The findings indicate that AR was significantly associated with overall workload, especially mental demand and effort, while VR had no significant effect on any workload sub-dimensions. There was a significant interaction effect between AR and VR on physical demand, effort, and overall workload. The results imply that the resources and cost of operating XR-mediated realities are different and higher than physical reality.Information technologies exist to enable us to either do things we have not done before or do familiar things more efficiently. Metaverse  enables novel forms of engrossing telepresence, but it also may make mundate tasks more effortless. Such technologies increasingly facilitate our work, education, healthcare, consumption and entertainment; however, at the same time, metaverse bring a host of challenges. Therefore, we pose the question whether XR technologies, specifically Augmented Reality (AR) and Virtual Reality (VR), either increase or decrease the difficulties of carrying out everyday tasks. In the current study we conducted a 2 (AR: with vs. without) × 2 (VR: with vs. without) between-subject experiment where participants faced a shopping-related task  to examine a proposed series of hypotheses. The NASA Task Load Index (NASA-TLX) was used to measure subjective workload when using an XR-mediated information system including six sub-dimensions of  Extended reality (XR) technologies are some of the most prominent new developments in information systems, processing, and management . In addition, workload has a multidimensional nature and can be influenced by various factors, for example an individual’s motivation, past experience and ability, and the specific characteristics of tasks Hart, . A numberesearch objective of this study is to develop an in-depth understanding of what kinds and how much workload users can experience when carrying out information processing tasks in XR environments. Accordingly, the current study design takes the form of a 2 (VR: with vs. without) × 2 (AR: with vs. without) between-subject experiment in a shopping context. A brick-and-mortar record store, as well as a 1-to-1 digital 3D “replica” of the store (using laser scanning and Unity 3D for modeling) were constructed as the experimental environments. The NASA Task Load Index (NASA-TLX) was applied as a subjective measure for evaluating six sub-dimensions of workload. The paper is structured as follows. The extant literature related to XR and workload is discussed for proposing hypotheses in the next section. The research method including participants, materials, measures and procedures is presented in section Thus, the In the field of information systems, individual workload or the lack thereof is often operationalized simply as the general ease of use of an information system in a given task (e.g. ‘freedom from difficulty’ according to the technology acceptance model (TAM): Davis, Workload is largely considered as the effort or cost  an individual devotes to accomplish a task Hart, . It can The practical necessity to assess the workload involved in human-computer interaction brought forth a number of different evaluation approaches, including objective measures based on performance indicators and psychophysiological cues, as well as measures based on subjective experiences  in academic and professional fields  AR has a positive effect, while b) VR has no effect on mental demand.In comparison, we can expect that in a pure VR setting, individuals only have to mentally process the virtual reality, and hence it should be similar to exclusively processing the perceived current reality. Individuals may perceive their surroundings through the VR interface as being natural, and therefore they do not have to exert additional cognitive effort Wickens, . MoreoveH2: a) AR has a positive effect and b) VR has a positive effect on physical demand.In addition to mental demand, prior studies postulate that using XR technologies can result in higher degrees of physical exertion  AR has a positive effect, while b) VR has no effect on temporal demand.While VR users might feel stressed from unnaturally interacting with digital objects and the environment with devices while performing tasks, the perception of time pressure might be the same as in an environment without VR . In the purely virtual world  that an individual has to expend in order to reach a certain level of performance Hart, , it seemH4: a) AR has a positive effect and b) VR has a positive effect on effort.H5: a) AR has no effect, and b) VR has no effect on performance.H6: a) AR has a positive effect, and b) VR has a positive effect on frustration.It should be noted that this does not necessarily mean that individuals’ performance will suffer. Rather, users may have to expend more effort to achieve the same level of performance in XR compared to the physical reality. According to theories of workload, performance may stay consistent even if individuals have to expend higher mental or physical effort, as long as the task demands do not reach a critical limit   × 2 (AR: with vs. without) between-subject experiment with a psychometric survey (see Table Procedure). Participants were randomly assigned to one of four groups. Two participants were omitted due to their inability to use the VR headset to complete the experimentFrom September to November 2019, a total of 165 student participants were recruited from a Finnish university to participate in a shopping-related experiment (see section 3.42) was built on the university campus, and functioned as the experimental setup for each condition. The shop floor plan, size, decoration and layout were identical in each condition. Fifty-four LP records were displayed on three different walls of the room, and each wall was equipped with three layers of shelves. For the Group 1 which is designed to control other groups, the shop functioned as a common style physical shop and the product information was presented on printed papers attached to the shelves. For Group 2, the shop functioned similarly except that the product information was displayed through an AR headset display (Microsoft HoloLens: see section 3.3Apparatus). For Group 3, the same room was used. The physical shop was scanned for 3D reconstruction by using an active scanning method, with a LiDAR sensor. Then the shop was modeled and refined in Blender with 1:1:1 scale and then imported to Unity. Thus, the shopping environment used in the “control” condition was reconstructed in virtual reality . For Group 4, the environment combined the condition in Group 3 with the superimposed product information as in the condition of Group 2. See Fig. A physical LP record shop . In addition, LP-record packaging is efficient to be modeled to match the physical versions of the products and the interactive quality for a computer-based environment. In the VR shop and AV shop, the front and back covers of the LP records were scanned in high quality and added as 3D objects into the shopping programs.Purchasing second-hand English LP records  was deemed as a suitable activity for the study as it represents a human activity that requires multimodal, multisensory interaction and effort. Moreover, students are the main consumers of music-related products. The average expenditure on a music hobby was 81.81 euros per year among the 157 participants in the current study. However, we wished to minimize any participant familiarity with specific artists/bands on display among the records in the shop. Therefore, all records in the shop were acquired second-hand and produced before the 1990s. To confirm our expectations of previous product knowledge, in the post-survey, we measured previous product knowledge by seven items based on 7-point Likert scale that were adapted from Awasthy et al. (2012), and the results indicate that the participants were not familiar with the products featured in the shop , track list, statistics, companies, and credits information were selected by the researchers. In the conditions without AR, all of the information pages were pasted either physically (Group 1) or virtually (Group 3) onto the edge of the shelf next to each record. In the conditions with AR Groups 2 and 4), the corresponding information about the LP record was displayed superimposed on the environment based on the image recognition algorithm from the Vuforia Engine. In Group 4, similar to the shop for Group 2 but without using image recognition, the head position of the participant was tracked to predict which record the participant was looking at , the Microsoft HoloLens version 1 AR headset was used for Group 2. Microsoft HoloLens is equipped with a 1280×720 display resolution (per eye) and weighs 579 grams. The digital information only “popped up” whenever the participant was looking directly at the same LP record (front cover) for more than 2 seconds. The straight-line distance between the participant’s head position and the targeted record was between 1 to 1.5 meters.For Group 3 and 4, the participants used the Valve Index headset and its controllers. The Valve Index headset is a fully-immersive headset weighing 809g and is equipped with dual 1440×1600 RGB LCDs, runs at 120Hz, and covers around a 130 degree field-of-view. The Valve Index is accompanied by two controllers (one for each hand) that enable the haptic interface to manipulate records naturally  via 87 sensors used to track hand and finger positions.mental demand , physical demand , temporal demand (how hurried or rushed was the pace of the shopping task?), performance (how successful were you in accomplishing what you were asked to do in the shopping task?), effort , and frustration , after the shopping task was complete. The second step is to obtain a weighted score for each workload factor for calculating the overall workload. Instead of pairwise comparisons, in the current study, participants proceeded to rank the six factors from 0 (least important) to 5 (most important), considering their contribution to the total load of their shopping experience. Thus, for each participant, the weighted score for each dimension was obtained by multiplying the rating score by the corresponding weight. The sum of the weighted ratings was then divided by 15 (the sum of the weights) for an overall workload score.In the present study, the independent variables were composed of the dummy-coded variables of AR  and VR . To examine the workload during shopping in extended realities, we employed the NASA-TLX (Task Load Index) . They were then linked to the online self-booking system on Doodle. A total of 165 students participated in the final experiment.We described the research purpose as “a study of shopping experience” without disclosing any experiment details in the online and offline advertisements posted in a higher education university in Finland. From September to November in 2019, 265 students successfully entered our recruitment system by scanning a QR code or opening the link presented on our advertisement page or flyer . For tutorial purposes, the same sample LP record and product information were presented to participants in all shopping conditions.After the pre-survey, participants got to practice before they started shopping. Experimenter 2 was responsible for the tutorial program, and Experimenter 1 guided the participant on how to wear a VR headset, move in the virtual space, read information, and interact with the record. The interpupillary distance (IPD) and eye relief for each participant were adjusted carefully during the tutorial.For Group 2, the experimenters introduced the Microsoft HoloLens and guided participants on how to wear and use the headset to recognize the physical sample LP record displayed outside of the shop room. Two tutorial shopping programs were developed for both Group 3 and Group 4 respectively, without revealing any details of the shops. EExperimenter 