Aortic regurgitation (AR) occurs commonly in patients with continuous-flow left ventricular assist devices (LVAD). No gold standard is available to assess AR severity in this setting. Aim of this study was to create a patient-specific model of AR-LVAD with tailored AR flow assessed by Doppler echocardiography.An echo-compatible flow loop incorporating a 3D printed left heart of a Heart Mate II (HMII) recipient with known significant AR was created. Forward flow and LVAD flow at different LVAD speed were directly measured and AR regurgitant volume (RegVol) obtained by subtraction. Doppler parameters of AR were simultaneously measured at each LVAD speed.We reproduced hemodynamics in a LVAD recipient with AR. AR in the model replicated accurately the AR in the index patient by comparable Color Doppler assessment. Forward flow increased from 4.09 to 5.61 L/min with LVAD speed increasing from 8,800 to 11,000 RPM while RegVol increased by 0.5 L/min (2.01 to 2.5 L/min).Our circulatory flow loop was able to accurately replicate AR severity and flow hemodynamics in an LVAD recipient. This model can be reliably used to study echo parameters and aid clinical management of patients with LVAD. One quarter to one-third of patients develop at least mild to moderate aortic regurgitation within the 1st year and these patients face reduced device durability, higher rates of hospitalization, and worse survival (Continuous flow left ventricular assist device (cf-LVAD) technologies for end-stage heart failure patients have become a long-term treatment strategy . Developsurvival , 3.Current guidelines recommend surgical correction of more than mild AR at the time of LVAD implant but the treatment strategy of significant AR that develops after LVAD implant is more complex , 5. In tin vitro, patient-specific, AR model that incorporates a cf-LVAD could represent a much-needed reference standard for dynamic flow and accurate AR volume quantification. Recently, 3D printing technology has been applied to the LVAD population for pre-implantation planning, but such patient-specific anatomic modeling has not been utilized for functional replication of this continuous flow/AR challenge  replicate the hemodynamic conditions of AR in the presence of a cf-LVAD; (2) directly measure AR volume (RegVol) in an experimental setting to isolate the impact of LVAD speed changes on AR severity; and (3) assess the performance of recommended quantitative echocardiographic parameters for the assessment of AR severity compared to a reference standard of flow.For our study, we selected an 81-year-old male with HM II LVAD and a history of ischemic cardiomyopathy. Appropriate Institutional Review Board approval and consent were obtained from the patient.2 (From computed tomographic (CT) image data, segmentation of the patient-specific model was performed using Mimics software  . The seg2  inset. TWe used a previously described process of segmentation to recreate a 3D patient-specific model. This model was outsourced for printing . Please Our group has previously described an echo-compatible flow loop that, coupled with 3D printing technology, was able to replicate patient-specific hemodynamic conditions in different clinical settings, and provide reference standard flow and pressure measurements , 13. TheA fixed beat rate of 60 BPM and a fixed forward flow volume was used for all experimental conditions. With this constant preload, afterload and heart rate, 6 progressively higher LVAD speeds, from a baseline speed of 8,800 RPM to 1,100 RPM  were tested for their impact on aortic and left ventricular pressure, forward systemic flow and regurgitant flow. Doppler echo parameters were assessed at each LVAD pump speed.in vitro pulse cycles and used for statistical analysis.For each LVAD speed setting, the systemic flow (L/min), cf-LVAD flow (L/min), aortic pressure (mmHg) and left ventricular pressure (mmHg) were directly captured. Aortic regurgitant volume  was calculated as the difference between the forward systemic flow measured directly within the flow loop through high fidelity transducers and cf-LVAD flow. An average of 3 measurements were recorded from the diastolic phase of 3 consecutive 2)] and Pulsed Wave Doppler interrogation of inflow and outflow cannula for determination of systolic and diastolic velocities (cm/sec) were recorded for every flow condition. To minimize variability, echocardiographic acquisitions were standardized for gain, filters, compression and rejects settings. TTE parameters describing AR severity and PISA-derived regurgitant Volume  were compared to the reference standard represented by the RegVol measured within the flow loop as described above. To enhance image acquisition the left heart model was placed in a water filled bath. A modified apical view was used for all Doppler evaluations.Echocardiographic acquisitions were performed using an IE33 machine  equipped with a S5 probe for 2-dimensional and Doppler acquisition. Standard echocardiographic parameters for the assessment of AR severity [Continuous Wave Doppler on AR jet with evaluation of peak Velocity (cm/sec), Velocity Time Integral (VTI) and Pressure half time ; color Doppler of regurgitant jet width with operator appraisal, measurements of vena contracta (cm) and proximal isovelocity surface area . Continuous variables were evaluated for normality using the Shapiro-Wilk test for normal distribution. To explore the relationship among the different indexes of AR, the Pearson coefficient of correlation was tested with linear regression analysis. Repeatability of hemodynamic data was assessed with repeated measurements on a second set of experiment under the same conditions and quantified by direct Pearson's correlation. Inter-observer variability for echocardiographic data was assessed by repeated measurement by independent readers for all echo parameters and quantified by direct Pearson's correlation.in vivo waveforms as reported by Rosenbaum et al. describing a left heart catheterization ramp protocol for hemodynamic optimization and variations in disease states  decreased, consistent with increased LV “unloading” with higher LVAD speed .AR created within the 3D patient-specific model replicated well the AR experienced by the patient with qualitatively similar continuous wave Doppler profile, peak velocities and event timing . In addiIncreasing the LVAD speed, the RegVol increased only by approximately 0.5 L/min (2.01 to 2.5 L/min) or approximately < 10 ml/beat (34.5 to 42.4 ml/beat). As such, the severity remained moderate across all tested flow conditions.in vivo  with the increases of LVAD speed (2 = 0.67) between PHT and RegVol that is inverse to what would be expected with worsening AR severity as suggested by higher peak velocities and AR VTI. Of note a similar pattern was seen in the in vivo echocardiographic clinical studies from the model patient .The evaluation of AR by continuous wave Doppler (CWD) demonstrated a trend toward increasing peak velocity (355 to 400 cm/sec) and regurgitant flow VTI (247 to 289) with increasing LVAD speed, pointing to a more significant AR. Moreover, a progressive increase in the systolic component of the flow was noted, that mirrored the pattern seen AD speed . PHT incColor Doppler analysis revealed that with increasing LVAD speeds there was a progressive increase in vena contracta diameter . As such, if the vena contracta diameter only appeared to classify the AR as moderate at 8,800 RPM, it reached measurements consistent with severe AR at higher LVAD speed. Similarly, a progressive increase in the PISA radius (0.4 to 0.94 cm) and thus PISA-derived regurgitation volume  was noted with increasing LVAD speed.AR_PISA resulted in an overestimation that was progressively more substantial at increased LVAD speeds , and resulted in classifying AR as moderate at low LVAD speed and severe at higher RPM , even though as described the RegVol only changed by approximately 10 ml.2 = 0.81) and its small range of variation was consistent with the small change in absolute RegVol.Peak systolic over peak diastolic velocity ratio (S/D) on pulse wave Doppler as measured at the outflow cannula a was measured for all flow conditions . The rat2 = 0.78, 0.95, 0.84 and 0.97 respectively for PHT peak velocity and vena contracta diameter). For S/D ratio the correlation was somewhat lower (R2 = 0.68).The interobserver variability was good for all tested classic echocardiographic parameters, as showcased by high correlations for PHT, peak velocities, vena contracta diameter and PISA radius  accurately replicate AR in the setting of cf-LVAD and (ii) directly measure the regurgitant volume at different LVAD speeds. We present preliminary data demonstrating that changes in LVAD speed change AR severity by only a small fraction as based on directly measured regurgitant volume when other hemodynamic parameters remained constant, and traditional echocardiographic parameters overestimated the severity of AR in the cf-LVAD patient. Our findings support the current clinical guideline recommendations to avoid use of PHT to assess AR severity in LVAD patients and suggest that other Doppler profile elements such as AR duration and systolic flow interruption may be important for AR quantification .In the current era of cf-LVAD, it is clearly established that AR is a common complication of long term LVAD use and is associated with worsening heart failure, poor end-organ perfusion, and decreased survival. Accurate quantification of aortic regurgitation remains difficult for cf-LVAD patients. Indeed, echocardiography lacks validation in this setting, and a reliable gold standard for regurgitant volume quantification is not clinically available. For instance, cardiac MRI cannot be performed due to the presence of a mechanical pump. Methods that combine echocardiography and right heart catheterization data to derive the aortic regurgitation volume have their own significant limitations .in vitro replication of dynamic flow conditions of cf-LVAD patients with AR that served as a gold standard against which standard TTE parameters for AR severity could be evaluated. Models simulating aortic regurgitation in cf-LVAD have previously been described. In a model of various regurgitant lesions by Shehab et al. they were able to successfully re-create the hemodynamic conditions of AR  did not change significantly. It can be therefore extrapolated that the AR is not always continuous but depends on LVAD speed and perhaps loading conditions. Therefore, we suggest that in clinical practice AR severity should be measured through TTE parameters at the lower LVAD speed.In our study, the traditional parameter for severity, vena contract, and PISA overestimated AR severity. While these findings need further exploration, we have clearly demonstrated that PHT is unreliable for AR severity assessment. Indeed, we found an inverse relationship between PHT on CWD and AR severity. In our model, PHT progressively increased at increased LVAD speed—that is to say, for slightly increased RegVol, a trend that is contrary to what is normally expected in non cf-LVAD related AR. Current guidelines recommend against using PHT alone to grade AR severity, recognizing its dependence on LV preload, afterload and aortic pulse pressure. Of note, all of these are affected by the presence of the cf-LVAD, which creates a continuous flow in the aorta and presence of recirculating blood in the LV, making the PHT likely even less reliable to grade AR severity in this subset of patients.As for the more recently proposed parameters to assess AR severity in cf-LVAD patients, the outflow cannula Doppler systolic to diastolic velocities ratio (S/D) remained <5.0 for all LVAD speed conditions in our model, correctly classifying AR as at least moderate (regurgitant fraction >30%) . The DopOur preliminary data from this analysis, within the limitation of a 3D printed model, suggests that traditional echo-based approaches (vena contracta diameter and regurgitant volume by PISA method) significantly overestimate the AR severity and might thus represent a fallacious tool in guiding clinical management of this population especially at higher cf-LVAD speed. This is in stark contrast to common clinical practice and assumptions as well as more recent data that consider AR in cf-LVAD generally underestimated by echo parameters , 21, 22.de novo AR in patients with cf-LVAD. Further analysis will have to consider AR severity grading by other TTE parameters, as well as different patient specific 3D printed models and different LVAD devices.Our circulatory flow loop was able to closely replicate the AR flow and hemodynamics of a LVAD recipient, providing a gold standard of direct flow measures against which TTE-derived parameters of AR severity could be evaluated. Preliminary results indicate that with increasing LVAD speed, the increase in AR regurgitant volume is small, and that standard TTE parameters tend to overestimate such increase, more significantly so at higher LVAD support. Combined, these data might indicate the need for a critical rethinking of the application of traditional TTE parameters to guide the device management of We replicated and tested flow conditions with only one of the available cf-LVAD devices, the HeartMate II ; however, although is currently unclear whether incidence and impact of AR in cf-LVAD is dependent on the type of device, the majority of available data relate to the HMII.The model replicated a small and constant contribution from the LV—provided in the model by the flow loop pump, that might not be the case for all cf-LVAD recipients but replicates the clinical scenario in which a residual LV function contributes to LVAD performances by augmenting VAD preload and providing some LVOT outflow. The right ventricle and pulmonary vasculature was not accounted for in the model, therefore the LV-RV interdependence as well as the effect on PA pressures could not be assessed. From an echo perspective, the model did not allow for insonation through a standard parasternal long axis view, therefore, a modified apical view was used for Doppler evaluation. Such a modified approach is however not uncommon in clinical practice, given the shadowing artifact produced by the LVAD inflow cannula. The 3D printed LV, although more compliant, still needs modification to simulate true diastolic function of the left ventricle.The 3D-printing process was outsourced and material properties were not independently tested given the clinical focus of this study.Finally, our model represents a single patient with heavily stiffened and remodeled aortic valve; further testing and modeling would be needed to confirm our findings on different patient specific modeling before fully being able to generalize our findings.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MK, SL, and SF participated in the study design and conception. EA, CA, SF, and SX equally participated in the acquisition, analysis, and interpretation of data for the work. EA, MK, KE-T, and SL participated in the drafting of the work and its critical revision for important intellectual content. All authors provided approval for the final version of this manuscript and for the publication of the content and agreed to be accountable for all aspects of the work here presented.


