The association between improved nutritional status and rehabilitation dose was assessed by a logistic regression analysis. Data were available for 145 patients . Daily rehabilitation dose was 109.5 (median 94.6–116.2) min and the MNA-SF score at admission was 5 (median 4–6). Nutritional status was improved in 97 patients and not improved in 48. Logistic regression analysis showed the following factors to be independently associated with nutritional status at discharge: Functional Independence Measure score , energy intake (OR 1.002 CI 1.000–1.004), daily rehabilitation dose , and length of hospital stay . The daily rehabilitation dose in malnourished patients with hip fracture may positively impact nutritional status at discharge.The object of this study is to determine the impact of the rehabilitation dose on the nutritional status at discharge from a convalescent rehabilitation ward in malnourished patients with hip fracture. This retrospective case-control study involved malnourished patients with hip fracture aged 65 years or older who had been admitted to a convalescent rehabilitation ward and whose data were registered in the Japan Rehabilitation Nutrition Database. The primary outcome was nutritional status at discharge. Patients were classified according to whether nutritional status was improved or not at discharge, according to the Mini Nutritional Assessment-Short Form The rapidly aging population in Japan has been accompanied by a steady increase in the number of hip fractures in the elderly ,2. TheseThe appropriate rehabilitation dose that takes nutritional status into account in patients with hip fracture is not known. Previous studies have reported that improved nutritional status in patients with hip fracture contributes to recovery of the ability to perform ADL at discharge and that an increased rehabilitation dose improves outcomes ,10. HoweTherefore, the purpose of this study was to clarify the effect of rehabilitation dose during convalescent rehabilitation on nutritional status at discharge in malnourished patients with hip fracture.® (MNA-SF), energy intake, dietary form, and route of feeding. Energy intake was evaluated as the mean over the first week after admission. The method for estimating energy intake was not specified when it was registered in the database. Most hospitals in Japan plan menus with commercially available software, incorporating the Standard Tables of Food Composition in Japan—2015 [This retrospective case-control study included patients with hip fracture whose data had been registered in the Japan Rehabilitation Nutrition Database (JRND). This database is managed by the Japanese Association of Rehabilitation Nutrition and was created to disseminate clinical research findings on rehabilitation nutrition worldwide. The database contains data on patients with pneumonia in acute care hospitals and those with stroke or hip fracture treated in convalescent rehabilitation wards. As of 31 March 2018, there were 11 participating centers and 1100 case registrations. Data recorded in the JRND at admission are age, sex, dates of onset and admission, diagnosis, type of surgery, Charlson Comorbidity Index (CCI) score , level opan—2015 . The amopan—2015 . Data reThis study involved 234 patients with hip fracture at nine centers who were admitted to a convalescent rehabilitation ward. Patients younger than 65 years, those with an MNA-SF score of ≥8 at admission, and those with missing data were excluded. Patients who had spent >60 days in an acute care hospital after hip fracture, and those with a convalescent rehabilitation stay of ≥91 days, were also excluded because they were not covered by public medical insurance for acute care or rehabilitation beyond these times.The primary outcome was nutritional status at discharge from a rehabilitation ward. The MNA-SF was used to assess nutritional status ,16,17. TIn the convalescent rehabilitation ward, public medical insurance covers individual rehabilitation for patients with hip fracture provided by physical therapists, occupational therapists, and speech–language–hearing therapists for nine units per day (1 unit = 20 min), 7 days per week, for up to 90 days. The time allocation for each type of rehabilitation can be tailored according to the needs of the individual patient. In this study, the daily rehabilitation dose was defined as the total amount of time spent on physical therapy, occupational therapy, and speech–language–hearing therapy during hospitalization, divided by the length of the hospital stay.t-test was used to examine continuous variables with a normal distribution, and the Mann–Whitney U test was used to examine continuous and ordinal variables that were not normally distributed. The chi-square test was used for nominal variables. Next, the effect of the rehabilitation dose on nutritional status at discharge was assessed by logistic regression analysis. For the dependent variable, the patients were assigned to two groups: one group with no change in MNA-SF status at discharge and another group with improved MNA-SF status at discharge. To account for multicollinearity, we checked whether each variable had a Spearman’s rank correlation coefficient with an absolute value of <0.7. All statistical analyses were performed using IBM SPSS Statistics version 18 . Statistical significance was set at 5%.The items surveyed were age, sex, CCI score, FIM score, MNA-SF score, FILS score, energy intake, daily rehabilitation dose, length of hospital stay, and the number of hospital beds in the convalescent rehabilitation wards at admission. The unpaired n = 6), those with missing data (n = 12), those who were transferred for rehabilitation after spending >60 days in an acute care ward (n = 1), those whose stay in a convalescent rehabilitation ward was >91 days (n = 11), and those with an MNA-SF score of ≥8 at admission (n = 59), data of 145 patients at nine centers were ultimately available for analysis  received a daily rehabilitation dose of 109.49 (median 94.61–116.21) min and had an MNA-SF score of 5 (median 4–6) at admission. There were 97 patients in the improved group and 48 in the not improved group. In the improved group, 88 patients (91%) were deemed to be at risk of malnutrition (MNA-SF score 8–11) and 9 (9%) were considered to have good nutritional status at discharge (MNA-SF score ≥12).p = 0.009), higher median FIM score on admission , higher MNA-SF score on admission , and higher FILS score on admission . This group also had a higher median energy intake , higher median daily rehabilitation dose , and higher median number of hospital beds . However, there was no difference in CCI (p = 0.225) or length of hospital stay (p = 0.696). All variables had a Spearman’s rank correlation coefficient with an absolute value less than 0.7 .The results of the logistic regression analysis are shown in In this study, there were two notable clinical findings regarding nutritional status and appropriate rehabilitation dose. First, in malnourished patients with hip fractures admitted to the convalescent rehabilitation ward, an increased daily rehabilitation dose was associated with improved nutritional status at discharge. Second, the nutritional status of 67% of malnourished patients with hip fracture admitted to the convalescent rehabilitation ward improved while in the hospital.Convalescent rehabilitation wards in Japan aim to prevent patients from becoming permanently bedridden and to allow them to return to home by improving their independence in ADL. Physical therapists, occupational therapists, and speech–language–hearing therapists provide up to 180 min per day of intensive rehabilitation from the first day of hospitalization onward. However, it has been reported that 26.2% of patients with hip fracture admitted to a convalescent rehabilitation ward have insufficient energy intake ,20 and iIn this study, 67% of malnourished patients with hip fracture admitted to the convalescent rehabilitation ward showed improved nutritional status during their hospital stay. This figure is lower than the 91% improvement in nutritional status documented in patients with stroke admitted to a convalescent rehabilitation ward . A possiThis study has several limitations. First, the rehabilitation dose was analyzed as a total quantity and did not examine the distribution of rehabilitation doses in terms of physical therapy, occupational therapy, and speech–language–hearing therapy. Second, the qualitative content of the rehabilitation could not be investigated and will need to be investigated in the future. Third, we have not collected and adjusted data on socioeconomic factors such as education, marriage status, number of family members, and household income that could affect nutritional status. Finally, daily protein intake is not included in the JRND; therefore, relevant survey results are not available. Given that protein intake may be directly related to improved nutritional status, further research in this area is needed.In malnourished patients with hip fracture, admitted to a convalescent rehabilitation ward, increased daily rehabilitation dose improved nutritional status at discharge in 67% of cases. It is essential to ensure a balance between nutritional therapy and rehabilitation dose to improve nutritional status in these patients.


