To evaluate the planned implementation of group based Cognitive Remediation therapy (CR) and Social Cognitive Interaction Training (SCIT) into routine psychosis care in a mental health service in Australia.The study was conducted over 3 years in a mental health service in a metropolitan city in Australia. Participants were 22 program facilitators and 128 patients attending the programs. Implementation outcomes were assessed using administrative data, staff surveys and program audits.There was fidelity to the particular therapies at a program level. Programs were assessed as being feasible within the study setting with each hospital district developing a capacity to run CR and SCIT. The establishment of new programs improved the reach, but waiting lists indicate a need to expand capacity. There was a relatively high dropout and several factors impacted on completion of the programs - notably, acute exacerbation of psychosis. Once initiated the therapies were acceptable with no-one ceasing SCIT due to loss of interest and only 10% of participants ceasing CR due to loss of interest. Annual audits of programs found programs established were maintained and facilitators were retained.SCIT and CR programs were successfully implemented in three hospital districts. Several factors impeded participants receiving the recommended “dose” of the programs. The maintenance of the programs in the short term is encouraging in regards to organisational fit. Dissemination of cognitive rehabilitation programs to a service population takes planning. An implementation plan is essential for guiding development and maintenance of programs. These therapies are best suited to people in a stable phase of illness. Service user co-production is recommended to improve recruitment in future studies.The online version of this article (10.1186/s12913-018-3240-5) contains supplementary material, which is available to authorized users. Comprehensive care for people diagnosed with psychosis includes access to adjunctive evidence based psychosocial interventions , 2. CognIn an implementation framework program implementation outcomes  are delineated as a distinct outcome category that influences service and clinical program outcomes . ImproveCognitive impairment is common in people diagnosed with schizophrenia , 9. The There is also evidence from a meta-analysis of the effectiveness of interventions remediating social cognitive deficits in schizophrenia . Social Four CR programs and four SCIT programs had been established in Brisbane’s Princess Alexandra Hospital (PAH), and the associated health district prior to this study commencing. These programs commenced following staff training in CR in 2008 and SCIT in 2010. In 2012 the service restructured into teams based on diagnosis or model of service (e.g. rehabilitation) to encourage the development of core diagnostically relevant evidence based practices and to more equitably distribute resources. This restructure gave organisational impetus to apply implementation theory to the issue of limited implementation and dissemination of cognitive therapies for psychosis.Staff from the service involved in psychosis care, Psychosis Academic Clinical Unit (ACU) and Rehabilitation ACU in which the study was carried out developed a plan to guide implementation of CR and SCIT in the 3 geographically defined hospital health districts of the service. The implementation plan was organised around the stages of implementation: exploration/adoption; initial implementation; full implementation and program maintenance . The expThis evaluation study was conducted over three years from 2013 to 2015 and collected information on the implementation outcomes  of CR and SCIT programs implemented.The study setting was the community mental health services of a metropolitan health service (MHS) with a population of around 920,000 in Brisbane, Australia. The health service comprises three hospital health districts . It has been estimated that approximately three thousand people residing in this area have severe and persistent mental illness with complex needs . The area has high rates of socioeconomic disadvantage with 19.8% of people postponing mental health care because of cost . The geographic area covers inner metropolitan suburbs to semi-rural areas. There are three hospitals in the geographically defined health districts.Both programs were facilitated by a multidisciplinary group of therapists who had identified an interest in becoming CR and SCIT therapists and had completed the requisite training and credentialing . The thePeople diagnosed with a schizophrenia spectrum disorder who were being treated in the community, aged between 18 and 65, and who did not have an intellectual impairment or active substance abuse disorder were eligible to participate. Service users could participate in the program and not consent to the evaluation research project.The characteristics of the programs implemented are listed in Table SCIT is based on a manual and uses some resources available freely online. SCIT is run as a closed small group of a maximum of 8 participants and 2 facilitators. The program is structured to build skills over 3 phases focussing in phase 1 on emotional processing; phase 2 focussing on thinking skills in social situations; and phase 3 integrating these skills into real life social situations experienced by participants.Implementation outcomes were assessed using data from multiple sources including yearly staff surveys, program audits, and measures of program feasibility.This survey asked questions about staff interest, training and current practice with 8 questions about cognitive behavioural therapy (with SCIT classified as a CBT derived therapy) and 5 questions related to CR. The survey was distributed yearly for the three years of the study to clinical staff in the community psychosis and rehabilitation teams.Program audits were conducted annually by the program co-ordinator VGJ. For CR the number of programs running in each service, the maintenance of programs established, the number and retention of facilitators, the number of computers and funding was recorded. For SCIT the number of programs conducted and number and retention facilitators was noted.No fidelity instrument had been developed for CIRCuiTS. Program fidelity was therefore measured using an instrument developed by Alice Medalia and colleagues for the Neuro psychological Educational Approach to Cognitive Remediation (NEAR) program which was adapted for the CIRCuiTS and SCIT programs . There were 12 criteria for CR programs: clinician credentials and training; supervision; software used and selection (NEAR only); referral process; assessment; personalised folders; application of assessment; specific CR techniques used; intrinsic motivation; time on each program (NEAR only); software features (NEAR only) and communication with referring team. The 7 criteria for SCIT program fidelity were: clinician credentials and training; supervision; manual adherence; referral process; assessment process; application of assessment; and demonstration of specific SCIT techniques. Each criterion was rated by FD on a 5 point scale: from not achieved; occasionally achieved; frequently achieved; infrequently not achieved; and 100% adherence.A three-item measure of transportability, acceptability and feasibility was used by the SCIT program facilitators at the end of the program. This measure had been used in a previous study by the SCIT program developers . The iteThis measure has not been previously used in CR studies and was not used to evaluate the feasibility or acceptability of CR in this study. The number of people referred to CR who commenced, whether consenting to the research or not, and reasons for discontinuing the program was used as a proxy measure of acceptability of the program.Cost outlays for the programs included labour and non-labour expenses over the three years. Labour costs were calculated based on an average therapist’s hourly award wage. Therapists spent 2 h per week conducting the groups with SCIT being held once a week for 2 h and CR 1-h sessions twice a week. The costs of acquiring new non-labour items were included in the calculation. For CR this included the cost of purchasing computers and internet connection.Descriptive statistics were used to report the implementation outcomes.n = 40) were aged between 26 and 60 with a mean age of 42 (SD = 10.4). They had between 6 and 15 years of education with a mean of 10.4 years (SD =2.0). More males (67%) than females (33%) consented to the study. Twenty-five percent were living in Department of Housing accommodation; 25% were living in private dwellings; 40% in residential rehabilitation units; and 10% where the accommodation was unspecified. Of the 19 participants who completed 10 or more sessions, 68% were male and 32% female.The consenting participants who commenced CR (n = 33) was 19 to 50 years with a mean of 30.9 years (SD = 8.6 years). They had between 6 and 15 years of education with a mean of 10.8 years (SD = 2.2 years). More males (67%) than females (33%) consented to the study. Fifty- 1 % lived in a residential rehabilitation unit; 46% in a private dwelling; and 3% accommodation was