Background: Epithelial ovarian cancer (EOC) is one of the most lethal gynecological malignancies and presents chemoresistance after chemotherapy treatment. Androgen receptor (AR) has been known to participate in proliferation. Yet the mechanisms of the resistance of this drug and its linkage to the AR remains unclear. Methods: To elucidate AR-related paclitaxel sensitivity, co-IP, luciferase reporter assay and ChIP assay were performed to identify that AR direct-regulated ABCG2 expression under paclitaxel treatment. IHC staining by AR antibody presented higher AR expression in serous-type patients than other types. AR degradation enhancer (ASC-J9) was used to examine paclitaxel-associated and paclitaxel-resistant cytotoxicity in vitro and in vivo. Results: We found AR/aryl hydrocarbon receptor (AhR)-mediates ABCG2 expression and leads to a change in paclitaxel cytotoxicity/sensitivity in EOC serous subtype cell lines. Molecular mechanism study showed that paclitaxel activated AR transactivity and bound to alternative ARE in the ABCG2 proximal promoter region. To identify AR as a potential therapeutic target, the ASC-J9 was used to re-sensitize paclitaxel-resistant EOC tumors upon paclitaxel treatment in vitro and in vivo. Conclusion: The results demonstrated that activation of AR transactivity beyond the androgen-associated biological effect. This novel AR mechanism explains that degradation of AR is the most effective therapeutic strategy for treating AR-positive EOC serous subtype. Epithelial ovarian cancer (EOC) is among the most lethal gynecological malignancies and has a variety of cellular origins, histological characteristics, and therapeutic responses . In termATP-binding cassette transporters (ABC transporters) are a type of transmembrane protein pumping drugs out of cells  by consuAndrogen, a type of steroid hormone, has been suggested to potentially play a role in ovarian pathophysiology ,16. AndrIn this study, we discovered a preference in AR expression in serous subtype EOC which causes the promotion of cancer growth. Specifically, we found that treating serous EOC cells with paclitaxel, the standard-of-care chemotherapy agent, surprisingly causes the activation of an AR–AhR complex →ABCG2 regulatory axis that in turn causes EOC cells to become resistant to paclitaxel.Given that previous studies have shown paclitaxel sensitivity influenced by ABCG2 expression and function ,27, we eTo identify AR-regulated ABCG2 expression, we surveyed a promoter of the ABCG2 genome (+336/−3628 kb) from the National Center for Biotechnology information (NCBI) database (accession no. NC_018915) . Accordihttp://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3 (Accessed on 2 April 2019) to find AREs and DREs. There were several putative AREs (−2642/−3110) and a core DRE (−194/−190) that were found , +336 ~ −1336 , and –3268 ~ −1898  on respective pGL3-basic luciferase plasmid F. We the50 of each set of manipulated-cells is shown in To study the potential impact of AR on paclitaxel therapy to suppress the EOC progression, we assayed their impact on the colony formation capacity. The results showed that increasing AR expression by adding AR-cDNA reduced paclitaxel-related colony forming inhibition A and cytWe then tested the effects of AR in the development of paclitaxel resistance. We observed the cell growth dynamics under a low-dose, long-term treatment with paclitaxel  (2 = 0.4641).To evaluate AR/ABCG2 expression in patients with different EOC subtypes, we performed a single cohort study with immunohistochemistry (IHC) using AR antibody. The review found that the tissue samples consisted of 16 serous, 20 clear-cell, 29 mucinous, and 49 endometrioid EOC subtype samples, and AR IHC was then performed on these samples B. We fouhttp://kmplot.com/analysis/index.php?p=background) to evaluate AR mRNA expression in relation to disease prognosis. We restricted our analysis to stage 3 and 4 serous EOC samples, we found AR expression to be a positive prognostic marker (hazard ratio (HR) = 1.48; p = 0.01; p = 0.0015; We further used the web-based gene survivor Kaplan–Meier Plotter  assay  was usedAs the paclitaxel → AR/AhR → ABCG2 regulatory axis had been elucidated, we then wanted to test the value of targeting AR for EOC therapy. There are two compounds available for interrupting this axis by interfering with AR function. One is the new anti-androgen enzalutamide (or MDV-3100), which was developed for the irreversible blockade of androgen–AR interactions, and which has already been proven to offer a survival benefit to relapsed prostate cancer patients . The oth50 of the combination therapy for each set of cells is shown in Thus far, the blocking of androgen/AR function with MDV3100  in EOC has been proposed ; however3, injecting the given intraperitoneal treatment two times/week for four consecutive weeks. The tumor sizes for the mice treated with vehicle vs paclitaxel vs ASC-J9 alone remained comparable. However, a significant reduction in tumor size was seen in the mice receiving the paclitaxel with ASC-J9 co-treatment (p < 0.05)  OVCAR3 cells using an add-on dosing and long-term treatment procedure ,27. The  < 0.05) E. After  < 0.05) F. Taken together, the data presented in Serous EOC is the subtype of EOC that affects the most women patients, has the poorest prognosis, and is the quickest to develop chemoresistance ,9,39. InSun et al. showed that AR and ABCG2 were upregulated under paclitaxel treatment and in paclitaxel-resistant EOC cells ,27. ThesExcept for ABCG, there is another ABC transporter gene family related to chemotherapy resistance, the ABCBs . Among tIn previous studies, Bailey et al. analyzed the ABCG2 promoter region and data showed none of the classical AREs existed in the promoter . Consistp < 0.001; data maturity, 58%) [The roles of androgen/AR signaling in ovarian cancer progression have been reported previously , and somty, 58%) ,50. The ty, 58%) ,51, (2) ty, 58%) ,53. In oThe paraformaldehyde-embedded EOC tissue samples analyzed in this study were obtained from patients diagnosed with EOC from 2008 to 2013 at the China Medical University Hospital , and clinical data for these patients was acquired from the Cancer Registry Database of CMUH. The subtypes of EOC patients were selected from ward charts. Patient tissue slides were confirmed by two boarded pathologists to exclude ambiguity or mixing histology between subtypes. A total of 114 tissue samples were collected . Access to the tissue samples was approved by the Internal Review Board of the China Medical University Hospital (#CMUH104-REC2-075). The histological studies were performed with modifications as described in previous studies ,54. For 2. The cells were then cultured in fresh DMEM/10% CD-FBS  for 24 h before the experiments. The ovarian cancer cell lines were provided courtesy of Dr. Min-Chie Huang , and the HEK293T cells were obtained from Dr. Yuh-Pyng Shyr . The chemotherapeutic drugs used in this study included paclitaxel, MDV3100  , and ASC-J9, which was kindly provided by Dr. Chawnshang Chang . The human EOC serous subtype cell lines  and the human embryonic kidney cell line (HEK293T) used in this study were grown in Dulbecco’s Modified Eagle medium (DMEM)  with 10% fetal bovine serum (FBS) (GIBCO) and 1% penicillin/streptomycin (GIBCO) at 37 °C in a humidified atmosphere of 5% COCells were first washed with 1× PBS and resolved in RIPA buffer  with protease inhibitors . Proteins were resolved by SDS-PAGE and then transferred to PVDF membranes. The blocking of non-specific binding was accomplished by adding 5% non-fat milk. After the application of primary antibody (AR (N-20), Santa Cruz, CA, USA; β-actin, Santa Cruz, CA, USA; AhR (H-211), Santa Cruz, CA, USA; BCRP/ABCG2 (BXP-21), Abcam, Cambridge, UK), secondary antibody  were applied for 1 h at room temperature. Signals were enhanced using an ECL chemiluminescence kit  and detected by ChemiDoc XRS+ .2 for 48 h. The medium containing the virus was collected by centrifugation and filtered through a 0.45 μm filter. Medium containing 0.8 mg/mL polybrene (Sigma-Aldrich) was then added to culture dishes containing 106 EOC cells. After 16 h of infection, the medium containing virus was replaced with fresh DMEM/10% FBS medium, and the cells were maintained at 37 °C in a humidified incubator in an atmosphere of 5% CO2 for 48 h. The infected cells were then collected and analyzed. Cells were transfected with the following lentivirus plasmids: psPAX2 packaging plasmid, pMD2G envelope plasmid , pLKO.1-shLuc (luciferase shRNA), pLKO.1-siAR , pWPI-vector ctrl, and pWPI-hAR (Addgen). Lentiviral plasmids were co-transfected with psPAX2 and pMD2G into HEK293T cells at a ratio of 3:2:4 by lipofectamine 2000  according to the manufacturer’s instructions. After 6 h, the medium was replaced with fresh DMEM/10% FBS medium, and the cells were maintained at 37 °C in a humidified incubator in an atmosphere of 5% CORNA was extracted from EOC cells. Briefly, cells that had reached 80%~90% confluence in 100-mm dishes were lysed with 1 mL Trizol (Invitrogen). Phenol/chloroform was then added, and RNA-rich layers were separated by centrifugation. Soluble RNA was precipitated with 2-propanol. The RNA was then rinsed with 75% ethanol and dissolved in RNase-free water. For first-strand cDNA synthesis, 5 μg of total RNA was used to perform reverse transcription PCR using the PrimeScript RT reagent kit . cDNA was synthesized according to the manufacturer’s instructions.−ΔΔCt) for the quantitation of gene expression. The quantitative real-time PCR primer sequences are shown in A real-time detection system  and the KAPA SYBR FAST One-Step qRT-PCR Kit  were used according to the manufacturers’ instructions. Relative gene expression was determined by normalizing the expression level of the target gene to the expression level of a housekeeping gene (β-actin). Threshold value (Ct) dynamics were used  in culture medium at 37 °C for 48 h. Then, 10 μL of WST-1 solution was added to each well, and the cells were allowed to incubate at 37 °C in an incubator for 1 h. Cell viability was quantified by colorimetric detection in an ELISA plate reader  at an absorbance of 450 and 690 nm to generate an OD proportional to the relative abundance of live cells in the given wells. The readings of the measured values of 50% inhibition concentration (IC50) [The water-soluble tetrazolium salt (WST-1) reagent  was used to assess cell viability. Briefly, 4 × 10n (IC50)  for eachn (IC50)   for 90 min at 37 °C, either alone or in the presence of 50 µM verapamil (Sigma-Aldrich). After certain incubation time periods, the cells were washed with ice-cold 1× PBS and then resuspended with 1× PBS supplemented with 2% FBS and 2 µg/mL propidium iodide (Sigma-Aldrich) at 4 °C for 5 min to exclude dead cells. The cells were analyzed using flow cytometry  with dual wavelength analysis  after excitation with 350 nm UV light.ABCG2 efflux analysis was performed as described previously by Goodell et al. . BrieflyGenomic DNA (gDNA) was purified from EOC cells using the Quick-gDNA MiniPrep kit . Extraction procedures were performed according to the manufacturer’s instructions. Before PCR amplification, gDNA was digested by the restriction enzyme BamHI and the digested gDNA fragments were used as the PCR template. Five ABCG2 promoter region fragments were amplified by PCR, namely the −3268/+362, −3268/−1396, −1396/+362, −959/+362, and −523/+362 fragments. The PCR product names and primers are shown in ABCG2 promoter region PCR reactions were as follows: denaturation at 95 °C for 5 min; 45 cycles of amplification ; and final extension at 68 °C for 10 min. ABCG2 promoter region fragments were completed using AccuPrim Pfx DNA polymerase, which allows for the high-fidelity amplification of DNA fragments for downstream plasmid construction (Invitrogen). All DNA fragments were purified and sequenced.The conditions for the All the ABCG2 promoter fragments were cloned into the SacI–HindIII sites of the pGL3-basic vector plasmids. A luciferase reporter assay was performed as previously described. Briefly, pGL3-ABCG2 promoter fragments and pRL-TK (thymidine kinase promoter-driven renilla luciferase plasmid) were transiently co-transfected into cells. After 6 h, the medium was replaced with fresh medium and 10% CD-FBS. The cells were then cultured for 48 h with or without DHT (10 nM). After 24 h, the cells were washed with 1× PBS, then incubated in the presence of 100 µL cell culture lysis reagent (CCLR)  at room temperature for 30 min. Cell lysates were then placed in a microtube and centrifuged at 14,000 rpm for 5 min. Supernatant (5 µL) was then mixed with 50 µL luciferase assay reagent. Luciferase activity was measured immediately using a luminescence microplate reader and presented as relative luminescence units.A ChIP assay was performed as described by Mulholland et al. . CultureEOC cells in test tubes were carefully washed twice with pre-chilled PBS. Non-denaturing lysis buffer , 2 mM EDTA) was then added to the tubes. The cells were scraped off and moved to clean 1.5-ml tubes. 20 µl protein A/G agarose was added to the tubes to eliminate non-specific binding proteins, and the tubes were then shaken using a horizontal shaker for 10 min at 4 °C. The tubes were then centrifuged at 1000 g at 4 °C for 5 min, and the supernatant was transferred to new tubes while the protein A/G-agarose beads were discarded. A total of 500 μg of cell lysate per IP was used to immunoprecipitate AhR using anti-AhR antibody  at 4 °C, with the tubes shaken overnight. Then protein A/G plus agarose beads  were added directly to the supernatant and the mixture was shaken for 2 h at 4 °C. Next, it was centrifuged at 1000 g at 4 °C for 5 min, and the supernatant was removed. The beads were then washed twice with ice-cold PBS. Finally, 50 µl RIPA buffer  and 6× sampling dye were added. The proteins were detected by western blotting using anti-AR and anti-AhR  antibody. Rabbit IgG was used as the control.Establishment of paclitaxel-resistant OVCAR3 (PTXR) cells was performed according to Sun et al. ,27 with scid/Cr1Nar1) were purchased from the National Laboratory Animal Center , and 5 × 106 PTXR cells were subcutaneously injected into both flanks of each mouse. When the tumor volume (TV) reached 400 mm3, the mice were randomized into four groups . All the treatments were given intraperitoneally three times a week for four weeks. The tumor length and width were measured by caliper twice weekly, as was the body weight of each mouse. When the mice were sacrificed, the tumor volume was calculated as follows: TV = (width2 × length)/2.The animal handling and experimental procedures were approved by the Animal Experimental Ethics Committee of China Medical University . Six-weeks-old female athymic nude mice (CB17/Icr-Prkdct-test. All experiments were repeated at least three times, and p-values < 0.05 were considered to indicate statistical significance.Statistical analyses were performed using a Student’s This is the first report demonstrating that paclitaxel induces AR transactivity that directly interacts with alternative ARE in the ABCG2 promoter region to regulate gene expression. Our study also provides a perspective therapeutic insight, specifically that the degradation of AR could provide the most effective therapeutic strategy for treating AR-positive serous subtype ovarian cancers.