Perioperative rehabilitation is crucial for patients receiving surgery in order to reduce complications and mortality. Conventional methods such as verbal instructions and pre-recorded video are commonly used, but several disadvantages exist. Therefore, we developed an augmented reality (AR) app that includes respiration training, resistance muscle training, and walking training for surgery preparation. The aim of this pilot study was to compare the effects of AR-based training rehabilitation programs with conventional (non-AR-based) programs considering the objective pulmonary function and subjective feasibility and potency in orthopedic patients. This prospective study was conducted in a medical center in Taiwan between 2018 to 2021. Sixty-six patients undergoing elective orthopedic surgery were allocated with a 1:1 ratio to non-AR or AR groups according to their wishes. After training, the inspiratory flow rate of the AR group was higher than that of the non-AR group pre-operatively. As for the subjective assessment, the feasibility (level of confidence and anxiety reduction) and potency (cooperation and educative effect) were superior in AR-based training, compared with the conventional training model. Our study showed that patients using our AR app had better subjective and objective outcomes compared with a conventional model for perioperative rehabilitation. The population of Taiwan is aging. Taiwan reached an aging society in 1993, aged society in 2018, and will reach super-aged society in 2025. In 2022, people aged 85 and over accounted for 10.5% of the total population [Studies have shown that the elderly population undergoing orthopedic surgeries experiences higher percentages of complications and morbidity compared with their younger counterparts ,5,6. In Perioperative rehabilitation is crucial for patients receiving surgery to reduce complications and mortality . In lighBecause of the pace of technological innovation, virtual reality (VR) and augmented reality (AR) have also been introduced to the medical and physical fields . VR immeAs for augmented reality (AR), it is a new technology involving seamless integration with virtual images in the real world. It could further align virtual objects with real world items, thus conducting a real-time experience . AR is gThe advantages of AR in the rehabilitation field are as follows. It could introduce gamification to rehabilitations, defined as “in non-game category using gaming design”, to arouse interest and motivation in patients . It has Therefore, we developed an innovative augmented reality (AR) app that includes respiration training, resistance muscle training, and walking training for surgery preparation. The aims of this study were as follows: (1) exploring the impact of AR-based training programs as perioperative rehabilitation on objective pulmonary function, and (2) exploring the subjective feasibility and potency of AR-based training programs in comparison with conventional perioperative rehabilitation programs among patients undergoing orthopedic surgery.This was a prospective, two-arm (AR group and non-AR group), non-randomized pilot study with a 1:1 allocation ratio. The objective outcome measure was repeated at three time points: preoperative day, postoperative day 1, and the day before discharge; subjective outcomes were assessed before and after the intervention. The study was conducted in a 2800-bed, 6000-staff medical center in northern Taiwan from April 2018 to December 2021.For patients in the AR group, an AR app was offered. It included 10 kinds of respiration training, 34 kinds of resistance muscle training (including 6 upper limbs strengthening exercises and 28 lower limbs strengthening exercises), and walking training. These programs were designed for individuals scheduled for elective orthopedic surgery, and the patients could present with or without chronic heart failure, myocardial infarction, degenerative joint disease, or chronic obstructive pulmonary disease. Healthcare providers  were trained to choose a suitable training program for the patients.The training program was led by an AR virtual teacher on the screen of mobile smart devices . Patients could check the accuracy of their exercise on the screen and monitor their heart rate by wearing a specific bracelet. Meanwhile, the AR virtual teacher would recommend either an intermittent or continuous mode of walking training, and remind patients to speed up or slow down according to the results and special positions of the patient derived from the app. The AR app assists rehabilitation in multiple modalities, including constructing interactive, entertaining, and automatic therapeutic tools for rehabilitation. In order to motivate patients to train regularly, the AR system will give incentive points according to their completeness of the pre-set program. As the incentive points accumulated, the AR virtual teacher would upgrade from the first level to the fifth level, thus inspiring the patients. Patients and physicians could receive instant feedback on the data analysis. The completeness and achievement of all the patients’ training programs would be recorded in their app. The patient information was all anonymous. All of the patients would log into the app with a corresponding number without entering personal information. All of the recorded data could be retrieved anytime, for patient self-evaluation and patient–doctor interaction. During the follow-up appointment, the physician could sign in and check the patient’s data, including the completeness of the program. The app is free and available on both Google Play (Android) and Appstore (Apple) and can be downloaded by anyone who needs it.As for the non-AR group, a pre-recorded video was provided to patients who chose conventional perioperative rehabilitation. The content included respiration training, resistance muscle training, and walking training. It was all pre-recorded, and the content was fixed, not personalized.Both interventions were offered preoperatively. Rehabilitation utilizing the designated tool before and after the surgery was required.Patients who met the following inclusion criteria were enrolled: (1) scheduled for elective orthopedic surgery, (2) needed enhancement of cardiopulmonary function, (3) aged 18 to 80 years, and (4) able to undergo spirometry measurement.The exclusion criteria were as follows: (1) visual impairment or hearing impairment; (2) poor self-care or in bed-ridden status; (3) had no mobile smart device, e.g., smartphone, tablet, or computer; and (4) already had other ongoing rehabilitation programs. The research flowchart is shown in Ethical approval (IRB no. 2018-07-030AC) was granted by the ethics committee of Taipei Veteran General Hospital, Taiwan. All of the enrolled patients were informed about the importance and advantage of this intervention for their safety. Oral consent was obtained from all patients. Questionnaire data were collected anonymously.Demographic data on age, gender, and years of education of the patients were collected at baseline. Objective and subjective outcomes of the perioperative rehabilitation were respectively assessed. The inspiratory flow rate was measured to objectively determine the pulmonary function using flow-oriented incentive spirometer (TriFlow device). This device had three chambers with one ball in each chamber. As air entered each chamber, the balls raised depending on the air flow inhaled per second. The corresponding inspiratory flow rate of each chamber was 600 milliliters per second (mL/s), 900 ml/s, and 1200 mL/s, respectively. The inspiratory flow rate was assessed at three time points: preoperative day, postoperative day 1, and the day before discharge from hospital, following one designated training program at each time point. The exact number of each patient’s inspiratory flow rate was recorded by the healthcare providers on a Google Sheet. The researchers then downloaded the whole google sheet for statistical analysis.As for the subjective assessment, we utilized a self-administered questionnaire  to obtaiFurthermore, any reporting of adverse effects was recorded. A descriptive feedback of the experience was also collected from the enrolled patients, as well as the involved healthcare providers.p value < 0.05 was considered statistically significant. Descriptive feedback was analyzed by identifying the themes from the contexts.Descriptive statistics and frequencies were used to characterize the study participants. A Chi-square test was used to compare the subjective assessment using a questionnaire, between AR and non-AR groups. The Chi-square test was used to compare the inspiratory flow rate at each of the three time points between the AR and non-AR groups. Statistical analyses were performed using IBM SPSS Statistics for Macintosh, Version 24.0 . A n = 33 AR group, n = 33 non-AR group). There was a higher percentage of female enrolled in both the AR and non-AR groups   a. The pactively) b,c.p = 0.009). The second time point (post-operative day one) showed no statistical differences between the two groups. As for the third (the day before discharge) time point, it showed a marginal statistical difference between them (p = 0.07).The incentive spirometry ranked the patients’ inspiratory flow rate as 600, 900, or 1200 mL/s, from low to high. Owing to its nonparametric distribution, we used the Chi-square test for the statistical analysis. In There was no statistical difference in the patients’ perceived familiarity with the rehabilitation program between AR-based and non-AR-based training, either before or after the training program a. As forThe study also showed that the AR group had a more educative effect than the non-AR group after the training, but the difference was not statistically significant b. The inThe following is feedback given by two of the patients and two of the healthcare providers. A 78-year-old woman, who had hypertension and type 2 diabetes mellitus, was enrolled in the program. After using the AR app, she mentioned “The app was very intriguing! I underwent an individual program and cooperated with my healthcare provider very well. My family and I strongly recommend this app to those with similar conditions”.Another patient, a 75-year-old man with COPD, was encouraged by the AR app training, stating “After discussing with my healthcare provider, a customized exercise plan including muscle strengthening and walking training seemed very suitable. The AR app made me more energetic and enthusiastic! I believe that continuous practice using it could greatly improve my cardiopulmonary function”.One of the healthcare providers mentioned the following, “After using the AR app, it seemed that the patients could understand how to appropriately inhale better, which made them more confident with further rehabilitation, and eventually lessened the staffs’ burden”. Another orthopedic ward staff commented “The AR app was convenient, user-friendly, and most importantly customized! Patients could select a suitable training program based on their own needs. The app could also