unspecified. Of the 22 participants who completed 16 or more sessions, 64% were male and 36% female.The age range of participants consenting and who commenced SCIT .All 3 hospitals in the geographically defined health districts participated. The estimated total population of patients diagnosed with schizophrenia spectrum disorders in the service at the time of the study was 1660. From this population 105 people were referred to the CR programs and 53 were referred for the SCIT programs. All sites had staff from multiple disciplines trained and competent to deliver both therapies. CR was run as an open group. The SCIT groups were run as a closed group once or twice a year. Three SCIT programs were conducted in the hospital district that had previously run pilot SCIT programs and 1 program ran at each of the other two hospital districts.Each hospital district was serviced by at least 1 CR and 1 SCIT program. The inner city hospital district had 4 CR programs established and 3 SCIT programs were conducted. CR was conducted at the residential rehabilitation units but accepted referrals from the community clinics in Logan and Bayside Hospitals and districts. The referring community clinics have made requests for access to programs that were at full capacity necessitating the creation of waiting lists. Based on this unmet demand a further 2 CR programs and 2 SCIT programs are estimated to be needed for the community teams.Of the 89 people referred to CR, 52 consented to participating in the evaluation research. Twelve of the 52 consenting failed to commence the program. Of the 40 participants consenting and commencing the CR program, 21 completed at least 10 sessions. Nineteen of the research participants discontinued CR before completing 10 sessions. Seven participants had time constraints, usually due to family responsibilities; 3 were hospitalised due to acute deterioration in mental state; 4 moved area and had trouble accessing the program and for one participant no reason for discontinuation was given. All cases of acute deterioration in mental health were considered by their clinical team as not relating to the programs. Where possible an interview of participants on exiting the program early was used to explore satisfaction with the CR program. Only 4 participants (10%) stopped attending CR due to loss of interest in the program.Of the 53 eligible people referred to SCIT, 39 consented to be involved in the study. Six of the 39 did not commence. Of the 33 commencing SCIT, 24 completed more than 16 sessions which was considered as having completed the program. Eight failed to complete the program due to acute deterioration requiring hospitalisation and no reason was recorded in one case. All cases of acute deterioration in mental health were considered by their clinical team as not relating to the programs. Program acceptability was recorded for SCIT using a three item measure (Table Over the 3 years of the study (2013–2015) the number of CR programs doubled to 8. The 3 programs running in the PAH district and 1 program running at Logan hospital district at baseline were still active at the end of the study period. In 2014 an additional 2 programs were established . Another 2 programs were established in Bayside in 2015. All established programs were maintained at the last audit in December 2015. In 2013 there were 10 program facilitators. These facilitators were retained and 3 new trained facilitators commenced in 2014. The 13 trained facilitators were retained at the end point audit in December 2015. In 2014 ongoing funding was secured to maintain internet connection and 12 new computers were purchased to replace old computers and to support the new programs.SCIT programs were run each six months in the PAH hospital district (2013–2015) and one program at Bayside and Logan in the last year of the project. The PAH had previous experience with SCIT and the Bayside and Logan programs were run at the residential rehabilitation services that had only opened in 2015. These programs are ongoing following the end of the study and scheduled to run every six months depending on demand. Ten therapists were trained in the mental health service and were still running groups at the last audit in 2015.The total expenditure on CR programs including labour and non-labour was $32,848 over the three years (2013–2015). The use of some donated computers lowered costs for purchasing computers. The SCIT programs involved only labour costs of $11,520  that had established the initial programs to the other two hospitals (Logan and Bayside) due to the large geographical area and the relative expense of public transport for this population. Support for these programs was provided from the central “hub” via supervision either face to face or via teleconferencing. Four participants who moved areas failed to complete CR implying that lack of local access can influence the feasibility of attending programs.For new programs to become embedded in routine care, the organisational fit, feasibility and acceptability of the programs to staff need to be considered . In thisReasons for discontinuing CR early were collected. The overall attrition rate for those consenting to the evaluation was high (55%). Wykes in a meta-analysis found the overall attrition rate for CR to be 11% with a range of 0–47.5% . In this study in relation to acceptability, once initiated only 10% ceased the CR program because of loss of interest. In both programs acute illness resulted in early exit from the programs reflecting the relapsing nature of schizophrenia. Accurate assessment of the utilization of the CR program was not possible due to the low rates of participants consenting to research . Previous pilot studies of SCIT found lower attrition in participants living in residential settings . A signiA formal economic analysis was not included in the protocol. Cost outlays are higher for CR than for SCIT due to more sessions being conducted and the use of computers and internet Table . PreviouImplementation studies are inherently context specific making extrapolation to other services difficult. These results are particularly relevant to the Australian public mental health sector. The higher program participation, while not consenting to research, also biases the results, limiting extrapolation to the population of people who attended the programs.The 3 year implementation timeframe for this study is brief given the complexity of the interventions. The study site had experience in the programs (exploration phase of implementation) and this needs to be considered in relation to the overall time required to implement these programs.The study did not involve a formal cost-effectiveness analysis. This information is needed to help prosecute the case for funding the implementation of these therapies in the context of increasingly constrained health budgets.Implementation theory and practice can help guide the implementation of evidence based therapies recommended into routine, comprehensive psychosis care. Having a staged plan over time is required to address the complex workforce, organisational and quality issues that are inherent in delivering cognitive therapies for psychosis. Service user involvement in the design and running of psychosocial programs is recommended.Additional file 1:SSPARS BMC Staff survey. (DOC 28 kb)


Phytosterols, found in many commonly consumed foods, exhibit a broad range of physiological activities including anti-inflammatory effects. In this study, the anti-inflammatory effects of ergosterol, β-sitosterol, stigmasterol, campesterol, and ergosterol acetate were investigated in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. Results showed that all phytosterol compounds alleviated the inflammatory reaction in LPS-induced macrophage models; cell phagocytosis, nitric oxide (NO) production, release of tumor necrosis factor-α (TNF-α), and expression and activity of pro-inflammatory mediator cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and phosphorylated extracellular signal-regulated protein kinase (p-ERK) were all inhibited. The anti-inflammatory activity of β-sitosterol was higher than stigmasterol and campesterol, which suggests that phytosterols without a double bond on C-22 and with ethyl on C-24 were more effective. However, inconsistent results were observed upon comparison of ergosterol and ergosterol acetate (hydroxy or ester group on C-3), which suggest that additional research is still needed to ascertain the contribution of structure to their anti-inflammatory effects. Inflammation is a natural response of the immune system to damage from physical, chemical, and pathogenic factors . A generInnotus obliquus indicated that phytosterols significantly inhibited the level of interleukin-1β (IL-1β), IL-6, and TNF-α in RAW246.7 macrophage cells [I. obliquus, and they indicated that ergosterol and ergosterol peroxide could inhibit NO production and nuclear factor-κB (NF-κB) luciferase activity in RAW246.7 macrophage cells [Currently, although nonsteroidal anti-inflammatory drugs such as aspirin and phenylbutazone are used in the treatment of inflammation induced by tissue damage, they were shown to have harmful side effects in clinical practice . Explorage cells . Ma et aStudies on the signaling pathway of phytosterols’ anti-inflammatory activity mainly involved the NF-κB and mitogen-associated protein kinase (MAPK) pathway. As for the NF-κB pathway, TNF-α increases NF-κB through enhancing the inhibitor of NF-κB (IκB) kinase β (IKKβ) function, and the ubiquitination of inhibitor of NF-κB 3 (IκB3), leading to the suppression of NF-κB, causes inflammatory responses ,26,27. RVillorita cyprinoides suggested that the anti-inflammatory activity of sterols is inversely related to their steric bulk, electronic, and hydrophobic features [Although the anti-inflammatory effects of phytosterols were investigated before, the role that their structure plays in modulating inflammatory responses is still obscure. A previous study of sterols from corbiculid bivalve clam features ,33. TakiPhytosterol compounds including ergosterol (purity: 98%), stigmasterol (purity: 97%), β-sitosterol (purity: 98%), campesterol (purity: 98%), and ergosterol acetate (purity: 98%) were purchased from Toronto Research Chemicals Inc. . The RAW264.7 cell line was purchased from the Type Culture Collection of Chinese Academy of Sciences . Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Hyclone . Cell Counting Kit-8 (CCK-8) was purchased from Japanese Dojindo Laboratories . Phosphate-buffered saline (PBS) was obtained from Chinese fir in Jinqiao . Lipopolysaccharide (LPS), fluorescein isothiocyanate (FITC)-dextran, and neutral red were purchased from Sigma Chemical Co. . NO Test Kit was purchased from Beyotime Biotechnology . A TNF-α enzyme-linked immunosorbent assay (ELISA) kit for mice was obtained from Wuhan Boster Biological Technology . COX-2 and iNOS activity assay kits were purchased from Nanjing Jiancheng Bioengineering Institute. Enhanced bicinchoninic acid (BCA) Protein Assay Kit, lysis buffer, phenylmethanesulfonyl fluoride (PMSF), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (5×), and Electrochemiluminescence (ECL) Kit were purchased from Beyotime Biotechnology . Bull serum albumin (BSA) was bought from Sigma-Aldrich . Rabbit anti-COX-2, iNOS, ERK, and p-ERK antibodies were purchased from Cell Signaling Technology (USA). The β-actin antibody, antibodies against rabbit immunoglobulin (Ig) horseradish peroxidase (HRP) and antibodies against mouse Ig-HRP were obtained from Chinese fir in Jinqiao . All other chemicals were of analytical reagent grade and were purchased from Shanghai Chemicals and Reagents Co. .2. Cells were used for study when they attained approximately 70–80% confluence.RAW264.7 cells were recovered from a liquid nitrogen tank, and then cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in an atmosphere containing 5% COAll phytosterol compounds were dissolved in complete medium to obtain 200-μM stock solutions. These phytosterols solutions were then filtered through a 0.22-μm membrane filter and stored in brown bottles at 4 °C for subsequent experiments.4 cells/well and cultured in an incubator maintained at 37 °C with 5% CO2 for 4 h. Afterward, the culture medium was removed and the cells were treated with 200 μL of culture medium containing 1 μg/mL LPS , or 200 μL of culture medium containing 1 μg/mL LPS  and phytosterols at different concentrations . Meanwhile, 200 μL of culture medium without phytosterols and LPS was added as a control group. After incubation for 24 h, the culture medium containing 10% CCK-8 was added, and the model, control, and experimental groups were incubated at 37 °C for 1 h. The absorbance value was measured at 450 nm on a microplate reader . Cell proliferation was calculated using the following equation:A1 is the absorbance of the control group, and A2 is the absorbance of the experimental or model group.The effects of tested compounds on the proliferation of RAW264.7 cells were simultaneously determined using CCK-8 assay. Briefly, RAW264.7 cells were seeded onto 96-well plates at a density of 105 cells/well, while wells with equal volumes of culture medium were set as the blank group. After 4 h of incubation, the culture medium was removed and cells were treated as mentioned above (v/v) in a fume hood at room temperature for 2 h. The absorbance value at 540 nm was recorded by a microplate reader  [bA is the value of the control, experimental, or model group, cA is the value of the model group, and A0 is the value of blank group.RAW264.7 cells were seeded in 96-well plates at a density of 1 × 10ed above . After iMA, USA) . Cell ph6 cells/well. After 4 h of incubation, the culture medium was removed, and the cells were treated with 2 mL of culture medium containing 1 μg/mL LPS , or 2 mL of culture medium containing 1 μg/mL LPS  and phytosterols at different concentrations . Meanwhile, 2 mL of culture medium without phytosterols and LPS was added as the control group. After incubation for 24 h, the culture medium was removed, and the cells were washed two times with PBS; then, 100 μL of FITC-dextran (1 mg/mL) was added, and the cells were covered with tin foil paper to protect from light and incubated at 37 °C for 1 h. After incubation, 1 mL of PBS was added. Then, RAW264.7 cells were harvested and washed three times with PBS, before being resuspended in 500 μL of PBS buffer. The stained cells were then analyzed by a BD FASCalibur flow cytometer  [RAW264.7 cells were seeded in six-well plates at a density of 1 × 10NJ, USA) ,36.6 cells/mL concentration in six-well culture plates. After 4 h of incubation, the cells were divided into control, experimental, and model groups, and were treated as mentioned above .The cytokine TNF-α secreted in the culture medium was measured using the ELISA method. RAW264.7 cells under exponential growth were seeded at a concentration of 1 × 10as above . After tg for 10 min at 4 °C; then, the supernatant was stored at −80 °C for protein detection and immunoblot analysis. Protein concentrations were determined by the Enhanced BCA Protein Assay Kit.RAW264.7 macrophage cells were treated as mentioned above . Then, tRAW264.7 macrophage cells were treated as mentioned above .. Then, Protein extracts were mixed with one-quarter volume of SDS-PAGE sample loading buffer (5×), and then were heated to 95 °C for 5 min. Proteins (30 μg) were separated by SDS polyacrylamide gel electrophoresis and were transferred electrophoretically onto an immobilon membrane  using a blotter  at 80 V for 30 min and then 120 V for 60 min. The membrane was blocked with Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and 3% BSA for 1 h at room temperature. After washing with TBS-T, the membrane was incubated for 1 h at room temperature in TBS-T containing 0.1% BSA, and then 1000-fold diluted anti-COX-2, iNOS, ERK, and p-ERK tag antibodies were added. For detection, the membrane was allowed to bind to 10,000-fold diluted antibodies against rabbit Ig-HRP in TBS-T containing 0.1% BSA for 1 h at room temperature. The protein expression was normalized by β-actin protein expression level; in other words, the gray value of β-actin bands was used as the internal standard to calculate the relative expression of target proteins. The protein expression was assessed using an ECL Plus Western Blotting Detection System  with a luminescent image analyzer (Bio-Rad).p < 0.05.Data of each group were expressed as means ± standard error of the mean (SEM). SPSS16.0 statistical software was used for statistical analysis. The statistical significance of the results was evaluated by one-way analysis of variance (ANOVA) followed by the Duncan’s multiple range test. Origin 8 software was used for graphic drawing. Win MID 2.9 software was used to analyze the results of flow cytometry. Quantity One software was used to analyze the results of Western blot. All means were calculated from six independent experiments, and the difference was considered significant when p < 0.05). As shown in p < 0.05). In addition, the cell proliferation of ergosterol, ergosterol acetate, and β-sitosterol groups at 200 μM was still not significantly different from that of the control group, while cell proliferation of the corresponding campesterol and stigmasterol groups was lowered and enhanced, respectively. LPS (1 μg/mL) could promote the proliferation of RAW264.7 cells, and 25–200 μM ergosterol, stigmasterol, β-sitosterol, campesterol, and ergosterol acetate exhibited little or no cytotoxicity on RAW264.7 cells (p < 0.05). It was reported that LPS stimulates and induces RAW264.7 macrophages, which in a short period of time can cause fluctuations in macrophage cell proliferation. The RAW264.7 macrophages were treated with LPS for 24 h, and the cell proliferation was obtained during the theoretical fluctuation period, and these results were in accordance with previous publications [To avoid cytotoxicity, the proliferation of RAW264.7 cells stimulated by LPS and treated with various concentrations of ergosterol, stigmasterol, β-sitosterol, campesterol and ergosterol acetate were detected by the CCK-8 method. The concentrations of phytosterols used in this study were based on our pre-experiment, in which the cell activity was tested at the concentrations of 12.5, 25, 50, 100, 200, and 400 μM, where the results showed that no anti-inflammatory effect was observed