabidopsis8. INDUCER OF CBF EXPRESSION 1 (ICE1) is the key transcription factor in the upstream of CBFs in cold responses, which could directly bind to MYC recognition sequences in the promoter of CBFs9. Under normal conditions, ICE1 is degraded by the E3 ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1), but under cold stress, ICE1 is phosphorylated by OPEN STOMATA 1 (OST1)/SUCROSE NON-FERMENTING 1 (SNF1)-related protein kinase 2.6 (SnRK2.6) for inhibiting its degradation, which in turn positively regulates CBF expression to enhance cold tolerance11.Cold response has been extensively studied in Arabidopsis, the understandings of cold tolerance for rice are fragmental. OsICE1/ BASIC HELIX-LOOP-HELIX PROTEIN 002 (OsbHLH002) could be phosphorylated and stabilized by MITOGEN-ACTIVATED PROTEIN KINASE 3 (OsMAPK3) to increase TREHALOSE-6-PHOSPHATE PHOSPHATASE 1 (OsTPP1) expression and enhance the chilling tolerance12. Cyclic nucleotide-gated channel 9 (OsCNGC9), a cyclic nucleotide-gated channel, could positively regulate rice chilling tolerance by mediating Ca2+ elevation in cytosol13. Under chilling stress, OsCNGC9 could be phosphorylated and stabilized by OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 8 (OsSAPK8), the closest rice homolog of OST1. The expression of OsCBF3/OsDREB1A is cold induced and oscbf3 mutant is chilling sensitive. OsCBF3 could directly bind to the DRE-box motif on the promoter of OsCNGC9 to upregulate its expression under chilling stress13. CHILLING TOLERANCE DIVERGENCE 1 (COLD1), different alleles of which conferred the chilling tolerance variation between indica and japonica species, could work with G-PROTEIN α SUBUNIT 1 (RGA1) to mediate the cold-induced Ca2+ influx to promote cold stress, and may function as a chilling sensor in rice16. Overexpression of CBL-INTERACTING PROTEIN KINASE 7 (OsCIPK7) could increase the chilling tolerance through Ca2+ influx17. The Ca2+ influx and the protein kinases have been revealed to play an important role in rice chilling sensing and tolerance. However, the cold signal cascade, especially the regulatory mechanisms underlying cold-induced CBF expression, was unclear in rice.Although the ICE1-CBFs-COR cold signaling cascade has been well-characterized for 18. IDEAL PLANT ARCHITECTURE 1 (IPA1)/ WEALTHY FARMER’S PANICLE (WFP)/OsSPL14 gene, encoding a SQUAMOSA promoter-binding protein-like (SPL) transcription factor, has been reported as a critical factor regulated by miRNA156/529 in shaping rice ideal plant architecture and substantially increasing rice grain yield22. IPA1 could also positively regulate disease resistance against Magnaporthe oryzae and bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo), conferring a promotion of both yield and disease resistance by sustaining a balance between growth and immunity25. Similar resources have been long-desired for abiotic stresses, and elucidating the mechanism underlying the tradeoff between growth and chilling tolerance will greatly benefit rice breeding.In plants, the activation of defense responses under biotic or abiotic stresses usually inhibit their own growth, and the genetic resources or tools that can enhance the resistance without harming crop yield are long desiredossapk6. Biochemical analyses revealed that OsSAPK6 could phosphorylate IPA1 and stabilize IPA1 under chilling stress. IPA1 could directly bind to the OsCBF3 promoter via the GTAC motif and induce OsCBF3 expression. Overexpression of OsSAPK6, IPA1, and OsCBF3 could each enhance chilling tolerance, and gain-of-function allele ipa1-2D promoted both rice yield and seedling chilling tolerance. Our study has revealed an OsSAPK6-IPA1-OsCBF chilling signaling cascade in rice, which has greatly expanded our understanding of chilling responses and provided important genetic resources for breeding high yield and chilling-tolerant rice varieties.In this study, we screened out and identified a chilling-sensitive mutant 26, we found one chilling sensitive mutant LBM3705. By sequencing the guide sequence and its target gene in LBM3705, we found one “A” insertion at the 42-bp site in exon 1 of OsSAPK6, which resulted in a frameshift and premature translation termination of OsSAPK6  in ZH11 background. Under normal conditions, 2-week-old seedlings of Ubi:OsSAPK6 was significantly taller than ZH11  backgroundlow Fig. , and theH11 Fig. . We thenent Fig. . Another6-1 Fig. . To furtH11 Fig. . All the27. To identify the substrates of OsSAPK6, we performed co-immunoprecipitation (co-IP) followed by mass spectrometry using the GFP-OsSAPK6 fusion protein transiently expressed in rice protoplasts. The result showed that IPA1 was highly enriched in candidate interacting proteins  results showed that chilling-stress could induce the expression of IPA1 in ZH11 as well as in OsSAPK6-OE and ossapk6 , we found 13 genes whose expression levels were further enhanced under chilling treatment in ipa1-3D compared to WT, including OsCBF2 and OsCBF3 in the top five list  using the recombinant IPA1-GST protein expressed in and purified from E. coli. The result showed that the GST-IPA1 protein was able to bind to the 42-bp GTAC-containing region P2 in the OsCBF3 promoter, but no signal was observed for the control GST protein , and OsSAPK6 proteins were purified from these plants using anti-Flag beads, which were then incubated with IPA1 for testing the phosphorylation levels of IPA1. Indeed, we found that the kinase activity of OsSAPK6 was increased in plants with 1 h chilling treatment and further increased in plants with 3 h