correctly instruct the patient to perform proper range of motion of the joints. It was also worth mentioning that health providers could monitor the patients’ condition and progress to enhance compliance. If there was any error in the app, the patient could contact the health provider via the app immediately to figure out the solution”.After analyzing the feedback from the patients and healthcare providers, three emerging themes around the advantages of the AR app were found, namely: (1) patients can repeat their training programs without the limitation of time and place, (2) the app could recognize whether the patient exercised in the right way, and (3) it could further facilitate regular exercise habits among patients.This prospective study found that pulmonary function inferred by the inspiratory flow rate was better in patients undergoing orthopedic surgery using the AR-based perioperative rehabilitation program compared with that of the conventional program, both on the preoperative day. The perceived feasibility (levels of confidence and anxiety) and potency (cooperation and educative effect) of AR-based training were superior to conventional training, although they were not statistically significant.The analysis of this study was comprehensive. We documented objective data regarding the inspiratory flow rate, as well as a subjective assessment of the perceived feasibility  and perceived potency . Descriptive feedback from both patients and healthcare providers was also recorded.Perioperative rehabilitation is gaining importance nowadays. Many studies have shown AR to be effective in stroke rehabilitation , physicaAlthough the subjective evaluation reported by the patients in the current study showed no statistical significance in both groups, the AR app could be regarded as a supplement therapy, aiding in ameliorating the rehabilitation process. Moreover, reports have shown that attentive functioning declines during aging , and theMoreover, because of the COVID-19 pandemic, the need for telemedicine is growing . UtiliziThere were several limitations in this study. The study sample in each group was relatively small; hence, further studies with a larger sample size are needed. Patients were self-allocated to each intervention group, which may have generated allocation bias, although there was no significant difference in characteristic distribution noted in this study. The subjective data were collected via questionnaires, which possibly presented questionnaire bias, especially when asking personalized and hypothetical questions. Moreover, blinding patients on the type of intervention they received was methodologically impossible. The assessors were also not blinded.Moreover, one study showed that it took months to years to improve through rehabilitation after the main surgery , but we Another limitation was that this study focused on pulmonary function indicated by the inspiratory flow rate. Indeed, the AR app had more options for patients, including upper and lower limb exercise, and bodyweight training. We look forward to focussing on strength training in the future.This prospective study suggests that our AR app may add benefit to perioperative rehabilitation in orthopedic patients by enhancing pulmonary function. The feasibility (confidence and anxiety reduction) and potency (cooperation and educative effect) of the app are also promising. Our findings indicate the potential that AR may be a potential tool for engaging patients undergoing orthopedic surgery in physical activity, even in older adults.


The first aim of this study was to investigate the impact of providing an additional four hours of Saturday occupational therapy to patients receiving Saturday physiotherapy in an inpatient setting on length of stay, functional independence, gait and balance. The second aim was to conduct an economic evaluation to determine if the introduction of a Saturday occupational therapy service in addition to physiotherapy resulted in a net cost savings for the rehabilitation facility.A prospective cohort study with a historical control was conducted in an Australian private mixed rehabilitation unit from 2015–2017. Clinical outcomes included the Functional Independence Measure , gait speed  and five balance measures . Economic outcomes were rehabilitation unit length of stay and additional treatment costs.p < 0.01) and total (p < 0.01) Functional Independence Measure scores. Participation in weekend therapy by the intervention group was 11% higher, attending more sessions (p < 0.01) for a greater length of time (p < 0.01) compared to the historical control group.A total of 366 patients were admitted to the rehabilitation unit over two 20-week periods. The prospective cohort  had 192 participants and the historical control group (receiving Saturday physiotherapy only) had 174 participants. On admission, intervention group participants had higher cognitive (p = 0.08) days. The economic evaluation identified potential cost savings of AUD1,536 per patient. The largest potential savings were attributed to neurological patients AUD4,854. Traumatic and elective orthopaedic patients realised potential patient related cost savings per admission of AUD2,668 and AUD2,180, respectively.After controlling for differences in admission Functional Independence Measure scores, rehabilitation length of stay was estimated to be reduced by 1.39 (Implementation of four hours of Saturday occupational therapy in addition to physiotherapy results in a more efficient service, enabling a greater amount of therapy to be provided on a Saturday over a shorter length of stay. Provision of multidisciplinary Saturday rehabilitation is potentially cost reducing for the treating hospital.The online version contains supplementary material available at 10.1186/s12913-022-07727-7. Rehabilitation aims to improve the functional status of people with health conditions leading to impairments, activity limitations or participation restrictions . Multidip = 0.1) for the multidisciplinary intervention [Randomised controlled trials have shown improvement in functional independence and health related quality of life with Saturday PT only  and for rvention ; these frvention . More rervention . One rearvention –13 and brvention , 15.Providing weekend rehabilitation will likely incur additional staffing, hospital overhead and ward costs for facilities. However, economic evaluations have suggested that multidisciplinary Saturday rehabilitation may reduce costs per quality-adjusted life year gained , 17 withA pragmatic prospective cohort study with historical control was performed on all patients admitted for rehabilitation at St Andrew’s War Memorial Hospital, a privately funded hospital located in Brisbane, Australia. Australia has a dual network of public and private hospitals. Public hospitals are managed and funded by government offering free services to eligible people. Private hospitals provide fee for service care including care provided by medical and supplementary ancillary services including OT and PT . PrivateThe rehabilitation unit serviced a mixed adult caseload. Participants in both groups received usual weekday (Monday to Friday) rehabilitation consisting of nursing, medical, and individualised OT and PT  care, with speech pathology and dietetic involvement as required. A Saturday PT rehabilitation service consisting of 3.5 h was available for the unit. The Saturday PT rehabilitation could be delivered as group or individual sessions in the therapy gym or ward and was staffed by a PT (3.5 h) and an Assistant-in-Nursing (3 h) who provided porterage and assisted the PT as required, as no additional allied health assistant coverage was available. Control group participants were deemed eligible by their treating physiotherapist if they were clinically assessed to be likely to deteriorate over the weekend without PT input, were making functional improvements and would benefit from weekend PT input, were admitted on a Thursday or Friday, or admitted for a stay of less than one week. Patients were excluded from Saturday therapy if they consistently refused usual weekday PT or did not meet the inclusion criteria. Total therapy time available in the control period was 6.5 h each Saturday .The intervention group were offered a Saturday OT service in addition to the PT service, consisting of four hours each of OT and PT, with an allied health assistant providing porterage, therapy assistance and cleaning associated with therapy spaces. The Saturday PT service was extended by half an hour in line with OT and allied health assistant service provision. No change was made to PT eligibility criteria. The intervention group were eligible to attend the Saturday OT service if they were admitted on a Friday, required an initial assessment , required compression therapy, were neurological patients who would benefit from weekend OT, or required additional OT prior to discharge. A maximum of two activities of daily living assessments could be scheduled each Saturday. OT was provided in group or individual sessions, in the therapy gym or ward. Participants could receive both OT and PT Saturday services. Total therapy time available for the unit during the intervention period was 12 h each Saturday .Motor), Cognitive (FIMCognitive) and Total  scores [Patient demographic data collected included age, sex, primary diagnosis, discharge destination, rehabilitation inpatient LOS and nine indicators of patient capability , measured on admission and discharge to the rehabilitation unit. Functional independence was recorded using Functional Independence Measure (FIM) Motor (FIM) scores , 20. Gai) scores , 22 Five) scores , 24 Step) scores , 26, Fun) scores , 28 maxi) scores , and the) scores , 31. DisThe economic evaluation was conducted with financial data obtained from St Andrew’s War Memorial Hospital’s human resources department to estimate the costs of providing 20 weeks of Saturday rehabilitation for both groups. Estimates of variable costs  and fixed costs  were included. Labour costs, which included allied health professionals who provided the rehabilitation treatments and nursing staffing who assisted with patient porterage and therapy in control period, were based on wage rates per hour (inclusive of weekend loading and on-costs). Estimates of average cost per bed-day published by the Hospital Pricing Authority  were useFirst, to explore the effect of a Saturday OT service in addition to a PT service on patient health at discharge, the following multivariate regression model was estimated:CapDC denotes one of nine indicators of patient capabilities measured at discharge, which were three measures of functional independence, gait speed and five indicators of balance. The explanatory variable of interest MSR (multidisciplinary Saturday rehabilitation) was a dummy variable that takes the value of one if the patient was enrolled in the intervention group and zero if enrolled in the control group. Controls for sex  and age (years) were also included. The vector D consisted of a set of dummy variables that control for admitting