at the concentration of 12.5 μM and that the phytosterols could not be dissolved completely at the concentration of 400 μM; thus, concentrations of 25, 50, 100, and 200 μM were chosen for this study. In our study, the cell proliferation of the model group was significantly higher than that of the control group when compared together with the experimental groups of 25, 100, and 200 μM, but no significant difference between model group and control group was observed when compared together with the experimental group of 50 μM . As for the experimental group of 50 μM, the inhibitory effects of ergosterol acetate, ergosterol, and β-sitosterol groups were at the same level, followed by the campesterol group, while the stigmasterol group showed almost no significant inhibitory effects (p < 0.05). Next, when treated with 100 μM phytosterol compounds, there was no significant difference in the inhibition of phagocytosis among ergosterol acetate, ergosterol, and campesterol groups, while weaker inhibitory effects could also be observed in the β-sitosterol and stigmasterol groups (p < 0.05). When treated with 200 μM phytosterol compounds, ergosterol acetate, ergosterol, campesterol, and β-sitosterol exhibited similar inhibitory effects on phagocytic activity, and they were more effective than stigmasterol (p < 0.05). Taking into consideration the minimum effective concentration of phytosterols and the inhibitory effects of phytosterols at the same level, it can be concluded that stigmasterol produced the weakest inhibitory effects on phagocytosis of neutral red, followed by ergosterol acetate. The inhibitory effects of β-sitosterol and campesterol were not significantly different and were stronger than those of ergosterol acetate but weaker than those of ergosterol.Phagocytosis is a self-protection mechanism of somatic cells, and the detection of phagocytic activity of macrophages is a method reflecting the immune status of the body . EffectsIn order to explore the structure–activity relationship of these phytosterol compounds, the anti-inflammatory activity of phytosterols with different structures were compared in pairs: ergosterol and ergosterol acetate ; β-sitosterol and stigmasterol (with or without a double bond on C-22); β-sitosterol and campesterol (methyl or ethyl on C-24). According to these results, the inhibition of phagocytosis in the ergosterol group was higher than that in the ergosterol acetate group; in other words, phytosterol compounds with a hydroxyl on C-3 were more effective than those with an ester on C-3 in the inhibition of phagocytosis. The inhibition of phytosterols on phagocytosis of neutral red was in the order of β-sitosterol (without a double bond on C-22) > stigmasterol (with a double bond on C-22), which suggested that, in the inhibition of phagocytosis of neutral red, phytosterols without a double bond on C-22 were more effective than those with a double bond on C-22. Moreover, the inhibitory effects of β-sitosterol and campesterol were not significantly different, which suggested that their inhibitory effects were not correlated with the functional groups (methyl or ethyl) on the C-24 position of phytosterols.p < 0.05). When RAW264.7 cells were treated with 50 μM phytosterols, similar inhibitory effects were observed. β-Sitosterol exhibited the strongest inhibitory effects, followed by ergosterol or ergosterol acetate. The inhibitory effects of campesterol were stronger than those of stigmasterol but lower than those of ergosterol acetate or ergosterol (p < 0.05). At the concentration of 100 μM, the inhibitory effects of ergosterol acetate, ergosterol, and β-sitosterol were similar, which were higher than those of campesterol; the lowest inhibition was observed for the stigmasterol group (p < 0.05). Next, at the concentration of 200 μM, ergosterol was the most effective, followed by β-sitosterol, ergosterol acetate, or campesterol. The lowest inhibition was observed for the stigmasterol group (p < 0.05). In The phagocytic activity of RAW264.7 macrophages in the control, experimental, and model groups was further explored and confirmed by the FITC-dextran method. In The inhibition on phagocytic activity of phytosterols was in the order of β-sitosterol (ethyl on C-24) > campesterol (methyl on C-24), β-sitosterol (without a double bond on C-22) > stigmasterol (with a double bond on C-22), and ergosterol (hydroxyl on C-3) > ergosterol acetate (ester group on C-3). It can be speculated that phytosterol compounds with a hydroxyl on C-3 may be more effective in inhibiting phagocytosis than those with an ester on C-3; these results were consistent with those detected by the neutral red method. Furthermore, the comparison between the inhibitory effects of β-sitosterol and campesterol suggested that phytosterols without a double bond on C-22 were more effective than those with a double bond on C-22, and that phytosterols with an ethyl on C-24 were more effective than those with a methyl on C-24.2 solutions, and the correlation coefficient (R2) was above 0.999. Then, concentrations of NO in the experimental or control group were calibrated and compared with those in the model group (as 100%) (p < 0.05). Also, their inhibitory effects on NO production exhibited dose dependence. When RAW264.7 cells were treated with phytosterols at the concentration of 100 μM, the NO production in experimental groups was reduced significantly, and no significant differences in the inhibitory activity were observed between ergosterol, ergosterol acetate, campesterol, and β-sitosterol groups. The stigmasterol group exhibited the lowest inhibitory activity in NO production (p < 0.05). As for the experimental group of 200 μM, campesterol, ergosterol acetate, and β-sitosterol had similar inhibition activity in NO production. The inhibitory activity of ergosterol and ergosterol acetate was stronger than that of stigmasterol but lower than that of β-sitosterol (p < 0.05). The general trend, as observed in It is reported that NO is an inflammometer which can modulate cellular signaling involved in inflammation. In RAW264.7 macrophages, detection of the NO production induced by LPS is considered as a convenient and credible method for anti-inflammation screening ,41. The as 100%) . ResultsAccording to these results, neither a hydroxyl or ester on C-3, nor an ethyl or methyl on C-24 affected inhibition of the production of NO in RAW264.7 macrophages. Also, the inhibition on NO production was in the order of β-sitosterol (without a double bond on C-22) > stigmasterol (with a double bond on C-22), which suggested that phytosterols without a double bond on C-22 may be more effective in inhibiting NO production than those with a double bond on C-22.R2) above 0.99. Then, concentrations of TNF-α in the experimental or control group were calibrated and compared with those in the model group (as 100%). In p < 0.05). As for the experimental groups of 50 μM, β-sitosterol exhibited the strongest inhibitory effect, followed by stigmasterol. The lowest inhibition was observed for the ergosterol or campesterol groups (p < 0.05). When treated with 100 μM phytosterol compounds, the β-sitosterol or stigmasterol groups exhibited the strongest inhibitory effects, followed by the ergosterol or campesterol groups (p < 0.05). Next, at the concentration of 200 μM, the TNF-α production was inhibited in all experimental groups, where the inhibition effects of β-sitosterol and stigmasterol were similar, higher than those of ergosterol or campesterol. The lowest inhibition was observed for the ergosterol acetate group (p < 0.05). The general trend, as observed in The production of cytokines, such as TNF-α, occurs in response to the inflammatory reaction induced by LPS in RAW264.7 macrophages. Therefore, the suppression of phytosterols on TNF-α production in inflammatory models was tested to indicate their anti-inflammatory activity ,45,46. TThe inhibition of phytosterols on TNF-α production was in the order of β-sitosterol (without a double bond on C-22) > stigmasterol (with a double bond on C-22), β-sitosterol (ethyl on C-24) > campesterol (methyl on C-24), and ergosterol (hydroxyl on C-3) > ergosterol acetate (ester on C-3), which suggested that, in the inhibition of TNF-α production, phytosterols with a hydroxyl on C-3 were more effective than those with an ester on C-3, phytosterols without a double bond on C-22 were more effective than those with a double bond on C-22, and phytosterols with an ethyl on C-24 were more effective than those with a methyl on C-24.p < 0.05)  .p < 0.05). At the concentration of 50 μM, the effect of ergosterol acetate was at the same level as ergosterol, while weaker inhibition was observed for campesterol, β-sitosterol, and stigmasterol groups (p < 0.05). At the concentration of 100 μM, ergosterol acetate showed the strongest inhibition on COX-2 expression, followed by ergosterol and stigmasterol, and the weakest effects were found for campesterol and β-sitosterol groups (p < 0.05). At the concentration of 200 μM, the suppression was in the order of ergosterol acetate, ergosterol, and β-sitosterol > stigmasterol > campesterol (p < 0.05). In general, the ergosterol group showed the strongest inhibition, while weaker