The effects of PM2.5 on oxidative stress and inflammatory response via the Nrf2/NF-κB signaling pathway in mice with or without AR indicating by histological, serum, and protein analyses were examined. PM2.5 administration enhanced allergic inflammatory cell expression in the nasal mucosa through increasing the expression of inflammatory cytokine and reducing the release of Treg cytokine in OVA-induced AR mice, although PM2.5 exposure itself induced neither allergic responses nor damage to nasal and lung tissues. Notably, repeated OVA-immunization markedly impaired the nasal mucosa in the septum region. Moreover, AR with PM2.5 exposure reinforced this impairment in OVA-induced AR mice. Long-term PM2.5 exposure strengthened allergic reactions by inducing the oxidative through malondialdehyde production. The present study also provided evidence, for the first time, that activity of the Nrf2 signaling pathway is inhibited in PM2.5 exposed AR mice. Furthermore, PM2.5 exposure increased the histopathological changes of nasal and lung tissues and related the inflammatory cytokine, and clearly enhanced PM2.5 phagocytosis by alveolar macrophages via activating the NF-κB signaling pathway. These obtained results suggest that AR patients may experience exacerbation of allergic responses in areas with prolonged PM2.5 exposure.Air pollution-related particulate matter (PM) exposure reportedly enhances allergic airway inflammation. Some studies have shown an association between PM exposure and a risk for allergic rhinitis (AR). However, the effect of PM for AR is not fully understood. An AR mouse model was developed by intranasal administration of 100 μg/mouse PM with a less than or equal to 2.5 μm in aerodynamic diameter (PM Acute adiseases ,4.Recently, increasing epidemiologic studies have demonstrated that PM exposure is closely associated with morbidity and mortality due to chronic respiratory diseases . PracticThe prevalence of allergic diseases, including asthma, allergic rhinitis (AR), and food allergies, has risen sharply in recent years . AR is aThe transcription factors involved in the oxidative stress response are nuclear factor erythroid 2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) . The Nrf2.5, there is no evidence to elucidate the clear molecular mechanism of PM2.5 mediated toxicity through the Nrf2/NF-κB signaling pathway. Therefore, the purpose of this study was to evaluate the direct impact of long-term PM2.5 exposure on the oxidative stress and inflammatory response through the Nrf2/NF-κB signaling pathway in an ovalbumin (OVA)-induced AR mouse model. The involvement of ROS-dependent MDA production and SOD activity were also evaluated. PM2.5-induced oxidative stress has been considered as a key molecular mechanism of PM2.5-mediated toxicity [Although many toxicological studies have been conducted to clarify the biological response to PMtoxicity .2.5/OVA group is strongly toxic to AR and can induce oxidative through MDA production. Evidence was also provided, for the first time, that the activity of Nrf2 signaling pathway decreased in PM2.5 exposed AR mice. Furthermore, the PM2.5 exposed group increased pathological changes, related to the inflammatory cytokine, and clearly enhanced PM2.5 phagocytosis by alveolar macrophages.The present study demonstrated that the PM2.5 exposure in AR, the frequency of allergic symptoms such as nasal rubbing and sneezing per groups was measured. The incidence of nasal rubbing and sneezing of mice in the AR group increased significantly compared with the Naive group, and more severe symptoms occurred in the AR/PM2.5 group  group . Therefore, we first investigated the effects of particulates on the alveolar macrophage function. There was no significant difference in the pattern of PM5) group .2.5 in OVA-induced AR, we monitored and tested the temporal accumulation of MDA and SOD production through the Nrf2/HO-1 pathway that was associated with antioxidant effects. The results revealed that the level of MDA significantly increased by AR with PM2.5 exposed group, followed by down-regulation of SOD activity and production of HO-1 in NALF, which subsequently induced the oxidative stress in AR  group increased the prevalence of more significant stained cells in nasal tissue . Mice were housed in a laminar air-conditioned room maintained at 23 ± 2 °C at a relative humidity of 55 ± 10% with a 12 h dark/light cycle. All experiments were controlled in accordance with the care and use of experimental animals and were approved by the Institutional Animal Care and Use Committee of Jeonbuk National University Laboratory Animal Center .2.5  was purchased from Merck, and concentrated and diluted with sterile saline. The PM2.5 was stored at 4 °C. The main polycyclic aromatic hydrocarbons (PAHs) and nitro-substituted PAHs in diesel particulate matter  are shown in PM2.5, and AR/PM2.5. PM2.5 samples were suspended in saline at 2.5 mg/mL . Mice were immunized with an intraperitoneal injection of 50 μg OVA with 1 mg aluminum hydroxide gel  on days 0, 7, and 14. Thereafter, prolonged continuous inflammation was maintained followed by a daily intranasal challenge with OVA diluted in sterile normal saline (400 μg OVA/20 μL per mouse) from days 21 to 27  once a day for a successive period of 35 days during days 7 to 27 . BALF was collected two times by flushing 1 mL of saline into the lung via a tracheal cannula. Lavage fluids were centrifuged at 1000× g for 10 min at 4 °C and the lavage supernatant was separated and stored for further analysis. Inflammatory cells were differentiated into macrophages, eosinophils, lymphocytes, and neutrophils according to standard morphology, and counted over 100 cells at ×200 magnification with a light microscope .After 24 h of the last OVA challenge, the trachea was opened and incised, and then 1 mL saline was flushed into the nasal cavity for collection of NALF. Lavage fluids were centrifuged at 1000× 2.5) in each macrophage was evaluated in BALF and lung tissue with H&E staining as previously described above [Airway macrophages were visualized by light microscopy. The area occupied by black material  was measured at 450 nm in accordance with the protocol provided by the manufacturer.Briefly, under deep ether anesthesia, the blood samples were collected via the retroorbital plexus. Serum samples were separated by centrifugation at 1000× v/v) paraformaldehyde, then the nasal samples were decalcified for 2 days in an ethylenediamine triacetic acid, and the specimens were embedded in paraffin wax.Nasal samples from the incisors to the mucosa of nasal septum were harvested on day 29 for histological analysis. The nasal samples and lung lobes were gently removed and, after 3 days, fixed in 10%  staining for evaluation of the airway inflammation, such as the nasal mucosa and lung tissue. Periodic acid-Schiff (PAS) staining was employed for visualizing the development of goblet cell hyperplasia. Granules in the cytoplasm and a two-lobed nucleus were counted under a microscope. Masson trichrome staining was used to reveal the sub-epithelial deposition of collagen in the lung tissue. Positive trichrome-stained areas were used to assess the degree of the sub-epithelial fibrosis. The nasal mucosa in the posterior part of nasal septum and lung tissue was determined under a light microscope in a blinded manner and observed at a high-power filed (×400).For the immunohistochemistry assay, paraffin sections were deparaffinized, dehydrated, and washed in PBS (10X) with 0.3% Tween-20 buffer (PBST), and aprotein block was applied and incubated for 10 min at room temperature to nonspecific background staining. Slides were microwaved with a citrate anhydrous buffer (pH 6.0) to facilitate antigen retrieval. Subsequently, slides were incubated with a primary mouse anti-rabbit NF-κB  antibody (1:400 dilution) overnight at 4 °C. After removal of primary antibodies, sections were washed and incubated with biotinylated secondary antibody at room temperature for an hour and a half, followed by an avidin-biotin-peroxidase complex . After the excess secondary antibody was removed, sections were washed with PBST and incubated with DAB solution for 1 min . Sections were rinsed in distilled water and dehydrated to terminate the reaction, and the cover was slipped for microscopic examination.The concentrations of Interleukin (IL)-4, IL-5, IL-10, IL-13, IL-17 and TGF-β1 in NALF were measured. In addition, supernatant was obtained for analysis of NF-кB, phosphorylation (p)-NF-кB, and the levels of Nrf2 and HO-1 in NALF were measured by ELISA in accordance with the manufacturer’s instructions . Supernatants were tested for lipid peroxidation by collecting and measuring the concentration of MDA. The activity of Superoxide dismutase (SOD) was analyzed and the OD value was read at a wavelength of 450 nm on a microplate reader. All assays were performed in triplicate. The concentration of each protein was calculated from the standard curve.t-test and ANOVA with Dunnett’s test. Statistical significance was defined at the 95% confidence level (p < 0.05).Results were analyzed using Graph Pad Prism software  and expressed as mean ± SEMs over the number of experiments. Statistical analysis was adjusted to Student’s 2.5 and OVA-induced AR. The present study suggested that PM2.5 presented a toxic effect to OVA-induced AR and induced oxidative stress by the Nrf2/HO-1-mediated signaling pathway. In addition, these results provided evidence that MDA production increased, and decreased the antioxidant enzymes such as SOD and HO-1 after PM2.5 exposure. Interestingly, PM2.5 can activate the NF-κB pathway and induce the inflammatory responses by increasing the inflammatory mediators.In summary, it is very meaningful to clarify the toxicity mechanisms caused by the co-exposure of PM2.5 could aggravate AR by targeting Nrf2/HO-1 and the NF-κB signaling pathway. These findings suggest that PM2.5-induced toxicological effects may contribute to increased oxidative stress and inflammatory response in AR. All these findings will help develop new strategies for the prevention and treatment of diseases associated with PM2.5 exposure.Taken together, these results indicated that PM