diagnosis  and p-value < 0.05. Specifications with continuous dependent variables were estimated using ordinary least squared and specifications with dependent variables that were count data were estimated using Poisson regression and the marginal effects (dy/dx) reported.The dependent variable Second, to test the effect of receiving multidisciplinary Saturday OT service in addition to a PT service on rehabilitation LOS, the following multivariate regression was estimated, to isolate the impact of the intervention on LOS:LOS was a count of the number of days the patient stayed in the rehabilitation unit, MSR, a binary variable that took the value of one if participants received multidisciplinary Saturday OT service in addition to a PT service. Variables, andThe dependent variable entclass1pt{minimaDx).Equation p < 0.01) more occasions of PT service. While there was no statistically significant difference in LOS for the acute admission, the average LOS in the rehabilitation unit was 2.4 (p < 0.01) days less for the intervention group compared to the control group.There was no statistically significant difference between the Saturday OT service in addition to PT (intervention) and Saturday PT service (control) groups in age, sex, medical diagnosis, acute inpatient care, or discharge destination (see Table p = 0.06), cognitive , and total  FIM scores, as well TUG , FR , FTEC  and BOOMER  test scores compared to the control group. The last column (Table p < 0.01), cognitive  and total  FIM scores when compared with the control group. The intervention group also had a larger decrease in their TUG test scores  compared to the control group. There were no other statistically significant differences in the indicators of recovery between the control and intervention groups  but there was no difference in the FIMTotal. The intervention group had a faster 10MWT  and a slightly better BOOMER score  compared to the control group.After controlling for differences in sex, age and medical diagnosis, some measures of patient function at discharge were marginally greater in the intervention group  indicated that the monetised value of reduced LOS was greater than the costs of providing additional OT on a Saturday in approximately 95% of simulations.After adjustment to AUD2020, the costs of providing 20 weeks of rehabilitation to the control and intervention groups were estimated to be AUD12,784 and AUD23,180, respectively Table . ControlModel 3, in Table This paper used a pragmatic prospective cohort study design to analyse the effect of Saturday OT service in addition to a PT service on patient outcomes admitted to a 20-bed rehabilitation ward in a private hospital located in Brisbane, Australia. The aim was two-fold. The first to analyse the impact on LOS and functional status, and second to conduct an economic evaluation from the perspective of the healthcare provider. Outcome measures of functional status included functional independence, gait and balance. LOS and hospital cost data were obtained for the economic evaluation.Controlling for age, sex and admitting diagnosis identified minor improvements in cognition and the composite balance measure (BOOMER) scores. After controlling for admission FIM, age, sex and days of admission and discharge, LOS for the intervention group was estimated to be 1.39 days less than the control group. This estimate corroborates results published by earlier research , 8. PublInterestingly, greater reductions in LOS have previously been found with facilities providing PT , 37 compMotor on admission but did not explicitly control for other measures of balance. It is therefore possible that our estimates slightly overestimated reduced LOS. At discharge, largely both groups had similar functional independence, balance and gait and both groups improved performance met minimum clinical important differences. It is reasonable to suggest that discharge is likely determined by patient readiness, functional performance, and preparedness of the home environment [ceteris paribus caveat, and causal inferences should be drawn with caution. Although our statistical models have controlled for some important observed differences between the control and the intervention groups, it is always possible that unobserved differences could confound our results.Participants in the intervention group in this current study had higher scores on some measures of functional independence, and some balance measures on admission. It is unclear why this was the case. The participating facility had no change in admission criteria or admission processes. Our regression models for LOS Table  did contironment . Previouironment , 41. IntPotential cost savings identified in the economic evaluation corroborate an evolving literature that suggests the provision of weekend rehabilitation services may deliver an economic dividend , 16, 17.This study was conducted in a private health service in Brisbane, Australia and as such findings may not be generalisable to other settings including publicly funded health services. Although private hospitals more commonly provide weekend rehabilitation services than publicly funded facilities  we belieper diem the benefit to the hospital would manifest as an improved throughput for the same cost. Alternatively, if the hospital was funded via a prospective payment model based on the admitting diagnosis the hospital would reduce their operating costs per treated patient.While utilizing hospital administration data to obtain individual patient level cost data can provide accurate cost estimates, this approach is resource intensive and was not a feasible option for this study. Instead, reduced LOS was used as a measure of the cost of treatment savings. However, a potential limitation of using the average cost of a rehabilitation bed-day as a proxy for the marginal cost of a rehabilitation bed-day is that this can result in an overestimation of cost-savings when the cost of the final day of admission is substantially less than the cost of an average bed-day . This caA final limitation was that this economic evaluation was restricted to the perspective of the healthcare provider and hence we have reported the results of a costs-analysis rather than a cost-effectiveness analysis. If our economic evaluation was conducted from a societal perspective, the inclusion of improvements in FIM and BOOMER scores would have captured benefits in patient health status not currently included in this analysis.The provision of a multidisciplinary Saturday rehabilitation service comprising OT and PT leads to a greater reduction in LOS compared a to a Saturday PT service, even when controlling for discrepancies in admission function. Providing a Saturday OT and PT rehabilitation service resulted in more patients receiving weekend therapy compared to Saturday physiotherapy only rehabilitation. The provision of a Saturday OT and PT rehabilitation service is potentially cost reducing for the treating hospital.Additional file 1: Figure A1.Distributions for nine measures of patient capability at discharge.


Mass spectrometry-based proteomic analysis of the VCaP prostate cancer cells treated with androgen and the androgen receptor (AR) signaling inhibitor darolutamide revealed a generally good agreement with transcriptomic responses. In some cases, however, the magnitude of changes induced in gene expression levels and in the corresponding protein levels differed, indicating post-transcriptional regulation mechanisms.Several inhibitors of androgen receptor (AR) function are approved for prostate cancer treatment, and their impact on gene transcription has been described. However, the ensuing effects at the protein level are far less well understood. We focused on the AR signaling inhibitor darolutamide and confirmed its strong AR binding and antagonistic activity using the high throughput cellular thermal shift assay (CETSA HT). Then, we generated comprehensive, quantitative proteomic data from the androgen-sensitive prostate cancer cell line VCaP and compared them to transcriptomic data. Following treatment with the synthetic androgen R1881 and darolutamide, global mass spectrometry-based proteomics and label-free quantification were performed. We found a generally good agreement between proteomic and transcriptomic data upon androgen stimulation and darolutamide inhibition. Similar effects were found both for the detected expressed genes and their protein products as well as for the corresponding biological programs. However, in a few instances there was a discrepancy in the magnitude of changes induced on gene expression levels compared to the corresponding protein levels, indicating post-transcriptional regulation of protein abundance. Chromatin immunoprecipitation DNA sequencing (ChIP-seq) and Hi-C chromatin immunoprecipitation (HiChIP) revealed the presence of androgen-activated AR-binding regions and long-distance AR-mediated loops at these genes. Prostate cancer is a leading cancer type in men worldwide, and about 268,490 new cases are expected in the United States in 2022 . InitialWe focused on mechanisms proximal to the AR, as this nuclear hormone receptor represents the essential driver at different stages of prostate cancer. The AR controls the expression of numerous genes essential for the normal physiological role of androgens, mainly dihydrotestosterone (DHT). This is, however, altered in prostate cancer, where the AR elicits an aberrant gene expression program due to gain or loss of binding sites ,9,10 andHere we determined the impact of darolutamide, an AR antagonist with significant clinical efficacy in non-metastatic CRPC and metastatic hormone-sensitive prostate cancer ,25 on thDarolutamide was synthesized at Orion Corporation . DHT and the synthetic androgen R1881 are available from Sigma-Aldrich . Details on R1881 synthesis have been published .TM) prostate cancer cell line was purchased from the ATCC . It has AR wild-type gene amplification and also expresses the splice variant AR-V7. DNA fingerprinting was performed at the DSMZ  for authentication. Mycoplasma contamination testing was carried out using MycoAlert .The CWR22Pc-R1-AD1 cell line is derived from the CWR22 xenograft, which originates from the University of Minnesota and contains a single copy of the AR gene with the mutation H875Y . The VCaCWR22Pc-R1-AD1 cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum, L-glutamine and penicillin/streptomycin. Prior to treatment, the cells were washed with TrypLE , harvested, washed twice in Hank’s balanced salt solution (HBSS) and resuspended to the desired cell density in HBSS or in complete medium.VCaP cells were grown for 2 days in hormone-deprived medium, after which they were treated either with dimethyl sulfoxide (DMSO) control, with 1 nM synthetic androgen R1881, or with 1 nM R1881 and 2 µM darolutamide. For the VCaP cells, samples for RNA isolation and further transcriptomic analysis were taken after 8 and 22 h of treatment from 5 replicates for the R1881 and darolutamide groups, and from 8–10 replicates for the DMSO or R1881 groups. Sequencing was