effects were observed for ergosterol acetate and β-sitosterol groups, and the lowest suppression of COX-2 expression was observed for stigmasterol and campesterol groups. The inhibition activity of ergosterol on COX-2 expression was higher than that of ergosterol acetate, while that of β-sitosterol  was higher than that of campesterol  and stigmasterol ; all these results suggested that ergosterol (hydroxyl on C-3) was more effective than ergosterol acetate (ester on C-3) in suppressing COX-2 expression, with an ethyl on C-24 may be more effective than a methyl on C-24, and the lack of a double bond on C-22 may be more effective than with the presence of a double bond on C-22.As shown in p < 0.05). At the concentration of 50 μM, stigmasterol was the most effective, followed by β-sitosterol, and the weakest inhibitory effects were observed for ergosterol acetate, ergosterol, and campesterol groups (p < 0.05). At the concentration of 100 μM, β-sitosterol and stigmasterol were the most effective, followed by ergosterol acetate and campesterol, and the weakest inhibitory effects were observed for the ergosterol group (p < 0.05). At the concentration of 200 μM, the inhibitory effects among campesterol, β-sitosterol, and stigmasterol were similar, and were stronger than those of ergosterol acetate, while the weakest inhibitory effects were observed for the ergosterol group. As for the relative expression of iNOS, the general trend was that the stigmasterol and β-sitosterol groups showed the strongest inhibition, while weaker effects were observed for the ergosterol acetate and campesterol groups, and the lowest suppression was observed for the ergosterol group. The inhibition activity of β-sitosterol (ethyl on C-24) on iNOS expression was higher than that of campesterol (methyl on C-24), whereas the inhibition activity of ergosterol (hydroxyl on C-3) was lower than that of ergosterol acetate (ester on C-3); all these results suggested that phytosterols with an ethyl on C-24 may be more effective in suppressing iNOS expression than those with a methyl on C-24, and those with an ester on C-3 may be more effective than those with a hydroxyl on C-3.As shown in Furthermore, the COX-2 and iNOS activity of RAW264.7 macrophages in the control, experimental, and model groups was further explored and confirmed by enzymatic activity assay kits. As shown in p < 0.05). As shown in p < 0.05). At the concentration of 50 μM, campesterol and β-sitosterol showed obvious inhibitory effects, while other phytosterols did not affect the expression of p-ERK. As for the experimental groups of 100 μM, ergosterol acetate and β-sitosterol produced the strongest inhibition of p-ERK, followed by campesterol, while ergosterol and stigmasterol still did not affect the expression of p-ERK (p < 0.05). As for the experimental groups of 200 μM, ergosterol acetate and β-sitosterol produced the strongest inhibition of p-ERK, followed by ergosterol, campesterol, and stigmasterol (p < 0.05). Above all, the general trend could be observed that β-sitosterol showed the strongest inhibition, followed by ergosterol acetate, while weaker effects were observed for the campesterol group, and the lowest suppression was observed for the ergosterol and stigmasterol groups. The inhibition activity of β-sitosterol  on p-ERK was higher than that of campesterol  and stigmasterol ; moreover, the inhibition activity of ergosterol (hydroxyl on C-3) was lower than that of ergosterol acetate (ester on C-3). All these results suggested that phytosterols without a double bond on C-22 may be more effective in suppressing p-ERK than those with a double bond on C-22, while an ethyl on C-24 may be more effective than a methyl on C-24, and an ester on C-3 may be more effective than a hydroxyl on C-3 .To further examine the functional importance of phytosterols on the phosphorylation of extracellular signal-regulated kinase (p-ERK), we investigated the expression of ERK and p-ERK in macrophages stimulated with LPS in the presence (different concentrations) or absence of phytosterols. As shown in p < 0.05). When treated with 50 μM phytosterols, the inhibition of p-ERK/ERK content was in the order of stigmasterol > β-sitosterol > ergosterol acetate > campesterol > ergosterol (p < 0.05). As for the experimental groups of 100 μM, ergosterol acetate and stigmasterol produced the strongest inhibition of p-ERK/ERK content followed by β-sitosterol, while weaker effects could be observed for the ergosterol group, and campesterol had the weakest inhibition on LPS-induced p-ERK (p < 0.05). As for the experimental groups of 200 μM, ergosterol acetate produced the strongest inhibition of p-ERK/ERK content, followed by β-sitosterol and stigmasterol, while weaker effects could be observed for the ergosterol group, and campesterol had the weakest inhibition on LPS-induced p-ERK (p < 0.05). Above all, the general trend could be observed that the ergosterol acetate, β-sitosterol, and stigmasterol groups showed the strongest inhibition, followed by the ergosterol group, while weaker effects were observed for the campesterol group. The inhibition activity of β-sitosterol (ethyl on C-24) on p-ERK/ERK content was higher than that of campesterol (methyl on C-24), whereas the inhibition activity of ergosterol (hydroxyl on C-3) was lower than that of ergosterol acetate (ester on C-3); all these results suggested that phytosterols with an ethyl on C-24 may be more effective in suppressing phosphorylation of ERK than those with a methyl on C-24, whereas an ester on C-3 may be more effective than a hydroxyl on C-3.The p-ERK/ERK content is shown in The LPS-induced RAW264.7 macrophage model is usually employed as a model for studies on the inflammatory response and inflammatory mechanisms. Different inflammatory mediators are secreted by LPS-induced macrophages, such as TNF-α and NO . The phap < 0.05), which means that the inflammatory response stimulated by LPS was recovered by these phytosterols. In other words, phytosterol compounds could relieve the inflammatory reaction induced by LPS.Results showed that the phagocytic activity was significantly inhibited by the tested phytosterol compounds, which demonstrated that the administration of phytosterol compounds may have a certain effect on the immune response caused by inflammation. To detect the phagocytosis of RAW264.7 cells, two methods were chosen, the FITC-dextran method and neutral red method, which were widely used in previous studies . The resThere are two types of cyclooxygenase (COX-1 and COX-2) in the body; COX-1 is commonly expressed in tissues, while COX-2 is only expressed after stimulation, which is often used as an indicator of inflammation as it also interacts with other inflammation-related factors so as to affect the inflammatory process . MeanwhiAccording to previous studies, we speculated that the anti-inflammatory activity of phytosterol compounds tested in this study are related to the MAPK pathway in macrophages . MAPK beTo the authors’ best knowledge, few previous studies were reported on the structure–anti-inflammatory activity relationship of phytosterol compounds. In this study, the anti-inflammatory activity of phytosterols with different structure were compared in pairs: ergosterol and ergosterol acetate ; β-sitosterol and stigmasterol (with or without a double bond on C-22); β-sitosterol and campesterol (methyl or ethyl on C-24). As shown in In this study, the anti-inflammatory effects of phytosterols with different structure in RAW264.7 macrophages induced by LPS were investigated, and the results showed that inflammation responses were largely mediated via a reduction of phagocytic capacity and NO/TNF-α production, inhibiting the expression and activity of pro-inflammatory mediators COX-2, iNOS, and p-ERK. In addition, the anti-inflammatory activity of phytosterols with different structure were compared in pairs: ergosterol and ergosterol acetate ; β-sitosterol and stigmasterol (with or without a double bond on C-22); β-sitosterol and campesterol (methyl or ethyl on C-24). It can be deduced that the anti-inflammatory activity of β-sitosterol (without a double bond on C-22) was higher than that of stigmasterol (with a double bond on C-22), suggesting that the anti-inflammatory activity is correlated with the functional group (with or without a double bond) on the C-22 position of phytosterols. Similarly, β-sitosterol was more effective than campesterol, suggesting that the anti-inflammatory activity is correlated with the functional group (methyl or ethyl) on C-24 of phytosterols. However, comparison of the anti-inflammatory activity of ergosterol and ergosterol acetate was controversial in this study, suggesting that the functional group (hydroxy or ester group) on C-3 may play a role in the anti-inflammatory activity of phytosterols, but additional research is still needed to ascertain the contribution of structure to the anti-inflammatory effects. This study provides a reference for further research on the structure–activity relationship of phytosterols, indicating that ergosterol, β-sitosterol, stigmasterol, campesterol, and ergosterol acetate have potential for developing anti-inflammatory drugs or functional food additives.