ZFHX3 transcription via androgen‐induced AR binding to the androgen‐responsive elements (AREs) of the ZFHX3 promoter. Androgen also upregulated ZFHX3 transcription in vivo, as castration dramatically reduced Zfhx3 mRNA and protein levels in mouse prostates, and ZFHX3 mRNA levels correlated with AR activities in human PCa. Interestingly, the binding of AR to one ARE occurred in the absence of androgen, and the binding repressed ZFHX3 transcription as this repressive binding was interrupted by androgen treatment. The enzalutamide antiandrogen prevented androgen from inducing ZFHX3 transcription and caused excess ZFHX3 protein degradation. In human PCa, ZFHX3 was downregulated and the downregulation correlated with worse patient survival. These findings establish a regulatory relationship between AR and ZFHX3, suggest a role of ZFHX3 in AR function and implicate ZFHX3 loss in the antiandrogen therapies of PCa.Both androgen receptor (AR) and the ZFHX3 transcription factor modulate prostate development. While AR drives prostatic carcinogenesis, ZFHX3 is a tumour suppressor whose loss activates the PI3K/AKT signalling in advanced prostate cancer (PCa). However, it is unknown whether ZFHX3 and AR are functionally related in PCa cells and, if so, how. Here, we report that in AR‐positive LNCaP and C4‐2B PCa cells, androgen upregulates  Interestingly, binding to AR to another ARE of ZFHX3 promoter occurred in the presence of androgen, and the binding repressed ZFHX3 promoter activity. Furthermore, an inhibition of AR signalling by an AR antagonist prevented androgen‐induced ZFHX3 transcription while causing excess degradation of ZFHX3 protein. These findings suggest that ZFHX3 is involved in AR function and implicate ZFHX3 loss in the treatment of PCa.This study examined the regulatory relationship between androgen/AR signalling and ZFHX3 in PCa cells. We demonstrated that the androgen/AR signalling upregulates the transcription of 22.12). For experiments involving the treatment of R1881 , cells were incubated in phenol red‐free medium containing 5% charcoal‐stripped FBS for 24 or 72 h, except the experiment involving a combined treatment of R1881 and enzalutamide (Beyotime), in which complete medium was used.Human PCa cell line LNCaP was purchased from ATCC, and C4‐2B was a gift from Dr. Leland Chung of Cedar Sinai Medical Center. Both cell lines were cultured in RPMI‐1640 medium supplemented with 10% foetal bovine serum  in a humidified incubator (37°C and 5% COAR (siAR) was 5′‐CAAGGGAGGUUACACCAAA‐3′, validated in a previous study.For gene silencing by RNA interference (RNAi), cells were transiently transfected with siRNAs using the Lipofectamine RNAiMAX reagent (Invitrogen). The siRNA sequence against human 2.2Cultured cells were lysed in the cell lysis buffer for Western and IP from Beyotime. Cell lysates were subjected to 4% (for ZFHX3) or 10%  sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and proteins were transferred to polyvinylidene fluoride (PVDF) membranes according to a standard protocol. The membranes were blocked with 5% nonfat milk for 30 min at room temperature and probed with primary antibodies overnight at 4°C. On the next day, the membranes were incubated with the secondary antibodies for 1 h at room temperature; and the WesternBright ECL HRP substrate (Advansta) was used with the Luminescent Image Analyzer  to capture images.Antibodies used in Western blotting included ZFHX3 , and cDNA was synthesized with the Moloney murine leukaemia virus reverse transcriptase (Promega). Real‐time qPCR was performed with the 2x SYBR qPCR Mix  using the qTOWER3/G system .GAPDH forward), 5′‐GTTGCTGTGTAGCCAAATTCGTTGT‐3′ (GAPDH reverse), 5′‐TGTTCCAGATCGAGATGGGAAT‐3′ (ZFHX3 forward), 5′‐CTTTCCCAGATCCTCTGAGGTTT‐3′ (ZFHX3 reverse), 5′‐GTGTGTGGACCTCCATGTTATT‐3′ (KLK3 forward), 5′‐TGCCCCATGACGTGATACCT‐3′ (KLK3 reverse), 5′‐GATCTGGCACCACACCTTCT‐3′ (Actb forward), 5′‐GGGGTGTTGAAGGTCTCAAA‐3′ (Actb reverse), 5′‐AGAGCAAGAGGGCAGCGTCATC‐3′ (Zfhx3 forward), 5′‐CGGTTCACGTCAGCGTTGCTATAC‐3′ (Zfhx3 reverse), 5′‐TGCCCGAATGCAAAGGTCTT‐3′ (Ar forward), 5′‐TTGGCGTAACCTCCCTTGAAA‐3′ (Ar reverse), 5′‐TTTGAAGATTCAGGCGTTATCCG‐3′ (Fkbp5 forward), 5′‐GGTGGACTTTTACCGTTGCTC‐3′ (Fkbp5 reverse), 5′‐GCTGTCTTGCTTTGGAGGTTC‐3′ (Tmprss2 forward) and 5'‐GACAATGTGCTACCCCGTCA‐3′ (Tmprss2 reverse).Primer sequences were as follows: 5′‐GGTGGTCTCCTCTGACTTCAACA‐3′ (2.4The promoter‐luciferase reporter plasmid pZFHX3‐Luc was constructed and named pATBF1‐Luc1 in our previous study.2.5ZFHX3 plasmids and pRL‐TK Renilla luciferase plasmid (Promega) using the Lipofectamine 2000 reagent (Invitrogen). Forty‐eight hours after transfection, including 24‐h R1881 treatment before cell collection, luciferase activities were determined using the Dual‐Luciferase Reporter Gene Assay Kit (Promega). The luciferase activity was normalized by the Renilla luciferase activity in each sample. Experiments were performed in quadruplication and repeated twice.C4‐2B cells were transiently transfected with indicated 2.6C4‐2B cells were grown in a hormone‐free medium for 1 day or 3 days and treated with 1 nM R1881 for 6 h or 12 h. The SimpleChIP Enzymatic Chromatin IP Kit  was used to pull down DNA‐protein complexes according to the manufacturer's instructions. Briefly, cells were cross‐linked with 1% formaldehyde for 10 min, quenched with glycine at room temperature for 5 min, and collected and digested with micrococcal nuclease for 20 min at 37°C. Digestion reactions were stopped by adding 0.5 M EDTA. Nuclear pellets were collected and incubated with protease inhibitors in the ChIP buffer for 10 min on ice. After sonication, chromatin extracts were immunoprecipitated using an anti‐AR antibody  or IgG.ChIP products were detected by regular PCR using the following primers: 5′‐ACTCAGGCCAATTCAGCTCCA‐3′ (P1 forward), 5′‐GTGCACCCTTCCGGGTTCTT‐3′ (P1 reverse), 5′‐CTGTCTAAACCCGCTGTACTGT‐3′ (P2 forward), 5′‐CTTACCCACTCTCCAAGCCAG‐3′ (P2 reverse), 5′‐TCTGGCTTGGAGAGTGGGTA‐3′ (P3 forward), 5′‐AAGGCAATTCTTCCCTCGCA‐3′ (P3 reverse), 5′‐GCAGAAGTTGCCAATTCCCT‐3′ (P4 forward), 5′‐GTCAAGCCTGCCTTTGTTCC‐3′ (P4 reverse), 5′‐AGATCACCTGCCTGTGGATT‐3′ (P5 forward), 5′‐TCTGCCCTAATACTGCCACTG‐3′ (P5 reverse), 5′‐GGACAGTGGCAGTATTAGGGC‐3′ (P6 forward), 5′‐ACAGGGGACAACCTCGTAAT‐3′ (P6 reverse), 5′‐CAGCAGGCCTTACCTATCCC‐3′ (P7 forward), 5′‐GGCACAAGCCCAATTCAGTC‐3′ (P7 reverse), 5′‐CCTGTTCTTGGGCCTGAAGT‐3′ (P8 forward), 5′‐CCTGTTGGGTACAGACAGCC‐3′ (P8 reverse), 5′‐GATTCCCTGGAGGCAGTCTT‐3′ (P9 forward), 5′‐CTCGGGAGAAAAAGGGATGCT‐3′ (P9 reverse), 5′‐GGGATGTGATGGTTTTCACC‐3′ (P10 forward), 5′‐CCGATTCTCACAGCACAGAA‐3′ (P10 reverse), 5′‐AGGAGCCTGGAGGCTTACAT‐3′ (P11 forward) and 5′‐GAGCCAATGTGGACAGGAAT‐3′ (P11 reverse).2.7g at 4°C for 10 min. Cytoplasmic proteins were in the supernatant and collected. The remaining nuclear pellets were added 50 μl of PMSF‐supplemented nuclear protein extraction reagent, vortexed for 15 s at maximum speed, incubated in an ice bath for 10 min and vortexed again for 10 s at full speed. The solution was then centrifuged at 14,000 × g at 4°C for 10 min, and nuclear proteins in the supernatant were collected for analysis.The Nuclear Protein Extraction Kit (Solarbio) was used to extract cytoplasmic and nuclear proteins. LNCaP and C4‐2B cells grown in complete medium or hormone‐free medium were washed with PBS, harvested with a scraper and collected via centrifugation. Cell pellets were resuspended in 200 μl of PMSF‐supplemented cytoplasmic protein extraction reagent, vortexed for 15 s at maximum speed and incubated in an ice bath for 10 min. Then, the cell lysates were vortexed for 10 s at full speed and centrifuged at 14,000 × 2.85 cells per well. After 24 h, cells were treated with 100 μg/ml of cycloheximide (CHX) with and without 10 μM enzalutamide treatment. At 0, 2, 4 and 6 h after CHX treatment, total proteins were collected, separated by SDS‐PAGE and subjected to Western blotting for ZFHX3 and α‐tubulin. Band intensities were quantified using the ImageJ program . ZFHX3 protein level was normalized by α‐tubulin level for each time point CHX treatment.C4‐2B cells were seeded into 12‐well plates at a density of 2 × 102.9Mouse experiments were approved by the Institutional Animal Care and Use Committee at the Southern University of Science and Technology. Twelve‐week‐old male C57BL/6 mice were purchased from Charles River, and castration was performed as described in our previous study.2O2 for 10 min, tissue sections for Zfhx3 were blocked with blocking buffer  and incubated with the anti‐ZFHX3 antibody at room temperature for 1 h. Sections for Ar were blocked with 10% normal goat serum and incubated with the anti‐AR antibody at 4°C overnight. Sections were then incubated with EnVision PolymerHRP secondary antibodies (MXB Biotechnologies) at room temperature for 2 h for Zfhx3 and 30 min for Ar, counterstained with DAB‐chromogen and haematoxylin (MXB Biotechnologies), dehydrated in ethanol and mounted. Slides were scanned using the Aperio VERSA 8 Scanner System . Image‐Pro Plus (NIH) was used to determine the integral optical density (IOD) of the positively stained signal (brown) and area of haematoxylin‐stained signal (blue) for each slide. The IOD/mm2 of the haematoxylin‐stained area was then calculated and compared between groups.Immunohistochemistry (IHC) staining was performed to detect protein expression with Zfhx3 antibody  and Ar antibody . Formalin‐fixed, paraffin‐embedded mouse prostates were sectioned at 4 μm, deparaffinized in xylene, rehydrated in graded ethanol (100%–75%) and boiled in 10 mM citrate buffer (pH 6.0) for 5 min (for Zfhx3) or 3 min (for Ar) using a pressure cooker. After treatment with 3% H2.10http://xenabrowser.net/). The dataset contained 499 PCa cancers with the following information: Gleason score, copy number variations (CNVs) estimated by the Genomic Identification of Significant Targets in Cancer (GISTIC) analysis, disease‐free survival status and genome‐wide mRNA expression