Triple-negative breast cancers (TNBCs) are aggressive, and they develop metastasis at earlier stages, relapse more frequently, and exhibits poorer prognosis than other subtypes of breast cancer. Due to the lack of estrogen receptor for endocrine therapy and HER2 for targeted therapy, new targeted therapies for TNBCs are urgently needed. Enzalutamide is a second-generation androgen receptor (AR) inhibitor, and HC-1119 is a new synthetic deuterated enzalutamide. Owing to the isotope effect, HC-1119 has many advantages over enzalutamide, including slow metabolism, high plasma concentration and low brain exposure. However, the efficacy of HC-1119 in inhibition of AR function in triple-negative breast cancer (TNBC) has not been studied. In this study, we found high-level AR expression in both Hs578T and SUM159PT TNBC cell lines. Activation of AR by dihydrotestosterone (DHT) in both cell lines increased AR protein, induced AR-nuclear localization, enhanced cell migration and invasion in culture, and promoted liver metastasis in mice. Importantly, cotreatment with HC-1119 of these cells efficiently abolished all of these effects of DHT on both Hs578T and SUM159PT cells. These results indicate that HC-1119 is a very effective new second-generation AR antagonist that can inhibit the migration, invasion and metastasis of the AR-positive TNBC cells.The online version contains supplementary material available at 10.1007/s11033-022-07749-8. According to the International Agency for Research on Cancer (IARC), breast cancer was the most commonly diagnosed cancer and the fifth leading cause of cancer death worldwide in 2020 . There aAndrogen receptor (AR) signaling regulates multiple bioprocesses such as the development and maintenance of male sexual organs, bones, and reproductive function, and also helps ovarian follicles in female to develop and ovulate normally , 7. AR iEnzalutamide is a 2nd generation AR inhibitor approved and extensively for treating metastatic castration-sensitive prostate cancer (mCSPC), non-metastatic castration-resistant prostate cancer (nmCRPC), and metastatic castration-resistant prostate cancer (mCRPC) , 24, HC-In this study, we performed both in vitro and in vivo experiments to evaluate how HC-1119 can effectively inhibit the growth, migration, invasion, and metastasis of AR-positive TNBC cells. We found that HC-1119 can significantly decrease the migration and invasion capabilities of the AR-positive TNBC cells by impeding the epithelial-mesenchymal transition (EMT) phenotype and inhibit the metastasis of these TNBC cell-derived tumors in mice. Our results demonstrate that HC-1119 may be useful for treating AR-positive TNBC.SUM159PT, Hs578T, T47D, MCF7, MDA-MB-231, MDA-MB-468, MDA-MB-453 and 22Rv1 cell lines were obtained from Chinese Academy of Medical Sciences. SUM159PT, Hs578T, T47D, MDA-MB-231, MDA-MB-468 and MDA-MB-453 cells were cultured in DMEM  with 10% fetal bovine serum (FBS) . 22Rv1 cells were cultured in RPMI medium 1640 containing 10% FBS, 1% glutamax and 1% sodium pyruvate. MCF7 cells were cultured in MEM medium with 10% FBS.Cells were lysed with RIPA buffer (25 mM Tris HCl (pH 7.6), 1% sodium deoxycholate, 150 mM NaCl and 0.1% SDS) containing protease inhibitor cocktail . Total cell lysates with 60–80 µg protein were boiled for five minutes in SDS-PAGE reducing buffer, and loaded to SDS-PAGE gels. Separated proteins in the gel were transferred onto a nitrocellulose membrane. Primary antibodies used for immunoblotting were against androgen receptor , vimentin , fibronectin , Keratin 8/18 , and GAPDH . Fluorescence-labeled secondary anti-mouse IgG antibody  was used to detect the primary antibodies. The fluorescence intensity of the secondary antibody was assayed using an Odyssey Imaging System (LI-COR).Total RNA was extracted by Trizol reagent (Invitrogen). One g RNA was used in reverse transcription. The relative concentration of a mRNA was measured by real-time quantitative PCR (qPCR) and normalized to GAPDH mRNA. The 5’ primer sequences, 3’ primer sequences, and the fluorescence-labeled probes were 5’-gccttgctctctagcctcaa, 5’-ggtcgtccacgtgtaagttg, and probe 14#  for AR, and 5’-agccacatcgctcagacac, 5’-gcccaatacgaccaaatcc, and probe 60# for GAPDH.Prior to drug treatment, all cells were cultured in phenol red-free DMEM medium  containing 10% charcoal-stripped FBS  for 48 h to exhaust androgen. Dihydrotestosterone (DHT) and HC-1119 were dissolved in DMSO. Hs578T cells were pretreated with 5 nM DHT and/or 0.5 µM HC-1119 or vehicle (DMSO) for 24 h. SUM159PT cells were pretreated with 5 nM DHT and/or 10 µM HC-1119 or vehicle (DMSO) for 24 h. For invasion assay, 5 × 104 pretreated cells in 200 µl of phenol red-free DMEM were seeded into the upper chamber with a membrane precoated with 20 µl of PBS-diluted Matrigel (1:1 dilution) . For migration assay, 3 × 104 pretreated cells in the same medium were seeded into the upper chamber without Metrigel on the bottom membrane. The lower chamber was filled with phenol red-free DMEM containing 10% charcoal-stripped FBS (CSS). After cultured in a 37 °C incubator with 5% CO2 for 24 h, cells on the upper surface of the membrane were removed with a cotton swab. Cells on the lower surface of the membrane were fixed in 500 µl methylal, washed in 1 ml PBS, dried, and stained in crystal violet solution  for 3 h. The stained cells were imaged under a microscope and quantitatively analyzed.Cells (4 × 104) were seeded on glass coverslips in a 24-well culture plates, treated with DHT and/or HC-1119 for one hour. The treated cells were washed with TBST, fixed in 4% formaldehyde solution and permeabilized with 0.3% Triton X-100 in TBST, and blocked with 5% BSA in TBST for 1.5 h. Cells on coverslips were incubated overnight with an androgen receptor antibody , followed by incubation with a goat anti-Rabbit IgG (H + L) secondary antibody  for 1 h, and DAPI staining. Finally, the stained cells on coverslips were examined with a confocal fluorescence microscope .Five- to six-week-old female BALB/c nude mice were purchased from Vital River Laboratory Animal Technology, Beijing, China. One week later, 2 × 106 SUM159PT cells were injected into one of the forth pair mammary gland fat pads of each mouse. When the volume of the tumors derived from these injected cells reached 100 mm3, mice in group 1 were treated with DHT  or equal volume of vehicle (dosing in 5% Dimethyl sulfoxide (DMSO), 30% Poly- ethylene glycol 400(PEG400), 5% Tween80 and 60% ddH2O). Mice in group 2 were treated with HC-1119  or equal volume of vehicle  sodium, 15% of 20% Vitamin E-TPGS in 20 mM citrate buffer (pH 4.0), 1% Polyvinylpyrrolidone (PVP)). Mice in group 3 were treated with both DHT and HC-1119 in the same doses given to mice in groups 1 and 2. Tumor length and width were measured 3 times a week by using a caliper. Tumor volume was calculated using the formulae: volume = 1/2×(length×width2). Mice were euthanized by carbon dioxide asphyxiation, and the tumors, lungs and livers were collected for histopathological examination and biochemical analysis.The harvested xenograft tumors and other tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm-thick sections. Sections on glass slides were deparaffinized using xylene and rehydrated in a series of ethanol dilutions and water. Some xenograft tumor and liver sections were stained with H&E for histopathological