1 instructed participants to a) enter the shop room (Group 1 and 2) or b) wait for the program to start (Group 3 and 4), and then start shopping.Each participant was also told that if he/she needed any help or felt uncomfortable during shopping, a short break could be offered during the experiment, and they were welcome to request additional breaks. After ensuring the participants understood the shopping tasks, A pilot study (N = 20) was conducted for all conditions to test the measurement items in the pre-survey and post-survey, as well as the experimental procedure, instruction, shopping program apparatus, and methods. In the actual experiment, participants were asked to spend 10 minutes in the shop and make their purchase decision independently and in accordance with their own preferences. The experimenter played the role of “cashier” in the four shopping conditions. When the shopping time had run out, the “cashier” counted the price of each record product on the check-out table and asked participants to pay with the gift card they had been given. When the shopping was completed and after making sure that the subjects had no physical problems, participants were guided into another room and filled out the post-survey. The 1-3 records that were “bought” by the participants were then restocked with new records of the same price category in the shop.Measures). Given that AR  and VR  were considered as the two categorical independent variables and overall workload as the (continuous) dependent variable, a between-subjects two-way analysis of variance (ANOVA) was employed. We then conducted a more granular analysis on the effects of AR and VR on each sub-dimension of workload for testing the proposed hypotheses. A two-way multivariate analysis of variance (MANOVA) was conducted to help protect against inflating the Type I error rate in the follow-up two-way analysis of variance (ANOVAs) and post-hoc comparisons.First, we calculated the overall workload by combining weights and ranks of the sub-dimensions according to the NASA-TLX scale (see section 3.3.1N = 157). It can be seen that the overall workload in the XR-mediated groups (Group 2&3&4) were higher than in the non-XR group (Group1). In addition, Group 2 had the highest overall workload (M = 40.081) and Group 1 had the lowest overall workload (M = 26.308). In order to test whether AR and VR have an effect on workload, a two-way ANOVA was employed. Even though the normality in one group cannot be assumed according to Shapiro-Wilk results, the two-way ANOVA is reasonably robust to violations of normality when group sizes are similar. Furthermore, the assumption of a homogeneity of variances was tested and satisfied based on Levene’s F test, F = 1.576, p = .197.The descriptive statistics related to overall workload among the four groups are reported in Table M = 37.249, SD = 2.019) had a higher overall workload compared to those groups without AR  = 6.173, p < .05), and AR explains 3.9% of the variance in the dependent variable. On the other hand, the groups with VR  did not reveal any significantly higher workload in comparison to those groups without VR  = 0.148 , p > .05), and VR only accounts for 0.1% of the variance in workload. The results also show that there was a significant interaction between AR and VR  = 5.713, p = .018) and the interaction explains 3.6% of the variance (see Table M = 34.150) lead to significantly higher workload than without VR . Similarly, when participants were in the groups without VR, AR (M = 40.081) led to significantly higher workload than those without AR .The results reveal that the groups with AR . The covariance matrices between the groups were assumed to be equal for the purpose of MANOVA. In addition, the absence of multivariate outliers was checked by assessing the Mahalanobis Distances among the participants . A two-way MANOVA was conducted to test the hypothesis that AR and VR have different effects on the various sub-dimensions of workload. A statistically significant MANOVA effect was obtained for the interaction between AR and VR, where Pillai’s Trace = 0.097, F = 2.643, p = .018. The multivariate effect size was estimated at .097, which implies that 9.7% of the variance in the dependent variables was accounted for by the interaction between AR and VR.A series of Pearson correlations were performed among the six dependent variables in each group for testing the MANOVA assumption. The results reveal no concern for multicollinearity, although a few variables were not highly correlated. In addition, the s-Vaughn  guidelinp > .05) - see Table F = 5.084, p = 0.026, Mean difference = 9.597) and effort  = 6.500, p = 0.012, Mean difference = 9.619), while all of the ANOVA’s for VR were statistically insignificant . Accordingly, regarding the effects of AR on each dimension of workload, H1a, H4a and H5a were supported, and H2a, H3a and H6a were rejected. Regarding the effects of VR on each dimension of workload, H1b, H3b and H5b were supported while H2b, H4b and H6b were rejected. In addition, the interaction effect between AR and VR significantly influenced physical demand, F = 11.405, p =.001 and effort, F = 5.647, p = .019.Prior to conducting the ANOVA tests, the homogeneity of variance assumption was tested for all six dependent variables. Based on a series of Levene’s F tests, the homogeneity of variance was considered as satisfied, as all six Levene’s F tests were insignificant (p = .052). In addition, only in the groups without VR did AR lead to a significant increase in effort compared to those without AR.Finally, a series of post-hoc analyses were performed to examine individual mean difference comparisons across different levels of AR and VR and the two subscales of workload see Fig. . AccordiOverall, users experienced higher workload in all three XR-mediated realities than non-XR. To be more specific, AR was significantly associated with overall workload, especially its sub-dimensions of mental demand and effort. In this study, AR was operationalized as “superimposed information”  influence the six dimensions of workload  and overall workload based on a 2 by 2 between-subject experiment in the retail context. The results from granular analysis indicate that AR was significantly associated with overall workload, especially mental demand and effort, while VR had no significant effect on any of the dimensions of workload. Additionally, the results of the interaction effects show that a combination of AR and VR compared to a single technology would not increase the difficultly of completing tasks , and may even decrease the difficulty . By filling the current gap in research on XR of information systems, human computer interaction and management science, the current study made a considerable research contribution as well as practical guidance for XR designers, developers and practitioners.The analysis of workload in the present study deepens the understanding of what factors affect usability and efficiency, and what factors are important to users in extended realities, contributing to the corpus of information system and technology-oriented information science. There is a lack of comprehensive understanding and accurate measurement relating to system efficiency and usage barriers in the extant XR-related literature. For a long time, usability has been considered as one of the determinant factors of efficiency in a system . In order to minimize the potential impact of all possible external factors on the study outcomes, we looked to ensure that all behaviors were to the greatest extent similar in the four shopping conditions by simplifying the shopping programs as close to non-XR shopping conditions as possible, and creating natural interactive ways to shop in each condition. For example, AR was simplified as a technology used to present information (the product information page) in an augmented way. Regarding the devices, given the consideration that consumers needed to interact with products by hand, head-mounted displays were used rather than hand-held devices . In addition, we also took the naturalness of interaction into consideration., and instead of using triggers or buttons as controls, two controllers that enabled haptic interfaces for simulating handling products naturally were used in the VR-mediated environments. The rigorous research design aims at ensuring the internal validity of the experiment results.The study findings have valuable implications, mainly for XR designers and programmers, and also for practitioners from different areas, especially those in the retail sector. Due to a lack of theoretical guidance and granular analysis of perceived workload, many program developers and designers face the challenge of finding good solutions of how to improve the perceived usability, ease of use, usefulness and efficiency extended towards XR technologies and systems . Therefore, we encourage retailers to bravely attempt to use VR technology, for example to replace online and offline shopping environments, given that VR can provide a possibility for consumers to shop online at any time and in any place  were highly realistic compared to a physical shop, some participants reported that their shopping experiences were a bit “odd” due to the lack of sound and touch experience when moving and interacting with the products. Given that visual experience has been considered as the most important dimension of human sensory experience, we expect that the visual experience itself is sufficient to create the perception of being “there”. Nevertheless, future studies should provide a more multi-dimensional sensory experience in their research design  or to create a virtual reality . Even though participants used these head-based devices and interacted with products in a similar way, there is still a possibility that the statistical differences seen in some areas of the results may be influenced to some degree by the different technical capabilities of the equipment, such as resolution, detection system, field of view, comfort, response time, etc. As pioneering research, the current study made a great effort to investigate comparisons between AR and VR, based on a rigorous experimental method. However, due to relatively irrevocable technical limitations, these potential technical factors might not have been fully controlled in this experiment. Therefore, we encourage future researchers to adopt more improved XR devices to control and reduce any meaningful influence brought by technological difference.In addition, the underlying mechanism and boundary conditions of how AR and VR influence different dimensions of workload could be investigated by future researchers. For example, our study did not reveal why AR would lead to high effort and mental demand and overall workload (compared to conditions without AR) while VR did not seem to affect any dimensions of workload. Therefore, there might be some important mediating variables (e.g., presence - see Lackey et al., As a final consideration, student participants were recruited in the current study for the reason of ready availability, less foreseeable difference within the sample, and an anticipated acceptance towards novel technologies. Therefore, the results might be influenced by the singularity of the sample. Particularly, the younger age groups have been seen to have a higher degree of flexibility, mobility, more tolerance of discomfort, and less difficulty in operating systems and devices. As for most younger users, wearing headsets and using controllers do not seem to restrict their movement and interaction. However, for specific groups such as elder adults, patients, children, or the disabled, there might be more challenges in operating extended realities. Therefore, future studies could investigate the workload of using AR and VR by recruiting participants from other demographic groups.