data. One of the 499 PCa samples did not have mRNA expression data and was excluded for analysis. Some of the PCa samples had information available for their corresponding normal prostate tissues. In the pre‐ranked gene list based on fold changes of RSEM normalized counts, gene set enrichment analysis (GSEA) was performed using the OmicStudio tools (https://www.omicstudio.cn/tool) to ascertain relationships between ZFHX3 mRNA levels and existing hallmark gene sets. An adjusted P‐value of 0.05 was set as the cut‐off point for significant enrichment.The TCGA prostate adenocarcinoma dataset was downloaded from the UCSC Xena public data hub (ZFHX3 higher and ZFHX3 lower) by the median ZFHX3 expression level. AR activities were defined by the sum of expression levels of 27 genes indicative of AR activities, as established in a previous study.2  relative to that in all PCa samples.A heatmap was built using the R package, in which the PCa samples were divided into two groups .The Pearson correlation analysis was conducted to determine the linear correlation between ZFHX3 deletion at one and both copies in PCa samples with different Gleason scores were tested using Fisher's exact test. The disease‐free survival was analysed using the Kaplan‐Meier analysis, and the statistical parameters were calculated using a log‐rank test.The differences between rates of t‐test was performed. For statistical comparison of more than two groups, one‐way ANOVA or two‐way ANOVA was performed. All statistical analyses were conducted using GraphPad Prism 6. p values <0.05 were considered statistically significant.All in vitro experiments were repeated at least twice. Statistical analysis was based on 3 or 4 replicates of one experiment. All experimental readings are expressed as mean ± SD. For statistical comparison between two groups, a two‐tailed Student's 33.1KLK3, a classical AR target gene also known as PSA , in which the consensus AR binding sequences are based on ChIP‐Seq studies.To determine whether androgen‐induced ZFHX3 expression is indeed mediated by the binding of AR to the ZFHX3, which contains the promoter sequence of ZFHX3 in Figure Then, the pZFHX3‐Luc promoter‐luciferase reporter plasmid for ZFHX3 transcription, including whether AR has different binding patterns for different AREs under R1881 treatment, we performed a ChIP‐PCR assay in C4‐2B cells undergone 24‐h hormone starvation and subsequent 6‐h R1881 treatment  for different times. The CHX assay demonstrated that enzalutamide significantly decreased the half‐life of ZFHX3 protein Figure , suggest3.5To determine whether the regulation of ZFHX3 expression by the androgen‐AR signalling also occurs in vivo, we castrated 12‐week‐old C57BL/6 mice to deplete androgen and detected Zfhx3 expression in mouse prostates. IHC staining demonstrated that the Zfhx3 protein was primarily expressed in the nucleus of luminal cells with a uniform staining intensity; Zfhx3 expression decreased at 14 days after castration but did not show a noticeable change at 1 day after castration, as indicated by the integrated optical density (IOD) Figure . As expeZfhx3 mRNA level decreased, and Ar mRNA level was increased, which is considered negative autoregulation.Fkbp5 and Tmprss2 were decreased by castration . Pearson correlation analysis further confirmed the positive correlation between ZFHX3 expression levels and AR activity scores regardless of tumour stage, grade, metastasis or age . Therefore, induction of ZFHX3 transcription by the androgen‐AR signalling also occurs in human prostatic epithelial cells.To evaluate whether transcriptional regulation of y Figure , left. P3.7ZFHX3 and its potential clinical relevance in PCa. We found that deletion of one or both copies of ZFHX3 was more frequent in PCa samples with higher Gleason scores  relative to that of all PCa samples, were lower in PCa samples, particularly those with Gleason scores greater than 7 . The gene set enrichment analysis (GSEA) was then performed using the pre‐ranked list of genes based on fold changes. The top five enriched hallmarks included androgen response genes, genes downregulated in response to UV radiation, protein secretion, mitotic spindle and early response to oestrogen. Those enriched in the ZFHX3‐low group included interferon‐gamma response, allograft rejection, E2F targets, MYC‐regulated genes, DNA repair, interferon‐alpha response, and oxidative phosphorylation , further supporting a role of ZFHX3 in AR function  in the ZFHX3 promoter  gene  is transcriptionally upregulated without androgen but repressed when androgen is present due to an enhancer element.Such a unique pattern of androgen‐induced reversal in gene transcription is unusual. Still, it has been reported that the prostate‐specific membrane antigen ; Investigation (lead); Writing – original draft (lead). Zhiqian Zhang: Writing – review & editing . Mingcheng Liu: Investigation (supporting); Methodology . Juan Li: Investigation (supporting); Methodology . Jun A: Data curation ; Software . Liya Fu: Project administration (supporting). Chenyang Huang: Project administration (supporting). Jin‐Tang Dong: Conceptualization (lead); Funding acquisition (lead); Supervision (lead); Writing – review & editing (lead).


Interpersonal rehabilitation games, compared to single-player games, enhance motivation and intensity level. Usually, it is complicated to restrict the use of the system to pairs of impaired patients who have a similar skill level. Thus, such games must be dynamically adapted. Difficulty-adaptation algorithms are usually based only on performance parameters. In this way, the patient’s condition cannot be considered when adapting the game. Introducing physiological reactions could help to improve decision-making. However, it is difficult to control how social interaction influences physiological reactions, making it difficult to interpret physiological responses. This article aimed to explore the changes in physiological responses due to the social interaction of a competitive game modality. This pilot study involved ten unimpaired participants (five pairs). We defined different therapy sessions: (i) a session without a competitor; (ii) two sessions with a virtual competitor with different difficulty levels; (iii) a competitive game. Results showed a difference in the physiological response in the competitive mode concerning single-player mode only due to the interpersonal game modality. In addition, feedback from participants suggested that it was necessary to keep a certain difficulty level to make the activity more challenging, and therefore be more engaging and rewarding. In the last report from the Stroke Alliance for Europe , around The effectiveness of rehabilitation in improving functioning and quality of life is higher in a high-intensity, reproducible therapy. Professionals have also described motivation as an important determinant of rehabilitation outcome . It has A promising way to maintain a high level of motivation during long-term rehabilitation therapies is to use interpersonal rehabilitation games in which patients cooperate or compete in a game. Several studies demonstrated that interpersonal rehabilitation games obtain better results in increasing motivation and exercise intensity compared to conventional rehabilitation exercises ,11,12,13One of the main research topics on interpersonal rehabilitation games is the development of difficulty-adaptation methods. To obtain good outcomes in a competitive or cooperative rehabilitation game, patients should be on an equal footing to ensure a proper level of competitiveness. Usually, it is complicated to limit the use of the system to pairs of impaired patients who have a similar skill level. That is why the system should be adapted to the condition of the patient.In competitive and cooperative rehabilitation games, the difficulty-adaptation methods are usually based on performance parameters ,15. EvalThis article aimed to study how the user’s physiological reactions are affected due to the interpersonal game modality. To perform this, we defined a single-player game modality with a virtual competitor with an intensity level very similar to the one of the competitive mode. In addition, we limited the social interactions during the competitive mode, preventing users from communicating with each other during the game. In this way, we measured differences in the physiological responses of the users only due to the type of competitor. Moreover, we also studied two single-player game modes, one without a competitor and another with an easy difficulty level. These served as a reference when studying the levels of motivation and intensity.A total of 10 subjects  with no motor or cognitive impairment were recruited for the study. They were between 23 and 50 years old (31.6 ± 9.5 years). All of them were right-handed. They were recruited from the staff of the Bioengineering Institute of Miguel Hernández University.Pairs of participants were recruited according to approximate age, gender, and handedness . In two-player game studies, it is common to match by gender ,20 becauFirstly, the player had to wait in the basket until an apple appeared a, then td, was less than or equal to the distance r  between two reusable electrodes placed on two fingers of the hand. GSR is a standard measure in psychophysiological paradigms and, therefore, often used in affective state detection. In this study, we placed the electrodes on the proximal phalanges of the index and middle finger of the hand not used to control the robot (non-dominant hand). The sample rate of the sensor unit was 50 Hz. From the GSR, we extracted the Skin Conductance Response (SCR).TM (Zephyr Technology Corporation) physiological monitoring telemetry device. The BioHarness transmits signals to be received via Bluetooth. This device also has a built-in signal-processing unit, so the received signal is already processed. The sampling rate of the sensor unit was 250 Hz. We extracted the Heart Rate (HR) evolution over each condition from the ECG signal. HR measurement is standard in rehabilitation games [We also recorded the Electrocardiogram (ECG) of each participant through the Zephyr BioHarnesson games .To study how the condition affected the user’s affective state, we obtained the value reached at the end of every condition for each physiological feature. We normalized all the physiological features by the min-max normalization method .