examination. Other tumor sections were soaked in 10 mM sodium citrate (pH = 6.0) and heated in a high-pressure cooker for 4 min for antigen retrieval, and treated with 3% H2O2. After blocked with 5% bovine serum albumin (BSA) for 1 h, sections were incubated overnight with AR antibody  at 1:200 dilution in TBS containing 5% BSA at 4 ℃. After washed and incubated with the biotinylated goat-anti-rabbit IgG secondary antibody, the immunostaining signal was visualized by using a DAB kit . The sections were further counterstained with Hematoxylin, dehydrated in a series of ethanol solutions and ethanol, and mounted with Permount for microscopy and imaging.Every experiment was repeated at least three times. Data analysis was conducted using GraphPad Prism Version 6.0. Student’s t-test and One-Way ANOWA were used to compare the differences between two and more groups, respectively. Statistical significance was accepted if P < 0.05.In order to choose appropriate breast cancer cell lines for this study, we examined ER, PR, HER2 and AR expression in multiple breast cancer cell lines. Among the TNBC cell lines, MDA-MB-231 and MDA-MB-468 cells do not express AR, while Hs578T and SUM159PT cells express a moderate-level and a high-level AR protein, respectively (Fig. To test the antagonistic effect of HC-1119 on DHT, we treated both Hs578T and SUM159PT TNBC cells with vehicle, DHT and/or HC-1119 and examined the expression levels of AR protein in these treated cells. Again, DHT treatment increased AR protein. However, HC-1119 treatment alone did not alter the levels of AR protein in these cells. Importantly, HC-1119 treatment abolished the DHT-induced AR expression in these cells Fig.  A and B.The translocation of AR from cytoplasm to nucleus is an essential step for the expression of AR-regulated genes, and block of AR nuclear-translocation is a key feature of the second-generation antiandrogens distinct from the first-generation antiandrogens . To deteWe first examined the effect of HC-1119 on the proliferation of Hs578T and SUM159PT TNBC cells, and found no obvious effect of HC-1119 treatment on the proliferation of these cells Figure S. Since DWe orthotopically inoculated SUM159PT cells into the mammary gland fat pads of female BALB/c nude mice to grow xenograft tumors, and treated these mice with vehicle, DHT, HC-1119 or both DHT and HC-1119 for 31 days. All of these treatments did not significantly affect the growth of the primary xenograft tumors derived from these cells (Figure SHC-1119 is a new second-generation AR antagonist that has been used in clinical trials to treat prostate cancer , 30. AltWe found that HC-1119 treatment could inhibit DHT-promoted migration and invasion capabilities of the AR-positive TNBC cells, and this inhibition might be a consequence of reduced EMT plasticity of these TNBC cells. Previous studies have shown that DHT-activated AR interacts with demethylase LSD1 to repress E-cadherin transcription and upregulate vimentin transcription in the ER and AR positive MCF7 breast cancer cells, leading to EMT that increases cell migration and invasion . FurtherHC-1119 at a dose of 7 mg/kg given to mice every 2 days could reduce DHT-promoted metastasis of TNBC cells in vivo. Early studies showed that testosterone levels are fluctuating, and plasma concentration of DHT is maintained in a dynamic balance during menstrual cycle, which is higher in premenopause women and lower in postmenopause women , 36. HenIt is known that DHT binds and activates AR, which in turn increases AR protein through multiple pathways –40. Our In this study, we have evaluated the efficacy of HC-1119 in inhibition of DHT-activated AR function in AR-positive TNBC cells. However, we have not compared the efficacy of HC-1119 with the regular enzalutamide in these cells, which will be planned in our future experiments. Furthermore, we only studied the response of DHT-treated TNBC cells, but accumulating evidence suggests that AR also plays an important role in ER-positive breast cancer cells , 42, 43.In summary, this study demonstrated that HC-1119 can effectively inhibit DHT-increased AR protein and DHT binding-induced AR-nuclear translocation, and thus block DHT-promoted migration, invasion and metastasis of TNBC cells. These results suggest that HC-1119 is a new second-generation AR inhibitor that might be used to treat AR-positive TNBCs. This is the first study showing that HC-1119 may be used to treat other types of AR-positive cancers such as AR-positive TNBC in addition to the prostate cancer.Supplementary material 1


Cardiac rehabilitation, which combines cardiology and preventive medicine, is an important part of treatment for cardiovascular diseases. Systematically, cardiac rehabilitation, including simultaneously inhibiting endothelial injury and promoting endothelial repair, is beneficial for physical and mental recovery and reduces the risks of recurrence and death in patients with cardiovascular diseases. Cardiac rehabilitation has developed rapidly in the last 50 years. A preliminary system for cardiac rehabilitation has been developed in China. The present article mainly focuses on the progress of cardiac rehabilitation from the aspects of goals, measures, and modes of research in the current scenario. Cardiac rehabilitation has a history of more than 200 years and has rapidly developed for nearly half a century. At present, approximately 54.7% of countries worldwide, mainly including the middle and high-income countries, have already started cardiac rehabilitation . In receIn 2007, the American Association of Cardiovascular and Pulmonary Rehabilitation/American Heart Association (AACVPR/AHA) defined cardiac rehabilitation as a comprehensive and coordinated long-term plan that includes medical care evaluation, exercise prescription, correction of cardiovascular risk factors, education, consultation, and behavioral intervention . At the Medication and surgery  are important modalities in the treatment of cardiovascular diseases. Both can ameliorate and stabilize most diseases within a short period and provide an important foundation for further long-term cardiac rehabilitation. Most patients under cardiac rehabilitation have chronic cardiovascular diseases and require long-term medication, which is another reason that medication prescription plays a basic role in cardiac rehabilitation. Certain guidelines should be followed during prescription. Currently, drugs with sufficient evidence for treatment of cardiovascular diseases include antiplatelet drugs, beta blockers, angiotensin-converting enzyme inhibitors (ACEI), angiotensin II receptor blockers (ARB), and statins . PrescriExercise prescription plays a key role in various methods of cardiac rehabilitation and is also the determining factor of treatment success or failure. It involves various aspects of exercise: mode, frequency, intensity, and venue. At present, the major modes of exercise are aerobic training, dynamic and static resistance training, flexibility training, and balance training, among others .Individualized exercise prescription guarantees exercise safety and effectiveness but is a difficult aspect of cardiac rehabilitation. However, there are no clear guidelines on creating exercise prescriptions. Presently, exploring various exercise modalities is the main direction that scholars focus on .While the pathophysiological