Molecular Dynamics simulations confirm allosteric transmission of the T850D mutation towards the AF2 site via extended hydrogen bond formation between coactivator peptide and AF2 site. This mechanistic study thus confirms the gain-of-function character of T850D and T850 phosphorylation for AR activity and reveals details of the allosteric communications within the LBD.Several key functions of the androgen receptor (AR) such as hormone recognition and co-regulator recruitment converge in the ligand binding domain (LBD). Loss- or gain-of-function of the AR contributes to pathologies such as the androgen insensitivity syndrome and prostate cancer. Here, we describe a gain-of-function mutation of the surface-exposed threonine at position 850, located at the amino-terminus of Helix 10 (H10) in the AR LBD. Since T850 phosphorylation was reported to affect AR function, we created the phosphomimetic mutation T850D. The AR T850D variant has a 1.5- to 2-fold increased transcriptional activity with no effect on ligand affinity. In the androgen responsive LNCaP cell line grown in medium with low androgen levels, we observed a growth advantage for cells in which the endogenous AR was replaced by AR T850D. Despite the distance to the AF2 site, the AR T850D LBD displayed an increased affinity for coactivator peptides as well as the  The androgen receptor (AR) is a ligand-activated transcription factor that belongs to the steroid receptor family . Similar23FQNLF27 motif of the NTD which has been proposed to stabilize hormone binding but also to affect co-regulator interactions  at 222 nm using the following equation, % helix = 100 )), where n is the number of total peptide bonds and [θ]222 has been corrected for background [Peptides  simulations. The structure of the AF2 peptide bound AR LBD; consisting of the AR LBD monomer, DHT and AF2 peptide; was extracted from the AR LBD crystal structure homodimer (PDB: 5jjm) . The T85ensemble ,64. Finaensemble . The lonensemble .Functional and structural studies pointed out that the AR T850D mutation is an exceptional gain-of-function mutation on the surface of the LBD in H10. While the T850D mutation does not affect ligand binding affinity, it does enhance co-regulator binding at AF2 by promoting the active conformation of the AR LBD. There is resemblance with an allosteric network in the heterodimeric NRs where the H10-11 dimerization interface transmits permissive or non-permissive signals from one LBD to the other. Via rotation of H5, signals at H10-11 are passed on to the LBP and to AF2. Modulation of this allosteric pathway could provide a new mechanistic explanation for the presence of AR-related diseases such as PCa and the androgen insensitivity syndrome. These findings provide new details on the allosteric pathways within the LBD of AR .


In Botswana, a total of 76 nurse anesthetists were in clinical practice during the study period. Sixty-six (86.9%) responded to the survey. Gender distribution was even, 50.0%. The overall JS was 36.4%. Males had significantly higher JS than females, p = 0.001. Significantly higher job satisfaction was found in married nurse anesthetists (p = 0.039), expatriate nurse anesthetists (p = 0.001), nurse anesthetists in non-referral hospitals (p = 0.023), and nurse anesthetists with ≥10 years' experience (p = 0.019). Nurse anesthetists were satisfied with security, social service, authority, ability utilization, and responsibility in ≥60.0% of the cases. They were not satisfied in compensation, working condition, and advancement in a similar percentage. The main reason to stay on their job was to serve the public in 68.2%. In Botswana, employers should make an effort to address the working conditions, compensation, and advancement of nurse anesthetists in clinical practice.Job satisfaction (JS) correlates positively with patients' satisfaction and outcomes and employees' well-being. In Botswana, the level of job satisfaction and its determinants among nurse anesthetists were not investigated. A cross-sectional study was conducted from January 2020 to June 2020 encompassing all nurse anesthetists in clinical practice in Botswana. A self-administered questionnaire was used that incorporated demographic data, reasons to stay on or leave their job, and a validated 20-item short form of the Minnesota Satisfaction Questionnaire which was pretested on five of our nurse anesthetists. Percentage is used to describe the data. The independence of categorical variables was examined using chi-square or Fisher's exact test.  The effectiveness of a functional healthcare delivery system depends to a large degree on human resource utilization and motivation , 2. Out Factors that improve the level of JS include job rotations, senior teaching or support to cope with difficult situations, non-salary incentives, job recognition, professional development, and a great sense of organizational justice , 13, 21.Job satisfaction which is based only on financial incentive is often temporary . EvaluatBotswana has a total population of about 2.3 million. The public health system serves about 85% of the population, while the remaining 15% subscribe to a private health sector. There are a total of 19 general  hospitals and 17 primary hospitals in the country . CurrentThis is a cross-sectional study involving all nurse anesthetists in clinical practice in Botswana. It was conducted from January 2020 to June 2020.All nurse anesthetists in clinical practice in Botswana at all levels of public and private hospitals were invited to participate in this study.All the 76 nurse anesthetists currently in clinical practice in Botswana were invited to participate in this study. All nurse anesthetists who consented to participate are included in the survey.Job satisfaction was assessed using self-administered questionnaire sent to all nurse anesthetists through a one-to-one e-mail or using the new end-to-end encrypted WhatsApp platform that blocks interception by a third party. The self-administered questionnaire has two parts. Part I contains basic demographic data , health sector (public or private), and reasons to stay or leave the profession . The overall satisfaction level was recorded on a separate scale as satisfied or not satisfied. Part II contains the validated English version of the 20-item short form of Minnesota Satisfaction Questionnaire (MSQ) which is based on 5-point Likert-type scale that ranges from 1 point, very dissatisfied, to 5 points, very satisfied. The clarity of the questionnaire was checked on five nurse anesthetists, and no modification was required. All 76 potential participant nurse anesthetists were informed about the objectives and the details of the study by the principal investigator. Nurse anesthetists who agreed to participate in this study gave written consent.p values <0.05 were considered statistically significant.The data were checked for completeness and entered into an Excel spreadsheet which was transported to IBM Statistical Package for Social Sciences Statistics 27 for analysis. Very dissatisfied and dissatisfied responses in MSQ were considered as not satisfied, while satisfied and very satisfied responses were considered as satisfied. Percentage was used to describe the data. Categorical variables were compared using chi-square or Fisher's exact test. Two-tailed In Botswana, a total of 76 nurse anesthetists were in clinical practice during the study period. Sixty-six of them, 86.9%, responded to the survey. Nurse anesthetists in the age group 40 years and older constituted 68.2%. The number of male and female nurse anesthetists who responded to the questionnaire was equal, 50.0% each .p=0.013. A majority, 92.4%, of our nurse anesthetists had diploma credentials; the remaining had a degree in anesthesia. The overall job satisfaction of the nurse anesthetists in our study was 36.4%. The JS of males was significantly higher than that of females, 57.6% vs. 15.2%, p=0.001. Statistically significant JS was found in nurse anesthetists who are married, 45.5% vs. 19.0%, p=0.039, expatriate nurse anesthetists, 68.4% vs. 23.4%, p=0.001, nurse anesthetists working in non-referral hospitals, 43.4% vs. 7.7%, p=0.023, and those nurse anesthetists with 10 years and more work experience, 46.5% vs. 17.4%, p=0.019 , p=0.645, health sector (public or private), p=0.103, relationship with surgeons (good or poor), p=1.000, the number of working hours per week (<40 hours or ≥ 40 hours), p=0.426, and senior staff backup (present or absent), p=0.715.The majority of nurse anesthetists were locals (Batswana) (71.2%); among the expatriates, 19.7% were Zimbabweans, 4.5% were Zambians, 3.0% were Malawians, and 1.5% were Tanzanians. The majority of Batswana nurse anesthetists were female (63.8%), while most of the expatriate nurse anesthetists were males (82.4%). Gender distribution among age groups, education levels, hospital types, relation with surgeons, working hours per week, years of experience, and senior backups was not significantly different. The marriage rate was 66.7%. Significantly more males were married than females, 81.8% vs. 53.1%,  p=0.019 . Job satp = 0.001  of the short form of MSQ for our study was 0.767, indicating an acceptable reliability. Our nurse anesthetists were satisfied in ≥50.0% of cases in independence, variety, supervision—technical, moral value, security, and social service, authority, ability utilization, responsibility, creativity, and achievement. In more than seventy percent of cases, they were not satisfied with compensation, advancement, and working condition. Our nurse anesthetists were more satisfied with extrinsic MSQ scale than intrinsic scale, 53.5% vs. 44.3%,  = 0.001 .p = 0.011, supervision—human relation, p = 0.008, supervision—technical, p = 0.047, and recognition, p = 0.042. More females were not satisfied than males in compensation, p = 0.038, and creativity, p = 0.004. In the remaining MSQ items, non-satisfied vs. neutral, non-satisfied vs. satisfied, and neutral vs. satisfied, there was no statistically significant difference  compensation, advancement, and working conditions, while nurse anesthetists in private hospitals were not satisfied in company policy and practice, compensation, and working conditions in >50.0% of the cases.There was no significant difference in job satisfaction among the two groups. None of the nurse anesthetists practicing in public hospitals were satisfied with compensation; 16.7% of the nurse anesthetists practicing in private health sector reported satisfaction with compensation, though it was not statistically significant, p = 0.016. Change of their position in their profession, further education, family