Arid1a in mice. We demonstrate that ARID1A is essential for maintaining the frequency and function of hematopoietic stem cells and its loss impairs the differentiation of both myeloid and lymphoid lineages. ARID1A deficiency led to a global reduction in open chromatin and ensuing transcriptional changes affected key genes involved in hematopoietic development. We also observed that silencing of ARID1A affected ATRA-induced differentiation of NB4 cells, suggesting its role in granulocytic differentiation of human leukemic cells. Overall, our study provides a comprehensive elucidation of the function of ARID1A in hematopoiesis and highlights the central role of ARID1A-containing SWI/SNF complex in maintaining chromatin dynamics in hematopoietic cells.Precise regulation of chromatin architecture is vital to physiological processes including hematopoiesis. ARID1A is a core component of the mammalian SWI/SNF complex, which is one of the ATP-dependent chromatin remodeling complexes. To uncover the role of ARID1A in hematopoietic development, we utilized hematopoietic cell-specific deletion of  Hematopoiesis is a tightly regulated process that constantly generates enormous numbers of mature blood cells through a cascade of differentiation stages. Self-renewal, quiescence and differentiation of hematopoietic stem cells (HSC), as well as subsequent lineage choices into various blood cell types are precisely orchestrated by stage-specific transcriptional and epigenetic machineries. Chromatin state is transformed dynamically during differentiation  and epigARID1A in a wide range of tumors including gynecological, liver, gastric, and breast tumors [ARID1A is the most frequently mutated member of the SWI/SNF family and high incidence of inactivating mutations in varied cancers along with emerging functional studies postulate ARID1A as a novel tumor suppressor [ARID1A is mutated in acute promyelocytic leukemia (APL) [ARID1A BAF250a) is a principal component of SWI/SNF (SWItch/Sucrose Non-Fermentable) family of evolutionary conserved, multi-subunit chromatin remodeling complexes. Rearrangements of chromatin structure mediated by SWI/SNF complexes are critical for modulation of gene expression, thereby, affecting a broad range of cellular processes, including proliferation and differentiation [50a is a t tumors . ARID1A ppressor –15. Amonia (APL)  and seveia (APL) –26. Noneia (APL)  observedIn this study, we have employed hematopoietic-cell specific deletion of ARID1A in mice to address systematically its function in the hematopoietic development. We demonstrate that lack of ARID1A results in a range of hematopoietic defects including highly diminished reconstitution ability in transplantation models. Our results illustrate that ARID1A maintains frequency and quiescence of HSCs, and regulates differentiation of both myeloid and lymphoid lineages in a cell-intrinsic manner. Depletion of ARID1A leads to extensive decrease in chromatin accessibility including loss of open chromatin at promoter/enhancer regions of several key regulators of hematopoietic development.Arid1a allele (Arid1af/f) in which exon 9 of Arid1a gene is flanked by loxP sites have been described before [Arid1af/f mice were backcrossed for five generations to C57BL/6 mice before crossing with either Vav-iCre or Mx1-Cre transgenic strains to generate hematopoietic cell-specific deletion of Arid1a. In Vav-iCre model, either Arid1af/f;Vav-iCre− or Arid1af/+;Vav-iCre− sex-matched littermates were used as controls. To induce deletion of Arid1a in adult Arid1af/f;Mx1-Cre+ mice (8–14 weeks old), five doses of 300 µg poly(I:C)  were injected intraperitoneally, every alternate day. Sex-matched Arid1af/f;Mx1-Cre− littermates were also administered poly(I:C) simultaneously and used as control in all experiments. All mice were maintained in the animal facility of Comparative Medicine Centre, National University of Singapore (NUS). All mice experiments were performed according to protocols approved by NUS Institutional Animal Care and Use Committee.Mice bearing floxed d before . Arid1afStained cells were acquired on FACS LSR II flow cytometer (BD Biosciences) and sorted on FACSAria cell sorter (BD Biosciences). Data were analyzed using FACSDIVA software (BD Biosciences). Antibodies used for flow cytometry are listed in Supplementary Table −Flt3− LSK) four weeks after poly(I:C) injection from Arid1af/f;Mx1-Cre+ and Arid1af/f;Mx1-Cre− mice. 150 LT-HSCs (CD45.2+) from either WT or Arid1a KO mice were mixed with 300,000 BM cells (CD45.1+) from Ptprca Pepcb/BoyJ (B6.SJL) competitor mice and injected intravenously into lethally irradiated (11 Gy) B6.SJL recipient mice. Reconstitution was assessed every four weeks in peripheral blood using flow cytometry. Blood leukocytes were stained with antibodies against CD3 (T cells), CD19 (B cells), CD11b, Gr1, F4/80 (granulocytes and monocytes) along with donor (CD45.2) and competitor (CD45.1) markers.For competitive reconstitution assay with purified HSCs, we sorted LT-HSCs (CD34Arid1a deletion), CD45.2-expressing BM cells from either Arid1af/f;Mx1-Cre+ or Arid1af/f;Mx1-Cre− mice were mixed in equal proportion with CD45.1-expressing (B6.SJL) competitor BM cells. Two million cells were injected into the tail vein of lethally irradiated B6.SJL recipient mice. Donor engraftment was assessed in peripheral blood 4 weeks after transplantation. Arid1a deletion was induced in recipient mice using poly(I:C) and reconstitution of different blood lineages was determined as described above.For assays with BM cells (prior to cDNA libraries from FACS-sorted LT-HSC were prepared using SMART-Seq v4 Ultra Low Input RNA Kit (Clontech Laboratories). poly-A selected RNA from purified CMP, GMP, and MEP populations was used for library preparation using TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer’s protocol.Libraries were sequenced on HiSeq 4000 and 100 bp paired-end reads were aligned to murine reference transcriptome  using Kallisto (version 0.43.1) . Further−Kit+ BM cell as previously described [http://broadinstitute.github.io/picard). The reads were filtered using samtools v1.5 for a minimum mapping quality of 10 and only properly paired reads were retained [https://sites.google.com/site/anshulkundaje/projects/blacklists) [ATAC-seq libraries were prepared from Linescribed . Transpoescribed . PCR dupretained . Additioretained . Peaks wcklists) . The bedcklists) , 36. Heacklists) . Peaks wcklists) . Genomiccklists) .Statistical analyses for all mice experiments were performed using GraphPad Prism 7 software.All sequencing data generated in this study were deposited in Gene Expression Omnibus database repository. The accession numbers are: GSE125846 and GSE125848 (RNA-seq), GSE125845 (ChIP-seq), and GSE125844 (ATAC-seq).Arid1a was expressed ubiquitously across different hematopoietic lineages with relatively lower expression detected in mature myeloid cells  and spleen of C57BL/6 mice showed that Arid1a floxed allele (Arid1af/f) [Arid1af/f;Vav-iCre+ mice were obtained at lower than expected frequency, both at birth (day 1) and weaning (day 21), suggesting perinatal mortality caused by Arid1a deletion in this model  caused rapid and complete