effects of exercise on the human body are still under continuous investigation, its benefits have been confirmed. Furthermore, most scholars believe that more advantages are seen with high-intensity intermittent exercise . Experimβ-adrenergic receptors [Exercise enhances cardiac function since it is conducive to improved myocardial remodeling . The poseceptors . These peceptors . Recent eceptors . The inceceptors . The inceceptors .Numerous studies have proven that exercise increases patients' 6-minute walking distance, prolongs the exercise time in cardiopulmonary exercise test, and significantly reduces the readmission rate, incidence of cardiovascular events, and mortality rate . AnotherExercise can regulate nerve function, inhibit sympathetic tension, and excite parasympathetic nerves through reduction of aldosterone secretion and inhibition of sympathetic nerve excitability . In addiPsychological factors share a close relationship with the development, progression, and outcome of diseases. Poor mental state is not only a causative factor in cardiovascular diseases but also affects disease outcomes through negative feedback. A survey showed that among patients in the cardiology department, the detection rate of mental and psychological disorders in outpatients and inpatients was 55% and 50%, respectively. Moreover, almost all patients undergoing stent implantation had different degrees of psychological problems . ExploriPsychological intervention aims to relieve tension, improve treatment compliance, and boost self-confidence. However, repetition is required to avoid the disadvantages of the simple biomedical model. Depressive symptoms were found to decrease from 17% to 6% after cardiac rehabilitation training. In addition, the mortality rate of patients with depression who completed rehabilitation treatment was 73% lower than those who did not . Sleep qSmoking is an independent risk factor for cardiovascular diseases. Several studies have outlined the significant causative effect of smoking on coronary heart disease, atherosclerotic peripheral vascular diseases, and stroke. Smoking cessation can reduce the risk of cardiovascular disease morbidity and mortality, and its long-term benefits are at least equivalent to those of commonly used secondary preventive drugs for coronary heart disease such as aspirin and statins. Therefore, medical professionals must explain the negative effects of smoking and the benefits of smoking cessation to the patients and their families. The optimal treatment plan for tobacco dependence is a comprehensive method combining drug therapy, psychotherapy, and behavioral therapy.Existing evidence-based medicine has proven that excessive intake of energy, saturated fat, and cholesterol and insufficient intake of vegetables and fruits increase the risk for cardiovascular diseases. Conversely, the intake of a scientific and reasonable diet was found to reduce this risk. Considering this, the purpose of nutrition prescription is to guide patients in forming healthy eating habits.The implementation of nutrition prescription is crucial to effective cardiac rehabilitation. Rehabilitation evaluation is an important starting point in the rehabilitation process. Current disease condition, exercise ability, nutrition, and psychological status of patients can be comprehended through rehabilitation evaluation. This enables the formulation of individualized, safe, and accurate rehabilitation plans. In America and developed European countries, the cardiopulmonary exercise test is considered the “gold standard” for clinical evaluation of cardiopulmonary function and formulation of exercise prescription . All patThe current focus of research is to explore cardiac rehabilitation modalities that offer easy facilitation and high patient compliance while ensuring safe and effective rehabilitation. However, rehabilitation should be conducted under the guidance of cardiologists regardless of the chosen modality.Cardiac rehabilitation can be classified into three modes according to location of implementation: inpatient rehabilitation (stage I rehabilitation), early outpatient rehabilitation or clinic rehabilitation (stage II rehabilitation), and long-term outpatient rehabilitation (stage III rehabilitation). These modes correspond to high-risk, medium-risk, and low-risk patients, respectively, with respect to disease condition.Stage I rehabilitation refers to cardiac rehabilitation under the guidance and supervision of medical staff in the hospital. The goals of this mode include shortening the length of hospital stay and promoting the recovery of patients' daily life and exercise ability while avoiding the adverse effects caused by bed rest (such as reduced exercise tolerance and thromboembolic complications). In addition, stage I also involves reminding patients of smoking cessation and providing comprehensive and complete information in preparation for stage II rehabilitation. It is generally implemented in high-risk patients. Stage II rehabilitation is generally carried out in the first 6 months after discharge. Patients continue to undergo rehabilitation treatment under the guidance and supervision of medical staff at the outpatient clinic. At the onset of disease stabilization, self-monitored exercises may be carried out at home, and remote network and electronic information technology can be used to supervise the entire process of their cardiac rehabilitation. Generally, it is adopted for medium-risk patients. Stage III rehabilitation involves long-term rehabilitation at home. Patients undergo in-hospital evaluation, and rehabilitation exercise is carried out at home under the guidance of rehabilitation personnel and self-management to cultivate a healthy lifestyle. It is generally utilized in low-risk patients –22.Selection of rehabilitation mode is influenced by factors such as income, education level, and the establishment level of tiered medical services. The United States implements hospital-based cardiac rehabilitation mode, with stage I rehabilitation being paid by medical insurance. Europe implements the mode of rehabilitation center combined with community, while Taiwan in China and Japan adopt the mode of family-driven rehabilitation clinic. In the Chinese mainland, the hospital cardiac rehabilitation mode, the family cardiac rehabilitation mode, or a mixture of both has begun to play a role in community cardiac rehabilitation . Most ofAnother difficulty in cardiac rehabilitation is improving patient compliance , 24. StuPresently, cardiac rehabilitation has been internationally recognized as a safe, effective, and cost-effective method for treating patients with cardiovascular diseases. Research on cardiac rehabilitation has shifted from the demonstration of various mechanisms and principles to the exploration of various patient-centered rehabilitation methods and modes. However, there is still a lack of an individualized and standardized cardiac rehabilitation mode with high patient compliance. In addition, the large-scale promotion of modes such as telerehabilitation, community rehabilitation, and family rehabilitation still requires further research and demonstration. The application of modern technologies such as the Internet and artificial intelligence in cardiac rehabilitation will actively promote the development of cardiac rehabilitation.