performed via single-end, 50 base-pair reads, with an average depth of 21 million reads per sample.VCaP samples were taken at 14 and 28 h after treatment start to account for the delay between gene transcription and protein translation. This delay is very close to the one reported to lead to the best correlation between global mRNA and protein fold-changes in a dynamic profiling study . For comFor AR ChIP-seq and HiChIP analysis, a total of 8 million VCaP cells were seeded in 15 cm plates and starved for 2 days. Treatment was with 1 nM R1881 only or with 1 nM R1881 and 2 µM darolutamide for 22 h before AR ChIP analysis . Treatme50 values determined at each antagonist concentration (A′) normalized to the corresponding EC50 value in the absence of antagonist (A) as [Log10((A′/A)-1)]. Positive values were then used to predict the pA2 , from which the Ki value can be calculated as 10−pA.The AR CETSA HT assay is based on the AlphaScreen fluorescence resonance energy transfer (FRET) technology  and was Transcriptomic analysis of VCaP cells treated with R1881 and darolutamide has previously been described . FASTQ rOver 6000 proteins were quantified in each condition with label-free quantification (LFQ). Only proteins with signals in all replicates of at least one condition were used further for the analysis. Missing values were substituted with a random value drawn from a normal distribution corresponding to the lower end of protein abundance values (lower 10%). Differential protein expression was analyzed using methodology from the limma expression analysis package  with fitThe joint analyses of proteomic and transcriptomic data were performed with the statistical program language R  using stats and base packages. The linear model was fitted to protein and gene expression log-fold changes in cells treated with androgen and darolutamide, compared to only androgen treatment. Outliers, that is, proteins with expression fold-changes not consistent with gene expression fold-changes, were defined based on standardized residuals larger than 3.VCaP cells grown in charcoal-stripped fetal calf serum medium were treated for 18 or 32 h with 1 nM R1881 and 2 µM darolutamide. Whole cell lysates were prepared and analyzed with the following kits: Human AMACR/P504S ELISA kit LS-F68619, LSBio ; Human FKBP51 ELISA kit, EH194RB, Thermo Fisher Scientific ; Human Hydroxymethylglutaryl-CoA Synthase, Mitochondrial (HMGCS2) ELISA kit, abx385022, Abbexa Ltd. ; Total PSA/KLK3 Sandwich ELISA kit, #14119C, Cell Signaling Technology .Treated VCaP cells were processed as described for genome-wide determination of AR peaks using the specific antibody ab74272  . The exphttp://aryeelab.org/hichipper) (accessed on 29 August 2019) on AR binding sites as anchor loops with default conditions. Visualization of the chromatin loops was performed using the diffloop R Bioconductor package (https://github.com/aryeelab/diffloop) (accessed on 29 August 2019).The protein-centric chromatin conformation method HiChIP was performed as previously described . Crossli50 values were plotted against the corresponding darolutamide concentrations as a Schild plot analysis. A linear regression was applied, and a Ki value of 119.5 nM determined  for darolutamide, indicating a strong interaction with the AR in the cellular context  demonstrated a clear separation of gene and protein expression according to treatments and short or long time points A. Along For an in-depth comparison of gene expression and protein level results, we initially focused on genes from the AR activity signature . In geneDespite the general concordance of expression changes on RNA and protein levels, we noticed a few cases of differential behavior, mainly at the later time points (22 h for RNA-seq and 28 h for proteomics). We therefore fitted a linear regression for RNA and protein changes upon R1881 plus darolutamide treatment, compared to R1881 treatment alone. The selected outlier genes where then analyzed using the MSigDB hallmark collection of gene sets to identify overlaps. A number of enriched gene sets, including hallmarks of androgen and estrogen response, was found; however, there was a small number of genes in each overlap .Examples of outlier genes constituted less than 1.5% of all proteins detected, and examples included alpha-methylacyl-CoA racemase (AMACR) and HMGCS2, for which there was little to no impact on protein levels following androgen or antagonist treatment, compared to the remarkable changes observed at the transcript levels A. AnotheExamination of MA plots of protein level changes following androgen or androgen plus darolutamide treatment indicated that there was no relationship between belonging to the outlier category and the measured protein expression level .Altogether, the differences in the effects on the transcription and protein expression levels observed for specific genes after androgen and antiandrogen treatment suggest that distinct kinetics of protein translation as well as differential post-transcriptional regulation of protein abundance occur in some instances and represent an additional important control mechanism.The outlier group is characterized by high androgen stimulation of mRNA levels but limited changes in the corresponding protein levels. In order to ascertain that the expression of these outlier genes was directly under androgen control, we examined the presence of AR peaks along the respective genomic regions. AR ChIP-seq analysis revealed in all cases several AR peaks that increased in intensity upon R1881 application and decreased again after additional darolutamide treatment A. We theCETSA was used to determine the interaction of darolutamide with the AR in a native context. This method offers the advantages of both the target and test compound being label-free. In this assay, agonist binding leads to thermal stabilization, and this is reversed by a competitive antagonist . The Ki Several papers report that proteomic data are generally more predictive for gene function than transcriptomic data ,41. The Our deep quantitative proteomic profiling of treated VCaP cells by label-free proteomic analysis allowed us to quantify 7427 proteins with signals in three replicates of at least one condition. We compared these results with transcriptomic data generated in the same cell line under similar treatment conditions. We synchronized the division of the VCaP cells by starvation and subsequent stimulation by androgen. We also introduced a time lapse between RNA and protein determination in order to account, in some measure, for the delay in translation and allow for steady-state status to be reached. We focused on intracellular proteins, as the cell secretomes reportedly show limited correlation to transcriptomes . We founThe stimulatory effects we observed following androgen application were potently reverted by additional darolutamide treatment, in line with the high anti-proliferative activity of this compound in androgen-dependent prostate cancer models and its efficacy in the clinic ,25,49. TWe furthermore supplemented our findings on expression regulation of the selected genes using AR ChIP-seq and AR-based HiChIP data. Both experiments pointed to a direct regulation of the selected genes by the AR, as androgen-dependent binding and looping was observed. Interestingly, there was no clear difference observed between those androgen-regulated genes for which there was a parallel shift in protein levels and those without this shift. This is in line with post-transcriptional translation regulation mechanisms or post-translational regulation of protein life cycle such as degradation, modification or subcellular localization, ultimately affecting protein levels. Additional studies are needed to determine the precise mechanisms involved.Previously published proteomic studies analyzing the impact of AR antagonists on prostate cancer cell lines mainly focused on early, first-generation compounds and used two-dimensional gel electrophoresis, so that far fewer proteins were analyzed than in the present study ,60. A moIn this study, we report that the integration of transcriptomic and of proteomic data from treated prostate cancer cells yield unique information on biological systems and reveal their complementarity when it comes to understanding the dynamics of how external stimuli and targeted drugs affect the cellular phenotype. These findings will potentially be strengthened by detailed protein turnover studies and subcellular localization analyses. Additional acquisition and integration of phosphoproteomic, glycoproteomic, and protein–protein interaction and metabolic perturbation data for a multi-omics analysis will lead to better characterized model systems and further increase our knowledge of the complex mechanisms involved, ultimately supporting the development of novel therapeutic treatments for prostate cancer .


The AMI model was established by ligating the left anterior descending arteries (LAD) of both the wild-type and α1A gene-knockout mice and treating them with EA for three consecutive days. A PowerLab 16 physiological recorder was used to collect the electrocardiogram (ECG) while the serum creatine kinase isoenzymes (CK-MB), lactate dehydrogenase (LDH), and norepinephrine (NE) levels in myocardial tissue were determined by using the enzyme-linked immunosorbent assay (ELISA) kit. Moreover, TTC staining was used to observe the myocardial ischemic area, while H&E and TUNEL staining determined the pathological morphology of the myocardium. Quantitative real-time PCR (qRT-PCR) was used to detect the α1A mRNA, and Western blot was used to detect the specific proteins, such as α1A, cleaved caspase-3, Gq, PLC, p-PKCα, and p-hERG. Our results showed that EA could effectively reduce elevated ST-segment, shorten the extended QT interval, and reduce the serum myocardial enzyme content and the degree of pathological injury in wild mice with MI. EA can also decrease the expression of α1A-AR, PLC, p-PKCα, and NE content in myocardial tissues of wild mice, while those of p-hERG increased in ischemic myocardial tissue. These findings suggested that α1A-AR is involved in the development of MI as well as LQTS. Additionally, EA treatment improves the cardiac function and ischemic long QT interval and plays an important role in reducing the hERG inhibition through the α1A-AR-mediated Gq/PLC/PKCα pathway and myocardial apoptosis. Hence, it is suggested that α1A-AR might become a potential target for EA in treating AMI treatment of myocardial ischemia injury and acquired long QT intervals caused by MI.Acute myocardial ischemia (AMI) is a condition caused by a decrease in blood flow to the heart that can sometimes predispose to acquired long QT syndrome (LQTS), thereby resulting in sudden cardiac death. Recent evidence indicates that electroacupuncture (EA) can alleviate MI injury, but its specific mechanism remains unclear. This study was aimed at investigating the efficacy of EA, which utilizes  Acute myocardial ischemia (AMI) is a leading cause of death worldwide while its prevalence and mortality rate have been on