Severe traumatic brain injury (TBI) may result in a disorder of consciousness (DoC) and lead to substantial long-term disability. While level of independence with activities of daily living, especially for persons who recover consciousness during inpatient rehabilitation, generally improves over time, the degree of change in participation remains unknown. We determined level of participation among persons with TBI between 2005 and 2017 who were admitted to inpatient rehabilitation unable to follow commands and subsequently enrolled in the TBI Model Systems National Database. Participation on the Participation Assessment with Recombined Tools-Objective (PART-O) Productivity, Social Relations, and Out and About subscales was evaluated at 1–5 years post-injury. We used a mixed-effects model to longitudinally compare participation between persons who did and did not regain command-following during inpatient rehabilitation. We further explored the level of participation associated with increasing levels of functional independence (FIM). The analysis included 333 persons . Participation across groups, at all follow-up time points, on all PART-O subscales, was remarkably low . Performance was highest on the Social Relations subscale and lowest on the Productivity subscale. Longitudinal analyses revealed no difference in level of participation or change in participation across time for persons who regained command-following during rehabilitation compared to those who did not. While productivity increased over time, social participation did not and participation outside the home increased more for younger than for older persons. Across all three PART-O subscales, FIM Motor scores positively predicted participation. FIM Cognitive scores positively predicted level of participation on the Productivity and Social Relations subscales. Exploratory analyses revealed that even persons who achieved independence on the FIM Motor and Cognitive subscales had low levels of participation across domains and follow-up years. In summary, persons with severe TBI who were admitted to inpatient rehabilitation unable to follow commands were found to be unlikely to participate in productive tasks, social endeavors, or activities outside of the home up to 5 years post-injury, even if functional independence was recovered. Medical advances have improved the ability of healthcare providers to prevent early death among persons with severe traumatic brain injury (TBI) . SurviviAlthough a precise operational definition of participation has not emerged from the literature, it is widely recognized to encompass varied life domains at the societal level, such as home activities, learning, social interactions –14, and Inability to return to work, attend school, or engage in other valued roles or routines, including those associated with leisure, is common during the years after moderate to severe TBI , 19–22. Despite improved knowledge of long-term clinical outcomes, participation among individuals who have experienced very severe TBI remains understudied. A more complete understanding of participation over time is necessary to develop or refine participation measures, inform interventions to improve participation, and potentially reduce monetary and emotional burden. This study aims to: (1) characterize the level of participation across the domains of productive, social, and outside activities at 1, 2, and 5 years post injury among persons admitted to acute inpatient rehabilitation without command-following; and (2) compare longitudinal levels of participation between persons who regained and those who did not regain command-following during inpatient rehabilitation. We hypothesized that persons who regained command-following during inpatient rehabilitation would demonstrate higher levels of participation compared to those who did not regain command-following during inpatient rehabilitation. We also explored the relationship between functional recovery and participation to determine whether individuals who regain independence become productive, socially integrated and able to participate in activities outside the home.The sample was drawn from the Traumatic Brain Injury Model Systems (TBIMS) National Database (NDB). The TBIMS NDB is a longitudinal multicenter study which prospectively enrolls and collects data from individuals with moderate to severe TBI hospitalized and later admitted to inpatient rehabilitation facilities in the United States. The TBIMS is currently comprised of 16 regionally and demographically diverse centers. Each center obtains approval to contribute data to the database from their local Institutional Review Board (IRB) and obtains consent from persons with TBI or surrogates as per IRB protocol. A model system must include a Level 1 Trauma Center, acute neurosurgical care, comprehensive inpatient rehabilitation services, and multi-disciplinary rehabilitation and follow-up care. TBI was considered moderate to severe if there was documented evidence of post traumatic amnesia for greater than a day, loss of consciousness >30 min, a Glasgow Coma Scale (GCS)  score <1The primary analysis divided the sample into those who regained command-following during inpatient rehabilitation [Rehab Command-following (RCF)] vs. those that did not [No Rehab Command-following (nRCF)]. In addition, we conducted a secondary analysis  that divided the cohort of individuals admitted to inpatient rehabilitation without command-following into those who did and did not regain command-following within 28 days post injury  as this timeframe represents the accepted definition of “prolonged” DoC .At the time of participant or proxy consent for TBIMS NDB enrollment, acute care charts were reviewed for demographic and injury characteristics . Additionally, the results of the following assessments were obtained from the TBIMS NDB.The presence of command-following was determined using two TBIMS variables at the time of rehabilitation admission, consistent with prior TBIMS DoC studies , 6. The The Participation Assessment with Recombined Tools-Objective The PART-O was the main outcome measure of participation. The PART-O is a 17-item self-reported or proxy-reported questionnaire developed specifically for use in persons with TBI. It is administered at every follow-up data collection point for the TBIMS NDB , 29. TheThe FIM is an 18-item standardized measure of self-care, mobility, and cognition, specifically examining level of assistance required in basic activities of daily living , 33. It p < 0.001). Therefore, these variables are reported as medians and interquartile ranges. Continuous demographic data were compared statistically using Mann-Whitney U test. Racial and gender compositions were compared using chi-squared tests of association. Cross-sectional analyses comparing the two rehabilitation groups on subscale PART-O scores  at each follow-up year were conducted with univariate analysis of covariance with the covariates of age, FIM Motor, and FIM Cognitive scores at time of follow-up. We also explored the relationship of functional status and participation at each follow-up where FIM Motor and Cognitive subscale scores were recoded into ordinal variables with three levels of performance. To better understand the relationship between functional independence and participation, we binned FIM Motor and Cognitive subscale scores into three levels and evaluated median PART-O scores at each FIM bin across subscales and follow-up years. The three FIM bins are based on categories of independence that reflect the underlying individual item rating definitions: Total Assistance Needed , Minimal/Moderate Assistance , and Complete Independence .Demographic data for the early and late recovery groups were analyzed separately. A Shapiro-Wilk test revealed that age, years of education, and injury severity measures  were not normally distributed (p < 0.05 cutoff) for each of the three PART-O subscales  as dependent variables. All three models began with the same fixed predictors and variances/covariances of random terms. Fixed terms consisted of our primary variable of interest, Command-Follow group (command-following recovered during rehabilitation [RCF] vs. command-following not recovered during rehabilitation [nRCF]), as well as year of follow-up (linear and quadratic function), age at injury, gender, length of stay in inpatient rehabilitation, and length of stay in acute care facility. Scores on the FIM Motor and FIM Cognitive subscales were included as time-varying covariates. Two-way interactions of group by year of follow-up, group by age at injury, and age at injury by year of follow-up as well as a three-way interaction of group by age at injury by year of follow-up were also included. The initial random terms were subject intercept, subject's linear term for year of follow-up, and their correlation. If the interaction between a fixed predictor and year of follow-up was significant, that would indicate that the linear trajectory of participation varied with the predictor variable. If a predictor term did not have a significant interaction with year of follow-up, but did have a significant main effect, then that would indicate that the predictor had the same influence on participation regardless of when it was measured, but that it did not predict the trajectory of participation over time. The backward elimination approach was a limited one intended primarily to pretest and remove, if non-significant, higher order interactions, quadratic terms, covariate terms, and random effects. Non-significant terms with the highest p value were systematically removed in a stepwise manner until only significant terms remained. By convention, non-significant lower order terms subsumed within significant higher order terms are permitted to remain in the model. Percent of dependent variable variance accounted for by fixed and random predictor terms were computed. Model residuals from fixed and random term predicted values were checked graphically for adherence to assumptions of normality and homoscedasticity, and to assess model fit. Only participants with at least two time points with non-missing dependent variable scores qualified for inclusion in any of the longitudinal analyses.Longitudinal analyses were conducted as separate mixed-effects backward elimination models  rather than those who did and did not regain command-following during inpatient rehabilitation. Analyses were conducted using SAS, version 9.4, and SPSS, version 24.n = 229) and nRCF (n = 104) groups. The RCF group had a shorter length of stay in acute care (p < 0.001). In addition, the RCF group was less disabled on rehabilitation admission and discharge, as measured by the DRS , and more independent at time of rehabilitation discharge , compared to the nRCF group. Demographic and injury characteristics for the groups determined by presence, or not, of command-following at rehabilitation discharge, are presented in As the primary measure of this study was introduced into the database in 