Castration-resistance of prostate cancer is one of the most challenging clinical problems. In the present study, we have performed proteomics and glycomics using LNCaP model. Growth differentiation factor-15 (GDF15) level is increased in androgen receptor (AR) inhibitor-resistant cells and the inhibitory effect of GDF15 on epithelial growth factor receptor (EGFR) pathway is relieved by GDF15 N70 glycosylation. Interference of GDF15 (siRNA or N70Q dominant negative) or EGFR pathway  decreases the resistant-cell survival in culture and tumor growth in mice. Our study reveals a novel regulatory mechanism of prostate cancer AR inhibitor resistance, raises the possibility of AR/SRC dual-targeting of castration-resistance of prostate cancer, and lays foundation for the future development of selective inhibitors of GDF15 glycosylation. Androgen deprivation therapy (ADT) is a common practice for the treatment of advanced prostate cancer (PCa) since its discovery by Huggins and Hodges in 1941 . DespiteClassical pathways of castration resistance have been studied extensively , 5. AddiGrowth differentiation factor-15 (GDF15)/macrophage inhibitory cytokine-1 (MIC-1) is a member of the TGF-β superfamily . GDF15 mHere, we have performed proteomics and glycomics in LNCaP culture model, discovered an important role of GDF15-EGFR pathway in the development of AR inhibitor resistance, and demonstrated its therapeutic potential. Our study may draw research interests in GDF15 and clinical efforts on CRPC using SRC/AR dual-targeting approach.50 values of cell survival rates in normal FBS culture medium  were 0.12 µM and 0.07 µM, respectively. Furthermore, with the artificial synthetic androgen methyltrienolone (R1881) manually added in c-FBS cultured medium, the IC50 values of R1881 + ENZ and R1881 + EPI were 8.5 µM and 10 µM  cell model, we treated LNCaP prostate cancer cells with enzalutamide (ENZ) and EPI-001 (EPI) for 9 days (short-term or ST) and for 33 days (long-term or LT), at concentrations of 10 µM (ENZ) and 8 µM (EPI), respectively, based on ICium Fig. , which w Figure S, respect Figure S. The sim Figure S.Fig. 1ARWe next examined markers of cell cycle checkpoint by Western Blot (WB) and cell cycle distribution by fluorescence activated cell sorter (FACS) analysis in ST and LT cells. WB results revealed that P21  was upregulated in ST and backed down in LT cells, and Cyclin E1 (G1-S phase marker), CDC6 (G1-S phase marker), and CDC2 (G1-S and G2-M phase marker) were downregulated in ST and up again in LT cells Fig. . ConcomiFurther, we determined ENZ/EPI effects on AR and its targets , AR-Vs, and its targets . WB results showed that all markers were downregulated to various degrees in ST and LT cells compared to NC Fig.  with excN-glycosylation glycomics analysis (Tables SN-glycopeptides (IGPs), and 79 N-glycan  glycoprotein was significantly upregulated in LT group, but not in ST group, compared to NC with both EPI and ENZ treatment Fig. . IncreasSubsequently we examined tryptic peptide glycotypes and 79 identified glycans were quantified. Levels of the majority of glycans, especially mannose-modified glycotypes, were downregulated, however, quantities of some complex glycan, such as N3H4F3, N6H3F2, N8H9F3S1 and N6H7S2, were upregulated in LT group compared to NC groups Fig. . There wN-linked glycosylation inhibitor tunicamycin (TM)  was employed. Glycans on glycosylated GDF15 (~38 kDa) was entirely removed, indicating GDF15 was modified by N-glycans  Fig. . To furtans Fig. . GDF15 ians Fig. .Fig. 3GDTo explore potential mechanism involving in the development of IRPC, pathway enrichment analysis of GO and KEGG was performed using quantitative glycoproteins data Table S. We founIt was reported that GDF15 could inhibit EGFR activity . We survTo validate its role in regulating EGFR activity, we knocked down GDF15 in LNCaP, ST, and LT cells Figure S with siRTo evaluate the role of GDF15 glycosylation in vivo, we used 22Rv1 cell (an ENZ-resistant cell line) in a xenograft model with male BALB/c nude mice castrated 21 days prior. Tumor growth in mice injected with cells expressing GDF15 N70Q mutant was significantly reduced compared to that of mice injected with control 22Rv1 cells Fig. . Thus, tSince EGFR pathway is activated in IRPC, we treated LT cells with EGFR, SRC, ERK inhibitor or siRNA. Pathway interference significantly reduced LT cell survival, and together with AR inhibition further decreased survival rate Fig. . InhibitTo further substantiate the therapeutic efficacy of SRC/AR dual targeting, we conducted in vivo experiments. AR inhibitor-resistant mice were generated using Hi-Myc mice  by contiTo further support our observation, we investigated GDF15 expression in cancer patients. GDF15 level was significantly higher in the serum of patients with localized PCa and metastatic castration-resistant prostate cancer (mCRPC) Fig. . AlthougTogether, our study indicates that EGFR pathway is inhibited by GDF15 in untreated and short-term AR inhibitor treated LNCaP prostate cancer cells. Long-term treatment, however, induces GDF15 N70 glycosylation which relieves its inhibitory effect on EGFR, consequently EGFR activation contributes to the development of castration resistance Fig. .Through proteomics and glycomics analyses, we identify GDF15 and show its regulation of EGFR pathway in PCa and importance in the development of AR inhibitor resistance. GDF15, a stress-induced cytokine, is known to have immunomodulatory functions and its high expression is often associated with cancer progression. However, whether GDF15 acts as a suppressor or promoter in prostate cancer remains unresolved . Some evetc.) are under the control of AR and link to the synthesis of cancer-associated glycans such as sialyl-Tn (sTn), sialyl LewisX (SleX) ).For hypodermic CRPC xenografts, male BALB/c-nude mice (age of 8–9 weeks) were anaesthetized and then 1*10Hi-Myc transgenic prostate cancer mice (gifted from George V. Thomas laboratory)  were useBriefly, after deparaffinization and rehydration, 5 µm thick longitudinal sections were stained with hematoxylin solution for 22 s, dipped in 1% hydrochloric acid ethanol, rinsed with distilled water, stained with eosin solution for 30 s, dehydrated with graded alcohol and cleared in xylene. Mounted slides were scanned using an Pannoramic Scanner .Human peripheral blood samples were collected from the Affiliated Hospital of Jiangnan University. All patients signed informed consent. The study was approved and authorized by the Ethics Committee of the Affiliated Hospital of Jiangnan University . The patients’ information is listed in Table St-test was used to compare means of two groups. One-way ANOVA was used to compare means of 3 or more groups . Turkey test was used to perform multiple comparison . Data were presented as mean ± std of biological repetition. P < 0.05 was considered as significant in all of the tests.Student’s checklistFigure S1Figure S2Table S1Table S2Table S3Table S4Table S5Table S6Table S7LNCaP-1LNCaP-222Rv1-122Rv1-2