treatment  NILIPA1 and NILipa1-2D in the Nipponbare (NIP) background35. After chilling stress and recovery, the survival rate of 2-week-old NILipa1-2D seedlings was significantly higher than that of NILIPA1 , a RING-finger E3 ligase, was previously reported to promote the degradation of IPA1 in panicles and stabilize IPA1 in shoot apexes to regulate plant architecture38. Whether IPI1-mediated degradation of IPA1 is inhibited in the chilling-induced phosphorylation of IPA1 by OsSAPK6 remains to be elusive.In es Figs.  and 6. Ies Figs.  and 7, aion Fig. . OsSAPK624. The stress-induced modification of IPA1 protein played important roles in stress responses24. For rice blast response, IPA1 was phosphorylated on Ser163 and in turn upregulated WRKY45 to enhance rice blast resistance23, whereas the responsible kinase was still unknown. In this study, we found that IPA1 was phosphorylated on Ser201 and Ser213 by OsSAPK6 to upregulate OsCBF3 to enhance rice chilling tolerance, and the phosphorylation of IPA1 was critical for its function in chilling stress responses  ZH11, ipa1-3D, ipa1-10, ossapk6-1/2, ossapk6-3 ipa1-3D double mutant, as well as OsSAPK6 overexpression (Ubi:OsSAPK6) lines were grown either in the controlled growth chamber  under 16-h day/8-h night at 25 °C/16 °C (day/night) cycles or in the experimental field of the Institute of Genetics and Developmental Biology in Beijing. Double mutant of ossapk6-3 ipa1-3D was generated by CRISPR/Cas9 using the vector VK005  from the ipa1-3D background. The ipa1S213N mutant line was generated by CRISPR/Cas9 using the vector pZRH-PBE from the ZH11 background. For OsSAPK6 overexpression lines, full-length cDNA of OsSAPK6 was cloned into pJL1460 vector and driven by Ubiquitin promoter to generate the Ubi:OsSAPK6 construct. Agrobacterium tumefaciens (strain EHA105)-mediated transformation was used to introduce the constructs into rice42. Positive lines were confirmed by PCR followed by sequencing, and then the homozygous T2 plants were used for the experiments. All primers and gRNAs used in the present study are listed in Supplementary Table SRice  maintained at 4 ± 0.5 °C for a chilling treatment. For heat treatment, the seedlings were moved into the plant growth chamber  maintained at 35 ± 0.5 °C for a heat treatment. The duration of the treatment varied from four to seven days. Subsequently, seedlings were returned to plant growth chamber conditions and allowed to recover for four to fifteen days. The survival rate (percentage of live seedlings) was determined. Each experiment was conducted independently at least three times.Rice seeds were soaked in water for 3 days at 37 °C. The germinated seeds were then placed into an incubator with Kimura B nutrient solution and grown in a plant growth chamber under 16-h day/8-h night at 25 °C/16 °C (day/night) cycles. Two-week-old seedlings were used for the subsequent tolerance assays. Chilling tolerance assays were performed as described11. Leave samples from 5 plants (0.3 g) were immersed in a 15-mL tube containing 5 mL deionized water and shaken at 200 rpm at room temperature for 1 h, and the electrical conductivity (S1) was determined. The samples were boiled at 100 °C for 20 min, shaken at 22 °C for 1 h, and then detected to determine the total conductivity (S2). The electrical conductivity of deionized water was defined as S0. Relative ion leakage was calculated as (S1‒S0)/(S2‒S0).The chilling-treated seedlings were harvested for ion leakage assays43. Total RNAs were extracted from rice tissues using TRIzol reagent , followed by reverse transcription using a QuantiTect Reverse Transcription Kit . qRT-PCR was performed using SYBR Green Kit  in a real-time PCR system . Three biological replicates were set up, and each sample was analyzed at least in triplicate. Primers used are listed in Supplementary Table SRNA isolation, reverse transcription and real-time PCR were performed as described previously201A213A, and OsSAPK6, the full-length coding sequences of the corresponding genes were amplified and fused to GFP in the plant expression vector pBI221 (HonorGene). Rice leaf protoplast isolation and transformation were carried out according to the protocol as previously described44. IPA1, IPA1201A213A, and OsSAPK6 together with 35 S:mCherry (marker for nucleus) were co-transformed into rice leaf protoplasts. 35S:GFP was used as a control. After incubation in the dark overnight, the localization patterns were assessed by visualizing GFP fluorescence using a confocal laser-scanning microscope (Zeiss LSM-710). The primers used are listed in Supplementary Table STo determine the subcellular localization of IPA1, IPA1IPA1 or IPA1201A213A was cloned into puc-SCYCE, and OsSAPK6 was cloned into puc-SCYNE. The plasmid mixtures were introduced into rice leaf protoplasts as described44. After incubation in the dark overnight, the fluorescence was observed with a confocal laser-scanning microscope (Zeiss LSM-710).The coding sequence of OsSAPK6 into the vector pGADT7 (Takara Bio Inc.) and IPA1 into pGBKT7 (Takara Bio Inc.). The truncation versions for IPA1 amino acids 1–103, 1–181, 104–417, and 182–417, were each inserted into pGBKT7. All different combinations of the prey and bait constructs were co-transformed into yeast strain AH109 by the lithium acetate method45. After transfection, strains were cultured on minimal medium/-Leu/-Trp, further selected on minimal medium/-Leu/-Trp/-His/-Ade. The primers used for the yeast two-hybrid assays are provided in Supplementary Table SYeast two-hybrid assays were