reasons, or personal reasons were not statistically significantly associated with either to stay on or leave their job.The main reason for our nurse anesthetists to stay on their post was to serve the public, 68.2%, and the other reasons were position change in 21.2%, further education in 24.2%, family related in 21.2%, and personal reasons in 13.6%. The main reasonsfor the nurse anesthetists to leave their job were family-related, personal reason, and change of profession in 6.1%, 15, 2%, and 15.2% of the cases, respectively. Non-satisfied nurse anesthetists had a significantly higher job-leaving response than satisfied nurse anesthetists, 74.4% vs. 36.8%, Like in any other profession, multiple factors affect nurse anesthetists' JS. These factors are generally classified as intrinsic and extrinsic. Intrinsic factors include individual achievements, accomplishment, and prestige, while extrinsic factors include elements of the work environment such as pay and benefits, working conditions, and resources , 19, 27.The response rate to our nurse anesthetists JS survey was 86.9%; this is comparable with similar studies that range from 83.6%–98.3% , 24, 28.The JS reports on gender are not uniform: absence of significant difference , 27, 29,Some researchers found that the age group younger than 40 years constitutes the majority, 53.4%–84.7% , 27, 28.The marriage rate in our study was 66.7%, and this agrees with similar studies that range from 64.0%–84.5% , 28–31, The majority of our nurse anesthetists, 92.4%, were diploma holders; this is higher than the reported range, 54.0%–58.8% , 24, 27.Most of our nurse anesthetists were working for more than 40 hours per week, and this is in agreement with reports from Iran and China , 31. OurIncreased JS is more influenced by the intrinsic factors than extrinsic factors , and thiOur nurse anesthetists in public hospitals were not satisfied in compensation, advancement, and working conditions in more than 50.0% of the cases. The overall poor job satisfaction in our nurse anesthetists working in public hospitals was similarly reported by others , 23. TheGlobally, anesthetists leave their jobs due to stress, fatigue, and long working hours . The mosThe cross-sectional and self-administered nature of the study may not demonstrate a causal relationship between variables. Although the Minnesota Satisfaction Questionnaire is a validated tool to evaluate the JS, it is not specifically designed for nurse anesthetists. The number of nurse anesthetists from the private sector was few, 12. A response rate of 86.9% of all nurse anesthetists currently in clinical practice in Botswana and involving all levels and types of hospitals are the strengths of this study.This study demonstrated the low JS of nurse anesthetists currently practicing in Botswana in both public and private hospitals. Most nurse anesthetists were satisfied with independence, variety, supervision—technical, moral value, security, social service, authority, ability utilization, responsibility, and creativity, but they were not satisfied in compensation, working conditions, and advancement. The health managers in Botswana need to develop strategies to improve the low JS level among nurse anesthetists currently in practice in the country. Many studies , 6, 22 d


Androgen receptor (AR) is critical to the initiation, growth, and progression of prostate cancer. Once activated, the AR binds to cis-regulatory enhancer elements on DNA that drive gene expression. Yet, there are 10–100× more binding sites than differentially expressed genes. It is unclear how or if these excess binding sites impact gene transcription.To characterize the regulatory logic of AR-mediated transcription, we generated a locus-specific map of enhancer activity by functionally testing all common clinical AR binding sites with Self-Transcribing Active Regulatory Regions sequencing (STARRseq). Only 7% of AR binding sites displayed androgen-dependent enhancer activity. Instead, the vast majority of AR binding sites were either inactive or constitutively active enhancers. These annotations strongly correlated with enhancer-associated features of both in vitro cell lines and clinical prostate cancer samples. Evaluating the effect of each enhancer class on transcription, we found that AR-regulated enhancers frequently interact with promoters and form central chromosomal loops that are required for transcription. Somatic mutations of these critical AR-regulated enhancers often impact enhancer activity.Using a functional map of AR enhancer activity, we demonstrated that AR-regulated enhancers act as a regulatory hub that increases interactions with other AR binding sites and gene promoters. Activat regions , 3. The  regions , 4, 5. O regions . This pr regions . There a regions , 9. We dEnhancers have traditionally been identified by correlating transcription factor binding sites with chromatin accessibility, RNA polymerase II, GROseq, or enhancer-associated histone modifications such as H3K27ac –13. ThesThere is increasing clinical evidence that non-coding mutations can act as oncogenic drivers in PCa –21. ReceTo provide the first locus-specific AR regulatory map, we functionally quantified the enhancer activity of all commonly observed clinical ARBS with STARRseq. We demonstrated that only 7% of ARBS have androgen-dependent enhancer activation, while 11% had enhancer activity that was independent of AR binding. Surprisingly, the vast majority of ARBS (81%) did not have significant androgen-dependent or constitutively active enhancer activity. These in vitro annotations strongly correlated with enhancer associated histone modifications in clinical PCa samples. To characterize the mechanism of AR enhancers, we then trained a machine learning classifier that successfully predicted active enhancers and identified key features of active enhancers. Integrating both the long-range chromatin interactome and transcriptomic data, we found that androgen inducible enhancers were significantly more enriched as anchors for gene looping and acted as “hubs” to activate AR-regulated genes. Finally, combining these results with whole genome sequencing of primary and metastatic PCa, we identified and characterized a non-coding somatic mutation that significantly impacted AR enhancer activity of a critical tumor suppressor.n = 7), primary PCa tumors (n = 13), or both. With this conservative selection criterion, we identified 262 ARBS present in only normal prostate tissue, 3225 ARBS present only in PCa tumors, and 652 ARBS in both normal and PCa tumors. Having selected these regions, we designed a custom DNA capture assay to enrich three different groups: common clinical ARBS , a positive control of previously identified strong enhancers that are not associated with AR [n = 500), and regions that contain an androgen response element (ARE) motif but there is no AR binding in either clinical samples or cell lines (n = 2783). With this, we then captured fragmented normal genomic DNA and cloned it into a second-generation STARRseq plasmid [Pearson correlation 0.84–0.99; Additional file KLK3  and tested with a luciferase reporter assay. Similar to previously published work, our results for the STARRseq enhancer activity correlated with a conventional luciferase reporter assay [n = 867; Additional file p < 2.1 × 10−16). As these regions did not have AR binding in our experimental model, they were separated in subsequent analysis (no AR). To confirm that our AR CRE annotations correlated with enhancer activity in vitro, we compared each group with published enhancer-associated features including H3K27ac, RNA polymerase II (Pol2), and bidirectional eRNA (GROseq) [To characterize AR CREs, we experimentally tested the enhancer activity of all commonly occurring clinical ARBS with STARRseq, a massive parallel enhancer assay Fig. a. In thimors n = 3, or botmors n = 3, or botmors n = 3, or boter assay , 26   Fig. 1ffn = 7, pn = 87), H3K27ac (n = 92), and H3K27me3 (n = 76) ChIPseq from primary PCa tissue [p-adj < 0.0001; Fig. p-adj < 0.05). However, as these primary PCa samples contain physiological levels of androgen, we could not separate induced and constitutive enhancers as the AR would be active in these tumors. Therefore, to further validate our in vitro classifications, we conducted H3K27ac ChIPseq on prostate tumors from patients enrolled in a neoadjuvant antiandrogen enzalutamide (ENZA) clinical trial (NCT03297385). ENZA is a classical antagonist that directly inhibits androgen binding and prevents translocation of the AR into the nucleus [.While histone modifications do not accurately identify individual AR enhancer CREs Fig. g, these  nucleus . Therefon = 90; Additional file n = 8) or inactive (n = 8) enhancers  interactions frequently occur within neighboring primary sequences , 31, we DBI) to a mixture of inactive, constitutive, and inducible AR enhancers   was inactivated, it also decreased the neighboring gene KLK2 [KLK3 promoter did not downregulate KLK2  to identify co-accessible AR CREs and determine the n = 196) [n = 101) [p > 0.5). To test the impact of these somatic mutations on AR enhancer activity, we focused on those metastatic SNVs that occur in inducible enhancers which looped to the TSS of AR-upregulated genes. Within the small set tested, we found that 19% (3/16) of SNVs significantly altered AR enhancer activity   and meta6  and men = 101) , 23 and n = 101) . We did  in CRPC , 40 . Of particular interest, we identified a somatic mutation in metastatic PCa that significantly reduced the activity of a critical AR enhancer required for the expression of ZBTB16 .In conclusion, we have created the first functional map of potential AR enhancer activity. By quantifying the activity of each ARBS, this provides mechanistic insight into mammalian gene regulation. Together, our data demonstrates that AR-driven inducible enhancers act as a regulatory hub that frequently cooperates with other ARBS to drive transcription. Identification of key enhancers can be used to stratify non-coding mutations for functional testing. These results have been integrated into an easy-to-use web app that is searchable by gene or genomic coordinates (Cell lines were purchased from ATCC and routinely tested for mycoplasma contamination. LNCaP cells were routinely grown in RPMI 1640 media (Gibco) with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). No activation of the IFNγ pathway by double-stranded DNA was observed in electroporated LNCaP cells which have been shown to systematically alter STARRseq activity  (Addition = 3) or independent PCa tumors (n = 13) [Common clinical ARBS were defined as those