a steep rise over the past few decades , 2. Vent+) channel protein hERG and further mediates the fast activation delay rectifier (K+) channel current (IKr), which plays a major role in cardiac action potential repolarization as well as QT interval adjustment [The KCNH2 gene encodes a voltage-sensitive potassium  activation during AMI in mouse cardiac tissue [α1A-AR activation inhibits cardiac repolarization of hERG/(IKr) current through the PKC pathway. Based on this, it is suggested that the α1A-AR-mediated PKC pathway induces sympathetic nervous system hyperactivity that decreases hERG/(IKr) current, delays ventricular repolarization, and prolongs QT interval, thereby leading to tip torsion and ventricular fibrillation. Therefore, inhibition of α1A-AR and associated signaling pathways and recovery of hERG (K+) channel activity might be effective for shortening the long QT interval after AMI.AMI is often accompanied by increased sympathetic nerve activity in conjunction with rhythmia –19. The c tissue . Many prc tissue , 22 have+ channel protein in mice with myocardial infarction [α1A-AR and potassium channels as well as shortening the long QT interval is still ambiguous.In recent years, the effectiveness and safety of acupuncture, as a component of traditional Chinese medicine (TCM), have been recognized for treating ischemic heart diseases , 5. Elecfarction , the rolα1A-AR in therapeutic mechanisms of EA for AMI and LQTS.Therefore, this study was aimed at exploring the protective effect of EA on the AMI and LQTS as well as at assessing a possible molecular basis for the role of α1A–/– mice  of C57BL/6 genetic background were bred by Jiangsu GemPharmatech Co., Ltd., Nanjing, China, and underwent genotyping using the PCR sequence: P1258 5′CCTGCC TGAATCCATCCG3′, P1259 5′TTCCCATCAGAATACA AACATCA3′, and P1260 5′AGCTCCACATGCTCGACTGC3′. All mice were kept in environmentally controlled rooms (22 ± 2°C) with a cyclic 12 h light/12 h dark lighting schedule. All mice had free access to food and water. The experimental protocol was approved by the Animal Ethics Committee of Anhui University of Traditional Chinese Medicine .Adult male C57 BL/6 mice  were obtained from Zhejiang Hangzhou Ziyuan Experimental Animal Science and Technology Co., Ltd., China. Adult male R500 small animal anesthesia machines and disposable acupuncture needles were obtained from Shenzhen Rayward Life Technology Co., Ltd. and Beijing Zhongyan Taihe Medical Instrument Co., Ltd., China, respectively. A PowerLab 16 conductive physiological recorder was from AD Instruments, Australia, while the SDZ-II EA instrument was procured from Suzhou Medical Supplies Factory, China. The following instruments were also used: RM2016 slicer , optical microscope , RT-600 enzyme marker analyzer , refrigerated centrifuge , electrophoresis apparatus , PS-9 rotary meter , and an imaging system .α1A antibody, anti-PLC antibody, anti-p-PKCα antibody, anti-p-hERG antibody, and β-actin antibody were purchased from Abcam  whereas anti-Gq and sheep anti-rabbit HRP-labeled secondary antibodies were obtained from Novus Biologicals, USA, and Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., China, respectively.The following reagents were utilized: isoflurane  and CK-MB, LDH, and NE ELISA kit from Shanghai Jianglai Biotechnology Co., Ltd., China. Additionally, the G1501 Tunel Kit was from Wuhan Servicebio, China; anti-cleaved caspase-3 antibody, α1A–/– mice, were randomly divided into seven groups; each group had 12 mice: sham (sham operation), MI, HT7-HT5 (MI+HT7-HT5), LU9-LU8 (MI+LU9-LU8), α1A–/–+sham, α1A–/–+MI, and α1A–/–+MI+HT7-HT5 groups.A total of 84 mice, including 48 C57BL/6 wild-type (WT) and 36 α1A–/–) and LU9-LU8 were treated with EA  the first day after modeling, and the needle was retained for 30 min once a day for three consecutive days. EA adopted the density wave with frequency and intensity of 2 Hz/15 Hz and 1 mA, respectively, while the sham and MI groups received no treatment. Furthermore, all mice were anesthetized by inhaling 5% isoflurane (1 L/min) and were euthanized after blood sampling through orbital venous plexus after three days. After the opening of the thoracic cavity, the hearts were harvested and washed with phosphate-buffered saline (PBS), out of which four hearts were fixed in 4% paraformaldehyde and embedded in paraffin for histological analysis, while the other hearts were stored at −80°C temperature in a refrigerator for subsequent TTC staining, ELISA, Western blot, and qRT-PCR analyses.The AMI model was established by ligating LAD as described in our previous studies –28, wherAs the mice were anesthetized with 2% isoflurane (1 L/min) and the limbs were fixed, needle electrodes were inserted subcutaneously into the limbs and connected to the PowerLab 16 physiological recorder. The standard II limb ECG of each group was recorded and observed followed by an analysis of the ST-segment and QTc values.The frozen heart was extracted and sliced into five 1 mm thick slices that were perpendicular to the long axis of the heart and were placed in the prepared 2,3,5-triphenyltetrazole chloride (TTC) staining solution. The oven temperature was maintained at 37°C for 20 min, in a dim environment. Subsequently, the MI area was white while the non-MI area appeared red. ImageJ software was used for processing images after photographic analysis. The myocardial infarct size was calculated by the MI area ratio (%) = white area/total myocardial area∗100%.The collected blood samples were allowed to stand at room temperature for one hour and then centrifuged for 15 min  to acquire the supernatant, which was further used for the detection of CK-MB and LDH markers in the mouse serum. The appropriate amount of myocardial tissue homogenate and supernatant was taken for NE detection. The ELISA test was conducted strictly according to the reagent and the kit's instructions.μm sections, stained with H&E, and observed under a light microscope.After fixation with 4% paraformaldehyde for 24 h, the heart was embedded in paraffin, sliced into 4 The TUNEL assay detection kit performed the apoptosis assay according to the kit's instructions, in which the positive cells expressed brown color. The percent of apoptotic cells was determined by counting cells in four random fields, in which the percentage of TUNEL positive cells was divided by the number of positive cells by the total number of nuclei in the field.α1A gene in the myocardium. Briefly, for purification of total RNA from all the hearts, an animal total RNA rapid extraction kit was used to synthesize cDNA. Real-time PCR was amplified by a 25 μL mixture of SYBR Green Mix (12.5 μL), Primer Mix (0.5 μL), downstream Primer (0.5 μL), ddH2O (9.5 μL), and template mixture (2 μL). The GAPDH antibody was used for normalization, and Premier 5.0 software designed the primer along with the utilized primer sequence: primer F 5′CAAAGCAGGGAGTTTAGTC3′ and primer R 5′GTGAGATGGCTCTGAGTGT3′′′.The qRT-PCR detected the expression of the μL lysate per 20 mg of tissue. The purified protein lysate was extracted, and the protein samples were quantified with the BCA protein quantitative reagent. After preparing separation and concentrated gels, an equal amount of total protein was added to each well for electrophoresis and transferred to PVDF membranes which were later immersed in 5% skim milk for one hour and incubated with primary antibodies α1A (1 : 1000), anti-Gq (1 : 1000), anti-PLC (1 : 500), anti-p-PKCα (1 : 1000), anti-p-hERG (1 : 10 000), anti-cleaved caspase-3 (1 : 500), or β-actin (1 : 5000) at 4°C overnight. Then, the membrane was incubated with a diluted HRP-labeled secondary antibody (1 : 10000) at 37°C for one hour. The protein bands were observed by using ECL luminescence solution and analyzed by ImageJ software.The myocardial tissue was cut into small fragments, and the lysate was added to the proportion of 150–250 t-test were used for comparison between the two groups. The difference was considered statistically significant at ∗p < 0.05 and ∗∗p < 0.01.All experimental data were expressed by mean ± SD and analyzed by SPSS 21.0 software. The histogram was drawn by GraphPad Prism 8.0. The statistical differences between the groups were evaluated by one-way variance analysis (ANOVA), while the post hoc least significant difference (LSD) multiple comparison tests and Student's α1A-AR in MI and long QTc induced by MI injury, the association between MI and QTc lengthening as well as altered α1A expression in the mouse heart was determined. As shown in Figures α1A mRNA in MI mice was significantly increased after the upregulation of α1A protein , which belongs to the α1-AR subtype and plays an important role in cardiac physiology and pathology. The α1A activation induced by high sympathetic nerve activity might be an important cause of arrhythmia after MI. A study found that ventricular tachycardia (VT) could be repeatedly stimulated by injecting the α1 adrenergic agonist phenylephrine into mice [α1A-AR activation [α1-AR activation during MI, the increased expression of α1A-AR is closely related to arrhythmia after MI [α1A-AR activation reduces cardiac repolarization K+ current, leading to acquired LQT [α1-AR blockers display antiarrhythmic effects in LQT2 animal models, thereby suggesting that α1A-AR might prove to be an effective therapeutic target for acquired LQTS [α1A-AR blockage is essential to reduce adrenaline-induced arrhythmias [α1A expression increased in mouse myocardial tissue after MI and was proportional to the prolonged QTc value. EA at the HT7-HT5 point can effectively reduce the prolonged QTc value and α1A expression of the myocardium in mice when compared to EA at the LU9-LU8 point. However, these effects are reversed in α1A–/– mice. Therefore, our results suggest that EA might shorten MI-induced long QT intervals by decreasing α1A-AR expression.Recent evidence indicates that arrhythmia induced by MI is related to abnormal innervation of cardiac sympathetic nerves , 33. α1Anto mice . Furthertivation . Since cafter MI , 36, 37.after MI , 38, 39.after MI . Recent ired LQT , while tred LQTS . Pytka ehythmias . Our res+ current (IKr) [+) channel protein encoded by the KCNH2 gene, which mediates fast delayed rectifying K+ current [+ current (IKr) plays a key role in cardiac repolarization, and reduced IKr can lead to arrhythmias [α1A-AR has been reported to inhibit cardiac repolarization of hERG/I(Kr) potassium current through the PKC pathway [α mediated the inhibitory effect of α1A on I(Kr) through the whole-cell patch-clamp technique [α1A-AR, in conjunction with Gq, activates downstream molecules and plays an important role in cardiac diseases such as cardiac hypertrophy and arrhythmia [2+ processing of cardiomyocytes but also directly changes the electrophysiological characteristics of the heart [α1A-AR [+ current (IKH) by α1A-AR stimulation in atrial fibrillation [α protein expressions increased while p-hERG expression decreased with an increased α1A-AR expression in MI mice. EA can inhibit α1A-AR and its downstream signals and upregulate hERG expression to increase