performed using the Matchmaker GAL4-based Two-Hybrid System 3 (Clontech) following the manufacturer’s instructions. Constructs were produced by cloning 35S:HA-IPA1/35S:GFP or 35S:HA-IPA1/35S:OsSAPK6-GFP constructs and immunoprecipitated with anti-IPA1 antibody. Proteins were detected with anti-GFP  antibody.The total proteins were extracted from rice protoplasts expressing IPA1-GFP transgenic lines . The IPA1 monoclonal antibody was produced by HUABIO Co., Ltd . The specificity of anti-IPA1 antibody was validated using wild-type and 46. For the in vitro phosphorylation assay, purified GST-IPA1 and His-OsSAPK6 were incubated in kinase buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 10 mM ATP at 30 °C for 30 min. The samples were then incubated with or without CIP at 37 °C for 30 min, and the proteins were separated in a 12% (w/v) SDS-PAGE gel containing 50 μM Phos-tag and 100 μM MnCl2. The gel was incubated in transfer buffer containing 10 mM EDTA three times and then washed in transfer buffer for 10 min. After transferring onto PVDF membranes, the GST-IPA1 protein was detected with an anti-GST  monoclonal antibody.Phos-tag reagent  was used for the phosphoprotein mobility-shift assay to detect phosphorylated IPA1 proteins as describedossapk6-2, or IPA1-GFP transgenic plants were treated with chilling stress and proteins were extracted with extraction buffer. The samples were incubated with or without CIP at 37 °C for 30 min, and then analyzed using 12% (w/v) SDS-PAGE gel containing 50 μM Phos-tag and 100 μM MnCl2. After transferring into PVDF membranes, the IPA1 protein was detected with an anti-IPA1 monoclonal antibody.For the in vivo phosphorylation assay, ZH11, 11 with some modifications. Total proteins were extracted from 2-week-old seedlings of wild-type ZH11, ossapk6-2, and Ubi:OsSAPK6 plants with extraction buffer. Equal amounts of above total proteins were incubated with equal amounts of recombinant GST-IPA1, GST-IPA1S201A, GST-IPA1S213A, or GST-IPA1S201AS213A protein and 10 mM ATP for the indicated time. The proteins were separated by SDS-PAGE and detected with anti-GST monoclonal antibody. Rice rubisco large subunit or actin was used as a loading control.Cell-free protein degradation assay was performed as describedg and 4 °C for 20 min. The suspension was transferred to a new tube. The concentration of proteins was measured using a BCA Protein Assay Kit  and finally the protein concentration of each sample was adjusted to the same level. For immunoblotting, proteins were separated by 10% (w/v) SDS-PAGE and transferred to supported nitrocellulose transfer membrane  by electro-transfer at 70 V for 30 min. The membrane was blocked in TBST buffer containing 5% skim milk powder and further incubated with primary antibody for 120 min at room temperature and then secondary antibody for 60 min. Finally, bands on the blot were detected using chemiluminescent HRP substrate . Relative amounts of proteins were determined by densitometry and normalized to loadings using Image J software.For protein extraction, rice tissues were ground in liquid nitrogen and proteins were extracted using the extraction buffer containing 100 mM Tris-HCl, pH 7.5, 100 mM KCl, 10% glycerol, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1% Triton X-100, and 1× protease inhibitor cocktail. The total extraction was mixed thoroughly and centrifuged at 12,000 × The antibodies used in this study included anti-GFP , anti-GST , anti-His , anti-Actin , and anti-LXRXX(pS/pT) .ProOsCBF3-LUC combined with 35S: IPA1-GFP, 35S: IPA1-GFP, or 35S: OsSAPK6-Flag were introduced into rice leaf protoplasts as described42. After incubation in the dark overnight, the luciferase activities were measured by the Dual-Luciferase Reporter Assay System  according to the manufacturer’s instructions.The plasmid pGreen0800-ProIPA1:7mIPA1-GFP transgenic seedlings were used for ChIP-qPCR assays, which were performed as previously described47. The enriched DNA fragments were analysed by qRT-PCR using the primers as listed in Supplementary Table Subiquitin (LOC_Os03g13170) promoter. No addition of antibodies (NoAbs) was served as a negative control.Two-week-old 29. The amplified CDS of IPA1 was fused in frame with the GST tags in the pGEX-4t-1 vector (Crgen). The GST-IPA1 recombinant protein was expressed in E. coli BL21 (DE3) cells at 16 °C for 10 h using 0.3 mM IPTG and purified with Glutathione Sepharose 4B . Oligonucleotide probes of OsOsCBF3 were synthesized and labeled with 5′-biotin (Invitrogen) by annealing of complementary oligonucleotides at 72 °C for 30 min. EMSA was performed using a LightShift® Chemiluminescent EMSA Kit  according to the manufacturer’s instructions. Probes used are listed in Supplementary Table SProbe labeling and EMSA were performed as described previouslyi BL21 DE cells at49. Fragment quantifications were computed with FeatureCounts (version 1.5.0) in paired-end mode, the features are exons50. Expression differentiation analyses were conducted using the R (version 3.3.1) package DESeq (version 1.26.0) with three biological replicates51.The paired-end clean reads of RNA-seq were aligned to the rice reference genome Os-Nipponbare-Reference-IRGSP-1.0 using STAR (version 2.4.2a)IPA1 (LOC_Os08g39890), OsSAPK6 (LOC_Os02g34600), OsSAPK8 (LOC_Os03g55600), OsCBF1 (LOC_Os09g35010), OsCBF2 (LOC_Os06g03670), OsCBF3 (LOC_Os09g35030), and OsCNGC9 (LOC_Os09g38580).Gene sequences used in this study can be found in the Rice Genome Annotation Project under following accession numbers: Supplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3