sites that were present in all normal prostate (n = 13) . Pooled  . Pooled8 cells/replica; 3 biological replicas) were electroporated with 266–300 μg (1 million cells:2 μg DNA) of the ARBS STARRseq capture library using the Neon Transfection System (Invitrogen). Electroporated cells were immediately recovered in RPMI 1640 medium supplemented with 10% FBS. After overnight recovery, the media were changed to RPMI 1640 medium supplemented with 5% charcoal-stripped serum (CSS) and 1% penicillin-streptomycin. Approximately 72 h after electroporation, the cells were treated with 10 nM DHT or EtOH for 4 h, washed with PBS, and then lyzed using the Precellys CKMix Tissue Homogenizing Kit and the Precellys 24 Tissue/Cell Ruptor (Bertin Technologies). Total RNA was extracted using Qiagen RNeasy Maxi Kit (Qiagen), and the mRNA was isolated using the Oligo (dT)25 Dynabeads (Thermo Fisher). Isolated mRNA samples were treated with Turbo DNase I (Thermo Fisher), synthesized into cDNA using the gene-specific primer . Inactive regions had both minimal DHT inducible activity and low plasmid to RNA ratios . Output of the DESeq2 was visualized with ggplot2 [Reads were mapped to reference genome (hg19) with BWA aligner (v0.7.17)  and all  ggplot2 .ClinicalTrials.gov #NCT03297385). The trial was approved by the IRB of the Netherlands Cancer Institute. Informed consent was signed by all participants enrolled in the study, and all research was carried out in accordance with relevant guidelines and regulations. Trial participants received 3 months of neoadjuvant ENZA treatment prior to robotic-assisted laparoscopic prostatectomy. Biopsy (pre-treatment) and prostatectomy specimens (post-treatment) were fresh-frozen and sectioned prior to immunoprecipitation. Tissue sections were examined pathologically for tumor cell content, and only those samples with a tumor cell percentage of ≥50% were used for tissue ChIPseq analysis.Clinical PCa tissue was collected before and after enzalutamide (ENZA) therapy from the Dynamics of androgen receptor genomics and transcriptomics after neoadjuvant androgen ablation study . Specific ARBS-promoter interactions were identified from the published AR ChIA-PET performed in VCaP cell line (GSE54946) by overlapping the loop end of either ARBS or gene’s TSS (± 5Kb) with GenomicInteractions R package (v1.20.3) [AR-regulated genes were identified from publicly available RNAseq data of LNCaP cells treated with 100 nM DHT for 6 h GSE64529) with DESeq2 (v1.26.0) . The dis529 with v1.20.3) .Previously published ChIPseq and GROseq data were downloaded from the GEO database and uniformly processed  for hg19 reference. The average conservation distribution of induced, constitutive, inactive ARBS enhancers and negative control regions were calculated with deeptools’ computeMatrix function (v3.1.3). Output distribution matrix then visualized with deeptool’s plotProfile function (v3.1.3).One hundred vertebrate species conservation track was obtained from UCSC golden path  and LNCaP ARBS (not tested for enhancer activity) as nodes and the chromosomal interactions from VCaP ChIA-PET as edges. The interactions between each enhancer class or ARBS were extracted from processed data and introduced as a graph with NetworkX v2.3). Only TSS of AR DEG were considered in the network. The annotated CRE network graph contained 2361 nodes, 1951 edges, and 830 separate components. The annotated CRE and non-annotated ARBS graph contained 10,484 nodes, 12,437 edges, and 1873 separate components. The relative density  was PCR amplified from pooled male human DNA (Promega) and cloned into a STARRseq luciferase validation vector_ORI_empty plasmid (Addgene plasmid #99298) with HiFi DNA builder (NEB). Primers used to amplify specific regions are described in Additional file FXN was also quantified to assess non-specific inhibition. Each experiment was done in triplicate with a minimum of 3 biological replicas.Multiple gRNAs per enhancer region were designed with CRISPR-SURF  and clon6 cells were isolated according to 10x Genomics recommended protocol (Nuclei Isolation for Single Cell ATAC Sequencing CG000169 Rev D). scATACseq libraries were prepared using 10x Genomics Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit v1.1. Libraries were sequenced on a NextSeq500 Illumina sequencer . scATACseq analysis was done with 10X Cell Ranger software. Deviation/z-score was calculated with chromVAR [S affinity matrices were calculated as described  was calculated (Eq. LNCaP cells were cultured in phenol red-free RPMI 1640 media supplemented with 5% CSS and 1% antibiotics for 60 h and then treated with 100% EtOH or 10 nM DHT for 4 h. Nuclei from 1 × 10chromVAR  and UMAPchromVAR  (v0.9.3)chromVAR . Random chromVAR . scATACschromVAR  with 10XchromVAR  and counchromVAR . The S av) was altered, the impact (w) was calculated as the RootED distance between corresponding row vector Sv1,  and Sv2,  calculated where v = j = 1 → nj and ni = 1. Otherwise, w = 0.If an edge adjacent to the node (N = 286), inactive (N = 2479), and constantly active (N = 465) categorization of the clinical ARBS, we trained a classifier to predict the groups using the bound factors in a given region as input. For each ARBS region, we extracted the ChIPseq signal scores over a 750-bp region for 90 different DNA binding factors (Additional file All ChIPseq data used in machine learning was analyzed with the standardized ChIP-Atlas bioinformatic pipeline . Based oAdditional file 1: Fig.S1- S14. Supplementary figures.Additional file 2. All the publicly available data used.Additional file 3: Table S1. Primers and gRNAs.Additional file 4. Review history.


Solanum nigrum L., has been found to exert inhibitory effect on cancers. However, the underlying anticancer mechanisms of solasonine, particularly in urinary bladder cancer (BC), remain unclear. In this study, we identified the potential targets and biological functions associated with solasonine activity using a bioinformatics approach. Ingenuity pathway analysis revealed that neuropilin-1 (NRP1) and other signaling pathways, such as PI3K/AKT and ERK/MAPK pathways, were potentially involved in the therapeutic effects of solasonine. The ability of solasonine in inducing apoptosis and inhibiting proliferation in BC cells was confirmed experimentally, and the inhibition of ERK/MAPK, P38/MAPK, and PI3K/AKT pathways was validated by Western blot. Mechanistically, solasonine suppressed the expression of NRP1 protein, but not that of mRNA. Further results of molecular docking and molecular dynamics simulation analysis indicated that solasonine could directly bind to the b1 domain of NRP1 protein with a reasonable and stable docking conformation. We previously found that targeting NRP1 is a potential antitumor strategy. Combined with these findings, it can be speculated that the binding of solasonine with NRP1 on the cell membrane could prevent the formation of NRP1/VEGFA/VEGFR2 and NRP1/EGFR complexes, resulting in the inhibition of downstream signaling, including ERK/MAPK, P38/MAPK, and PI3K/AKT pathways. Additionally, intracellular solasonine could inhibit the membrane localization of NRP1 and provoke its cytoplasmic retention, facilitating the degradation of NRP1 protein in the cytoplasm. The dual effects induced by the binding of solasonine to NRP1 extracellularly and intracellularly could account for the antiproliferative and proapoptotic effects of solasonine on BC.Solasonine, a steroidal alkaloid extracted from  Urinary bladder cancer (BC), one of the leading causes of cancer-associated death, is the tenth most common malignancy worldwide. According to the Global Cancer Statistics, approximately 570,000 new cases and 210,000 BC-related deaths were recorded during 2020 –5. Metasα (HIF-1α), that are important for clinical applications [Emerging immunotherapeutic approaches such as checkpoint inhibitors and tumor-targeted CAR-T cells have shown efficacy in improving the prognosis of patients with advanced BC, but only a fraction of patients can achieve durable clinical responses –9. Moreoications , 10. Canications . These rSolanum nigrum L. (S. nigrum) is an annual herbaceous plant that has been used as a folklore medicine in China for BC therapy since ancient times [S. nigrum activity against BC. It was found that it exerted its therapeutic effects via multiple signaling pathways, including the MAPK, PI3K/AKT, and HIF-1 pathways [S. nigrum contains two main kinds of effective components, namely, solanine and Solanum nigrum polysaccharide [Solanum by Oddo and Cesaris in 1911, solasonine has been isolated from over 100 species and has shown beneficial pharmacological effects, such as anticancer and antimicrobial activities [Natural plants are an important source of anticancer agents. nt times , 13. In ccharide . Solasonccharide , 15. Sintivities , 16. Acctivities –19. Solativities . The acttivities . In additivities . Howeverin vitro. Our results demonstrated that solasonine significantly inhibited BC cell proliferation and promoted cell apoptosis by inhibiting the MAPK and PI3K/AKT signaling pathways. NRP1 may be a potential effector of solasonine in mediating anti-BC responses.Neuropilin-1 (NRP1) is a novel target and immunomodulator for cancer therapy –24. Our https://jsme-editor.github.io/) [https://pubchem.ncbi.nlm.nih.gov/). The molecular structure was converted to a 3D image using the Yinfo Cloud platform (http://cloud.yinfotek.com/).The molecular structure of solasonine was generated using the JSME platform (hub.io/)  by imporhttps://tcmsp-e.com/) is a systems-level pharmacology database, which can calculate pharmacokinetics properties for naturally occurring compounds based on ADME  principle. The TCMSP server was used to evaluate the pharmacokinetics characteristics of solasonine on ADME-related properties, such as human oral bioavailability (OB), drug-likeness (DL), Caco-2 permeability (Caco-2), blood-brain barrier (BBB) permeability, and Lipinski's rule of five  [The Traditional Chinese Medicines for Systems Pharmacology (TCMSP) database and analysis platform server  .http://www.lilab-ecust.cn/pharmmapper/; updated on January 01, 2016) [http://targetnet.scbdd.com/, updated on February 25, 2014) [http://sea.bkslab.org/, updated on March 26, 2019) servers [https://www.genenames.org/) symbols to avoid confusion across platforms.Potential targets of solasonine were predicted using an integrative application of PharmMapper  , 28, Tar5, 2014) , and sim servers . These dThe potential target genes of solasonine were analyzed by IPA, which can predict downstream effects and identify new targets or candidate biomarkers and can obtain data