deletion at Arid1a locus in peripheral blood and BM cells, while the deletion was ∼90% in spleen cells, which was also reflected in the residual protein levels observed in western blot analysis  treatment   were croid1af/f)  or Mx1-Cid1af/f)  strains.sis Fig. . Loss ofent Fig. ; and a sArid1a early in hematopoietic development resulted in markedly reduced number of BM leukocytes; and cellularity was also decreased significantly when Arid1a was deleted in adult mice using the Mx1-Cre model  cells, which contains HSCs, was not significantly altered  injection), compared with the control mice  was significantly reduced in ARID1A-deficient BM, indicating loss of quiescence  injection  and a decrease in proportion of mature granulocytes (CD11b+Gr1+) in both deletion models  and granulocyte monocyte precursors  were unchanged in ARID1A-deficient BM; however, these populations exhibited reduced surface expression of CD34 compared with the control mice  was observed in the BM of Arid1af/f;Mx1-Cre+ mice  treated Arid1af/f;Mx1-Cre+ mice  and altered frequencies of subsequent stages of erythrocyte maturation in the BM of mice lacking ARID1A . Collectively, these data demonstrate that ARID1A plays a pivotal role in myeloid and erythroid differentiation.To understand how ARID1A regulates production of mature blood cells, we carefully analyzed differentiation towards myeloid and lymphoid lineages in ARID1A-deficient mice. ARID1A deficiency caused defects in granulocytic differentiation in BM of both Vav-iCre and Mx1-Cre models. This was evident by increased frequency of immature granulocytes/monocytes , was increased in Arid1a KO BM, in both Vav-iCre and Mx1-Cre models  to Fraction B (CD24+BP1−) proB cells , immature B (CD43−IgM+B220+), and mature B (CD43−IgM+B220hi) cells in ARID1A-deficient BM  compartment comprises of immature thymocyte populations, and immediately precedes the CD4+CD8+ double positive (DP) stage in development. We observed a striking reduction in DP cells with a concomitant increase in the proportion of DN and CD4+ SP cells in the thymi of Arid1af/f;Vav-iCre+ mice compared with the control mice  and DN2 (CD44+CD25+) cells were elevated, while DN3 (CD44−CD25+) subpopulation was reduced in KO thymi, suggesting early arrest of thymocyte development . These data demonstrate that ARID1A is required for normal T cell maturation in mice.We noted that Mx1-Cre mediated deletion of ARID1A was incomplete in thymoctyes 4 weeks after administration of poly(I:C) -treated lls Fig. . This ilice Fig. . Four weice Fig. . Deficieice Fig. . Similar−Kit+ BM cells from poly(I:C) treated Arid1af/f;Mx1-Cre+ and Arid1af/f;Mx1-Cre− mice. We observed that the biological replicates for WT and Arid1a KO samples were highly concordant  was performed on sorted LinArid1a-deficient cells, suggesting global loss of open chromatin in KO cells compared with the WT cells   in both ARID1A sg1- and sg5-expressing cells compared to control cells  Fig. . Loss ofox) Fig. . To assells Fig. . This inlls Fig. . Importalls Fig. . GSEA ofGenetic studies using mouse models indicate that ATP-dependent SWI/SNF chromatin remodeling complex is a crucial regulator of hematopoiesis. While BAF53a, BAF47/Snf5, and BAF45a/PHF10 regulate HSC activity –48, otheArid1a (during embryogenic hematopoiesis) in Vav-iCre model, while in Mx1-Cre model, mice were administered poly(I:C) at 8–14 weeks of age and hematopoietic parameters were analyzed four weeks later. Indeed, an efficient Mx1-Cre mediated deletion of Arid1a in BM following poly(I:C) treatment phenocopied largely the Vav-iCre model. Conversely, poly(I:C) administration caused an inefficient deletion of Arid1a in the thymus of Mx1-Cre mice which resulted in less dramatic effect on T cell maturation compared with the Vav-iCre mice.In the current study, the function of ARID1A in normal hematopoiesis was elucidated by utilizing two different Cre transgenes, driven by either Vav or Mx1. Vav-iCre mediated deletion of floxed allele commences in fetal HSCs and persists through adult hematopoiesis. Mx1-Cre exerts its effect in mice following stimulation with poly(I:C). Owing to a possible developmental role of ARID1A in fetal/neonatal hematopoiesis, a large majority of ARID1A-deficient mice did not survive at weaning in our Vav-iCre model. Analysis of the hematopoietic compartment revealed largely overlapping phenotypes in both models of ARID1A deficiency, although the consequences of its loss were clearly enhanced in the Vav-iCre model. We believe that this is possibly caused by earlier deletion of Arid1a KO embryos [Arid1a KO HSCs possessed poor multilineage reconstitution ability compared with the WT HSCs and were outcompeted in the transplantation settings. Apart from its vital role in maintaining HSC frequency and function, we uncover that ARID1A is also required for normal maturation of both myeloid and lymphoid lineages.Loss of ARID1A resulted in accumulation of LTHSCs in the BM, indicating requirement of ARID1A in maintenance of HSC pool size. This is reminiscent of increased HSC frequency observed in fetal liver of constitutive  embryos . ResultsCd34, protein encoded by which is expressed on the surface of HSCs and its knockout in mice leads to hematopoietic defects [Cebpa and Csf1, which are crucial regulators of myeloid differentiation [ATAC-seq data demonstrated that chromatin remodeling mediated by ARID1A-containing SWI/SNF complex is critical for hematopoiesis in mice. Deficiency of ARID1A led to a global decrease in open chromatin in myeloid precursors, including at several gene loci, which encode for key regulators of hematopoiesis. Notably, loss of ATAC-seq signal at these loci also correlated with reduced gene expression in myeloid precursors. One of the important downstream targets of ARID1A deficiency is GATA2, which plays an essential role in proliferation and maintenance of hematopoietic stem/progenitor cells, as well as differentiation towards multiple hematopoietic lineages . Another defects . Similarntiation , 56 alsoARID1A were previously described [ARID1A in NB4 cells did not affect significantly their growth but impaired the ATRA-induced granulocytic differentiation. Further studies are warranted to investigate whether the impaired differentiation in many hematological disorders including B-cell malignancies and APL is probably in part caused by loss-of-function mutations of ARID1A.Evidence is emerging that ARID1A is vital for differentiation of various cell types –59. Our escribed . In factARID1A, several approaches to therapeutically target ARID1A-deficient cancer cells have been proposed. These include increased sensitivity of ARID1A-mutated cells to inhibition of either polycomb activity, the PI3K/AKT pathway, the DNA damage checkpoint or the immune checkpoint [ARID1A, similar strategies can be extended potentially to hematological malignancies harboring mutations of ARID1A. For instance, in APL cases with mutation of ARID1A, standard therapy (either ATRA or arsenic trioxide) could be combined with an approach directed at ARID1A deficiency, thus leading to potentially improved and personalized therapeutic approaches. Experimental models developed in this study including hematopoietic cell-specific Arid1a KO mice and ARID1A-deficient NB4 cells are valuable to explore these therapeutic strategies. For example, mice lacking ARID1A can be crossed with murine models of hematological malignancies for preclinical studies to test precision therapies.Following the discovery of frequent inactivating alterations of eckpoint . AlthougOverall, this study enhances our understanding of the function of SWI/SNF complex in hematopoiesis. It demonstrates that nucleosome remodeling mediated by ARID1A is fundamental for normal hematopoietic development.Supplementary dataSupplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7