We also emphasized the effects that both other hormone receptors and the newly emerging androgen-inducible non coding RNAs may have on AR function in BC pathology and the putative implementation in the clinical setting. Moreover, we pointed out the latest results by clinical trials and we speculated about the use of anti-AR therapies in BC clinical practice.Androgen receptor (AR) is expressed in 60-70% of breast cancers (BCs) and the availability of anti-AR compounds, currently used for treating prostate cancer, paves the way to tackle specifically AR-positive BC patients. The prognostic and predictive role of AR in BC is a matter of debate, since the results from clinical trials are not striking, probably due to both technical and biological reasons. In this review, we aimed to highlight WHAT is AR, describing its structure and functions, WHAT to test and HOW to detect AR, WHERE AR should be tested (on primary tumor or metastasis) and WHY studying this fascinating hormone receptor, exploring and debating on its prognostic and predictive role. We considered AR and its ratio with other hormone receptors, analyzing also studies including patients with ductal carcinoma  Breast Cancer (BC) still remains the leading cause of cancer-related deaths for women, with an estimated 5-year prevalence in Europe in 2012 of about 1.8 million cases –6. ER exSince BC is highly heterogeneous, it is usually classified in five intrinsic molecular subtypes determined by a 50 gene-expression profile (PAM50), that have different clinical and biological characteristics affecting patient outcome  . Howevere.g. in ER-positive BC AR may play an unfavorable prognostic role  in BC pathology is gaining clinical interest also in relation to the development of drugs that can modulate AR activity. Androgen receptors are expressed in 60%–90% of BCs, mainly in ER-positive tumors (about 70%) and in about 40% of triple negative tumors –10. Howetic role ; in TNBCtic role , but dattic role . In thisAR belongs to the steroid receptor superfamily and is made up of 919-aminoacids encoded by a 180 kb gene localized on human chromosome Xq11-12. The AR is expressed in a diverse range of tissues including bone, muscle, prostate, adipose tissue and the reproductive, cardiovascular, immune, neural and hematopoietic system . The recvia phosphorylation and/or interaction with co-activators promoted by a number of different growth factors has been widely demonstrated. For instance, IL-6, commonly expressed at high circulating levels in patients with different cancers, increases AR activity in a ligand-independent manner via the protein kinase A (PKA), PKC and MAPK pathways  pathway, promoting an agonist effect of tamoxifen on ER pathway and this mechanism could be blocked by a combination of enzalutamide and gefitinib . A crossNon-coding RNAs (ncRNAs) are regulators of intracellular and intercellular signaling and control different cellular processes, cell proliferation, invasion, migration, apoptosis, and stemness. MicroRNAs  are 20-25 base pair long, single-stranded, non-coding RNAs which primarily bind to the 3’-untranslated region (3’-UTR) of messenger RNAs (mRNA) to suppress their transcription with the following reduction of target protein levels . Beside +/CD24-/low tumor-initiating cells expressing a stemness phenotype and patients with these features showed a better outcome . H score was also proposed to define AR positivity, a semiquantitative parameter that considers both the staining intensity and the percentage of positive cells (in situ hybridization (FISH) to analyze the gene copy number . Recently, it has been reported that it is important to test AR by IHC to evaluate the utility of AR antagonists for chemoprevention in patients with AR+ and ER- DCIS (in situ tumors are routinely treated with surgery and radiotherapy (and Tamoxifen in some cases). In this context, the prognostic and predictive role of specific markers, including AR, for clinical outcome in this population was investigated by Ravaioli et al.  in ER expressing tumors , 84, 85,In line with this finding, Kraby and colleagues demonstrated an independent favorable prognostic role of AR, in particular for grade 3 and Luminal A BCs .in situ ductal carcinoma, independently of treatment  is a non-steroidal selective androgen receptor modulator (SARM) that has demonstrated preclinical and clinical activity in AR positive BC . PalmierProstate cancer growth and progression are sustained by AR signalling, hence androgen deprivation therapy is the gold standard treatment in PCa. AR upregulation is the most common event underlying the progression from hormone sensitive to castration-resistant PCa. AR overexpression can occur caused by different mechanisms, including mutations, amplifications, gene rearrangements, that produce truncated AR variants . Severalin vitro and in vivo experiments had the principal aim to test the dose, efficacy, safety, and tolerability of anti-AR therapies. A phase 1 study tested the anti-tumor activity of Seviteronel, a selective CYP17 lyase and AR inhibitor. The safety, tolerability, pharmacokinetics and activity of daily Seviteronel administration were evaluated in women with ER-positive tumors or TNBC and showed to be well tolerated (in vivo experiments confirmed that S1 blocking could be a valuable anti-AR strategy. Lehmann’s group showed that AR enriched TNBC cell lines carrying PI3KCA mutations acquire sensitivity to PI3K/mTOR inhibition, promoting cancer cell growth (in vitro models (Abiraterone is