analysis and interpretation to understand the experimental results within the context of biological systems . In thishttp://db.idrblab.net/ttd/; updated on June 01, 2020) [https://www.pharmgkb.org/) [https://www.drugbank.com/; version 5.1.8) [The genes associated with BC were collected from five sources, including the databases of GeneCards  , Online 1, 2020) , Pharmackb.org/) , and Drun 5.1.8) . We searAs described in our previous research , GO analhttp://www.genemania.org/) [The gene-gene interaction network was generated using GeneMANIA prediction server (ia.org/) , which cia.org/)  to consthttps://www.rcsb.org/). The protein structures were then prepared by adding hydrogen atoms and merging nonpolar hydrogen atoms using AutoDock Tools  [Molecular docking analysis was conducted to evaluate the binding affinity of solasonine towards NRP1 protein. The 3D structure of solasonine was optimized with energy minimization in the MMFF94 force field. The crystal structure of human NRP1  was directly retrieved from the RCSB protein data bank (PDB) database (n 1.5.6)  and saven 1.5.6)  was utilThe protein-ligand docked complex structure of NRP1-solasonine was prepared for molecular dynamics simulation (MDS) using Visual Molecular Dynamics  software . The TIPμg/mL streptomycin, and 10% fetal bovine serum at 5% CO2 in a 37°C humidified culture environment, which was as previously described [The human bladder cancer cell lines T24 and 5637 were purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences . Solasonine was purchased from Chengdu Must Bio-Technology Company . The cell lines were cultured in RPMI-1640 medium with 100 U/mL penicillin, 100 escribed . Short-t4 cells/well in quintuplicate and treated with increasing concentrations of solasonine or vehicle (DMSO) for 48 h. The cell counting kit-8 (CCK8) assay was used to measure cell viability. Lastly, optical density (OD) was measured at 450 nm to detect viable cells.T24 and 5637 cells were seeded in 96-well cell culture plates at an initial density of 0.2 × 104 cells/well in triplicate. After treating with solasonine or vehicle (DMSO) for 24 h, the medium was discarded and the cells were cultured in a resuspended RPMI-1640 medium at a volume of 100 μL/well. At different time points, the cells were incubated with 10 μL CCK8 reagent  at 37°C for 4 h. Finally, the OD of each well at 450 nm wavelength was measured to evaluate cell proliferation.T24 and 5637 cells were seeded in 96-well cell culture plates at an initial density of 0.2 × 10μL of 1× binding buffer to each tube. Next, 5 μL of Annexin-V-APC  and 5 μL of propidium iodide (PI) (Sigma) were added to each tube. The tubes were then incubated for 15 min at room temperature and shielded from light. Then, a BD FACSCalibur flow cytometer (BD Biosciences) was used to assess the cell apoptosis.As performed in our previous study , T24 andµL permeabilization solution  were treated and the tubes were incubated at room temperature for 30 min in the dark. Finally, cell cycle detection was performed using a NovoCyte 2060R flow cytometer  and analyzed by FlowJo software .As described previously , cells wμg) were loaded into each lane. The primary antibodies for Western blot are listed below: NRP1 , extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) , China), phosphorylated ERK (p-ERK) , p38 mitogen-activated protein kinase (P38) , phosphorylated P38 (p-P38) , mitogen-activated protein kinase kinase 1 (MEK1) , phosphorylated MEK 1 and 2 (p-MEK1/2) , PI 3 kinase (PI3K) , phosphorylated PI3K (p-PI3K) , threonine-protein kinase (AKT) , phosphorylated AKT (p-AKT) , EGFR , vascular endothelial growth factor A (VEGFA) , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , and β-actin . After washing with TBST, the membranes were further incubated for 2 h with a secondary anti-mouse , China) or anti-rabbit  antibody, as appropriate. The presentation of target protein bands was enhanced using chemiluminescence (Millipore) and then quantified by densitometry  and normalized to β-actin levels.Total protein was extracted from T24 and 5637 cells, and Western blot were performed as described previously . BrieflyP < 0.05 was considered statistically significant.Statistical analyses were conducted using SAS 9.43 statistical software . One-way ANOVA was carried out to perform significance tests for multiple groups. Significant differences for two groups were evaluated using Student's t-test. A The molecular structure of solasonine is shown in The canonical pathway analysis generated by IPA revealed that the potential target genes of solasonine were significantly enriched in 494 canonical pathways, including Hippo signaling (−logP = 6.66), cell cycle (−logP = 6.44), HIF-1 signaling (−logP = 3.28), PI3K/AKT signaling (−logP = 1.72), and ERK/MAPK signaling (−logP = 3.13) . The disBy searching the databases of GeneCards, OMIM, TTD, PharmGKB, and DrugBank, we obtained a total of 9288 genes related to the occurrence and development of BC after eliminating the duplicated ones . IntegraP-value ≤ 0.05), including proteoglycans in cancer, HIF-1 signaling pathway, and Yersinia infection. In addition, the results of GO term analysis indicated that a total of 692, 16, and 48 terms  were remarkably enriched in biological process (BP), cellular component (CC), and molecular function (MF), respectively , which could stabilize the NRP1 protein and mediate the anchorage of solasonine in the binding site of NRP1 to form a stable complex Figures . FurtherThe binding free energy for the NRP1-solasonine complex was calculated to be −97.744 kcal/mol . ConsistSolasonine treatment resulted in a significant inhibitory effect on NRP1 protein expression , but no We further constructed hypothetical solasonine-NRP1 interactions, showing that extracellular solasonine binds to NRP1 at the cell membrane and thereby prevents the binding of VEGFA/VEGFR2 and EGFR to NRP1, resulting in the inhibition of the subsequent activation of downstream pathways, including ERK/MAPK, P38/MAPK, and PI3K/AKT pathways; in addition, intracellular solasonine bound to NRP1 to block the membrane localization of NRP1 and then downregulated NRP1 protein expression by facilitating its degradation . We hypoAs single-pass transmembrane, non-tyrosine kinase surface glycoproteins, neuropilins (NRPs), are unique to vertebrates and highly conserved across species . NRPs inα [α and PI3K/AKT through a positive feedback loop [We previously showed that NRP1 was upregulated in BC, and NRP1 silencing could induce cell apoptosis and suppress proliferation, invasion, metastasis, and angiogenesis in BC cells. We also found that inactivation of the MAPK signaling pathway was involved in mediating the anti-BC effects of NRP1 . In thisα , which iack loop . Overexpack loop , 58, andack loop . This obAccording to the causal regulatory network analysis by IPA, NRP1 was identified as a master upstream regulator involved in solasonine treatment. We then detected the protein and mRNA expression levels of NRP1 in BC cells treated with solasonine. The results showed that treatment with solasonine suppressed NRP1 protein expression but had no effect on NRP1 mRNA expression. Further molecular docking analysis indicated that solasonine could directly target the NRP1 protein with a reasonable docking conformation. The stability and reliability of the tight binding results were validated by the binding free energy of the docked complex calculations through molecular dynamics simulations. These findings suggest that solasonine is not responsible for the transcriptional regulation of NRP1 but may downregulate the stability of NRP1 protein by direct binding to alter the molecular conformation of NRP1. In general, as a membrane protein, NRP1 needs to be inserted into the cell membrane to exert biological functions. By binding to NRP1, solasonine might inhibit the membrane localization of NRP1 and provoke its cytoplasmic retention, leading to its degradation in cells. This mechanism could be among the contributors to NRP1 protein downregulation, which requires further experimental verification.The NRP1 protein consists of a long N-terminal extracellular domain, followed by a transmembrane region and a short cytosolic tail of 43–44 amino acids . Its extBased on these results, we inferred that solasonine binding with NRP1 on the cell membrane could prevent the formation of NRP1/VEGFA/VEGFR2 and NRP1/EGFR complexes, resulting in the inhibition of downstream pathways, including ERK/MAPK, P38/MAPK, and PI3K/AKT pathways; meanwhile, intracellular solasonine could inhibit the membrane localization of NRP1 and provoke its cytoplasmic retention, facilitating the degradation of NRP1 protein in the cytoplasm. The subsequent dual effects induced by the binding of solasonine to NRP1 extracellularly and intracellularly could be potential contributors to the solasonine-dependent antiproliferative and proapoptotic effects on BC cells.S. nigrum in various disorders [There were some limitations in this study. First, although the target prediction for solasonine against BC was performed in several online databases, it lacked accuracy and specificity compared with mass spectrometry and sequencing experiments. Second, we did not construct the silenced and overexpressed NRP1 groups to observe the anticancer effect of solasonine, which would weaken the evidence that solasonine targeting NRP1 induces apoptosis and inhibits proliferation in BC cells. Finally, immunoprecipitation experiments should be performed to confirm the binding of solasonine to NRP1 and to validate the mechanism of solasonine activity against BC observed in this study. These issues can be addressed in subsequent experiments. Our findings suggest a potential novel mechanism by which solasonine inhibits the growth of BC cells and indicate an important role of NRP1 in the activity of solasonine. Considering the widespread application of isorders , exploriIn this study, the mechanisms and candidate targets of solasonine in the treatment of BC were examined using bioinformatics analyses and verified using experimental models. Molecular docking predicted that solasonine could directly bind to NRP1. The inhibition of NRP1 protein expression was observed in BC cells following solasonine treatment. We suggested that solasonine could inhibit the MAPK and PI3K/AKT pathways by preventing the formation of NRP1/VEGFA/VEGFR2 and NRP1/EGFR complexes on the membrane surface, inducing NRP1 protein degradation by restricting its membrane localization. The dual effects induced by solasonine-NRP1 binding extracellularly and intracellularly could be potential contributors to the antitumor effect of solasonine.