(1)xnorThere are currently several evaluation tools available for assessing patient motivation and satisfaction during technology-assisted rehabilitation. One of the instruments most used is the Intrinsic Motivation Inventory (IMI) . This suTM on the chest and a Shimmer3 GSR sensor unit placed on the non-dominant hand. Finally, they sat in front of the robot.Two instances of the game were executed, one on each of the rehabilitation platforms and one for each player.Free mode: In this mode, there was no competitor, which means that there was no time limit;t was set as 2 s. In this case, players had a virtual competitor;Low-difficulty mode: The time t parameter was set as 0.7 s.High-difficulty mode: In this game mode, it was challenging for the player to beat the virtual competitor since the Each participant performed the different single-player game modes in the same order :Free modEach single-player game mode was performed simultaneously and separately. In this way, both participants were ready at the same time to perform the competitive mode at the end.It is important to note that no difficulty level was configured in the free mode and the competitive mode. Therefore, the intensity level was only self-imposed by the participants.For this study, it was decided not to randomize the conditions due to the small sample.The number of apples was set to 21 in all cases to prevent a tie in the competitive mode. The robotic rehabilitation platform allowed us to configure the range of motion to match it with the patient’s range of movement. In this study, subjects did not have any motor or cognitive impairment, so the range of movement was set to a 10 cm diameter circle, so they all traveled the same distances.In the protocol, the duration of each game mode was not determined. The number of points a subject earned per apple was not influenced by the amount of time required to catch it. Therefore, the only goal of the participant was to be able to catch the 21 apples.Before each session, there was a 5-min rest period where the physiological signals were recorded to compute the baseline . This peIn the statistical study, we carried out a normality test through the Shapiro–Wilk test. There is evidence to suggest that some parameters were not normally distributed.For the purposes of data analysis, all 10 participants were treated as independent.One-way repeated-measures Analysis of Variance (ANOVA) was employed for normally distributed parameters. We used Mauchly’s test of sphericity to evaluate whether the sphericity assumption was violated. If sphericity was violated, repeated-measures ANOVA was corrected using Greenhouse–Geisser correction when epsilon was On the other hand, the Friedman test was used for not normally distributed parameters. In the post hoc analysis, the Holm–Bonferroni method was used to adjust for familywise error rate correction.Finally, the magnitude of Spearman’s rank correlation coefficient (In The root mean squared velocity value showed significant differences between conditions (one-way repeated-measures ANOVA We also obtained similar results for the case of the mean velocity value.The analysis also showed significant differences between the game modes for the maximum velocity value (Friedman test Regarding the reaction time, analysis also showed significant differences between conditions (Friedman test In the result of the score a, we sawIn the competitive mode, the participants’ score was around 50%, which means that couples were well defined since there was not much difference between winners and losers, so it was a close game.On the other hand, the results showed that the interest/enjoy parameter from the IMI increased with the difficulty level, reaching the highest values in the high-difficulty mode and the competitive mode.Post hoc analysis suggested that the interest/enjoy parameter was weakly correlated with the root mean squared velocity value (Physiological response signals are illustrated graphically in GSR a showed Post hoc analysis reflected a weak correlation between the GSR and the score (Regarding HR a, resultPost hoc analysis suggested that HR was moderately correlated with the root mean squared velocity value (In In the post hoc analysis, a strong correlation with the root mean squared velocity value was found with pressure/tension (We also note that effort/importance was strongly correlated with GSR (The differences between conditions were largely due to the defining of the different game modes. Therefore, although importance was given to statistically significant differences among groups, in the discussion of the results, special attention was given to the trend of the parameters among modes, although these were not statistically significant.As previously mentioned, the intensity level was estimated mainly through the root mean squared velocity value. Firstly, we observed that modes in which an intensity level was established were significantly different . That meAnother parameter directly related to the intensity level is the reaction time. In Comparing the high-difficulty mode and the competitive mode, although we did not obtain a statistically significant difference, despite both modes having an equal intensity level, results suggested that the reaction time was longer in the competitive mode. However, this effect was not due to a different intensity level, but rather due to the game mode. We observed that as the participants could see each other inside the game, influenced how they reacted when a target appeared. In single-player modes, where the competitor was virtual, participants learned when the apple would appear, so they reacted faster. In competitive mode, the target appeared at the same time as in single-player modes, so prior learning should have made them react at least as fast as in the high-difficulty mode. However, seeing the opponent within the game made the participants look at his/her reaction, making them react slower.Based on these results, we can conclude that despite some differences due to the game modality, the intensity levels of the high-difficulty mode and the competitive mode were very similar.In the competitive mode, the participants’ scores were around 50% , which mIn the high-difficulty level, the score was around 80%. To obtain the competitiveness level of the competitive mode with a virtual competitor in a single-player game, we would need to increase the difficulty level even more. However, in the high-difficulty mode, users reported that the intensity level was very high, so increasing the difficulty level could be counterproductive.In light of the results for the interest/enjoy parameter from the IMI, participants preferred a challenging game b.Considering that the intensity level was the same both in the high-difficulty mode and in the competitive mode, it would not be strange to obtain a similar physiological reaction in both modes. However, the GSR results suggested that the GSR value was lower than the high-difficulty level in the competitive mode. We could also observe a similar effect in the HR results. Therefore, it was a measurable effect through different physiological features.Regarding the effort/importance parameter from the IMI, results suggested that the participants’ perceived exertion had a similar behavior as the intensity levels . In addiThis finding agreed with the notion that the observed difference in the physiological reactions between the competitive mode and the high-difficulty mode was not due to the intensity level. However, this could be due to the interaction with a live player through the game.While our results showed an appreciable difference on physiological responses, a few study limitations should be discussed. Our study involved only 10 unimpaired participants, and therefore, the results may not be generalized beyond the conditions of this study. However, the results suggested that the competitive game mode presented differences that should be validated in a more extensive study.In this article, a study of how the user’s physiological reactions were affected due to the interpersonal game modality was performed. To perform this, we defined a single-player game modality with a virtual competitor with an intensity level very similar to the one of the competitive mode. In addition, we limited all social interactions, preventing users from communicating with each other during the competitive game. In this way, we measured the differences in the physiological responses of the users only due to the type of competitor.Due to the current study design, the only difference between the high-difficulty mode and the competitive mode was the type of competitor, since we did not allow the participants to communicate with each other during the game. Knowing that they were competing with a real player seemed to modify their physiological reaction. However, we considered that this change in the physiological reaction could also be due to other aspects, such as the relationship between the two players or a more introverted personality when playing with another person, among others. Therefore, we believe that it is difficult to conclude why the physiological reaction was lower in the competitive mode. Even so, we can affirm that the results suggested that there was a difference in the physiological response concerning the single-player game mode only due to the interpersonal game modality.In addition, it is interesting to note that according to the IMI questionnaire results, participants found the most challenging game more enjoyable. This result suggests that it is necessary to maintain a certain difficulty level to make the activity more challenging, and therefore more engaging and rewarding.