2007, only subjects with an injury date of 2005 or later were included as they could complete the PART-O measure at the 2nd and 5th year follow-up. Of 12,343 participants with a 2005 injury date or later in the TBIMS-NDB , 11,358 regained command-following before rehabilitation admission and were excluded. After further controlling for DRS motor scores on admission to rehabilitation and number of completed follow-up visits, 333 subjects met inclusion criteria for this study . DemograCross-sectional analyses comparing PART-O subscale performance at each year showed that, although descriptively the RCF group had higher levels of participation, there were no significant differences in levels of participation between groups in any subscale at any year despite moderate effect sizes . ParticiCategorized performance on the FIM Motor and FIM Cognitive subscales was examined in relation to performance on the PART-O subscales and revealed that participation levels remained low even among subjects who reached ceiling levels of the FIM. In fact, the highest levels of participation across subscales  were attained by fewer than 10 persons across domains and follow-up years. These data were plotted as box and whisker plots in Contrary to our hypothesis, there were no between-group longitudinal differences on any of the participation subscales. The FIM Motor subscale was the only variable independently positively associated with participation across subscales.p < 0.0001 and p = 0.0003 respectively, p = 0.0003, β = −0.006) with older individuals having less productivity, as well as a positive main effect of year of follow-up  indicating that productivity improved over time. The random terms of subject intercept and subject's linear term for year of follow-up were also retained (uncorrelated). All the retained fixed effects in the model accounted for 33% of the variance in PART-O productivity (random and fixed together accounted for 81%).In the backward elimination model for the PART-O Productivity subscale, both motor and cognitive FIM scores at follow-up predicted level of productivity . The percent variance of social participation accounted for by all of the retained fixed effects in the model was 32.189% (random and fixed accounted for 88%).Follow-up motor and cognitive FIM scores  and an interaction between age at injury and year of follow-up (p = 0.041), whereby the trajectory of increase in follow-up years was sharper for younger participants than for older participants. The random terms of subject intercept and subject's linear term for year of follow-up were also retained (uncorrelated). The percent variance of the PART-O Out and About subscale accounted for by all the retained fixed effects in the model was 42.764% (random and fixed together accounted for 83%).On the Out and About subscale, higher scores on the follow-up FIM Motor subscale were associated with higher levels of participation  there were no significant effects of interest involving group except for an interaction between group and age for the Out and About subscale and a complex three-way interaction of group, age, and linear time for the Productivity subscale .For all analyses, residuals from values predicted by the fixed effects as well as from values predicted by the combined fixed and random effects were reasonably normally distributed in accordance with significance test assumptions and indicated good model fit.We found that persons admitted to inpatient rehabilitation without command-following have profoundly impaired levels of participation years later, even when compared to normative participation levels of individuals currently treated in inpatient rehabilitation . AlthougFunctional independence was the strongest independent contributor to levels of participation. However, even individuals who regained functional independence demonstrated low levels of participation. This is consistent with prior studies showing that although mobility and participation are related, persons who are independent in this domain have low levels of participation . FurtherThe current reimbursement model of health care in the United States focuses on independence in mobility and activities of daily living rather than societal engagement. This focus affects what practitioners prioritize as treatment goals, and therefore may result in failure to address the needs of individuals recovering from a traumatic DoC, especially in post-acute stages. The results of our study suggest that regaining functional independence may be a necessary but insufficient milestone for returning to participatory activities. Rehabilitation aimed at improving participation could be more precisely targeted toward individual's current abilities and the goals deemed valuable for the individual with DoC and their caregiver. For example, clinicians may focus on recovery of basic functions and mobility in some situations and on supported community engagement or independent participation in others. In that latter case, providing access to day-programs, specialized transportation, or supportive social internet sites may be appropriate for higher functioning persons with ongoing cognitive impairments , 37. AltAlthough participation does increase in the 5 years post-injury, this increase is not substantial. This finding may be the result of the constraints of the PART-O as a measurement tool for persons with the most severe injuries. Prior studies reporting functional improvement among persons with a DoC have typically employed the FIM, which was developed to monitor the ability to complete basic activities of daily living in inpatient settings . HoweverIn interpreting and applying the study findings, several limitations are relevant. Only individuals treated at an acute care hospital and transferred directly to acute inpatient rehabilitation for specialized brain injury care were studied. Clinical services may not be provided in a uniform manner, participants may drop out of the study over time, or inpatient rehabilitation may not be offered for some persons who are not following commands. This may lead to a selection bias or confounding in outcome measures and group assignments. Groups differences in demographic characteristics and data on important pre-injury characteristics  were lacking, which may have confounded the analysis , 40. TheIn conclusion, participation remains extremely limited among persons who have experienced a traumatic DoC and are admitted to inpatient rehabilitation with persistent impairments in level of consciousness. This is the case even for persons who regain command-following during rehabilitation and for those who recover functional independence. The incongruity between performance on functional and participatory measures suggests that social or psychological factors as well as the complex integration of behavior, cognition and mobility may be meaningful determinants of levels of participation in the years after severe brain injury.https://www.tbindsc.org/Researchers.aspx).The datasets generated for this study can be found in the Traumatic Brain Injury Model Systems National Data and Statistical Center (The studies involving human participants were reviewed and approved by Partners IRB. The patients/participants provided their written informed consent to participate in this study.YB, KE, CM, JW, JG, FH, and RN-R contributed conception and design of the study. CM organized the database. JL performed the longitudinal statistical analyses. CM, YB, KE, and JL wrote the first draft of the manuscript. All authors contributed to manuscript revision, read 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.


The involvement of microglia in neuroinflammatory responses has been extensively demonstrated. Recent animal studies have shown that exposure to either acute or chronic stress induces robust microglial activation in the brain. In the present study, we investigated the underlying mechanism of brain microglial activation by acute stress.We first looked at the spatial distribution of the noradrenaline (NA)-synthesizing enzyme, DBH (dopamine β-hydroxylase), in comparison with NA receptors—β1, β2, and β3 adrenergic receptors —after which we examined the effects of the β-blocker propranolol and α-blockers prazosin and yohimbine on stress-induced microglial activation. Finally, we compared stress-induced microglial activation between wild-type (WT) mice and double-knockout (DKO) mice lacking β1-AR and β2-AR.The results demonstrated that (1) microglial activation occurred in most studied brain regions, including the hippocampus (HC), thalamus (TM), and hypothalamus (HT); (2) within these three brain regions, the NA-synthesizing enzyme DBH was densely stained in the neuronal fibers; (3) β1-AR and β2-AR, but not β3-AR, are detected in the whole brain, and β1-AR and β2-AR are co-localized with microglial cells, as observed by laser scanning microscopy; (4) β-blocker treatment inhibited microglial activation in terms of morphology and count through the whole brain; α-blockers did not show such effect; (5) unlike WT mice, DKO mice exhibited substantial inhibition of stress-induced microglial activation in the brain.We demonstrate that neurons/microglia may interact with NA via β1-AR and β2-AR. Stress, both physical and psychological, is a risk factor for neurodegenerative disorders such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) , 39, 50,One intriguing feature of microglia is their rapid response to stressful events such as acute , 76 or rIn our previous study, we demonstrated that stress-induced microglial activation occurs within 30 min of exposure to restraint/water immersion stress . This inNA is the best documented neurotransmitter in stress experiments. For instance, NA has been reported to increase in the brain in response to various types of stresses including immobilization, foot shock, tail pinch , 44, 62.Guide for the Care and Use of Laboratory Animals, aiming to minimize the number of animals used and their suffering. Fischer rats , known to be a stress-sensitive strain, were purchased from Japan Laboratory Animals, Inc. . For stress experiments, the animals were restrained with wire nets for 1–4 h. Restraint stress (RS) was started at 10:00 a.m. and ended at 11:00 a.m., 12:00 a.m., and 2:00 p.m.  and were performed in accordance with the National Institutes of Health .m. Fig. . The rattm1Bkk Adrb2tm1Bkk/J; stock no. 003810) for the Adrb1 and Adrb2 mice were obtained from the Jackson Laboratory and genotypes were confirmed as described by the Jackson Laboratory. Briefly, two separate PCR reactions were set up for each genomic DNA sample, one for adrb1 and one for Adrb2. Both PCR reactions were performed at 95 °C for 2 min, , 35 cycles, 72 °C for 5 min, hold at 4 °C. Primers for Adrb1 include WT forward (5′-CTG ATC TGG TCA TGG GAT TG-3′), WT reverse (5′-GCG ATG ACA CAC AGG GTC T-3′), Mutant forward (5′-CGC TGT CCA CAG TGG TTG T-3′) Mutant reverse (5′-TGG CTA CCC GTG ATA TTG CT-3′), Primers for Adrb2 include common reverse (5′-CCG GGA ATA GAC AAA GAC