cardiac repolarization IKr potassium current and shorten the long QT interval. When α1A-AR was knocked out, the signal regulation of EA weakened, leading to decreased hERG protein expression and the long QTc interval, thus leading to insufficient improvement in MI. This suggests that the improvement of long QT interval by EA treatment depends on the reduction of inhibition of the hERG potassium channel by α1A-AR and downstream Gq-PLC-PKCa signal. Our results are similar to the results from previous studies, which suggested that in HEK293 cells, the intracellular pathways linking α1-AR to hERG include the activation of Gq, PLC, PIP2, and PKC [α mediates hERG inhibition when α1A-AR is activated [The pathogenesis of acquired long LQTS involves hERG channels and fast delay rectifying Knt (IKr) –45. hERG current . Fast dehythmias , 47. Thehythmias , 48. The pathway , 22. Liuechnique . Howeverrhythmia –52. It nhe heart , which h [α1A-AR . The Gq  [α1A-AR . PLC-medillation . Our resctivated .2+ and Na+ both inside and outside the cells, leading to cell apoptosis and affecting cardiac structural abnormalities [α1A protein expression was increased, whereas the hERG protein expression decreased in MI mice, and the apoptosis rate was significantly higher than that in the sham group. Furthermore, due to inhibition of α1A by EA, hERG was activated, leading to a reduced expression of cleaved caspase-3 and myocardial cell apoptosis. Previous studies have reported that α1A–/– in mice leads to increased apoptosis and fibrosis of uninfarcted distal cardiomyocytes [α1A-AR is involved in cardiomyocyte survival. In contrast, sustained activation of α1A-AR induced by catecholamines does not benefit the cardiac functions after MI [α1A-AR may be an important molecular target for EA treatment and prevention of MI-induced acquired LQTS and can provide a theoretical basis and experimental support for further studies on EA treatment and rehabilitation of such diseases.It has been reported that hERG mutations inactivate cell membrane potassium channels and affect the imbalance of Camalities . It has myocytes , 58, thuafter MI , 59, 60.α1A gene-knockout mice were used to test the effect of EA on MI injury and the relation of LQT interval to α1A-AR expression. The potential limitation of this study is that there was no drug-positive or a negative control group. Considering that EA was only used as in vivo stimulation, the observation of IKr current in mouse cardiomyocytes was not carried out. In future studies, drug control trials and whole-cell patch-clamp techniques should be considered for positive patient outcomes.In this study, α1A-AR plays an important role in the EA therapy for MI. EA may upregulate hERG expression by inhibiting the Gq/PLC/PKCα pathway mediated by α1A-AR, shortening the MI-induced LQT interval, reducing the occurrence of ventricular arrhythmias, and ultimately playing a protective role in cardiac function (In conclusion, our results suggest that function . Our fin


KDM6B, a gene encoding a histone H3 lysine 27-specific demethylase, is one of the highest ASD risk genes. However, the relationship between KDM6B mutations and neurodevelopmental diseases remains unclear. Here we use an animal model to show that genetic deletion of one Kdm6b allele in mice leads to autistic-like impaired sociability and object recognition memory. In addition, the mutant mice display markedly increased locomotor activity and impulsivity, two ADHD-like behavioral traits that are ameliorated by methylphenidate treatment. Thus, our study not only uncovers a potential causal link between disruptive KDM6B mutations and ASD/ADHD-like behavioral deficits but also provides a new mouse model for studying the cellular and molecular mechanisms underlying the Kdm6b-mutation-related neurodevelopmental diseases.Autism spectrum disorder (ASD) is a neurodevelopmental disease that has intellectual disability (ID) and attention-deficit/hyperactivity disorder (ADHD) as its common comorbidities. Recent genetic and clinical studies report that  Autism spectrum disorder (ASD) is a clinically heterogenous neurodevelopmental disorder that has impaired sociability and stereotyped behaviors as its core clinical manifestations, and intellectual disability (ID), attention deficit and hyperactivity disorder (ADHD) as its common comorbidities , a transcriptional silencing-related histone modification deposited by Polycomb repressive complex 2  mice to show that Kdm6b haploinsufficiency is sufficient to induce the ASD-like deficit in social preference and impaired cognitive memory. Moreover, the heterozygous Kdm6b-KO mice show high locomotor activity and impulsivity, two core ADHD-like behavioral deficits that are ameliorated by the methylphenidate treatment. Thus, our behavior-genetics study not only reveals a potential causal link between disruptive KDM6B mutations and ASD/ADHD behaviors but also provides a new ASD/ADHD-related animal model for studying the cellular and molecular mechanisms underlying the pathogenesis of KDM6b-mutation-associated neurodevelopmental diseases.In this study, we used the Kdm6b-cKO mice were obtained from the Mutant Mouse Resource and Research Centers at UNC  with food and water ad libitum. The data obtained from all embryos were pooled without discrimination of sexes for the analysis. All mouse experiments were performed with the approval of the Michigan State University Institutional Animal Care & Use Committee.The sperms of Kdm6b-KO mice (Kdm6b+/1f) were obtained by crossing the wild-type Kdm6b+/2f mice with CMV-Cre mice . The wild-type (Kdm6b+/+) and heterozygous Kdm6b-KO (Kdm6b+/1f) mice were generated by Kdm6b+/1f × Kdm6b+/1f mating.All mice were backcrossed to C57BL/6 mice for at least five generations to reach a pure C57BL/6 background. The heterozygous Kdm6b-KO mice. The mice were labeled with ear tags. All the behavioral tests were performed by the researcher who was blinded to the genotypes of animals during the behavioral tests. The same animal cohorts were used in the behavioral tests. To minimize the interference effect due to the same experimental condition repeatedly used in consecutive tests, we performed the behavioral tests according to the following order: (1) open field test; (2) novel object recognition test; (3) three-chamber sociability test; (4) marble burying test; (5) elevated plus maze test; and (6) cliff avoidance reaction test. One month after the test (6), open field tests and cliff avoidance reaction tests with saline treatment were performed (7). One week after the test (7), open field tests and cliff avoidance reaction tests with methylphenidate treatment were performed (8).All behavioral tests were performed on littermates of wild-type and heterozygous The open field test was used to assess locomotor activity and anxiety-like behaviors in an open field. The method was adapted from the previously reported protocols  dissolved in saline was intraperitoneally (i.p.) injected at the dose of 2 mg/kg body weight 30 min prior to behavioral tests  were used if ANOVAS showed significant main or interaction effects. Data are presented as mean ± SEM.All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software). Parametric data were analyzed by a two-tailed KDM6B mutations and ASD genesis, we mated the Kdm6b conditional knockout (Kdm6b-cKO: Kdm6b+/2f) mice with CMV-Cre mice to generate a global heterozygous Kdm6b-KO (Kdm6b+/1f) progenies. The heterozygous Kdm6b+/1f × heterozygous Kdm6b+/1f mating produced a normal number of neonates. Consistent with the previous report  neonates died within 24 h after birth. Therefore, we used wild-type (Kdm6b+/+) and heterozygous Kdm6b-KO (Kdm6b+/1f) littermates for all behavioral analyses in this study. To exclude any undue effects of strain backgrounds on behaviors, we backcrossed the heterozygous Kdm6b-KO mice to the C57BL/6 mice for at least five generations to reach a pure genetic background. We did not observe any obvious differences in postnatal growth or gross body development between wild-type and heterozygous Kdm6b-KO littermates.To examine the relationship between disruptive Kdm6b-KO mice develop the core autism-like deficit in sociability, we performed a three-chamber test to examine the voluntary exploration of a social vs. a non-social stimulus . Planned comparisons revealed that both male and female wild-type controls spent more time with the social stimulus than with the object , representing normal sociability. In contrast, the heterozygous Kdm6b-KO showed either no preference in males or reduced preference in females  for the social stimulus  and time  compared to wild-type controls  and immobile time  into the arena center  test to examine the anxiety-like behaviors under a highly stressful condition by measuring the number of entries into the open arms and the total time for mice running on the open arms. The results showed that compared to wild-type controls, male heterozygous Kdm6b-KO mice had a significantly increased number of entries into the open arms  and spent more time in running along the open arms , while wild-type and mutant female mice had a similar number of entries into the open arms  and running time , suggesting that the Kdm6b haploinsufficiency reduced anxiety-like behaviors in male mice under a highly stressful condition  test to examine whether heterozygous Kdm6b-KO mice display impaired CAR by measuring the number as well as the latency for the mice to fall off from an elevated platform. The results showed that compared to wild-type controls, both male and female heterozygous Kdm6b-KO had high number of falling from the platform in a 60-min testing period  to fall was much shorter than that of wild-type mice , a reagent clinically used to ameliorate ADHD symptoms, to examine whether the hyperactivity and impulsivity observed in mutant mice responded to the MPH treatment. The results showed the main interactive effects of genotype and treatment on running distances . Compared to wild-type controls, Kdm6b mutant mice did not respond to the saline treatment and maintained locomotor hyperactivity in the open-field testing chamber . In contrast, the Kdm6b mutant mice treated with methylphenidate largely reduced their running distances to a similar level as wild-type controls . Planned comparisons revealed that mutant mice treated with saline still maintained a higher number of falling . In contrast, methylphenidate treatment ameliorated the CAR of mutant mice by reducing the number of falling to a similar level as wild-type controls  the behavioral deficits in male Kdm6b mutant mice appear to be more severe than that in female mice, which is consistent with the high prevalence of ASD and ADHD in males (Faraone et al., Kdm6b-KO mice.Although the heterozygous Kdm6b-KO mice reported in this study are the first mice displaying the core ADHD-like behavioral traits that are induced by the deletion of a disease risk gene identified in human patients (Satterstrom et al., Kdm6b-KO mice recapitulate a variety of core clinical manifestations observed in patients, they provide an invaluable tool for further