The Last of Us Part II, a game that has elicited highly polarized reactions in relation to how players responded to a switch of the PC in the game. Through thematic analysis, three themes were found, illustrating the connection between aspects of the player-character relationship and the overall game satisfaction. The themes are “Tolerance of forced character switch”, “Malleability of character image” and “Flexibility of character attachment”. We discuss how those findings should be taken into consideration when designing diverse and meaningful gaming experiences.While player characters (PCs) are the key element in engaging players in narrative games, the experience and relationship of the player with the PC have received scarce attention from the perspective of the subjective player experience. The diversity of players and the importance of the PC in the game suggests meaningful connections between how players relate to their PC and the resulting satisfaction with the game. We, therefore, investigated in this study how the player-character relationship influences satisfaction of the player with the game. We performed semi-structured in-depth interviews with 12 players of  Video games are continuously gaining popularity not only as an entertainment medium, but also in fields such as art  killed in the previous game. Then, the player plays as Abby for the same 3 days until the face-off with Ellie, and continues controlling Abby in fighting Ellie, but after gaining the upper hand in the fight Abby spares Ellie and Dina. Back in Jackson, the player controls Ellie again, who hears about Abby's location and decides to go after her once more, against Dina's will. She finds Abby worn out and captured by another group, and after the last fight, lets her go and returns to an empty home.We illustrate our findings in the following sections. We first illustrate the found differences in overall game satisfaction as well as the more specific player reactions to the game. We then present our themes concerning three aspects of the player-character relationship which were found to be related to the differences in game satisfaction and reactions. The Last Of Us Part II. The game satisfaction of the player was often either very high or very low with only a few mixed cases, as illustrated in My overall feeling is this is one of the greatest games I've ever played. It's one of the bravest narratives I think I've ever interacted with.” On the contrary, other players outed their frustration similar to P12: “I played this emotionally abusive trash game for nothing.” Two players had mixed feelings about the game and would acknowledge positive and negative aspects like P4 did: “It was really well done but, it already came with a baggage right, expectations from players (…) it was, narratively speaking, (…) badly structured.” To understand the differences in the overall game satisfaction better, we looked at more specific player reactions toward the game, as described in the next section.Players varied greatly in whether or how much they were satisfied with The differences in game satisfaction were visible in how the players reacted to the game. There were some clear commonalities in how players who did or did not like the game reacted to certain game events and elements. Those events and elements are (1) the reactions of the players to being forced to play as the Abby character and (2) the perceptions of the players of the different PCs. First, almost all players initially showed great reluctance, confusion, and anger when having to control the Abby character, as she was the character who brutally killed the previous PC Joel. However, the way in which players coped with the continuous obligation to play Abby differed across players. Players with low game satisfaction continuously showed great resistance and either finished the game very reluctantly or all over quit the game. The players who later voiced great appreciation for the game mentioned that they slowly accepted this game-mechanic and started to engage with the new PC. Second, the way players would perceive and describe the three main PCs in the game varied greatly. Players who did not like the game would critique how the actions of the character are illogical, not human-like, and how Abby appears as a tool of the game developer to convey a message. On the other hand, players who greatly valued the game mentioned how the PCs were interesting, complex, and human. This contrast in player's reactions points to underlying factors in how players interacted with the game. In the following, the three themes related to the differences in game satisfaction and player reactions are presented.We now present the three themes which were found in relation to how the participants related to their PCs. We present each theme in the following section illustrating how each aspect of the player-character relationship was related to the players' reactions and overall game satisfaction. The Last Of Us Part II is that the player is forced to play as the Abby character midway during the game. The player has no choice but to play as her, to keep progressing in the game. This led to furious reactions in our participants, but not everyone was as resistant to this game mechanic. Some players who had a high game satisfaction valued this forced change of perspective in hindsight, as it enabled them to change their view on the Abby character.The probably most controversial part about All but two participants were initially very reluctant to being forced to play as Abby. Of the reluctant participants, five did not appreciate the part as Abby until the end of gameplay. Those players showed a strong reluctance to being forced by the game to play in a way they did not want and having no choice. Particularly, the role of the developer who made them do this was strongly emphasized and the anger was also directly aimed at the development. P8 explains how the Abby character felt like the tool of the developer with which he wants to teach the player something. This created great reluctance in the player so that any engagement with the game was made impossible as P8 stated.P8: “Every time I played as Abby, it was really tormenting. (…) She was Neil Druckmann's avatar. The character, Abby. So, I keep feeling that Neil Druckmann is trying to convey the game's message through Abby, but it doesn't feel somehow natural like in [part]1, I always felt like he was trying to artificially force it onto me. So I couldn't get any positive feelings for the Abby character even if I wanted to. That's why it was tormenting and exhausting to play her. No sense of immersion at all.”P8 further mentioned that particularly being forced to directly play Abby made it impossible for him to relate to the character, whereas through other means of story-telling an engagement would, theoretically, have been possible:P8: “If Abby came up not as a playable character, but always on the other side and the situation would have played out in a cinematic way or so I think I would have been able to relate at least a bit (…) but because the user was forced directly I felt repulsive.”When you don't want to do what the game wants you to do it's just not a good game.” In broader terms, the players did not accept forced gameplay as a narrative tool, as it seemed to go against their understanding of what a game is or should be. This is in contrast to the experiences of the remaining participants of our study, as explained in the following section.It can therefore be understood that the forceful play itself, the removal of the agency of the player as a separate element was rejected strongly by the dissatisfied players. The theme is therefore closely related to the continued resistance to playing Abby. This rejection also correlated strongly with the evaluation of the player and eventual satisfaction of the game, as P1 put it: “Most of the participants who had high satisfaction with the game were first also very reluctant to being forced to control the Abby character. However, other than the dissatisfied players, the participants started to change their view on the Abby character and the decision to make the player play Abby against their will was appreciated as a bold and effective narrative device, as explained by P5.P5: “I was furious with Abby up until I had to play as Abby, I mean even through that, the first few scenes of playing as Abby I felt I don't really want to play as her, I hate her, she's awful, she's ruining the life of the person I've really come to enjoy and now I have to play as her. But I think that's genius storytelling. I think it is a risk to tell a story like that to kind of force the player into a perspective that they don't really want to be in. That's gutsy and I appreciate that, not everyone likes that but I really like that. But that way of telling it, by kind of rewinding the clock, loved it I think that's a really smart way of doing it.”Another participant expressed fascination with how the game medium can be used to make players understand and experience characters differently than in other media, such as literature.P6: “I was really surprised. Of course in literature there is also attempts like that, but it's really hard to create stories where A kills B and then make them understand that from A's perspective. That's really hard but I thought oh so but through a game that's possible, I often thought that's somehow the game's unique ability. (…) The story isn't like good or bad but it's just those two people's unavoidable encounter and mismatch. How the story was resolved like that was really surprising and great.”The Last Of Us Part II is, they had already bought it. The reaction to the forced game play also likely had an influence on the subsequent perception of the PCs by the player, as will be described in Theme 3 and is illustrated in In summary, the forceful deprivation of the agency of the player was not appreciated immediately, by almost any of the participants. However, the satisfied players started to value this forced character play as an effective narrative tool to make them change their minds about the PC, which they considered a meaningful playing experience. For the dissatisfied players, however, their agency seemed a more central part of their gaming experience, which is in direct conflict with the freedom of the developer to use the PC as a story-telling device. Therefore, in their eyes, the developers took not only the agency of the player, but also their game enjoyment to convey their own message. This resulted in strong anger against the developers. An additional factor adding to the players' frustration was that since the game was a sequel to a game without such harsh deprivation of agency, many players did not expect such an experience, and by the time they realized what kind of game This theme concerns how participants formed attachments to the PCs. We show how dissatisfied players fixated on their strong attachment to the PCs, Joel and Ellie, from part one and how other players created an additional attachment to the new PC, Abby. This provided the latter players with a meaningful emotional playing experience which led to high game satisfaction.While all participants reported having a close attachment to the PCs of the first game, for some of the players this attachment directly interfered with their willingness to form a new attachment to the Abby PC. First, participants reported that they connected to the PCs of the first game through a gradual built up of an emotional bond