CA-3′), WT forward (5′-ACC AAG AAT AAG GCC CGA GT-3′), and Mutant forward 5′-CAC GAG ACT AGT GAG ACG TG-3′).Homozygous null mice , DBH (1:200), β1-AR (1:200), β2-AR (1:200), and β3-AR (1:200).E. coli transfer RNA, 600 mM NaCl, and 1 mM ethylenediaminetetraacetic acid [EDTA]). Digoxigenin- (DIG-) labeled sense and antisense RNA probes were synthesized from the template cDNA (tyrosine hydroxylase (TH), β1-AR, β2-AR, and β3-AR), which was subcloned into pGEM-T Easy Vector  using a DIG RNA labeling kit . SP6 RNA polymerase was used for antisense probe labeling with SphI (restriction enzyme) cut plasmid, whereas T7 RNA polymerase was used for sense probe labeling with SpeI (restriction enzyme) cut plasmid.ISH was performed as previously described, with minor modifications , 59. TisTotal RNA was isolated from the brain tissues using the RNA extraction buffer ISOGEN . cDNA was synthesized from 2 μg of total RNA using SuperScript III First-Strand Synthesis System  according to the manufacturer’s instructions. PCR amplification was performed with Taq PCR polymerase , and the amplified products were dissolved by agarose gel electrophoresis. The sequence of primers used for PCR is as follows: rat GAPDH: 5′-CCT TCA TTG ACC TCA ACT ACA TGG T-3′ and 5′-TCA TTG TCA TAC CAG GAA ATG AGC-3′; rat β1-AR: 5′-CAT CGT GGT GGG TAA CGT GCG G-3′ and 5′-AAA TCG CAG CAC TTG GGG TC-3′; rat β2-AR: 5′-ACC TCC TTT TTG CCT ATC CA-3′ and 5′-TAG GTT TTC GAA GAA GAC CT-3′; rat β3-AR: 5′-TCC CCT CCT TGT GAT GCT-3′ and 5′-AAC GGA CGC GCA CCT TCA-3′. PCR assays comprised an initial 10 min, 94 °C step to activate Taq polymerase, followed by 36 cycles of denaturation at 94 °C for 10 s, annealing at 55 °C for 10 s, and extension at 72 °C for 25 s.In order to quantify immunohistochemical signals and ISH, the mean optical densities (ODs), defined as the average of the ODs within the target area, were measured from each section using the image analysis software WinROOF  .The Iba1-immunoreactive (Iba1-ir) microglial cells of the hippocampus (HC) , the thalamus (TM), and the HT were counted in a 200 × 200 μm square using WinROOF. For the cell size measurement, all pixels with gray level values below the threshold value were treated as belonging to cell image, and other pixels were treated as background. The appropriate threshold value was determined as the level at which the binary overlay completely covered the entire cell body and processes. Cell surface area was measured using binary images of cells, microglia and astrocyte, with image analysis software (WinROOF). To further evaluate the morphological changes, we performed Sholl analysis for microglial cells as well as astrocytes . BrieflyBlood was collected into a tube containing EDTA. After 30 min of centrifugation, the plasma samples were stored at − 80 °C until used for the measurements. Corticosterone levels were measured using an ELISA kit .t test and one-way and two-way analysis of variance (ANOVA). ANOVA was followed by Bonferroni’s test for multiple comparisons. A level of p < 0.05 was considered statistically significant. All results were obtained from four rats and experiments under each condition.Results are presented as the mean ± standard error of the mean (SEM). All statistical analyses were performed with SPSS software . Statistical significance was determined by Student’s First, the stress levels of Fischer rats exposed to acute RS were monitored via the quantification of plasma corticosterone, following the stress procedure Fig. .n = 4, p < 0.05) and to 554.7 ± 88.8 ng/mL at 2 h RS , slightly decreasing to 253.9 ± 28.7 ng/mL at 4 h RS   = 22.479, p < 0.001; one-way ANOVA), suggesting that 1–4 h exposure to acute RS effectively induces stress responses in Fischer rats.Plasma corticosterone levels significantly increased from 24.0 ± 7.9 ng/mL (under control conditions) to 278.7 ± 49.3 ng/mL at 1 h RS (05) Fig. . Consistn = 4, F = 17.355, p < 0.001; one-way ANOVA), HC  = 3.962, p < 0.05; one-way ANOVA), and TM  = 3.561, p < 0.05; one-way ANOVA)  = 4.079, p < 0.05; one-way ANOVA), but not in the HT  = 3.066, p = 0.069; one-way ANOVA) or HC  = 3.066, p = 0.069; one-way ANOVA)  Fig. . This moVA) Fig. . This inVA) Fig. . With reVA) Fig. . These rMicroglia possess ARs , 61, sugIHC revealed that DBH-ir fibers extended to the HT, TM, and HC Fig. . DBH-ir Double IHC, using OX-42 and DBH, revealed that the fibers of DBH-ir neurons were extended, surrounding microglial cells in the HC, TM, and HT (data not shown). Furthermore, confocal immunofluorescence revealed that, in the HC, OX-42-ir microglia were meticulously surrounded by DBH-ir fibers Fig. . This fin = 4, F = 21.236, p < 0.001; one-way ANOVA)  = 1.126, p = 0.377; one-way ANOVA)  Fig. . In contVA) Fig. . These rAlthough both β-ARs  and α-ARs (α1-AR and α2-AR) are expressed in cultured microglia, the former has been well demonstrated to induce c-AMP elevation , 47, 61.RT-PCR analysis showed a clear band of β1-AR and β2-AR in each brain region, including the HC, TM, and HT. However, β3-AR was not detected in any brain region Fig. . ISH shoLaser scanning microscopy showed that no immunoreactive β3-AR cells were co-localized with microglia Fig.  or astron = 4, F = 27.401, p < 0.001; two-way ANOVA), and no significant effect of drug treatment was observed  = 0.575, p = 0.456; two-way ANOVA)  was administered intraperitoneally 1 h before each stress procedure Fig.  72]. Co. Co72]. VA) Fig. .Fig. 5an = 4, F = 193.082, p < 0.001; two-way ANOVA). Propranolol significantly suppressed the microglial density at CTRL , 1 h RS , 2 h RS , and 4 h RS , compared with saline-treated rats  = 374.025, p = 0.001; two-way ANOVA) in that the HT of propranolol-treated rats had a significantly lower microglial count at CTRL , 1 h RS , 2 h RS , and 4 h RS  than that of saline-treated rats  = 0.060, p = 0.809; two-way ANOVA) or cell count  = 0.053, p = 0.819; two-way ANOVA)  Fig. Fig. 6A n = 4, F = 18.000, p < 0.001; one-way ANOVA). However, in propranolol-treated rats, there was no significant change between control and stressed conditions in propranolol-treated rats based on Sholl analysis  = 0.425, p = 0.739; one-way ANOVA)  Fig. . In factVA) Fig. . The decn = 4, F = 0.284, p = 0.836; one-way ANOVA) or propranolol  = 0.393, p = 0.760; one-way ANOVA) on the intersections of GFAP-ir astrocytes , 1 h RS , 2 h RS , and 4 h RS , as compared to saline-treated rats   and the n = 4, F = 1.311, p = 0.286; two-way ANOVA)  = 22.303, p < 0.001; two-way ANOVA)  Fig. . HoweverVA) Fig. .n = 4, F = 10.447, p < 0.001; two-way ANOVA), and the morphological activation of saline-, prazosin-, and yohimbine-treated rats differed significantly  = 7.638, p < 0.001; two-way ANOVA). In prazosin-treated rats, microglial activation occurred in a similar pattern to that in saline-treated rats based on cell size and dendrite intersections  = 4.138, p < 0.05; two-way ANOVA), differing significantly among groups  = 24.203, p < 0.001; two-way ANOVA)  Fig. . These rLastly, we compared acute stress-induced microglial activation between WT and DKO mice lacking β1-AR and β2-AR Fig. .Fig. 11n = 4, F = 20.382, p < 0.001; two-way ANOVA), with no significant difference between WT and DKO mice  = 2.670, p = 0.120; two-way ANOVA)  Fig. .n = 4, p < 0.01), HC , and TM   = 221.618, p < 0.001), TM  = 108.529, p < 0.001), and HC  = 108.315, p < 0.001)  Fig. . In cont01) Fig. . Two-way01) Fig. .Fig. 12n = 4, p < 0.01), TM , and HC   Fig. .The main finding of the present study was that microglial activation, as represented by enlarged Iba1-ir cell surface areas in the HT, HC, and TM, was significantly inhibited by pretreatment with the β-blocker propranolol. This finding was further confirmed by impaired, stress-induced, microglial activation in DKO mice, which contrasted sharply with that of WT mice.Regarding the mechanism of neuronal–glial interaction, we demonstrated that fibers immunoreactive to DBH extend into wide brain regions, including the HC, TM, and HT. This result is consistent with those of previous studies which demonstrated extensive branched axons providing the main source of NA throughout the brain including neocortex, amygdala, cerebellum and spinal cord , 40. In In the present study, we investigated the role of β-ARs using the β-blocker propranolol prior to acute RS and observed that microglial activation triggered by acute RS was substantially inhibited in the HT, HC, and TM. In addition, pretreatment with propranolol significantly decreased the number of Iba1-ir microglial cells in those regions. These findings are consistent with those of previous studies demonstrating that propranolol inhibits microglial activation following various stresses, such as social disruption stress  and inesOn the other hand, and against our expectations, microglia were further activated by pretreatment with the α2-AR blocker yohimbine. This effect was stronger than that of the α1-AR blocker prazosin. In this study, it was also found that the levels of plasma corticosterone were elevated in animals treated with those α-AR blockers, which supports the fact that the hypothalamic pituitary adrenal (HPA) axis may be activated by pretreatment with α-AR blockers. In fact, this finding is consistent with those of previous studies showing upregulated corticosterone levels in prazosin- or yohimbine-treated rats , 55. ImpAlthough the involvement of α-ARs in stress-induced microglial activation cannot be excluded, the effect of propranolol, a β1-AR and β2-AR blocker, was more predominant than those of blockers for α1-AR and α2-AR. Therefore, we used DKO mice lacking β1-AR and β2-AR in order to study the functional involvement of β1-AR and β2-AR in microglial activation. Microglial activation was first confirmed in WT mice exposed to 2 h RS. IHC demonstrated that, in DKO mice, the intensities of CD11b immunoreactivities, as a marker of morphological microglial activation in mice, were significantly suppressed in the HT, HC, and TM following acute RS. The level of corticosterone was relatively decreased in DKO mice, as compared to WT mice. Since corticosterone limits microglial activation , it is uIn several studies, the anti-inflammatory effects of NA have been reported, which contradicts the results of our study. For instance, in cultured microglia, NA and isoproterenol, a β-AR agonist, inhibit proinflammatory markers, such as IL-1β, IL-6, and iNOS mRNA, following LPS treatment (1.0 μg/mL), through the inhibition of NF-κB translocation . In addiMicroglia play distinctive roles depending on their morphologies. Under resting conditions, microglia with ramified, long, thin processes play constructive roles, such as debris clearance, neuronal support, and synaptic monitoring and remodeling , 15. In In the present study, we demonstrated that neurons/microglia may interact with NA throughout the brain via β1-AR and β2-AR. NA may therefore be one of the neurotransmitters regulating microglial activation in the brain. β-blockers may effectively treat neurological disorders associated with microglial activation.