The increase in female participation in athletics over the past decade has been accompanied by an increase in injury rates as a result of higher demands placed on athletes. Although previous studies have shown that anatomic, biomechanical, hormonal, and psychological factors may play a role in differences between men and women that can influence injury risk in athletes, there is still a lack of understanding of sex-related mechanisms of injury, guidelines, and prevention strategies. This article provides an overview of common injuries affecting female athletes. We present guidelines for upper- and lower-extremity injury rehabilitation, focusing on considerations specific to the female athlete with the goal to facilitate a safe return to sports.Level V, expert opinion. Female participation in athletics has increased over the past decade, with a rapid rise in participation in sports previously deemed “male dominated.”,Increased sports participation has led to an increased rate of injury in female athletes, and previous studies have shown an increased risk of certain injuries in women when compared with men.,ACL tears are 2 to 8 times more common in female athletes than in their male counterparts.,Previous studies have shown sex differences in the epidemiology of ankle injuries, particularly ligament sprains and instability.,Although traumatic shoulder instability is more prevalent in male athletes, female athletes often present with multidirectional instability and studies have shown that female athletes have poorer outcomes after surgical management of shoulder instability.,Several innovative therapeutic modalities have been developed and used to reduce rates of injury and minimize the amount of rehabilitation required for athletes to return to play (RTP) after an injury. One such modality is cryotherapy—a well-established and widespread therapy used in athletes in an attempt to treat soft-tissue injuries and reduce post-training inflammation and pain.,,Another often-performed therapeutic technique is iontophoresis, whereby a direct current of low amperage is applied to the skin by means of bipolar electrodes to increase the permeability of skin and enhance the delivery of transdermal drugs or prodrugs into the deeper tissues such as the muscles or tendons or the synovial fluid.Blood-flow restriction (BFR) therapy is a more recently developed technique often used in the athletic population. BFR involves localized restriction of venous blood outflow from a specific portion of the body using an occlusive pressure device such as a tourniquet or inflatable cuff placed at the proximal end of an extremity. The restriction of venous outflow results in an anaerobic environment, which promotes muscle hypertrophy through cellular signaling and hormonal changes similar to those seen during higher-intensity training with more resistance.Finally, there are several injectable therapies used in the athletic population including platelet-rich plasma therapy, autologous conditioned plasma therapy, mesenchymal stem cell exosome therapy, mesenchymal stem cell–Wharton jelly therapy, gold-induced cytokine therapy, and prolotherapy. These and several other recently developed therapeutic techniques have been seen more prevalently in the recent sports medicine literature and have been gaining popularity with athletes, coaches, physiotherapists, and physicians alike.Injury rehabilitation typically involves several sequential phases . The iniOnce pain-free ROM and mobility have returned to or are near baseline, the athlete can advance to the strength redevelopment phase of rehabilitation. An athlete’s baseline strength level and sport are integral factors to consider when developing a strength training program. A strength training program should maximize the use of compound movements if the athlete’s injuries allow for it. Compound movements facilitate functional strength development by having the athlete perform complex movements that involve multiple joints and muscle groups . These movements are more neurally demanding and allow for larger loads to be lifted, leading to greater neuromuscular development. Compound training has also been proved more effective for increasing overall muscular strength, promoting hypertrophy, and reducing rates of injury and reinjury in athletes when compared with isolation movements.,Once these initial phases have been completed, sport-specific exercise selection then allows for adaptations tailored to the athlete’s sport. Given that the risk factors for sustaining an injury vary based on sport, tailoring the rehabilitation program to the athlete’s sport can result in better outcomes.,For the upper extremity (UE), there are several devices that can allow for self-myofascial release. The Graston technique, massage balls, and massage guns are the primary methods by which UE self-myofascial release is performed because foam rollers are generally larger and more suited for the lower extremity (LE) and posterior chain.Once ROM is re-established, the strength training phase of rehabilitation should start with low-intensity exercises. Single arm–throwing athletes typically have significant asymmetry in shoulder strength and flexibility, which can frequently cause injury.Although there are some compound exercises that allow for shoulder development , these can be challenging to perform, especially by female athletes with pre-existing shoulder injuries. Instead, shoulder strength can be developed by performing isolation exercises with lighter loads such as light dumbbells, resistance bands, or cable machines. Such exercises include lateral raises, internal and external shoulder rotation, and the Y-T-W-L exercise (a popular shoulder exercise performed while prone with the UE taking the shape of each letter).Elbow and wrist training plays an important role, particularly in participants in throwing sports, in reducing the risk of medial and lateral epicondylitis. Resistance training has proved to be an effective method in strengthening the muscles of the forearm and reducing the incidence of epicondylitis.30Finally, grip training is widely recognized as a crucial part of UE development and rehabilitation. Many recently developed creative techniques target this specifically, including the use of handgrip tools, kettle bells, farmer’s walks, and TheraBand Flexbars. Grip training can also be incorporated into general strength training by using handles that are more difficult to grasp, forcing the development of strength and dexterity in the muscles responsible for grip. This can be further extended into the specific development of the intrinsic muscles of the hand by using finger exercise methods, such as finger extension resistance bands and finger exercisers.31,UE orthotics and taping can be a very useful tool during the RTP phase because they can provide an extrinsic support that reduces the risk of reinjury. Splints and braces are particularly useful in the treatment of elbow, wrist, and finger injuries because they can effectively limit ROM and protect against hyperextension and valgus-varus stresses. Kinesio Taping has been shown to be effective in improving shoulder proprioception and reducing the risk of shoulder injury.LE rehabilitation follows many of the same principles as UE rehabilitation. The added difficulty, however, is that effective strength training in the LE, unlike in the UE, is heavily dependent on compound movements , which often involve the UE and the core. This can be challenging because injuries involving other joints in the UE or core might limit an athlete’s ability to adequately perform LE strength rehabilitation.,,To some degree, this can be overcome by performing isolation resistance training that focuses on monoarticular movements, but unfortunately, this is not a very effective method of building LE strength or functional rehabilitation when used as a monotherapy. In addition, addressing lower-limb coordination and proprioception can be challenging during rehabilitation. However, it is of utmost importance to avoid reinjury, particularly in female athletes with knee and ankle injuries , which cannot be addressed with isolation training.,,,Dynamic stabilization of the knee is primarily achieved via the hamstrings and quadriceps. The functional hamstring-to-quadriceps (H/Q) ratio—the functional ratio of eccentric hamstring to concentric quadriceps moments (extension) and concentric hamstring to eccentric quadriceps moments (flexion)—has been described as one of the integral principles that determine the degree of strain on the ACL.,At high knee flexion angles, a low H/Q ratio may represent a compromised ability of the hamstrings to stabilize the knee joint throughout the full ROM. Near full knee extension, shifts in favor of the knee flexors may represent an attempt to stabilize the knee at the angle of greatest ACL strain.,Given the crucial protective value of the hamstrings and quadriceps in reducing the risk of ACL injuries, developing functional strength is at the core of LE rehabilitation. A highly cited study by Renström et al.Similarly, rehabilitation after an acute ankle sprain or chronic ankle instability includes early functional rehabilitation, which focuses on increasing ROM, resistance strength training, proprioception exercises, and sport-specific training.,,Anatomic differences, hormonal differences, biomechanical factors, neuromuscular factors, genetic predisposition, and even psychological assessment of competitive ability have all been suggested to play roles in the differences between male and female athletes.,Although there is a paucity of literature evaluating outcomes of various gender- and sex-specific rehabilitation strategies, many protocols address the 4 following deficits typically seen in the injured female athlete: (1) ligament dominance, (2) quadriceps dominance, (3) leg dominance, and (4) trunk dominance.Psychological factors including self-efficacy, identity, and fear of reinjury may all play a role in an athlete’s ability to return to sport.Increased female sports participation has been accompanied by an increase in injury rates. Understanding how female athletes differ from their male counterparts regarding the mechanism of injury and recovery is important to tailor rehabilitation strategies to the individual female athlete. This article shows the importance of rehabilitation considerations specific to female athletes to more effectively return them to their sports efficiently and safely.