The number of patients with allergic rhinitis (AR) has exceeded 500 million worldwide due to the unstable curative effect that can easily produce mental and psychological disorders. However, most of the relevant existing literature is one-on-one retrospective analyses or targeted meta-analyses of AR with psychological disorders like irritability, depression, and anxiety, while “multi-hospital + interdisciplinary” multiple regression analyses are scarce.This study aims to precisely identify the psychological disorders of patients with AR who were diagnosed and treated in the five most renowned hospitals in Chengdu, China over the past 5 years using 10 classification methods so as to attract attention and care from otolaryngologists.The Symptom Checklist 90 (SCL-90) was used to group and score the mental state of 827 strictly screened patients with AR according to 9 classification criteria. The scores were then compared within groups. Intergroup comparisons were made between the study group and the Chinese norm, and the positive factors for psychological disorders were extracted. Four symptoms in the study group, that is, nasal itching, sneezing, clear discharge, and nasal congestion, were scored on a visual analog scale. Partial correlation analysis was performed between the extracted positive factors for psychological disorders and the symptom scores by the multiple regression statistical method.Among 827 patients, 124 (15%) had no mental health impairments, 176 (21.3%) had mild impairments, 474 (57.3%) had mild to moderate impairments, 41 (5%) had moderate to severe impairments, and 12 (1.4%) had severe impairments. The average score of the SCL-90 for all 827 patients was 2.64 (SD 0.25), which corresponded to mild to moderate mental health impairments. The 827 patients scored significantly higher for the 4 positive factors: depression, anxiety, psychosis, and other . Depression was positively correlated with sneezing and clear discharge, anxiety was positively correlated with nasal itching and congestion, psychosis was positively correlated with nasal itching and sneezing, and other  was positively correlated with clear discharge and nasal congestion.Patients with AR have mild to moderate mental health impairments, with women and those with abnormal BMI, aged ≥45 years, with a monthly salary <¥5110 (US $700), with a disease duration <13 years, residing in urban areas, with a high school or above education, or who are indoor laborers being at high risk and requiring more care, follow-up, and comprehensive therapy from otolaryngologists. According to the guidelines of the World Health Organization (WHO) and Allergic Rhinitis and its Impact on Asthma, the number of patients with allergic rhinitis (AR) has exceeded 500 million globally . Due to However, most of the relevant existing literature are one-on-one retrospective analyses or targeted meta-analyses of AR with psychological disorders like irritability, depression, and anxiety , while “A total of 1536 patients with AR who received treatment at 5 hospitals from July 2013 to January 2018 were selected. A routine questionnaire survey was conducted strictly following the inclusion and exclusion criteria for the AR group, rejecting 709 cases and enrolling 827 cases.All selected patients signed the informed consent and questionnaire. The study has been certified by the ethics review system of the World Federation of Traditional Chinese Medicine Societies (CAP2013 (A) 0038).We will strictly follow the latest AR diagnostic criteria promulgated by China in 2018 . The folThe following exclusion criteria will be used: failing to complete the questionnaire carefully or completely; having abnormal nasal anatomy ; having autoimmune diseases; having systemic and chronic diseases; having any history of psychogenic illness; or having an incomplete family structure, such as separation, divorce, widowhood, and loss of child or children.There are 90 items on the Symptom Checklist 90 (SCL-90) , each ofThe following will be used for the classification of the symptom : a factoA total of 4 AR symptoms  were evaluated separately on a 10-point VAS, with 10 representing the severest, and 0 indicating the mildest.The 10 SCL-90 factor scores and whole average scores were compared between the AR group (study group) and the Chinese norm  , from whThe 4 AR symptoms were evaluated with the VAS and recorded for scores. A partial correlation analysis  was madex̅ ± s, and all data will accept the normality test and homogeneity test of variance in a single-sample Kolmogorov-Smirnov test. The normal distribution data was tested by independent sample t or paired sample t test, and the nonnormal distribution data was tested by Mann-Whitney U. A one-way and orderly contingency table will accept the rank sum test of Kruskal-Wallis H. Using the multivariate regression statistical method, the control variables were first established, and then a partial correlation analysis with a partial correlation coefficient was performed. SPSS 19.0 software (IBM Corp) was used to carry out the statistical analysis of the data, and the difference has statistical significance if P<.05.If the measurement data is consistent with the normal distribution, they will be expressed by The SCL-90 was used to group and score the mental state of 827 patients with AR -4. The iThe intragroup comparison of BMI is shown in The intragroup comparison by living environment  is shown in A comparison was made between the AR group and the Chinese norm, and the positive factors of the SCL-90 were extracted . Among 8Partial correlation analysis was performed on the 4 positive factors of the SCL-90 and the 4 symptoms of AR . First, Zheng et al  pointed In the existing literature, despite many in-depth studies, there are few comprehensive studies in this regard due to the following reasons. First, part of the literature only made one-on-one retrospective analysis or targeted meta-analysis of AR with depression, anxiety, hostility, and other psychological disorders , while fTherefore, this study addresses the above 3 shortcomings and makes the following 3 optimizations on the research design. First, the SCL-90 scale was introduced systematically, which has a good discriminating function as it includes 90 items such as consciousness, emotion, diet/sleep, sensation, and lifestyle, and comprehensively reflects the distribution characteristics of 10 psychological symptoms with 10 factors . Second,To our knowledge, this is the first multicenter and interdisciplinary study of patients with AR. However, there are some limitations in this study. First, a few patients inevitably pretend to cooperate when filling out the questionnaire, which will affect the accuracy of the data. Second, psychological factors are greatly influenced by subjective factors, and the psychological state of patients at the time of filling in the form cannot reflect their later psychological state. Third, this study took place before the COVID-19 pandemic, but unfortunately, epidemic factors were not included in the study as a single factor. Since the outbreak of COVID-19 in Chengdu, China, the number of patients with mental diseases has increased greatly.The intergroup comparisons in this study were performed on 827 patients with AR in China’s Chengdu City versus the Chinese norm, which reveal mild to moderate psychological disorders. The scores for 4 factors, namely, depression, anxiety, psychosis, and other , are statistically higher than the Chinese norm, indicating that the psychological disorders develop mainly into these 4 types among the patients with AR. Therefore, otolaryngologists should pay close attention to the above four psychological states of patients when assessing the curative efficacy of somatopathies. Specifically, this study comprises 9 intragroup comparisons and 2 intergroup comparisons. Among the 9 classification criteria for intragroup comparisons, the living environment  yielded 8 positive results; working environment (indoors or outdoors) yielded 7 positive results; gender yielded 4 positive results; age, marital status, and education level yielded 3 positive results each; and BMI, monthly salary, and disease duration yielded 2 positive results each. This demonstrates the influencing factors of AR patients’ physical and mental health and their proportions, as well as the importance of the “biological-psychological-social” medical model. Meanwhile, these results also remind the otolaryngologists in Chengdu to offer more care and follow-ups to the following target positive populations: women and those with abnormal BMI, aged ≥45 years, with a monthly salary <¥5110 (US $700), with a disease duration <13 years, residing in urban areas, with a high school or above education, and who are indoor laborers.