that was not felt for the Abby character.P7: “I think it's because I can't ignore the attachment I got for the first game. And as I said before, you can really emphasize with the small emotional changes of Joel who is first just annoyed at Ellie and then gradually starts opening up and getting warmer. But I didn't get that feeling of delicate emotional development from Abby.”Furthermore, in contrast to satisfied players who would describe their relationship to the character as one of a close friend or movie character, most players who were very dissatisfied with the game mentioned that they immersed or projected themselves into the PC or saw the PC as their alter-ego as described by P12.P12: “Joel is my alter ego while playing this game. Since I need to occupy that character while playing and since it moves as I do, it's my alter ego which I feel like I can control directly and it gives me the ability to act within the game even though I can't make choices about the story in the game.”This close connection to the PC could subsequently not be transferred to another PC from another side, which P8 elaborates.P8: “Anyways since I always have an emotional bond while playing from the perspective of Joel, even if I play Ellie, since Ellie is on Joel's side you can play as her and share that emotional bond. But if you go to the complete opposite side you can't share that emotional bond anymore. Why should I play as her? I don't see any justification for playing as her.”For the remaining participants, it seemed possible to keep the attachment to Ellie and Joel and still form a new attachment to the Abby character. It was clear that after playing the game the players had an attachment to both main PCs Ellie and Abby at the same time, and this conflicting attachment was one of the core meaningful experiences for which the players reported to appreciate the game for. The degree to which the attachment to Abby was achieved varied; however, a commonality between satisfied players was that they all had some degree of attachment to Abby. P9 explains how this inner conflict of attachment solidifies in the final fight of the game between Ellie and Abby.P9: “I think this fight might be the real climax of the game. At that time I can really emphasize with Ellie's sadness but I also can roughly understand Abby's life and her changed ideology, so it would be so hard to watch any of the two die”The ways through which the participants related to the character varied, but frequently mentioned topics were the relationships of Abby with the Yara and Lev character as well as moments where Abby appeared flawed, or vulnerable as illustrated in the following two quotes.P3: “But then in the second game, once I think the turning point really was once you meet Yara and Lev because that just, brings out Abby's humanity (…) So when then these extra people come and save her and then in turn she goes like she owes them.P5: “One thing that really struck me as Abby and I love this about the game, is that Abby is terrified of heights. I am also terrified of heights and the game did this so well where they would illustrate her fear when you know she looked down (…) As someone who's afraid of heights I have a similar reaction, so to hear that and to see it depicted so realistically, was another moment of empathy for me and her.”To summarize, dissatisfied players only had one strong attachment to Joel and Ellie, while the satisfied players had all formed an attachment to the Abby PC to some degree. The former participants immersed or projected themselves strongly into PCs of the first part and were therefore unable to form new attachments while the latter players connected to Abby through her new relationships or hardships.The last theme concerns how the players construct and adjust their own mental image of the PC. We found interesting differences in how some participants would see certain actions of the character as “not making sense” and based on that, they criticized the realism or logic of the game, while other players would actively create their own interpretations of why characters acted in the ways they did.The Last Of Us Part II compared to the first part.A frequent criticism of participants who were disappointed in the game was how illogical the story was, particularly, how the actions of the characters do not make sense. Especially when that character in question was the player's favorite PC, and the actions related to a less positive character representation, this led to strong negative reactions and a negative evaluation of the game as a whole. This is illustrated by P12 who was strongly criticizing the logic of how Joel changed in P12: “What I couldn't understand the most was that originally in part 1 Joel was more wary of people than zombies and if he was so strong and able to survive under any circumstances, then the fact that he was too weak to do anything in 2 and gets killed by Abby's group and he was beaten so helplessly to the degree that it made me think it might have been a setting error, made me so angry. (…) It wasn't the Joel I know.”Note how the last sentence “It wasn't the Joel I know” implies that the representation of the character was deviating from a mental image she had constructed about the PC. Also, inconsistencies in actions of negatively evaluated PCs were criticized, as seen when P8 explains how he got upset by the inconsistent actions of Abby when she spares the NPC Dina.P8: “Why is she [Abby] acting like this here? If she would act like what is common sense and what she showed us last time, it would have been normal for her to make a massacre ending, but now you don't kill that thing? This feeling of estrangement, based on the first impression she was a terminator who would kill everything, but actually she's a nice person.”The participant goes on to explain that he felt this inconsistency was due to the developers wanting to bring across their point of Abby actually being a nice character, but this being represented unnaturally in the game.On the other hand we had participants who would flexibly adjust their character image based on new information provided in the game, and create their own interpretation for the underlying reason for the actions of the PC. A great example is P6, who contrary to P12 as discussed above, interpreted Joel's change in behavior as him starting to gain the ability to trust people thanks to Ellie and thereby saw it as character progression.P6: “In Joel's case, it's just my interpretation, but in Joel's case, it seems that he eventually started to believe in humanity. He believed in humans and built a community. Originally, starting as a very distrustful person, he met Ellie, started to trust Ellie, and while starting to depend on Ellie got to join a community. I understood that this is some kind of growth from his point of view, and that's why I interpret it as him starting to trust people. Of course, looking at the game's realism you can interpret it as Joel just becoming so weird. But Joel now got a deep trust, got to be a person who deeply trusts people.”P6 continues to explain how he never questions the “logic” or “realism” of a character and his way of approaching game or literary character is that he likes to reason about the underlying motives which move a character.P6: “Rather than thinking from the outside, in this piece of work this person doesn't make sense, it's not realistic, I'm the type who thinks more about the inside of the person, why did this person have to act in that way? That's why I never really thought things like what's wrong with this person. I don't think those things and there is also a lot of hidden meaning, it's the same with real humans. Why is person A acting like that, that person will have a reason, you don't say that person's action is unrealistic. That's kind of how I see it. (…) I think I treat it like they are real human beings.”Overall, this can be seen as a stark contrast in how the character image is constructed in players. It is mainly a difference in how players create and adjust their own mental image of the character in contrast to what is represented in the game. Participants who were part of the former type of players were usually the ones with a negative evaluation of the game representation of the game characters, mentioning how the characters do not make sense and exhibited an overall dissatisfaction with the game, while the latter type of players praised the game for it is interesting and complex characters and had high overall game satisfaction. This tendency is likely dependent on individual differences in the player. However, we can also assume that the willingness of the player to create narratives in their mind justifying the characters actions, is strongly influenced by the players' reaction to the forced gameplay and resulting overall attitude toward the game.The Last Of Us Part II, this can explain why the players who only had affection for Ellie and Joel until the end did not have a high enjoyment of the game, since those characters did not have good outcomes in the narrative. However, this theory does not explain why some participants formed an affective disposition to Abby and some did not. Similarly, the concept of allegiance from Lankoski .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In recent years, we have witnessed a growing number of people needing locomotion rehabilitation (e.g. stroke). The inability to walk has tremendous effects on the individuals' wellbeing and quality of life, making locomotion rehabilitation a vital component of physiotherapy. Virtual reality (VR), a term used to describe a technological system that creates a simulated world or environment, is a promising technology that has proven significant benefits in rehabilitation . Our resLocomotiver consists in a VR environment that can be used by therapists and patients, simultaneously. The patient experiences a 360° immersive environment using an HTC Vive headset and four trackers. Kondo et al.  inspiredPhysiotherapists agreed that Locomotiver would be an innovative solution to their interventions and to increase patients’ engagement. They agreed that Locomotiver is more proper for patients with musculoskeletal disorders than for neurological patients. Therapists also stated that exercises need to allow further customisation and collect additional performance data. Participants praised both the instruments and prototype, namely how fast and easy they were to set up, compared to other conventional systems. Overall, professionals show high interest in adopting Locomotiver, pointing benefits such as optimisation of their methods, ease of customisation, and improved diagnosis.We presented Locomotiver, an immersive VR system for locomotion rehabilitation that includes three customisable exercises. We verified that Locomotiver an engaging and motivating experience for the users. We concluded that professional therapists would be interested in adopting Locomotiver as a rehabilitation tool. This research contributes to highlight key challenges and opportunities when introducing immersive VR technologies in clinical rehabilitation practices. We believe that this research contributes to establish this a baseline to develop and introduce immersive VR technology that significantly increases the motivation of patients, optimises intervention processes and improves the overall locomotion rehabilitation. As future work, we propose that physiotherapists use Locomotiver with patients for a certain period, in a real-life scenario.