exploring the cellular and molecular mechanisms underlying the pathogenesis of Kdm6b-mutation-related ASD/ADHD behavioral deficits, as well as for developing and testing new therapeutic approaches to treat neurodevelopmental diseases caused by Kdm6b mutations.Currently, a variety of ADHD rodent models have been developed by genetically targeting putative neural signaling pathways or molecules Wilson, . To our The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by Michigan State University Institutional Animal Care & Use Committee.JH conceived the project. YG performed the behavioral tests. MA maintained the mouse colonies. YG, MA, and JH interpreted the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Frontal fibrosing alopecia (FFA) is a primary patterned cicatricial alopecia that mostly affects postmenopausal women and causes frontotemporal hairline regression and eyebrow loss. Although the incidence of FFA has increased worldwide over the last decade, its etiology and pathology are still unclear. We cover the latest findings on its pathophysiology, including immunomodulation, neurogenic inflammation, and genetic regulation, to provide more alternatives for current clinical treatment. A persistent inflammatory response and immune privilege (IP) collapse develop and lead to epithelial hair follicle stem cells (eHFSCs) destruction and epithelial-mesenchymal transition (EMT) in the bulge area, which is the key process in FFA pathogenesis. Eventually, fibrous tissue replaces normal epithelial tissue and fills the entire hair follicle (HF). In addition, some familial reports and genome-wide association studies suggest a genetic susceptibility or epigenetic mechanism for the onset of FFA. The incidence of FFA increases sharply in postmenopausal women, and many FFA patients also suffer from female pattern hair loss in clinical observation, which suggests a potential association between FFA and steroid hormones. Sun exposure and topical allergens may also be triggers of FFA, but this conjecture has not been proven. More evidence and cohort studies are needed to help us understand the pathogenesis of this disease. BioRender.com (accessed on 7 February 2022). Different hypothetical triggers, such as facial soap, cosmetics, sunscreen, or stressful conditions, can cause FFA in potentially genetically susceptible individuals. PPAR-γ/TGF-β pathway plays an essential role in EMT and fibrosis. mTOR signaling works as a modulator of PPAR-γ activity and lipid homeostasis. Arrows and lines indicate a regular relationship between two elements mTOR: mammalian target of rapamycin; PPAR-γ: peroxisome proliferator-activated receptor γ; DHEA: dehydroepiandrosterone; HF: hair follicle; IFN: interferon; TGF-β: transforming growth factor-β; IP: immune privilege; EMT: epithelial-mesenchymal transition; SP: substance P; NK-1: Neurokinin-1. Created with  Frontal fibrosing alopecia (FFA) is a type of primary lymphocytic cicatricial alopecia that leads to irreversible alopecia. In 1997, Kossard described FFA as an uncommon frontal variant of lichen planopilaris (LPP) . PreviouFrontal fibrosing alopecia (FFA) is a systemic skin disease with a variety of clinical manifestations and characterized by receding frontal and temporoparietal hairlines . The recThe slow progression of FFA results in inaccuracies in estimating the age of onset, leading to errors and interference in clinical retrospective analysis. Currently, there are few reports on the pathogenesis of FFA. We reviewed the current literature for immunomodulation, neurogenic inflammation, hormonal and metabolism, genetic and external factors to help clinicians better understand and explore the pathogenesis of this disease.Many reports have revealed the role of immune cell and substance-mediated inflammatory responses in the pathogenesis of FFA . The curHistological findings in FFA show inflammatory cell infiltration around lesioned LPP hair follicles (HFs), especially near the bulge area and infundibulum , which iMoreover, LPP has shown a reduction in Ki-67 labeling and downregulation of keratin 15, a marker of hair follicle epithelial progenitor cells in the bulge area , 10. PreEpithelial-mesenchymal transition (EMT) is considered a normal component of embryogenesis, tissue reconstruction and wound healing, but it also occurs in pathological processes, including epithelial cell malignant transformation, cancer metastasis, and other fibrosis-related illnesses  is involved in lipid metabolism and the differentiation and maturation of sebocytes . The redin vitro , 23. PPAA previous study revealed that PPAR-γ signaling was critical for the survival of eHFSCs in mice, suggesting that the loss of this receptor targeted by the keratin 15 (K15) promoter in these stem cells leads to cicatricial alopecia-like manifestations . HoweverDue to the unique role of PPAR-γ in fat metabolism and hair follicle growth, another pathway, the mammalian target of rapamycin (mTOR) signaling pathway, also plays a role in these two pathways. The mTOR signaling pathway plays various roles in the immune system and stem cell proliferation . Previouex vivo. In this model, PPAR–γ agonists have been found to inhibit or even reverse EMT to some extent , which may be involved in lipid metabolism, has attracted the attention of some researchers . In manyIn addition to PPAR-γ, SP induces the production and release of the proinflammatory cytokines IL-1, IL-6, and TNFα through the neurokinin (NK) 1 receptor signaling pathway. The upregulation of substance P expression around the bulge can mediate neurogenic inflammation and EMT. The expression of substance P was different between LPP/FFA lesional and non-lesional HFs . In geneDue to the age of onset of FFA, it is vital to explore the role of steroid hormones in the pathogenesis of FFA. The mechanism of androgen action in hair loss has been previously described, while the exact mechanism of estrogen in hair growth and alopecia remains controversial. After menopause, circulating estrogen levels drop, and androgen levels rise. In the vast majority of reported cases, the onset of FFA occurs after menopause, but more recent cases of FFA have been reported in premenopausal women and men, prompting researchers to focus on the role of high androgen levels vs. estrogen in the pathogenesis . A recenMany women with FFA also have female pattern hair loss (FPHL), and similarly, many men with FFA have male androgenetic alopecia (MAGA). A large retrospective study of 343 women and 12 men in Spain showed that 40% of affected women had concurrent FPHL and 67% of affected men had concurrent MAGA. FPHL/MAGA is a non-cicatricial alopecia disorder that is androgen-dependent in genetically susceptible individuals . AndrogeIn previous studies, we could not establish a genetic link to FFA, although there were some reports of familial clustering in FFA patients. In 2019, Christos Tziotzios et al. conducted a genome-wide association study that found associations with FFA at four genomic loci: 2p22.2, 6P21.1, 8Q24.22, and 15Q2.1. Within loci 6P21.1 and 2p22.1, fine mapping showed associations with the HLA-B*07:02 allele and a presumed casual missense variant in CYP1B1, encoding homologous heterospecifics and hormone-processing enzymes. CYP1B1 is a widely expressed gene that codes for the cytochrome P450 1B1 microsomal enzyme . This enzyme participates in the oxidative metabolism of estradiol and estrogen into the corresponding hydroxylated catechol estrogen. Therefore, it can be speculated that increased female exposure to CYP1B1 substrates may have an active role in the development of FFA .Human leucocyte antigen (HLA) profiling of a familial cluster (7 FFA members and 4 unaffected members) and 7 sporadic cases revealed two susceptible haplotypes in familial cases (C*17:01:01:02/B*42:01:01 and C*07:02:01:03/B*07:02:01:01), and three unaffected family members also had the haplotype . This prIn the past 10–15 years, the incidence of FFA has increased year by year. It remains to be seen whether this increase is related to the improvement of the diagnosis level of dermatologists and the gradual increase in understanding of the disease. The rising incidence has suggested that FFA may be related to some exposure factors, similar to basic chronic diseases such as hypertension and diabetes. The relation of Frontal fibrosing alopecia (FFA) with environmental factors is still controversial. A recent multicentre case–control study used the odds ratio (OR) to express the effect size of different factors. It showed that FFA and formalin (OR 3.19), the use of ordinary  facial soap (OR 2.09) and cream (OR 1.99), thyroid disease (OR 1.69), and alcohol scum nose (OR 2.08) are related. Smokers (OR 0.33) and anti-residue/cleaning shampoo users (OR 0.35) were negatively correlated with FFA, and FFA was not correlated with sunscreens use . This stDue to a proven correlation between LPP and light exposure, daylight exposure has also been speculated to be an environmental trigger for FFA. One hypothesis is that the light protection provided by many facial care products reduces the synthesis of the tryptophan light product 6-formylindolo  carbazole (FICZ), which is a potent endogenous ligand of AHR, from the photooxidation of the essential amino acid L-tryptophan. It shows proinflammatory effects at lower concentrations and anti-inflammatory effects at higher concentrations . HoweverFrontal fibrosing alopecia (FFA) is a type of primary cicatrical alopecia. At present, many studies have shown that FFA is associated with many autoimmune diseases, further illustrating the role of complex immune regulatory networks in the pathogenesis of the disease.From a clinical and pathological point of view, FFA is a regional disease dominated by localized eHFSC pathology as previous study have previously described . HoweverAlthough the pathogenesis of FFA is complex and not completely clear at present, the incidence of FFA worldwide has been increasing over the past decade, and it is important to understand the pathogenesis of FFA to target the direction of future treatment. The results of the current study suggest that the complex interaction between immune-mediated neurogenic inflammation, genetics, hormones, and possible external stimuli provides strong evidence for the pathogenesis of FFA, and the lack of PPAR-γ plays a vital role in all stages of the disease. Genome-wide association analysis explains that FFA is an immune inflammatory disease with a genetic predisposition, which is driven by HLA-B*07:02. The role of sex steroids and external stimuli in FFA is derived from clinical studies of disease behavior, and the potential influence of both on the pathogenesis of FFA is speculative. More extensive studies are needed to verify the actual pathogenesis of FFA to provide more ideas and help solve this complex problem.Y-JM gathered the information and wrote the original draft. Z-FL and JJ were responsible for funding acquisition and modification. All authors are contributed to the article, participated in resources, wrote the review and edited, and approved the submitted version.