Selection criteria were met by 50 primary sources reporting the effects of 79 treatments. Experimental conditions were grouped into categories targeting mechanisms mediated by nitric oxide (NO), prostanoids, purines, potassium, amongst others. Blockade of neuronal NO synthase was found to have the largest effect of inhibiting any individual target, reducing the neurovascular response by 64% (average of 11 studies). Inhibition of multiple targets in combination with nNOS blockade had no further effect. This analysis points to the existence of an unknown signalling mechanism accounting for approximately one third of the neurovascular response.The mechanisms of neurovascular coupling contribute to ensuring brain energy supply is sufficient to meet demand. Despite significant research interest, the mechanisms underlying increases in regional blood flow that follow heightened neuronal activity are not completely understood. This article presents a systematic review and analysis of published data reporting the effects of pharmacological or genetic blockade of all hypothesised signalling pathways of neurovascular coupling. Our primary outcome measure was the percent reduction of the neurovascular response assessed using  CO2) are ensured by several mechanisms controlling cerebral blood flow. Control of global cerebral circulation is provided by metabolic factors, cerebral autoregulation, and autonomic mechanisms  functional MRI (fMRI) imaging . Regiona2The physiological significance of the neurovascular response is predicated on the fact that neuronal activity requires copious amounts of energy substrates. Oxygen and glucose are used to generate ATP, which is required to maintain ion gradients and provide energy to fuel the mechanisms of neurotransmitter release/recycling that make neuronal communication and brain information processing possible . As leveFeedback mechanisms are fundamental for the operation of all physiological systems, including those which maintain energy homeostasis. A metabolic feedback hypothesis of brain blood flow regulation was proposed by −1 demonstrated no effect of excess glucose on the neurovascular response    and neur-5 mmHg)  or hyper-5 mmHg) . In cond2, protons and lactate have powerful effects on cerebral vasculature and are generated proportionately with increased brain tissue metabolism.Could a build up of metabolic waste products be mediating the neurovascular response? CO2 or proton concentrations responding to these stimuli with increases in intracellular [Ca2+] .3+ accumulating as a result of neuronal activity initiate and maintain the neurovascular response  or natural  stimulation, evoked the neurovascular response measured by recording changes in cerebral vessel diameter, cerebral blood flow, BOLD fMRI or local brain tissue PO2 in experimental animals. While it is accepted that not all of these measures are exclusively dependent on changes in blood flow only, all have been used as measures of the neurovascular response. Our secondary measure was percent reduction of the cerebral vessel response studied in brain slice preparations in vitro. Studies of neurovascular responses evoked by application of receptor agonists  outcome measure.Initial searches returned hundreds of relevant studies published since the feedforward hypothesis was proposed. In an attempt to distil critical information, meta-analysis was performed using the selection criteria outlined next. Our primary outcome measure was the percent reduction of the cerebrovascular response induced by increased neuronal activity assessed using ts (e.g. ), high K(+ (e.g. ), directpression ) were nontervals , Fig. 3.in vivo and 36 experimental conditions in vitro. Upon aggregation, blockade of neuronal NOS (nNOS) was found to have the largest effect of blocking any individual target, reducing the magnitude of the neurovascular response by 64% (average of the results of 11 in vivo studies) (Nω-nitro-l-arginine (L-NNA) attenuated neurovascular response in both the wild type and eNOS-deficient mice  . Attemptstudies) . A potenstudies) . The temstudies) . Howeverent mice  but had ent mice . Furtherstration ). Dependl-arginine was reported to inhibit 95 ± 4% of NOS activity when superfused on the cortical surface of anesthetised rats. However, this treatment was associated with a mere 37 ± 4% inhibition of the neurovascular response in the area  of the response remains after pharmacological blockade of nNOS. This assumes complete blockade, which is likely as genetic nNOS deletion was found to have an effect of similar magnitude as pharmacological intervention . Almost the area . Another2 = 0.63 . It is i2 = 0.63 . In the 2 = 0.63 . One stu3 (l-NNA . This susubjects . In cont2+-dependent production of arachidonic acid and PGs from membrane phospholipids are expressed in astrocytes (2 and PGI2 in particular) are potent vasodilators. Recent evidence has implicated PGE2 as the dominant prostaglandin mediating neurovascular coupling in the rodent cerebral cortex  brain arterioles of rats and mice  had no effect on CO2-induced dilations  of certain products of arachidonic acid metabolism including prostaglandins (PGs) and epoxyeicosatrienoic acids (EETs). All the enzymes required for the Catrocytes , cells wtrocytes . PGs  receptors  and excitatory  adenosine receptors, all sensitive to blockade by caffeine, which is also a weak inhibitor of phosphodiesterase activity, important for the maintenance of vascular tone and reactivity (Released ATP is rapidly broken down to adenosine by the activities of extracellular nucleotidases . Seven sactivity .2+-activated K+ (BK) channels on astroglial end-feet release K+ which in turn activates inward-rectifying K+ channels (KIR) expressed by smooth muscle cells, causing hyperpolarisation and relaxation  resulted in 40% inhibition of the neurovascular response  of neurovascular coupling which accounts for approximately a third of the neurovascular response. There are other precedents in the history of physiology illustrating the fact that the signalling mechanisms underlying activity-dependent changes in blood flow are sometimes difficult to identify. For example, the nature of the mediator(s) responsible for exercise-induced coronary vasodilation and increases in coronary blood flow  remains unclear despite similar research interest and significant effort. The involvement of common pathways was experimentally tested, but prostaglandins and Kood flow . On the ood flow .Considering the importance of maintaining brain energy balance it is likely that there are several parallel signalling pathways evolved to work in concert to shape the neurovascular response and in conditions when one pathway is blocked experimentally, recruitment of the other pathway(s) compensates. However, this raises an obvious question of how the alternative pathways are engaged to maintain  the vascular response when the targeted pathway is no longer functional. Some form of a feedback mechanism which can recruit these redundant pathway(s) when the need arises would be required. Alternatively, if the effects of parallel pathways are not simply additive, then a feedback mechanism may not be needed. It is not inconceivable that some form of negative synergy limits the effects of one signalling pathway when it operates in conjunction with the others in the intact system. Finally, in the opinion of the authors, full understanding of the complex mechanisms underlying the development and maintenance of the neurovascular response may emerge if some of the original ideas of metabolic feedback hypothesis proposed by The authors declare that there is no conflict of interest.


Augmented reality (AR) technologies for the consumer market are nowadays mature for many potential fields of applications. In the healthcare sector, as demonstrated by the increasing number of publications on AR for surgery, medicine, and rehabilitation, there is a great demand for solutions that are able to improve current clinical practice. The aim of this special issue is to offer to engineers, computer scientists, and final users an overview of the potentials of AR technologies in fostering the development of useful applications in the early future and to steer the academic research towards overcoming the technological and human-factor issues still present among the current devices and among the most popular modalities for enriching the visual sensation with computer-generated elements.Sixteen papers were submitted for this special issue. Our distinguished reviewers from respective research fields narrowed the field down to six papers which were finally accepted.In this special issue, the reader can find useful examples of applications in the healthcare domain from doctor-patient communication up to surgery, rehabilitation, and phobia treatments.Even if AR devices and applications are to date mostly devoted to augmenting the sense of sight, and the augmentation of different senses has not yet reached the same widespread diffusion, Z. Qin et al. show us in their work the potential of haptic feedback towards increasing the user's accessibility and allowing an intuitive and natural interaction with computer-generated elements.From a technological standpoint, it is important to outline that, as confirmed in R. Touati et al., video-based tracking can be done through feature detection on the patient with a marker-less tracking approach.Overall, it is often difficult to decide where exactly within the reality-virtuality continuum a specific AR application should be located. This is especially true for medical AR, where a lot of patient-specific data and images are available and sometimes it is almost impossible to clearly define to what extent a digital content shown on a display is real or virtual. In some research works in the healthcare sector, this debate becomes a pure comparison between VR and AR while the final goal of the application is lost. In this special issue, the reader can see that there are many ways by which the real and virtual information can be acquired and merged in a useful way for the user. M. Melero et al. show that, for some applications, the visualization of both VR and AR modalities can be an added value for the patient, while C.-F. Tsai et al. prove that the VR and AR visualization modalities stimulate different physiological reactions.In almost all proposed applications, the AR view is shown on a traditional stand-up monitor. In the case of endoscopic procedures, as in V. Mamone et al., this choice is the best in terms of ergonomics since the users usually see the endoscope images directly in front of them. In principle, in the case of manual procedures, the optimal choice should be the use of a wearable display but, as confirmed by S. Condino et al., there are still perceptual limits to take into account, such as a parallax error and/or a focus rivalry between real and virtual, that can compromise their efficacy in a real scenario.