We wanted to investigate whether parasympathetic inhibition affected the expression of type 2 innate lymphoid cells (ILC2s) in the nasal mucosa of a mouse model of allergic rhinitis (AR).Thirty male C57BL/6 mice were randomly divided into 3 groups: control group, AR group, AR-treated group. AR nasal symptoms were assessed on a semi-quantitative scale according to the frequencies of nose rubbing and sneezing and the degree of rhinorrhea. The expression of cytokines protein in serum was detected by enzyme linked immunosorbent assay (ELISA). The number of ILC2s in nasal mucosa was detected by immunofluorescence double staining assay. Quantitative real-time Polymerase Chain Reaction (qPCR) was used to detect the expression of ILC2-associated factor in nasal mucosa.The symptom scores of the AR group were significantly higher than those of the control group and AR-treated group. The expression levels of mouse ovalbumin (OVA) specific IgE, IL4, IL5, and IL13 in the serum of AR group were significantly higher than those in the control group and AR-treated group. The number of ILC2s and the gene expression of ILC2s related factors GATA3, CD25 and CD90 (Thy1) in the nasal mucosa of the AR group were significantly higher than those of the control group and AR-treated group.We found that parasympathetic inhibition relieved AR symptoms and inhibited immune response of AR mice. ILC2s play an important role in the occurrence and development of AR, and parasympathetic nerve inhibition reduced the number of ILC2s and inhibited the cytokines expression by ILC2s. Our data provide information on the mechanism of action of parasympathetic inhibition in AR. In this study, a mouse model of AR was established and treated with ipratropium bromide solution as a parasympathetic inhibitor. Nasal symptom scoring scale, immunofluorescence double staining, quantitative real-time PCR (qPCR), and enzyme-linked immunosorbent assay (ELISA) were used to investigate whether parasympathetic inhibition affected the expression of ILC2s in the nasal mucosa of the AR mouse model.Ipratropium bromide was purchased from yuanye Bio-Technology Co., Ltd . Ovalbumin (OVA) and aluminum hydroxide were obtained from Sigma-Aldrich . Antibodies against CD90 (Thy1) (ab3105), ST2 (ab25877) were obtained from Abcam . Secondary antibodies were obtained from Invitrogen . IL-4, IL-5, IL-13 and mouse OVA specific IgE ELISA kits were purchased from Shanghai Xitang Biotechnology Co., Ltd . Trizol was obtained from Thermo Fisher scientific . SYBR green was obtained from Bio-RAD Laboratories .A total of 30 specific pathogen-free male C57BL/6 mice, aged 5 weeks, were purchased from Nanjing University Nanjing Biomedical Research Institute. The animal experiment in this study was approved by Animal Experimentation Ethics Committee. The 30 mice were randomly divided into 3 groups (n = 10 for each): control group, AR group, AR-treated group. The establishment of the AR model and the time of treatments are described below A. In briThe expression of mouse OVA specific IgE, IL4, IL5, and IL13 were tested by employing an ELISA method. Briefly, 0.5 mL sample blood was collected from eye socket of each mouse. The blood was centrifuged at 4 °C and 1000 g for 10 min. The supernatant and pre-prepared standard solution were added into well plate. The immunosorbant assay was performed at 37 °C for 40 min. The absorbance value at 450 nm was measured by an enzyme labeling instrument. The concentration of mouse OVA specific IgE, IL4, IL5, and IL13 was calculated according to the standard curve.Five mice from each group were randomly selected for immunofluorescence double staining to detect the number of ILC2s. The nasal mucosa wrapped by nasal bone was fixed in 4% neutral buffered formalin, then decalcified in EDTA solution. Three-micrometer slices were obtained and dewaxed in xylene, followed by ethanol gradient dehydration and washed in phosphate buffer solution (PBS). CD90 (Thy1) and ST2 antibodies  were employed to stain the aimed protein according to the manufacturer protocol. Photo capture was performed using upright fluorescence microscope .−ΔΔCt method.The nasal mucosa of other 5 mice in each group were used to detect the expression level of ILC2s associated factors by qPCR according to the manufacturer protocol. Total RNA from mouse nasal mucosa tissue was extracted and reverse transcription reaction was performed. As shown in t-test, and one-way ANOVA was used in three groups. When 2 variables existed, two-way ANOVA analysis was performed, followed by Tukey's multiple comparison test. When P < 0.05, the difference was considered to be statistically significant.All the experimental data were analyzed by GraphpadPrism9 statistical software. Pairwise comparison was carried out by two-sample ####P < 0.0001) (AR symptoms were scored according to the frequency of sneezing and wiping the nose and the degree of runny nose . Compare 0.0001) B.ELISA was used to detect the protein expression levels of IL-4, IL-5, IL-13, and mouse OVA specific IgE in mouse serum . CompareDouble immunofluorescence staining for CD90 (Thy1) and ST2 showed that the number of double positive cells in the AR group was significantly higher than that in the control group. In contrast, the immunoreactivity of CD90 (Thy1) and ST2 was greatly attenuated after the treatment of ipratropium bromide A. We calThe mRNA expression levels of CD25, CD90 (Thy1), GATA3 in mice nasal mucosa were detected by quantitative real-time PCR. Compared with the mice in control group, the expression of CD25, CD90 (Thy1), GATA3 in the nasal mucosa of the mice in AR group was significantly increased in mRNA level, and the difference was statistically significant. Compared with the mice in AR group, the expression of CD25, CD90 (Thy1), GATA3 in the nasal mucosa of the mice in AR-treated group was significantly decreased in mRNA level, and the difference was statistically significant .Fig. 4Th,AR is one of the most common diseases in otorhinolaryngology, head and neck surgery. But its pathogenesis remains to be clarified, and the clinical symptoms of some patients can not be effectively relieved. Therefore, there is an urgent need to further study the mechanism of AR. In recent years, the study of ILC2s, innate lymphocytes that can participate in adaptive immunity, provides a new research scope for the pathogenesis of AR. ILC2s were first discovered in 2001, Fort et alAlthough AR is an immune disease, neuroregulation plays a very important role in occurrence and development of AR. In recent years, some research groups proposed that nerve cells and immune cells can interfered with each other in specific anatomical sites to form a neuroimmune cell unit (NICU) to play a regulatory role.+CD45+CD127+KLRG1+Thy-1+ cells.,However, few studies on whether parasympathetic inhibition affects the expression of ILC2s in AR mice. In this study, we established a C57BL/6 mouse model of AR and treated the mice with parasympathetic inhibitor ipratropium bromide to explore whether parasympathetic inhibition affects the expression of ILC2s in nasal mucosa of AR mice. The ILC2s were defined as Lin-ST2To sum up, our experimental study confirmed that ILC2s play an important role in the occurrence and development of AR. Ipratropium bromide, an inhibitor of parasympathetic nerve, relieved the symptoms of AR, inhibited the number and function of ILC2s and immunity of mice with AR, which provided a new therapeutic basis for the treatment of AR by parasympathetic inhibition, but the specific mechanism of parasympathetic inhibition on ILC2s still needs to be further explored.AR, allergic rhinitis; ILC2s, Type 2 innate lymphoid cells; OVA, ovalbumin; DC, dendritic cells; IL, interleukin; TSLP, thymic stromal lymphopoietin; qPCR, Quantitative real-time Polymerase Chain Reaction; TH, helper T cell; CADM1, cell adhesion molecule 1; PBS, phosphate buffered saline; IgE, immunoglobulin E; ELISA, enzyme linked immunosorbent assay; VIP, vasoactive intestinal peptide; NMU, neuropeptide neuromedin UThe animals used in this study were approved by the Animal Experimentation Ethics Committee of Wenzhou Medical University, Wenzhou, China. The living condition and experimental procedures were conducted in accordance with the National Institutes of Health Guideline concerning the Care and Use of Laboratory Animals.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Liyan Ni and Hailin Zhang conceptualized and designed the study, and approved the final manuscript as submitted. Binbin Hu, Yan Wang, Guotong Zheng collected the sample, performed the experiment, data collection and statistics, drafted the initial manuscript, reviewed and revised the manuscript, and approved the final manuscript as submitted.All authors agreed to publication of the work.Science and Technology Plan Project of Taizhou (1902ky75), 10.13039/501100007194Wenzhou Science and Technology Bureau (Y20190154), the Special Project for Significant New Drug Research and Development in the Major National Science and Technology Projects of China (2020ZX09201002), 10.13039/501100001809National Natural Science Foundation of China (81973382).This work was supported by grants from the The authors declare that they have no relevant conflicts of interest.