not an AR antagonist, since it is a selective inhibitor of the cytochrome P450 involved in androgens biosynthesis. It causes a decrease of circulating testosterone levels . In 2018olerated . As Abirolerated  alone orolerated , 117. Giolerated . The in l growth . Regardio models . These fo models  (Table PCa studies suggest AR as prominent prognostic and predictive marker. However, the prognostic and predictive role of AR in BC is still matter of debate, since the results from clinical trials are not striking, probably due to both technical and biological reasons. Among the former, no companion diagnostic test to assess AR status and select eligible patients that could benefit from an anti-AR treatment is available. Disparities emerged in its evaluation due to different types of tests to detect AR, antibodies used, scoring systems and positivity cut offs. However, the prognostic role of AR expression detected by IHC and the ratio AR/ER in DCIS patients, could be worthy of further investigations. The differences in AR expression between primary and metastatic tumors suggest that AR should be detected in all patient biological materials, also considering the different role of this biomarker in the different subsets of disease. Although the real role of AR in predicting the response to endocrine therapy has to be defined yet, the ratios with hormone receptors should be taken into account, given their importance for patient risk assessment.in vivo efficacy of the combination of anti-AR strategies.The possibility to treat AR positive TNBC patients with new anti-AR compounds, opens new perspectives in this prognostically unfavorable subset. However, additional studies are needed to verify the SB conceived the study. SR analyzed and interpreted the data. SR and SB were the major contributors in writing the manuscript. All authors read and approved the final manuscript.This work was partly supported thanks to the contribution of Ricerca Corrente by the Italian Ministry of Health within the research line 3, including genetics and environment in the development and progression of tumors and inhibitory mechanisms, exposomics and primary and secondary prevention.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver. Long non-coding RNAs (lncRNAs) play important roles in the occurrence and development of HCC through multiple pathways. Our previous study reported the specific molecular mechanism for sulfatide regulation of integrin αV expression and cell adhesion in HCC cells through lncRNA AY927503. Next, it is necessary to identify more sulfatide-related lncRNAs, explore their clinical signifcance, and determine new targeted treatment strategies.Microarrays were used to screen a complete set of lncRNAs with different expression profiles in sulfatide-treated cells. Sulfatide-related lncRNAs expression data and corresponding HCC patient survival information were obtained from the The Cancer Genome Atlas (TCGA) database, and the prognosis prediction model was constructed based on Cox regression analysis. Methylated RNA immunoprecipitation with next generation sequencing (MeRIP-seq) was used to detemine the effect of sulfatide on lncRNAs m6A modification. Tumor Immune Estimation Resource (TIMER) and Gene set nnrichment analysis (GSEA) were utilized to enrich the immune and functional pathways of sulfatide-related lncRNAs.P<0.05) were screened in sulfatide-treated HCC cells. As a result, 24 sulfatide-related lncRNAs were highly expressed in HCC tissues, six of which were associated with poor prognosis in HCC patients. Based on thses data, a sulfatide-related lncRNAs prognosis assessment model for HCC was constructed. According to this risk score analysis, the overall survival (OS) curve showed that the OS of high-risk patients was significantly lower than that of low-risk patients (P<0.05). Notably, the expression difference in sulfatide-related lncRNA NRSN2-AS1 may be related to sulfatide-induced RNA m6A methylation. In addition, the expression level of NRSN2-AS1 was significantly positively correlated with immune cell infiltration in HCC and participated in the peroxisome and Peroxisome proliferator-activated receptor (PPAR) signaling pathways.A total of 85 differentially expressed lncRNAs (|Fold Change (FC)|>2, In conclusion, sulfatide-related lncRNAs might be promising prognostic and therapeutic targets for HCC. ITGAV transcriptional chromatin modification and was a potential molecular marker of HCC metastasis or poor prognosis (Hepatocellular carcinoma (HCC) is the most common liver malignancy worldwide and is one of the top five deadliest cancers, with high morbidity and mortality rates . The levrognosis . Long norognosis . Therefo2 at 37°C. For the sulfatide treatment, cells were incubated at the initial density of 0.5×105 cells/mL and treated with 2 μM galactocerebroside  or sulfatide .SMMC-7721 cells were obtained from Cell Bank of Type Culture Collection of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Science. They were cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum (FBS) (Gibco). SMMC-7721 cells were identified by their morphological characteristics which were consistent with the establishment report . Cells wMicroarray profiling was conducted in the laboratory of Aksomics Inc. . The microarray was analyzed using the nrStar™ Functional LncRNA PCR chip software, version 1.0 . The hierarchical clustering analysis was carried out using a platform-independent software TBtools (version x64_1_09867) .https://xenabrowser.net/) (The TCGA Liver Cancer project (TCGA-LIHC) (N=423) data