The increase in life expectancy is linked, among other factors such as genetic factors, environmental factors, factors of medical conditions, socio-demographic factors, self-management, and access to care, to an increase in non-communicable diseases (NCDs) , which pBenin is currently facing the double burden of acute and chronic diseases with only limited resources allocated to the healthcare system . HoweverAccording to the WHO, one of the most significant constraints on the rehabilitation process is lack of access to specialist facilities or healthcare personnel . The useIn the remainder of this article, we highlight our strategy and collaborative efforts regarding the implementation of affordable technology-supported rehabilitation services, including mHealth, in the region of Parakou, Benin, with the aim to improve individual and public health.Despite the obvious need for EBP in LMICs, the development and implementation of EBP are currently applied more in HICs . This diEcole Supérieure de Kinésithérapie) thanks to the support of the so-called B4 initiative . However, since there is a lack of training in other French-speaking African countries, more than half of students who graduated (approximately 20 students are graduating every year) are international students who return to practice in their country after their training; therefore, the number of graduates, as reported by the Benin Physiotherapy Association, is still very low approximately 220 PT are working in Benin, among them 97 are member of the association (n = 6). There is thus an urgent need to develop more training facilities in western African countries to catch up to the level of training offered in Benin. It is also of utmost importance to create a training program for other rehabilitation discipline, as the number of other specialists is poor, i.e., there is no educational program for occupational therapy  in all of French-speaking western Africa is in Benin (bachelor and master levels). This physiotherapy program was established in 2000 in Cotonou  in high income countries (HICs) to the local specificities and realities of LMICs. This is a highly multidimensional problem starting from the limited support and integration of the rehabilitation services within the healthcare system, leading to suboptimal infrastructure and facilities and lack of integration within the cares . This problem is particularly relevant for patients suffering from neurological diseases, representing, however, a growing part of the patients and is responsible for the majority of the burden of the disability in LMICs . Here, sAnother limitation is the lack of information on EBP from health professionals themselves. Among the numerous barriers to EBP, the lack of specific academic training, which prevents rehabilitation specialists from comprehending the research language, is indeed one of the most significant obstacles in implementation of EBP in LMICs. Other factors currently limiting the knowledge translation include the lack of skills and the English language, motivation, and the ability to make decisions. Positive attitudes and motivation served as facilitators of transfers. Lack of time, lack of financial resources, limited access to scientific journals (despite program to support free access to publications), and applicability of research to rural settings were identified as organizational barriers .Different directions need to be taken simultaneously to drive sustainable changes.Educational programs and dedicated workshops should be developed to inform the local healthcare professionals about the concept of level of evidence and clinical recommendations. The utilization of new affordable technologies  may provide an alternative to the above-mentioned restrictions of rehabilitation or may supplement the present rehabilitation programs . mHealthDespite these educational efforts, the lack of direct and easy translation of the validating studies performed in HIC may currently limit the applicability of EBP in LMICs. It is therefore necessary that evidence be developed locally by researchers in the country collaborating directly with researchers from HICs.The Hasselt University and the University of Parakou signed a memorandum of understanding to strengthen the cooperation between the two institutions. The project is supported by both the rector and the vice rector of the University of Parakou for the cooperation between universities in Benin. Besides individual institutions, the collaboration is supported by the Ministry of Health and the Ministry of Education, thus showing the strong commitment of the country to further develop the quality of rehabilitation services. The bilateral collaboration also aims to expand the educational programs to include inland regions, such as central Benin in this sphere, too.Based on collaborative research between UHasselt and UParakou in recent years, different axes of research and development have been identified to ease the implementation of the rehabilitation services in Benin. After mutual visits of (assistant) professors of both universities, 3 doctoral students were enrolled in a bilateral program including a 3-month research stay in Belgium yearly. The stay was focused to cover scientific training with the aim to perform data collection and interpretation in Benin. These activities could potentially lead to internationally respected results along with direct impact in the regional clinical and academic services as well.In an attempt to further develop EBP in Benin, various research projects are currently underway. The first set of research are patient centric and focus on running qualitative studies to document the current (lack of) rehabilitation services that were attended during and after hospital discharge . These studies were run in the subacute and chronic phases after an event of stroke. Patients' requirements and expectations about the rehabilitation were also evaluated, after which further studies need to investigate the level of acceptance of the different types of interventions . As key players in the process, the healthcare professionals should be involved in this process, and therefore interviews, questionnaires, and focus groups also need to be performed to fully understand the needs of the clinicians. Currently, the modalities  and formats  of supervised exercise therapy as well as physical activity (PA) practice in community-dwelling chronic stroke survivors in Parakou are being explored. The patients' perceived barriers and facilitators to PA practice are also assessed. The content of physical therapy (PT) practice within inpatient rehabilitation facilities for stroke survivors in Benin, at the acute and sub-acute stages, is described using a standardized taxonomy . These insights are believed to contribute to opening the “black-box” of stroke inpatient rehabilitation in Benin .The initial results of these research lines will determine the extent to which interventions need to be adapted  before being tested in the field . For exaOther research focuses on the validation of the (modified) intervention. Pilot and feasibility studies are envisioned first, and later on validation is done in bigger studies . At this moment, a feasibility and effectiveness study of an intensive rehabilitation exercise program at the acute stage of stroke is being prepared. The outcomes of this pilot study should guide the design of a randomized controlled trial comparing conventional care and an intensive exercise program at the acute phase of stroke on their clinical outcomes. Additionally, the feasibility and effects of a 10-week community-based PA program using the WalkWithMe app  on walking performance in stroke survivors in Benin are also being investigated. WalkWithMe is a personalized mobile application that has been previously tested in Belgium in persons with multiple sclerosis to motivate patients to walk more during the rehabilitation process . In the It is strongly suggested that the discussion with the policymakers should start in parallel with the development of the scientific evidence to determine the best way to integrate this intervention in the care paths . It is also significant to ensure the accessibility of these interventions to as many patients as possible, despite the limited budget. The consortium already had several meetings with the Beninese Ministry of Health to prepare this next step and to integrate technologies and mHealth solutions in the provision of care.Despite the level of evidence supporting various rehabilitation interventions still being sparse in HIC, developing strong local research capacity and knowledge in LMICs is the best solution to increase the availability of rehabilitation services and their implementation in the long term.By developing local research and using culturally and locally adapted interventions, scientific evidence can be progressively built, thanks to the collaborations between the universities of Parakou and Hasselt. The ultimate result of this collaboration is expected to not only increase the availability of rehabilitation services, including technology-supported interventions, but also increase the quality of such services.BB wrote the first version of this paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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.