Acute hypoxemic respiratory failure (HRF) is associated with high morbidity and mortality, but its heterogeneity challenges the identification of effective therapies. Defining subphenotypes with distinct prognoses or biologic features can improve therapeutic trials, but prior work has focused on ARDS, which excludes many acute HRF patients. We aimed to characterize persistent and resolving subphenotypes in the broader HRF population.2-to-FIO2 ratio ≤ 300 on cohort enrollment . We classified patients as persistent HRF if still requiring mechanical ventilation with PaO2-to-FIO2 ratio ≤ 300 on day 3 following ICU admission, or resolving HRF if otherwise. We estimated relative risk of 28-day hospital mortality associated with persistent HRF, compared to resolving HRF, using generalized linear models. We also estimated fold difference in circulating biomarkers of inflammation and endothelial activation on cohort enrollment among persistent HRF compared to resolving HRF. Finally, we stratified our analyses by ARDS to understand whether this was driving differences between persistent and resolving HRF.In this secondary analysis of 2 independent prospective ICU cohorts, we included adults with acute HRF, defined by invasive mechanical ventilation and PaOn = 386) and validation (n = 1032) cohorts. Persistent HRF was associated with higher risk of death relative to resolving HRF in both the discovery  and validation cohorts , after adjustment for age, sex, chronic respiratory illness, and acute illness severity on enrollment . Patients with persistent HRF displayed higher biomarkers of inflammation  and endothelial dysfunction (angiopoietin-2) than resolving HRF after adjustment. Only half of persistent HRF patients had ARDS, yet exhibited higher mortality and biomarkers than resolving HRF regardless of whether they qualified for ARDS.Over 50% developed persistent HRF in both the discovery (Patients with persistent HRF are common and have higher mortality and elevated circulating markers of lung injury compared to resolving HRF, and yet only a subset are captured by ARDS definitions. Persistent HRF may represent a clinically important, inclusive target for future therapeutic trials in HRF.The online version contains supplementary material available at 10.1186/s13054-021-03755-7. Acute hypoxemic respiratory failure (HRF) is associated with extended hospital length of stay, functional disability, and increased mortality, but care remains supportive –5. One c8One area of heterogeneity in HRF is the early clinical trajectory. The majority of patients with respiratory failure requiring mechanical ventilation are weaned off within three days , while opersistent HRF). The primary objective of this study was to estimate mortality of patients with persistent HRF compared to patients whose respiratory failure and need for mechanical ventilation resolves (resolving HRF). The secondary objective was to compare circulating biomarkers previously linked to severity and outcomes in lung injury and profile the early biologic differences between patients with persistent and resolving HRF. In exploratory analyses, we examined relationships between persistent/resolving HRF and ARDS. In particular, we were interested in characterizing the clinical and biologic features of patients with acute HRF who do not fulfill ARDS criteria,  a population that is relatively understudied [Our overall goal was to define and characterize a novel subphenotype of acute HRF using persistence of respiratory failure at day 3 following the initiation of mechanical ventilation . The discovery cohort was enrolled from medical and surgical ICUs at Harborview Medical Center  between 2006 and 2010. Patients were enrolled within 24 hours of ICU admission if meeting criteria for the systemic inflammatory response syndrome –30. Excl2-to-FIO2 ratio (PaO2:FIO2) ≤ 300 at study enrollment. If PaO2:FIO2 was unavailable, we used SpO2-to-FIO2 ratio ≤ 315 [We restricted analyses to patients with acute HRF, defined as new invasive mechanical ventilation and PaOio ≤ 315 .  were still classified as persistent HRF as long as they met criteria on day 3. Patients who were extubated or who had PaO2:FIO2 > 300 on day 3 were classified as resolving HRF. All PaO2 measurements were obtained as part of clinical care, and we used the lowest value of PaO2:FIO2 for each ICU day. To understand how classifying persistent HRF at different early time points altered our results, we performed sensitivity analyses redefining persistent HRF on day 2 after admission  and day 4 (which we hypothesized may better delineate differences in underlying biology) [Patients with acute HRF were further classified as persistent or resolving. Persistent HRF was defined by ongoing need for invasive mechanical ventilation and PaObiology) –35. PatiARDS was defined by the Berlin criteria . Each chIn both cohorts, plasma samples were collected at enrollment for measurement of biomarkers associated with the development of acute lung injury and ARDS , 31. In We reported demographics, baseline comorbidities, ICU conditions, and outcomes by persistent and resolving HRF. Continuous variables were reported as medians with interquartile ranges and compared using Mann–Whitney U tests, while categorical variables were reported as counts with percentage and compared using Chi-squared tests.2:FIO2 or modified acute physiology and chronic health evaluation (APACHE) score as prespecified confounders. We used APACHE-III in the discovery cohort and APACHE-II in the validation cohort [2:FIO2 and APACHE score in the same model to avoid multicollinearity. To further select for a population of comparable HRF severity at baseline, we performed analyses restricting to patients with PaO2:FIO2 < 150 and another excluding patients with chronic lung disease.Our primary analysis estimated relative risk (RR) of mortality associated with persistent HRF compared to resolving HRF using generalized linear models with Poisson distribution and robust standard errors . In adjun cohort , 39. We 2-transformed biomarker measurements due to right skew.In our secondary analysis, we used linear regression with adjustments as above to estimate the fold difference in geometric mean concentrations of each biomarker between persistent and resolving HRF. We used geometric means and logWe performed two exploratory analyses. First, we examined whether ARDS, identified at any point by ICU day 3, was driving differences between persistent and resolving HRF. We stratified persistent/resolving HRF by ARDS (± ARDS) to create 4 groups: (1) resolving HRF/-ARDS, (2) resolving HRF/+ARDS, (3) persistent HRF/-ARDS, and (4) persistent HRF/+ARDS. We compared RR of mortality in each group to the reference of resolving HRF/−ARDS using the methods from our primary analysis. In a second exploratory analysis, we examined whether previously described hyperinflammatory and hypoinflammatory subphenotypes have any relationship to persistent HRF , 40–43. Observations with missing covariates were excluded from each model. Analyses were conducted in STATA (version 16.0).2 than patients with resolving HRF in the discovery cohort, and all patients in both cohorts were on at least 5 cm H2O positive end-expiratory pressure.We identified 768 patients with acute HRF in the discovery cohort, and 1715 patients in the validation cohort . Adjusting for age, sex, chronic respiratory disease, and APACHE-III, persistent HRF carried a 1.68-fold higher  risk of death in the discovery cohort  and endothelial injury (higher Ang-2) compared to resolving HRF in the validation cohort Fig. . Only sT2:FIO2 < 150 magnified biologic differences between groups.Sensitivity analyses produced similar results , followed by persistent HRF/-ARDS (15%), resolving HRF/+ARDS (10%), and resolving HRF/-ARDS (7%) Table . PersistPatients with persistent HRF, both with and without ARDS, exhibited similar biomarker profiles characterized by inflammation and endothelial injury  and validation cohorts (79% vs. 63% P < 0.001). However, since few patients were classified as hyperinflammatory, far more hypoinflammatory patients than hyperinflammatory developed persistent HRF.Patients with hyperinflammatory HRF were more likely to develop persistent HRF compared to patients with hypoinflammatory HRF in the discovery (61% vs. 45%, In two independent cohorts of acute HRF, we found that patients with persistent HRF 3 days following ICU admission had a markedly higher risk for subsequent in-hospital mortality compared to patients with resolving HRF, even after adjustment for initial illness severity. Cumulative incidence of persistent HRF was over 50% in both cohorts, which included a large, diverse population of patients from medical, surgical, and trauma ICUs. Patients with persistent HRF had evidence of greater systemic inflammation and endothelial dysfunction/activation on enrollment compared to resolving HRF. Finally, we saw that these findings were not driven only by patients with ARDS; rather, patients with persistent HRF who did not meet ARDS criteria represented a prevalent group with poor prognosis and elevated markers of lung injury. Overall, this work illustrates that ARDS is not merely one end of the spectrum of disease severity in acute HRF and that characterizing clinical and biologic features associated with persistent HRF regardless of ARDS diagnosis may generate new insight into what drives outcomes in respiratory failure.2:FIO2 and APACHE score. Furthermore, our sensitivity analysis of patients with PaO2:FIO2 < 150 diminished differences in initial illness severity while strengthening the effect of persistent HRF on subsequent mortality. In fact, over half of the resolving HRF patients in our study had PaO2:FIO2 < 150 at baseline, indicating that many patients who begin their ICU course with serious respiratory failure improve quickly. The prognostic value of this early trajectory, and the disconnect between initial severity of respiratory failure and subsequent outcomes, has not previously been investigated in acute HRF but has been described in ARDS [2:FIO2 at onset of respiratory failure is not as consistently associated with overall mortality as PaO2:FIO2 measured at later time points, and in our study too, we saw the overall mortality among patients with enrollment PaO2:FIO2 < 150 was similar to that of the entire cohort [We observed significant differences in mortality, VFD, and length of stay between persistent and resolving HRF. While the findings may in part reflect the milder illness among patients with resolving HRF at enrollment, we hypothesize patients with persistent and resolving HRF have other underlying reasons to explain differing trajectories. Supporting this hypothesis, the risk of death was different even after adjusting for baseline PaO in ARDS , 45–47.  in ARDS . A study in ARDS . In genee cohort . These dEnrollment of patients with persistent HRF into clinical trials of therapies for respiratory failure could help select for patients at higher risk for disease-related outcomes, a strategy known as prognostic enrichment . There iAnother important strategy to improve therapeutic trials is selecting patients with shared biologic mechanisms that are likely to respond to a specific therapy, known as predictive enrichment. Although we do not directly examine whether persistent and resolving HRF have differential responses to therapy, we do show that patients with persistent HRF have higher systemic inflammation and endothelial dysfunction. These patients could be targeted for such emerging therapeutic strategies as early immune modulation and endothelial stabilization/repair . AdditioMost notably, we showed that 48% of persistent HRF patients in the discovery cohort and 54% in the validation cohort have high mortality and elevated circulating markers of lung injury, yet are not captured in ARDS definitions. Data on these patients with acute HRF who do not meet ARDS criteria remain limited. One large epidemiologic study of acute respiratory failure from Northern Europe also showed patients who did and did not meet ARDS criteria had similar mortality, although this study was done before the dissemination of lung-protective ventilation and the development of the Berlin definition . In our 2:FIO2 is an established measure of the severity of respiratory failure, it is subject to change with ventilator management independent of changes in clinical status [This study has several limitations. First, while we used a broad range of baseline clinical information to control for differences between groups, there may be residual confounding due to unmeasured differences in early clinical care. Second, while the biomarkers measured have been associated with ARDS outcomes and severity, they may not be lung-specific and could reflect processes outside of the lung. Third, the platforms used to measure these biomarkers were not the same in both cohorts. While we would expect this difference to bias our results to the null, it could partially explain discrepancies between cohorts in the relationship between sTNFR-1 and persistent HRF. Fourth, while PaOl status , 56, andl status . AlthougAmong mechanically ventilated patients with acute HRF, the subphenotype of persistent HRF on ICU day 3 identifies patients with high mortality and prolonged respiratory failure. Patients with persistent HRF have distinctive features early in the course of illness, such as elevated markers of inflammation and endothelial dysfunction. Patients with persistent HRF, both with and without ARDS, may warrant inclusion in trials of targeted therapeutics for respiratory failure.Additional file 1. Supplemental methods, figures, and tables.