were downloaded from the UCSC Xena database (er.net/) . Log2  in HCC. The patients were divided into two groups according to the best cutoff value for each sulfatide-related lncRNA, which calculated by the R package maxstat. The OS significance map in HCC was evaluated using the Kaplan-Meier plotter  .The least absolute shrinkage and selection operator (LASSO) regression analysis was conducted on the sulfatide-related lncRNAs. The LASSO regression algorithm was used for feature selection with 10-fold cross-validation. The R package glmnet was used for the analysis. For Kaplan-Meier curves, p-values and hazard ratios (HRs) with 95% confidence intervals (CIs) were generated using log-rank tests and univariate Cox proportional -hazards regression. Finally, six sulfatide-related lncRNAs were selected for incorporation into the risk score. The regression coefficient β for multivariate Cox regression model and lncRNA expression were used to construct the risk score formula as follows:http://timer.cistrome.org/) (The Tumor Immune Estimation Resource (TIMER) database (me.org/)  analyzesme.org/) , 17. Theme.org/) , an R soTotal RNA samples were extracted, ragmented to 100bp, and immunoprecipitated using anti-m6A antibody (abcam). Then, eluted RNA and MeRIPed RNA were analyzed using deep sequencing with an Illumina Novaseq™ 6000 platform on the CLOUDSEQ Bio-tech Ltd  following the vendor’s recommended protocol l.NRSN2-AS1 expression (P) <0.05, and false discovery rate (FDR) q <0.25 were considered to have significant enrichment.Pathway enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed using the Gene Set Enrichment Analysis (GSEA) database in TCGA-LIHC, which classified the data into high- and low- expression groups based on their pression . Gene sevia Kaplan-Meier plots. The significant correlations between sulfatide-related lncRNAs expression and immune cell infiltration scores in HCC were determined by analyzing Spearman’s correlation coefficients. Pearson correlation analysis was performed between the expression level of NRSN2-AS1_(ENSG00000225377) and gene set expression level of RNA m6A methylation-modifying enzyme. The level of significance was set at P < 0.05. The bioinformatics analysis platform Sangerbox, version 3.0 (http://vip.sangerbox.com/)  , was useP < 0.05)  based on their Ensemble IDs . Subsequently, an X-tile diagram was used to generate the optimal cutoff point for the risk score. The TCGA-LIHC patients were divided into high- and low-risk groups based on this cutoff risk score value. A prognostic curve and a scatter plot were used to indicate the risk score and survival status of each HCC patient . In addition, the heat map of the expression profiles for candidate lncRNAs demonstrated that they were all highly expressed in the high-risk group  = (2.0727) × LRRC52-AS1 + (0.3691) × RNASEH1-AS1 + (0.2646) × NRSN2-AS1  in sulfatide-treated HCC cells based on their Ensemble IDs. Tthe TCGA-LIHC database 27 sulfatide-related lncRNAs that were differentially expressed in HCC and adjacent tissues, of which 24 were highly expressed in HCC tissues. RP11-122M14.1, RP11-280O1.2, AC079354.5, AC005037.3, AC108488.3 and RP5-1103G7.4 are six sulfatide-related lncRNAs with abnormally high expression that were significantly associated with poor prognosis in HCC patients. When selecting specific variables to build the prognosis evaluation model, overfitting often occurs if too many variables are present (P<0.05).The present study screened 85 DE-lncRNAs  belong to the nuclear hormone receptor family. They are divided into α, β, and γ subtypes, and participate in the metabolism of various energy substances and tumor immunity. PPARα was found to respond to the fatty acids delivered by tumor-derived exosomes (TDEs), resulting in excess lipid droplet biogenesis and enhanced fatty acid oxidation (FAO), culminating in a metabolic shift toward mitochondrial oxidative phosphorylation, which drives tumor-infiltrating DCs (TIDCs) immune dysfunction . It was In this study, we described the influence of sulfatide on lncRNA expression in HCC cells and found that these sulfatide-related lncRNAs serve as a good prognostic marker for HCC patients. In addition, we showed that NRSN2-AS1 may be an indicator of TIME characterization in HCC. These results help to improve the understanding of the comprehensive characteristics and role of sulfatide in the development and progression of HCC and will help to optimize immunotherapy regimens.https://www.ncbi.nlm.nih.gov/geo/, GSE151111.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The concept of the project was proposed by FF and QC. XH and QC collected the data from the databases and performed data analysis. The MeRIP-seq cell samples were cultured and prepared by LF. XH wrote the manuscript, and QC and FF contributed to editing and participated in manuscript-related discussions. All authors contributed to the article and approved the submitted version.