Endothelial activation plays an essential role in the pathogenesis of sepsis-induced acute lung injury, however, the detailed regulatory mechanisms remain largely unknown. Here, we reported that TRIM47, an E3 ubiquitin ligase of the tripartite motif-containing protein family, was highly expressed in vascular endothelial cells. TRIM47-deficient mice were effectively resistant to lipopolysaccharide (LPS)-induced acute lung injury and death by attenuating pulmonary inflammation. TRIM47 was upregulated during TNFα-induced endothelial activation in vitro. Knockdown of TRIM47 in endothelial cells inhibited the transcription of multiple pro-inflammatory cytokines, reduced monocyte adhesion and the expression of adhesion molecules, and suppressed the secretion of IL-1β and IL-6 in endothelial cells. By contrast, overexpression of TRIM47 promoted inflammatory response and monocyte adhesion upon TNFα stimulation. In addition, TRIM47 was able to activate the NF-κB and MAPK signaling pathways during endothelial activation. Furthermore, our experiments revealed that TRIM47 resulted in endothelial activation by promoting the K63-linked ubiquitination of TRAF2, a key component of the TNFα signaling pathway. Taken together, our studies demonstrated that TRIM47 as a novel activator of endothelial cells, promoted LPS-induced pulmonary inflammation and acute lung injury through potentiating the K63-linked ubiquitination of TRAF2, which in turn activates NF-κB and MAPK signaling pathways to trigger an inflammatory response in endothelial cells. Leukocyte adhesion to endothelial cells and migration across endothelial cells are mediated by the interaction of complementary adhesion molecules on leukocytes and endothelial cells. The increased expression or release of endothelial cell adhesion molecules is a hallmark of endothelial cell activation.4 Upon the access of leukocytes into the lung parenchyma, they can release inflammatory mediators to destroy pathogens, but the over-activated immune response has the potential to result in the imbalance of the pro-inflammatory and anti-inflammatory mechanisms, triggering “cytokine storm” and subsequent tissue damage.5 Considering the critical role of endothelial response during ALI, strategies that try to target endothelial components including cell surface receptors, signaling pathways, transcriptional networks, and endothelial cell gene products, have been recently proposed to attenuate endothelial activation and improve endothelial dysfunction.7As a main target of injury-related circulating cells and humoral mediators, the pulmonary endothelium is critically implicated in the pathogenesis of ALI.8 Multiple TRIM proteins have been found to participate in innate immunity through positive or negative regulation of cytokines, toll-like receptors, pattern recognition receptors, intracellular signaling pathways, and transcription factors.9 Although numerous studies have focused on the roles and regulatory mechanisms of TRIM proteins in immune cells,14 fewer studies have focused on their regulatory functions on endothelial inflammation. It has been reported that TRIM28 was abundant in endothelial cells, and interfering with TRIM28 expression has anti-inflammatory and anti-angiogenic phenotypes.15 However, the precise mechanism of TRIM family members in regulating endothelial activation remains largely unknown.Tripartite motif-containing (TRIM) proteins, a subfamily of E3 ubiquitin ligases, are involved in many physiological processes including cell proliferation and differentiation, innate immunity, and autophagy.16 It was primarily located in the nucleus and its LXXLL motif was thought to be closely related to nuclear receptor binding.16 Emerging evidence indicated that TRIM47 played the roles in tumorigenesis and progression,19 viral resistance processes,20 and cerebral ischemia–reperfusion injury.21 A recent genome-wide association study showed that the SNPs of TRIM47 and TRIM65 which were located in the adjacent position of human chromosome 17, were closely related to white matter hyperintensities, a result of the ischemic damage of the small deep cerebral vessels.22 These findings suggested that TRIM47 and TRIM65 may coordinately or independently participate in the regulation of cerebrovascular injury. Our previous work demonstrated that TRIM65, as an E3 ubiquitin ligase, selectively promoted the ubiquitination and degradation of VCAM-1, reducing lung inflammation and damage caused by sepsis.23 However, the role of TRIM47 in endothelial inflammation remains to be elucidated.TRIM47 was initially found in brain astrocytomas and was named as GOA (gene overexpressed in astrocytoma).In this study, a global TRIM47 knockout mouse was constructed to investigate the roles of the protein in ALI. The effects of TRIM47 on endothelial activation were also assayed in an in vitro model of inflammation induced by TNFα. Furthermore, the effects of TRIM47 on signaling pathways associated with endothelial activation were examined. Finally, we demonstrated that TRIM47 promoted pulmonary inflammation and injury through endothelial activation by potentiating the K63-linked ubiquitination of TRAF2, a key component of the TNFα signaling pathway.We first examined the distribution and expression of TRIM47 in various tissues of mice. As shown in Fig. –/−) mouse was generated by using CRISPR/Cas9 to remove all exons of the TRIM47 gene  of TRIM47 was found by cNLS Mapper or NLStradamus Software. Whether TRIM47-mediated endothelial activation depends on its subcellular location needs further investigation in the future.Many TRIM proteins have been proved to be implicated in innate immunity and inflammatory response. Our previous work showed that TRIM47 was down-regulated upon TNFα exposure in THP-1-derived macrophages.23 TRIM47 promotes inflammatory response both in vitro and in vivo. TRIM47 knockdown showed strong inhibition of adhesion molecules expression and monocyte adhesion in HUVECs. These effects were further confirmed in pulmonary microvascular endothelial cells from TRIM47 KO mice, indicating that TRIM47-mediated endothelial activation mainly contributed to the pathological process of acute lung injury. Since the previous GWAS results suggested that both TRIM47 and TRIM65 are closely related to the cerebral vessels injury,22 our results provided strong evidence that although TRIM47 and TRIM65 were critically involved in the regulation of endothelial inflammation and injury, their mechanisms might differ.Different from the anti-inflammatory effect of TRIM65, another constitutively expressed protein in endothelial cells,9 Recent work showed that TRIM proteins positively or negatively mediated NF-κB activation through K48- or K63-linked polyubiquitin, respectively. For example, TRIM5 interacted with the TAK1-containing kinase complex to positively regulate NF-κB activation by mediating K63 polyubiquitin chain synthesis.12 TRIM25 promoted NF-κB activation by enhancing the K63-linked ubiquitination of TRAF2 and bridging the interaction of TRAF2 and TAK1 or IKKβ.27 TRIM14 enhanced NF-κB activation in endothelial cells via directly binding to NEMO and promoting the phosphorylation of IκBα and p65, which was dependent on its K63-linked ubiquitination.32 Several target proteins of TRIM47 have been found in recent years. For example, TRIM47 induced ubiquitination of PPM1A and decreased the expression level of PPM1A in human embryonic lung fibroblast.33 TRIM47 interacted with CYLD and induced its degradation in proportion to non-alcoholic steatohepatitis severity.34 As an oncogene, TRIM47 promoted cancer proliferation through ubiquitination and degradation of multiple targets such as FOXO1,35 P53,36 SMAD418 and FBP1.37 TRIM47 was also implicated in innate immunity through K48-linked ubiquitination of NF90.38 Structurally similar to TRIM25, a reported E3 ligase facilitating K63-linked ubiquitination,8 TRIM47 also interacts with TRAF2 and promotes MAPK and NF-κB activation through K63-linked ubiquitination. Taken together, we provided evidence that E3 ligase TRIM47 aggravated TNFα-induced endothelial activation and acute lung injury through selectively promoting the K63-linked ubiquitination of TRAF2, a key component of the TNFα signaling pathway.Proteins covalently modified with K48-linked polyubiquitin are targeted for proteasomal degradation, whereas proteins covalently modified with K63-linked polyubiquitin generally become functionally activated.8 TRIM47 may be an attractive target for drug development in endothelial