To explore the expression level and clinical significance of decoy receptor 3 (DcR3) in patients with acute-on-chronic liver failure (ACLF). Serum DcR3 levels were measured by enzyme-linked immunosorbent assay (ELISA) in 76 patients with ACLF and 41 non-ACLF patients with chronic liver disease. Blood routine and liver functions were accessed for their correlations with DcR3. Serum DcR3 in ACLF patients was significantly higher than that in non-ACLF patients. It was positively correlated with neutrophilic granulocyte, aspartate aminotransferase, prothrombin time, and international standardized ratio, but negatively correlated with platelet and serum albumin. At the early stage, the level of DcR3 was not significantly different between the survival and nonsurvival group of ACLF. However, at the late stage, DcR3 increased in nonsurvival and gradually decreased in survivals. The baseline DcR3 could not sufficiently predict the outcome of ACLF, while the change of DcR3 within the first week displayed a better predictive value than model for end-stage liver disease (MELD) score. DcR3 was highly expressed in patients with ACLF and correlated with several clinical indices. Dynamic change of DcR3 might predict the prognosis of ACLF. For patients with liver failure, those indices were also collected at 3, 7, and 14 days after admission.A quantitative ELISA was used to measure DcR3 as previously reported . The DcR t-test in the case of normal distribution or the Mann-Whitney U test in the remaining cases. Categorical variables were expressed as counts (percentages) and evaluated by Chi-squared test or the Fisher's exact test when the number of samples was limited. The correlations of PCT levels with the other indicators were analyzed using the Pearson correlation test and the association between the two variables was measured as Pearson's correlation coefficient(r). The diagnostic accuracy for DcR3 and other indicators for the outcome of ACLF were expressed as the area under the receiver operating characteristic curve (AUROC). A p value<0.05 was considered statistically significant.Statistical analyses were performed by the SPSS software, version 18.0 . The normality of the distribution was estimated by the Kolmogorov-Smirnov test. Continuous variables were represented as mean ± standard deviation or median (interquartile range)  and compThe study was approved by the institutional ethics review board of First Affiliated Hospital of Fujian Medical University (protocol# 2015-084) and was in compliance with the Declaration of Helsinki.A total of 117 patients with available blood samples were included in this study, 76 (65.0%) patients with ACLF (ACLF group) and 41(35%) with liver cirrhosis (non-ACLF group). The details of clinical characteristics and laboratory data are shown in γ-glutamyl-transferase (γ-GT), WBC, N%, plasma ammonia, prothrombin time (PT), and international normalized ratio (INR) levels than non-ACLF patients, while the albumin and platelet levels were significantly lower in ACLF patients.ACLF patients had higher TBIL, PCT, alanine aminotransferase (ALT), aspartate aminotransferase (AST), The DcR3 level was significantly higher in ACLF patients than in non-ACLF patients .γ-GT  and negatively correlated with platelet  and albumin , indicating that the DcR3 levels reflected the severity of liver damage.Pearson correlation test was used to explore the correlations of PCT levels with the other indicators in overall population. The results showed that DcR3 levels were positively correlated with TBIL , N% , PT , INR , AST , and Among 76 patients with ACLF, 38 (50%) died within 3 months. There was no difference in patients' age and sex between the survival group and nonsurvival group. But, the TBIL, N%, PT/INR, and model for end-stage liver disease (MELD) score in nonsurvival group were significantly higher than those in survival group . AlthougAccording to previous report, DcR3 level correlated with sepsis , 18. We The predictive accuracies of DcR3 and MELD score for 3-month survival in ACLF patients were compared. The AUROC of baseline MELD score : 0.521-0.769, p=0.030) was significantly higher than baseline DcR3 , indicating a poor predictive value of baseline DcR3. Then we compared the changes of DcR3 and MELD score within the first week in patients who had serial DcR3 data. Of 76 ACLF patients, 43 patients have serial collections of serum samples for DcR3 tests. On the 7 days of admission, the patients in nonsurvival group had a steady increase of serum DcR3 (0.15±0.20 ng/mL), while the DcR3 was declined in the patients of survival group (-0.45±0.21 ng/mL) . The chaγ-GT, but negatively correlated with platelet and albumin, (3) DcR3 was not significantly different between the survival and nonsurvival groups of ACLF at the early stage of the disease. However, it steadily increased in nonsurvival group, while it gradually decreased in the survival group, and (4) the steadily increased serum DcR3 level in patients with ACLF indicates a poor outcome.This study revealed for the first time that (1) serum level of DcR3 in ACLF patients was significantly higher than that of no-ACLF, (2) DcR3 level was positively correlated with TBIL, N%, PT, INR, AST, and While the elevated DcR3 in ACLF is not reported previously, an increased DcR3 has been observed in patients with chronic HBV infection and is also suggested to be a useful noninvasive biomarker for discrimination of active hepatitis B from inactive HBV carriers  and a maThe pathological feature of ACLF is the massive hepatocyte death resulting from an excessive immune response targeting liver cells . This inIn terms of mechanism, DcR3 reduces apoptosis in ACLF by blocking Fas signaling. The elevation of DcR3 in nonsurvived patients might be the response to overactivated Fas signaling. Higher DcR3 levels may indicate a greater magnitude of inflammation and poor outcome. However, endogenous DcR3 might not be enough to overcome the overwhelming effect of proapoptotic factors in ACLF patients with poor outcome. Exogenous DcR3 analog has been shown to attenuate Fas L-induced apoptosis in fulminated liver injury and reduce the death rate . TherefoTaken together, DcR3 levels are elevated in ACLF patients. Continuous increase of DcR3 levels might be a sign of poor outcome.The major weakness of this study was that it was a single-center retrospective study with small-size samples. Multicenter studies with larger samples are needed to address the significance of DcR3 in ACLF and the possible therapeutic potential of DcR3 or its analogs.