The designed monomer was obtained via a two-step procedure in a good yield. This norbornene derivative with a rigid and a bulky binaphthyl group was successfully polymerized over the 1st generation Grubbs catalyst, affording high-molecular-weight products (Mw ≤ 1.5·106) in yields of 94–98%. The polymer is amorphous and glassy (Tg = 161 °C), and it shows good thermal stability. Unlike most, polyNBi is a classic low-permeable glassy polymer. The selectivity of polyNBi was higher than that of polyNB. Being less permeable than polyNB, polyNBi unexpectedly showed a lower value of dielectric permittivity (2.7 for polyNBi vs. 5.0 for polyNB). Therefore, the molecular design of polynorbornenes has great potential to obtain polymers with desired properties in a wide range of required characteristics. Further tuning of the gas separation efficiency can be achieved by attaching an appropriate substituent to the ester and aryl group.Polymers from norbornenes are of interest for applications in opto- and microelectronic . Norbornenes with ester motifs are among the most readily available norbornene derivatives. However, little is known about dielectric properties and the gas-transport of polynorbornenes from such monomers. Herein, we synthesized a new metathesis polymer from  Strained cycloalkenes have attracted much attention as building blocks in organic chemistry and as monomers in the synthesis of various polymers. There are several significant benefits of cycloalkenes in the synthesis of polymers. First, there is their increased reactivity in polymerization processes due to the high strain energy of a carbocycle. This feature allows involvement in polymerization monomers, bearing two, three, or four substituents, which is often impossible for a polymerization of most olefins. Second, there are at least two ways for polymerization of cycloalkenes (ring-opening metathesis polymerization and vinyl (addition) polymerization ,2,3,4,5)Norbornene derivatives are among the most available and highly reactive cycloolefins, which are synthesized from petrochemical and bio-feedstock. Polymers from norbornenes are of interest for applications in opto- and microelectronic  ,9,10,11,exo-5-norbornenecarboxylic acid and (S)-1,1′-bi-2-naphthol. The gas-transport and dielectric properties of this polymer have been studied to elucidate the effect of the incorporation of ester groups in side-chains on the characteristics of the polymer. The results obtained are considered with data of TGA, DSC, and WAXD analysis, and they are compared with earlier described data for the related polymers.In this work, we have designed and synthesized a new polymer based on motifs widely used in organic chemistry and polymer science, Exo-5-norbornene-2-carboxylic acid was synthesized according to the procedure published in the literature -2-ol as a colorless solid .(S)-2′-Methoxy--2-ol was prepared according to the published procedure . K2CO3 ((S)-2′-Methoxy--2-ol1H NMR : 8.03 , 7.93–7.81 , 7.46 , 7.40–7.31 , 7.34 , 7.31–7.28 , 7.27–7.25 , 7.21–7.14 , 7.07–7.01 , 4.91 , 3.78  (in  CH3) in . (S)-2′-Methoxy--2-yl bicyclo [2.2.1]hept-5-ene-2-carboxylateexo-5-norbornene-2-carboxylic acid  in 25 mL of dry CH2Cl2, containing 3 drops of DMF. The mixture was stirred at rt for 8 h. The resulting solution was evaporated to dryness. The residue was dissolved in 10 mL of dry CH2Cl2 and added dropwise to the ice-cooled mixture of triethylamine  and (S)-2′-methoxy--2-ol  in 50 mL of dry CH2Cl2. After the reaction mixture was stirred for 16 h at room temperature, it was quenched with water. The mixture was extracted with CH2Cl2. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (hexane/EtOAc = 40:1 to 10:1) to afford the desired product  as a colorless oil . [α]D21 = −17.7o in THF (C = 1). HRMS (ESI): calcd. for C29H24O3 [M+nNH4]+438.2064, found 438.2065.Oxalyl chloride  was added to the suspension of 1H NMR : 8.03–7.88 , 7.82 , 7.50–7.35 , 7.34–7.07 , 5.94 , 5.82 , 3.76 , 2.57 , 2.10–1.97 , 2.04–2.00 , 1.52–1.41 ), 1.05–0.89 , 0.78–0.64 , 0.44 .13C NMR : 174.01 (C = O), 154.96 (Ar-OMe), 146.97 (Ar-CO2R), 138.07 (C = C), 135.40 (C = C), 133.76 (Ar), 131.80 (Ar), 129.97 (Ar), 129.05(Ar), 128.14 (Ar), 127.67 (Ar), 126.58 (Ar), 126.44 (Ar), 126.10 (Ar), 125.52 (Ar), 125.40 (Ar), 125.36 (Ar), 123.72 (Ar), 122.08 (Ar), 118.02 (Ar), 113.68 (Ar), 56.87 (OCH3), 46.30 (C(4)), 45.47 (C(7)), 43.05, 41.43, 29.37.1H NMR : 8.03–7.88 , 7.82 , 7.50–7.35 , 7.34–7.07 , 5.94 , 5.82 , 3.72 , 2.57 , 2.27 , 2.10–1.97 , 1.25–1.14 , 1.05–0.89 , 1.01–0.93 , 0.78–0.64 .13C NMR : 174.07 (C = O), 155.00 (Ar-OMe), 146.93 (Ar-CO2R), 137.89 (C = C), 135.40 (C = C), 133.74 (Ar), 131.80 (Ar), 129.97 (Ar), 129.05 (Ar), 128.14 (Ar), 127.72 (Ar), 126.63 (Ar), 126.44 (Ar), 126.10 (Ar), 126.06 (Ar), 125.45 (Ar), 125.36 (Ar), 122.13 (Ar), 122.08 (Ar), 118.02 (Ar), 113.68 (Ar), 56.71 (OCH3), 45.81 (C(4)), 46.03 (C(7)), 42.96, 41.39, 29.70 (C(5)).−3 mmol, 3.36·10−3 M) was added to the monomer solution at stirring. The stirring continued for 30 min. The polymerization was terminated by the addition of ethyl vinyl ether followed by stirring for 5 min. Then, the polymer solution was precipitated by methanol containing the inhibitor ). The polymer was separated, washed by several portions of methanol, and dried in a vacuum. The polymer was twice reprecipitated by methanol from the chloroform solution and dried in a vacuum to a constant weight. The polymer yield was 0.91 g (94%). Mw = 1.5·106, Mw/Mn = 3.4. [α]D21 = −28o in CHCl3 (C = 1).The monomer  was dissolved in 44 mL of absolute dichloroethane, and the solution of the 1-st generation Grubbs catalyst 0.97 mL : 8.15–6.80 , 5.51–4.52 , 3.93–0.62 .13C NMR : 173.63–173.32 (C = O), 154.99–154.95, 146.84–146.80, 133.8–122.05, 117.76–117.71, 113.66–113.60, 56.72–56.67, 50.10–49.70, 46.83–46.72, 41.95–41.76, 36.71–35.63.exo-5-norbornenecarboxylic acid as starting compounds (The target monomer was readily prepared in a good yield by the usage of (S)-1,1′-bi-2-naphthol and ompounds . (S)-1,1The obtained monomer was successfully involved in ring-opening metathesis polymerization in the presence of the 1-st generation Grubbs catalyst