The androgen receptor (AR) regulates male sexual development. We have now investigated AR homodimerization, hormone-dependent monomerization and nuclear translocation in PC-3 and COS-1 cells, by utilizing mutations associated with the androgen insensitivity syndrome: Pro767Ala, Phe765Leu, Met743Val and Trp742Arg. AR wild type (WT) was expressed as a homodimer in the cytoplasm, while none of these mutants formed homodimers. Unlike AR WT which responded to 1 nM dihydrotestosterone (DHT) to dissociate and translocate into the nucleus, AR Pro767Ala and Phe765Leu mutants remain as the monomer in the cytoplasm. In the crystal structure of the AR LBD homodimer, Pro767 and Phe765 reside closely on a loop that constitutes the dimer interface; their sidechains interact with the Pro767 of the other monomer and with the DHT molecule in the ligand-binding pocket. These observations place Phe765 at a position to facilitate DHT binding to Pro767 and lead to dissociation of the AR homodimer in the cytoplasm. This Pro-Phe Met relay may constitute a structural switch that mediates androgen signaling and is conserved in other steroid hormone receptors. Androgen directly binds AR in the cytoplasm and translocates it into the nucleus, conferring its physiology by activating various genes3.The androgen receptor (AR), a member of the nuclear steroid hormone receptor (NR3C) subfamily, regulates male sexual development4. Polymorphic mutations associated with the androgen insensitivity syndrome (AIS) are observed in this D3L interface of AR molecule4. However, it remains elusive as to how these mutations affect androgen sensitivities of AR through its homodimerization.AR is believed to form a homodimer for binding and activating its target genes. Recently, an X-ray crystal structure of AR LBD homodimer was determined, in which three loops constitute the dimer interface 8, . Similarly, estrogen receptor α (ERα) and hepatocyte nuclear factor 4α (HNF4α) are also reported to form their homodimers through this H10/11 interface10. The D3L interface was first observed in the X-ray crystal structure of the glucocorticoid receptor (GR) homodimer11. The D3L interface resides on an area of opposite surface of the H10/11 interface of the AR molecule10. However, a recent confirmation that the AR LBD utilizes the D3L interface to homodimerize strengthens the notion that this interface may be physiologically relevant4. In addition, the nuclear receptor CAR (NR1I3) has been found to form a homodimer through its D3L interface in the cytoplasm which dissociates upon activation and translocation into the nucleus13. To advance the biology of the D3L interface, we have now investigated whether (how) AIS-related mutations affect AR homodimerization and nuclear translocation in cells.Nuclear non-steroid hormone receptors heterodimerize with retinoid X receptor (RXR) through a well-established interface that includes helices 10 and 11 in their X-ray crystal structures14, Phe765Leu15, Met743Val16 and Trp742Arg15 were further examined by employing various experimental methods including site-directed mutagenesis, co-immunoprecipitation of GFP- and FLAG-tagged proteins ectopically-expressed in cells, 2-dementional electrophoresis, cell-based reporter assays and confocal analysis of GFP tagged proteins in cells. Pro625 of GR corresponds to Pro767 of AR was previously investigated as a critical residue in GR homodimerization11. In this study, we demonstrated that AR WT formed a homodimer that dissociated and nuclear translocated in response to dihydrotestosterone (DHT) in COS-1 cells. With this background, AR mutants  were expressed in COS-1 cells to examine their roles in AR homodimerization. In addition, these residues are mapped to AR molecules based on its X-ray crystal structure to understand structure-function relationships. This mapping was then extended to other steroid hormone receptors. Here we present experimental observations in support of the hypothesis that the AR homodimer responds to androgen in the cytoplasm by dissociation and nuclear translocation, and, additionally, that an intramolecular Pro-Phe-Met relay regulates the androgen signal.The AIS-associated mutations, Pro767Ala13.Dexamethasone (DEX), 5α-dihydrotestosteron (DHT), R1881 and HRP-conjugated anti-FLAG M2 were purchased from Sigma-Aldrich . HRP conjugated antibodies against rabbit IgG and SP-1 (sc-059) and histone H1 (FL-219) antibodies were purchased from Santa Cruz Biotechnology . The antibody against HSP90 was purchased from Cell Signaling . The antibody against GFP (HRP-conjugated) was purchased from Abcam . An enhanced chemiluminescence reagent WesternBrighTM was from GE Healthcare . The α−tubulin antibody (21445) was obtained from Cell Signaling Technology. Anti-GFP agarose beads for immunoprecipitation was provided by the Protein Expression Core Facility in NIEHSPSA promoter (region -430 to +12) were inserted into XhoI and BglII sites of pGL3 plasmid (Promega). 3xGRE-TK-pGL3, GR-pCR3.1, pEGFP-GR and pECFP-GR were constructed previously17. Using a PrimeSTAR Max DNA Polymerase (Clontech) and appropriately mutated primers, we constructed all the mutants and confirmed them by nucleotide sequencing. Plasmids were transfected into cultured cells with Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions.Human AR cDNA (NM_000044.6) was cloned into pEGFP-c1, pECFP-c1, pEYFP-c1  and pcDNA3.1 . A DNA fragment of FLAG tag was placed at the 5′ end of AR in pcDNA3.1. human 2). The cell suspensions were centrifuged at 2000x g for 5 min. Resulted supernatants were collected as crude cytosolic fraction which was centrifuged at 20000x g for 30 min and its supernatant was collected as cytosolic fraction. Cell pellets were resuspended in 1 mL of wash buffer (10 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 0.075% NP-40), centrifuged at 2000 × g for 5 min. Resulted pellets were resuspended in 400 μL of nucleus extraction buffer (10 mM HEPES (pH 7.5), 100 mM KCl, 3 mM MgCl2, 0.1 mM EDTA), incubated for 30 min on ice and centrifuged at 20000 × g for 30 min, supernatants from which the nucleus fraction was collected. A portion of nuclear fraction was resuspended in 400 μL of DNA-binding protein extraction buffer (10 mM HEPES (pH 7.5), 400 mM NaCl, 100 mM KCl, 3 mM MgCl2, 0.1 mM EDTA), incubated for 30 min on ice and centrifuged at 20000 × g for 30 min to obtain the DNA-binding protein fraction.Cells were cultured on 100-mm culture dishes to about 70% confluency with serum free DMEM high glucose medium with or without 10 nM DHT for 24 hr. Whole cell extracts were prepared by centrifuging cell homogenates in lysis buffer containing Tris-HCl (pH 8.0), 0.5 mM EDTA, 100 mM NaCl, 1% Triton X-100 and 10% glycerol. For fractionation, cells were rinsed with PBS and suspended in 1 mL of cytoplasm extraction buffer (10 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgClTwenty-four hours after seeding, COS-1 cells were co-transfected with expression plasmids for GFP- and FLAG-tagged ARs and cultured in serum free DMEM media for additional 24 hours. These cells were treated with 0.1%DMSO or DHT for 1 hour in serum free media. Then, cells were lysed with the lysis buffer containing Tris-HCl (pH 8.0), 0.5 mM EDTA, 100 mM NaCl, 1% Triton X-100 and 10% glycerol. Cell lysate was incubated with an anti-GFP agarose at 4 °C for 12 h and, subsequently, washed with lysis buffer for three times, and eluted with SDS-PAGE sample buffer and subjected to Western blot analysis. In addition, FLAG-ARs and GFP were co-expressed as a control group. We confirmed that FLAG-ARs wasn’t precipitated by GFP co-expressed samples in all experiments.Western blot samples were subjected to SDS-PAGE on 10% polyacrylamide gels following transblot to an Immobilon-P Transfer Membrane . Membranes were blocked for 1 h with 5% non-fat dried milk in 0.05% Tween 20 containing Tris-buffered saline (TBST). The membranes were incubated for 16 h with the 1000-fold diluted primary antibody in TBST and incubated for 1 h with 10000-fold diluted HRP-conjugated secondary antibody. After incubating with secondary antibody, proteins were detected by an enhanced chemiluminescence reagent Western Bright ECL . PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was run as a molecular weight marker. Uncropped images of blots are shown in Supplemental Figs 12. Cells were homogenized in buffer containing 20 mM HEPES (pH 7.6), 150 mM NaCl, 15% Glycerol, 1% Triton X-100 and 1 mM EDTA. Cell lysates were subjected to electrophoresis on a 4–16% gradient Native gel (Invitrogen). The gel was placed onto 10% SDS-PAGE gel and western blot analysis was carried out.Blue native/SDS-PAGE