to produce high-molecular weight polymers in 94–98% yields .The prepared metathesis polymer was soluble in common organic solvents . High-molecular weight polyNBi exhibited good film-forming and mechanical properties. The values of Young’s modulus (E), tensile strength (σ), and elongation at break (ε) were consistent with the properties for conventional glassy polymers .exo-5-norbornenecarboxylic acid and 1,1′-bi-2-naphthol were analyzed with help of NMR spectroscopy (S-isomer), the resulting monomer and the corresponding metathesis polymer were optically active compounds. The specific optical rotation for both compounds was different from zero (The structure and the purity of the metathesis polymer based on the ester of troscopy . Since wrom zero .The synthesized polyNBi is an amorphous polymer. Its WAXD pattern is represented by two broad peaks a. Most aThe structure of metathesis polynorbornene (polyNB).g for polyNBi is much higher than that of the related polymer without substituents, polyNB . PolyNB2, N2, O2, CO2, CH4, and C2H6) in polyNBi were experimentally measured  > P(He) > P(H2) > P(O2) > P(CH4) > P(N2) > P(C2H6). The change in diffusivity coefficients for polyNBi corresponds to the kinetic diameter of gas molecules: D(O2) > D(N2) > D(CO2) > D(CH4). At the same time, the highest solubility coefficients of gases in polyNBi were for the most easily condensable gases—CO2 and CH4—correspondingly: S(CO2) > S(CH4) > S(O2) > S(N2) (The gas-transport characteristics of polyNBi were studied by the Daynes–Barrer method. Permeability (P) and diffusivity (D) coefficients of various gases  .Interestingly, it has been earlier shown that the introduction of bulky groups in side-chains of polynorbornenes usually leads to an increase in gas permeability . Herein,2H6 was noticeably lower than CH4 permeability. Thus, this study shows that the influence of the ester and aryl fragment on gas permeability is stronger than the influence of the hydrocarbon group in the ester moiety.Another interesting feature is the diffusivity-controlled permeability of hydrocarbons through polyNBi. Usually, such type polymers with hydrocarbonic or organosilicon-side groups possess solubility-controlled permeability of hydrocarbons ,39. NeveA/PB). The corresponding values of α for various pairs of gases are given in 2/N2 and H2/CH4). Interestingly, these selectivities are much higher than those of polyNB (3–6 times higher). At the same time, for pairs of gases with similar molecular sizes, the selectivity of polyNBi was only 1.4–2.8 times higher than that of polyNB. The high selectivity for the O2/N2 and the He/CH4 gas pairs for polyNBi also draws attention. The value of O2/N2 selectivity is one of the highest for all studied polynorbornenes [2/N2 gas pair compared to the data of more permeable polyNB (Permeability selectivities for polyNBi were estimated as the ratio of permeability (P) coefficients of gas A to gas B  for polyNBi was approximately 8.0 at temperatures above Tg and at low frequencies.In −2) and for non-polar polymers (tanδ~10−4). This can be explained by the presence of large aromatic groups in side-chains of polyNBi. Furthermore, the value of tanδ for polyNBi did not depend on the frequency of the electric field and temperatures below Tg. At the same time, at higher temperatures the value of tanδ was much higher, and it increased with temperature  and tanδ are also very important properties of a dielectric material. Unexpectedly, polyNBi exhibited lower values of ε″ and tanδ than are typical for polar polymers . Thus, tperature . AnotherThe review of the literature data describing the dielectric properties of metathesis polymers allows valuable comparisons to be made. Fang et al. synthesized metathesis polynorbornenes, bearing fluorinated eugenol groups. These polymers showed dielectric permittivity below 2.65 . Metatheexo-5-norbornenecarboxylic acid and 1,1′-bi-2-naphthol. The designed monomer was prepared according to a two-step procedure in a good yield using conventional organic tools. The metathesis polymerization of this monomer over the 1-st generation Grubbs catalyst gave high-molecular-weight polymers with yields close to quantitative. The polymer exhibited good mechanical and film-forming properties. It was an amorphous high Tg polymer (161 °C). Despite the presence of bulky and rigid binol-groups in side chains, gas permeability of polyNBi was lower than that of metathesis polynorbornene without substituents (polyNB). Shown, unlike most, polyNBi is a classic low-permeable glassy polymer. This is unique to polynorbornenes with large side substituents, which tend to be highly permeable glasses. It was found that the selectivity of polyNBi was higher than that of polyNB. The high selectivity for the O2/N2 and the He/CH4 gas pairs for polyNBi also draws attention. At the same time, being less permeable than polyNB, polyNBi unexpectedly showed a lower value of dielectric permittivity (2.7 for polyNBi vs. 5.0 for polyNB). Therefore, the nature of the aryl group in the ester motif of side-chains can have a strong influence on dielectric permittivity through various kinds of interactions . Therefore, the molecular design of polynorbornenes has great potential to obtain polymers with desired properties in a wide range of required characteristics. Further tuning of the gas separation efficiency can be achieved by attaching an appropriate substituent to the ester and aryl group.A new norbornene-type monomer with ester moiety (NBi) and its metathesis polymer (polyNBi) were synthesized from