was performed as previously describedPC-3 cells were transfected with reporter plasmid (pGL3), phRL-TK control plasmid (Promega) and pcDNA3.1 expression plasmid for ARs or GRs with Lipofectamine 2000 and treated with ligands in the serum free media for 24 h. Cell lysate was subjected to Dual Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity.5 cells/well) were grown on 35 mm glass coverslips dishes  and transfected with a given expression plasmid for GFP, CFP or YFP-labeled proteins. Twenty-four hours after transfection, media was changed to serum free DMEM media and cells were treated with DEX or DHT for 1 h. Cells were fixed with methanol and fluorescence was visualized using a Zeiss LSM 710 confocal microscope PC-3 or Huh-7 cells  100-ps belly dynamics runs with fixed peptide, (2) minimization, (3) low temperature constant pressure dynamics at fixed protein to assure a reasonable starting density, (4) minimization, (5) step-wise slow heating molecular dynamics at constant volume, and (6) constant volume unconstrained molecular dynamics for 10 ns. All final unconstrained trajectories were calculated at 300 K under constant pressure  using the PMEMD module of Amber.18 to accommodate long range interactions19. The parameters were taken from the FF14SB force field for proteins and the DHT parameters were from the gaff2 force field in the AMBER.18 package. Partial charges on DHT were calculated using Gaussian.0920 at the B3LYP/6–31 g level. Ligand binding free energies were calculated with the help of the MMPBSA module of AMBER.18 using default parameters and equally spaced 200 configurations selected from the last 200 ns of the molecular dynamics trajectory. MD trajectories of dimers  were extended for 1 microsecond and averaged Cα deviations were calculated from 100 configurations selected at 1 ns intervals from the last 100 ns of these extended trajectories. Several final snapshots were created using the program VMD-1.9.121.Using molecular dynamics (MD), solution structures of the androgen receptor, its dimer form, and several mutants were generated. The initial structure of androgen receptor for simulations was taken from the homodimeric X-ray crystal structure from the PDB code 5JJM. Desired mutations  were introduced using the program CootStatistical analysis was performed using GraphPad Prism . All experiment was repeated at least 2 times to confirm reproducibility. Data are provided as the means ± S.D. The significance of difference between control and treated groups was assessed using ANOVA followed by Dunnett’s test for data from multiple groups. The Tukey–Kramer test or Bonferroni’s correction was used to compare multiple groups.et al., reposted a crystal structure of DHT-bound AR LBD homodimer structure and performed FRET assays to show ectopically expressed AR LBD formed this homodimer in the nucleus of Hep3B cells4. Pro767 resides in the AR homodimer interface and these residues of two monomers interact  simulations were performed on AR homodimer with and without DHT binding Fig. . In the Mutations Trp742Arg, Met743Val and Phe765Leu have been reported to be associated with AIS Fig. . These T11. The Pro625Ala mutation was previously examined as a regulator of GR homodimerization in the 1990’s. Recombinant GR and its mutant proteins were analyzed in vitro by density gradient ultracentrifugation25. In addition to Pro767, Phe765 and Met743 of AR are superimposed with the conserved residues, Phe623 and Met601, respectively, in GR  in the GR Fig. . As Pro7 GR Fig. . To exam GR Fig. . Moreove GR Fig. .Figure 626. Among these former mutations, Pro767Ala and Phe765Leu are intriguing since both co-localize in a loop that constitutes the homodimer interface. Pro767 directs its sidechain towards a surface of the other monomer, while the sidechain of Phe765 interacts with the DHT molecule in the LBP. Phe765 may be a molecular switch endowing DHT binding to Pro767, the regulator of AR homodimerization. Our present works showed that AR is expressed as a homodimer in the cytoplasm and that DHT binding induces translocation of the AR monomer into the nucleus. The homodimerization and nuclear translocation events appear to link the DHT response in AR activation.Ligand-bound homodimerization in the nucleus has been a major focus of AR research (1). On the other hand, AR in the cytoplasm was not well-studied. A recent structure of AR LBD homodimer revealed that AIS-associated mutations are found on a surface of the homodimer interface (D3L), although many of them are located within the ligand-binding pocket (LBP)The reported AR LBD homodimer crystal structure contains the DHT molecule (4). In FRET assays, ectopically- expressed AR LBD molecules interacted through Pro767 in the nucleus (4). Consistent with a general knowledge, this AR homodimer was considered as a DHT-liganded homodimer that binds DNA. However, the FRET assays of AR lacking the DBD could not detect a DNA-bound form of AR homodimer. On the other hand, our present study utilized a full-length AR molecule that is properly retained in the cytoplasm, translocated to the nucleus and activated genes in response to DHT in cells. This reported AR LBD homodimer is partially restrained through the D3L interface by Van der Waals interactions between the Pro767 residues of the two AR monomers. In fact, weakening of this interaction by replacing Pro with Ala disables the AR to homodimerization. On the other hand, the AR Pro767Ala mutant was co-immunoprecipitated with AR WT co-expressed in COS-1 cells. Thus, a subtle change of free binding energy associated with a conformational alteration of Pro767 may be sufficient to support dimerization. MD simulation was performed to display molecular motions of AR WT homodimer with or without DHT and of AR Pro767Ala mutant with or without DHT , which is reported to form homodimer in solution without DNA binding motif33, utilizes different interface from ER or HNF4α36. Thus ligand-induced monomerization maybe a regulatory process common to steroid hormone receptors. Our co-immunoprecipitation experiments also suggest that, once DHT-liganded AR binds DNA, it does so as a homodimer. Whether this AR homodimer utilizes the D3L interface remains as an important subject for future investigations. However, it is not unreasonable to think that the AR homodimer which dissociates by DHT in the cytoplasm is stabilized by DNA binding with transcription coregulators, in the nucleus.Unlike the AR LBD that has been suggested to form a homodimer in response to DHT in the nucleus37. We demonstrated that nuclear translocation of AR depends on its ligand-induced homodimerization in the cytoplasm. Similarly, cytoplasmic GR homodimer was reported to dissociate in response to by a plant delivered ligand compound A38.Homodimerization/monomer conversion appears linked to proper nuclear translocation of AR. Among ARs examined, only AR WT underwent dissociation and translocation. All four mutants  neither formed a homodimer nor properly translocated; AR Met743Val spontaneously translocated while the other three mutants remained in the cytoplasm at 1 nM DHT. In support of this proposed link, AR Pro767Ala co-expressed with AR WT formed a heterodimer and dissociated at 1 nM DHT, translocating into the nucleus. Thus, heterodimerization with AR WT enabled AR Pro767Ala to translocate in the nucleus in response to ligand and induce transcription of its target reporter gene. Similar to AR, GR was reported that it mutant restored nuclear translocation capability by heterodimerizing with wild type MRThus, ligand-free homodimerization appears to be prerequisite for AR translocation. Heterodimerization with AR wild-type restored AR P767A’s lost-functions. Thus, heterozygous mutants might not be defective in AIS patient. However, AR is encoded by a single copy X-chromosomal gene, suggesting heterozygous AR mutants shouldn’t exist in male sex characterized AIS patient.13.GR also underwent ligand-free homodimerization in the cytoplasm and DEX response monomerization and subsequent nuclear translocation. Consistent with AR and GR, the nuclear receptor CAR formed a homodimer via the D3L interface in the cytoplasm, dissociation of the homodimer and subsequent translocation to the nucleus11. Now, our present study has found that the corresponding mutations  disabled GR from responding to DEX as observed with AR mutants to DHT. Moreover, the Pro-Phe-Met axis is conserved in mineralocorticoid receptor (MR) and PR (Supplemental Fig. GR conserves these residues corresponding to Pro767, Phe765 and Met743 of AR as Pro625, Phe623 and Met601 Fig. . Pro625 Supplemental Figure1-8Supplemental movie