inflammation and ALI. Certainly, further elucidating the underlying mechanism of TRIM47 in endothelial activation should provide an insight into the development of effective therapeutic agents clinically.To sum up, we identified TRIM47 is a novel activator of endothelial cells, in which it mediates inflammatory response and promotes inflammation and tissue damage during ALI through activating endothelial TRAF2-MAPK/NF-κB pro-inflammatory axis Fig. . At presEscherichia coli O111:B4) was purchased from Sigma. VCAM-1 (sc-13160), ICAM-1 (sc-1511-R) and β-actin (sc-47778) antibodies were from Santa Cruz Biotechnology. TRIM47 antibody (26885-1-AP) was purchased from Proteintech. Phospho-p65 (3033), p65 (8242), IκBα (4812), phospho-IκBα (2859), phospho-IKKα/β (2078), IKKα (11930), IKKβ (8943), phospho-JNK (4668), JNK (9252), phospho-ERK1/2 (4370), ERK1/2 (4695), phospho-p38 (4511), p38 (8690),TRAF2 (4724), TRAF6 (8028), TAK1(5206), IKKγ (2685), TBP (44059), Flag (8146 and 2368) and α-tubulin (2125) antibodies were purchased from Cell Signaling Technology. VE-cadherin (ab33168), ubiquitin (ab7780), ubiquitin , and ubiquitin  antibodies were purchased from Abcam.Recombinant Human and murine TNFα were obtained from Peprotech. LPS  to induce lethal endotoxic shock. The control group received injections of the equivalent volume of 0.9% NaCl solution. After injection, the mice were closely monitored for general condition and survival for 7 days.Age-matched mice (7–9 weeks) were randomly assigned to control or experimental groups. Wild-type and TRIM4739 Photomicrographs were taken by a light microscope (Olympus BX51). Lung injury was evaluated by an independent pathologist who was blinded to the grouping, including hemorrhage in the lung tissue, alveolar congestion, edema, infiltration of macrophages and neutrophils, and morphological changes in the alveolar wall. For immunostaining, the paraffin tissue sections were incubated in 3% H2O2 to inactivate endogenous peroxidases. Antigen retrieval was performed by heating the sections in citrate buffer. Endogenous biotin was blocked with the Biotin-Blocking Kit . Sections were then incubated with the anti-TRIM47 antibody for 60 min at a 1:200 dilution, followed by HRP-conjugated secondary antibodies for 30 min. Detection was performed using DAB substrate kit (Maixin Biotechnologies).The right upper lungs were removed after 24 h of LPS challenge and were fixed in 4% phosphate-buffered paraformaldehyde. The 4-μm paraffin tissue sections were cut and stained with H&E as previously described.To evaluate the magnitude of pulmonary edema, the wet-to-dry weight ratios at 24 h after LPS challenge were determined. The left lung tissue samples were weighed immediately after removal (wet weight) and then subjected to desiccation in an oven at 50 °C until a stable dry weight was achieved after 72 h. The ratio of the wet/dry weight was then calculated.4/ml) were prepared and infected at a Multiplicity of Infection (MOI) of 50 with negative control, TRIM47 siRNA1, TRIM47 siRNA2 or TRIM47 overexpression lentiviruses for 16 h at 37 °C in the presence of 5 μg/ml polybrene. The cultures were then washed and cultured in fresh medium for 72 h. GFP expression was detected to calculate the infection efficiency. Then, cells were treated with 10 ng/ml TNFα for indicated times, and mRNAs or proteins from those cells were extracted and detected.Human Umbilical Vein Endothelial Cells (HUVEC) and THP-1 cells were purchased from Lonza Walkersville Inc. HUVECs were cultured in EGM medium according to the manufacturer’s instruction, and used for the experiment in less than eight passages. THP-1 was cultured in RPMI-1640 medium supplemented with 10% FBS and 2-mercaptoethanol to a final concentration of 0.05 mM. RAW264.7, EA.hy926, bEnd.3, and HEK293 cells were purchased from ATCC and cultured in DMEM supplemented with 10% FBS. U251, HeLa, MDA-MB-231, 3T3-L1, and A549 cell lines were from National Collection of Authenticated Cell Cultures  and cultured in DMEM supplemented with 10% FBS. The hCMEC/D3 cell line was purchased from BeNa Culture Collection  and cultured in RPMI-1640 medium supplemented with 10% FBS. HL-1 cardiac muscle cell line was obtained from Sigma-Aldrich and cultured in Claycomb medium supplemented with 100 μM norepinephrine, 4 mM l-glutamine and 10% FBS. Two siRNA target sequences were selected in Human TRIM47 gene (GenBank accession NM_033452.2): siRNA1: 5′-TGAAGCTCCCAGGGACTATTT-3′, and siRNA2: 5′-TACTGGGAGGTGGAGATTATC-3′. TRIM47 siRNA was constructed into the lentivirus expression vector pLV[shRNA]-EGFP:T2A:Puro-U6. A universal sequence was used as a negative control for RNA interference. Human TRIM47 gene was constructed into pLV[Exp]-EGFP:T2A:Puro-EF1A vector to obtain the expression lentiviral vector. The viral particles were produced by third-generation packaging in 293T cells and Lentiviral stocks were concentrated using ultracentrifugation. HUVECs  with positive selection by the CD31 MicroBeads  according to the manufacturer’s instructions. Enriched primary endothelial cells were incubated with 10 μl of mouse CD31 Microbeads at 4 °C for 15 min and were centrifuged at 300×g for 10 min. CD31-positive cells were collected on a MACS LS column and washed three times with PBS containing 10% FBS. The cells were re-suspended and cultured in endothelial cell basal medium-2 . The pulmonary microvascular endothelial cells were treated with 10 ng/ml murine TNFα for 8 h. The protein levels of ICAM-1 and VCAM-1 were detected by western blot.The pulmonary microvascular endothelial cells were isolated from wild-type and TRIM47 KO mice, respectively, according to the previously described method.−ΔΔCT method. The results were from three independent experiments performed in triplicate.Total tissue or cellular RNA was isolated using TRIzol reagent, according to the manufacturer’s instructions . One microgram of total RNA was reverse-transcribed using a One Step PrimeScript™ RT-PCR Kit  with a thermocycler. The mRNA levels were determined by SYBR Green dye using an ABI 7500 sequence detection system with a reaction mixture that consisted of SYBR Green 2×PCR Master Mix , cDNA template, and the forward and reverse primers. Primer sequences were listed in Supplementary Tab. Tissue extracts and whole-cell lysates were prepared in radioimmunoprecipitation assay buffer (Thermo Scientific) supplemented with 1 mM PMSF. Nuclear and cytoplasmic protein fractions from cells were extracted by Nuclear-Cytosol Extraction Kit , according to the manufacturer’s instructions. Fifty micrograms protein per sample was loaded in each lane and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes  in Tris-glycine buffer  containing 20% methanol. The membranes were blocked with skimmed milk for 1 h, washed in Tris-buffered saline containing 0.1% Tween-20 (TBST), and incubated with primary antibodies overnight at 4 °C. After washing in TBST for three times, nitrocellulose membranes were incubated for 1 h at room temperature with the horseradish peroxidase-conjugated IgG . The bands were visualized by the SuperSignal West Pico Chemiluminescent Substrate Trial Kit . The immunodetected protein bands were then analyzed using ChemiDoc XRS system with Quantity One software .At the end of the treatment, cells were rinsed with phosphate-buffered saline (PBS) three times, fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized in 0.1% Triton X-100 for 10 min. An incubation in 5% bovine serum albumin (BSA) in PBS for 1 h was performed to prevent antibody non-specific binding. The cultures were incubated with primary antibodies overnight at 4 °C. After incubation with primary antibodies, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG , and the nuclei were stained with DAPI. Immunostained cells were examined under a fluorescence microscope .23 The control, TRIM47 knockdown, or TRIM47 overexpressed HUVECs were cultured on a six-well plate overnight, and then were treated with 10 ng/ml TNF-α for 8 h. THP-1 cells were labeled with a Zombie Red fluorescent staining kit  according to the manufacturer’s instructions. The labeled THP-1 cells were added into HUVECs at a density of 5 × 105 per well and were co-cultured for 1 h at 37 °C. Non-adherent cells were removed by gently washing with cold RPMI-1640 medium. The images of adherent THP-1 cells and the number were determined under Cytation 3 Cell Imaging Multi-mode Reader (Biotek Instruments).The adhesion assay was performed as previously described.The concentrations of TNFα, IL-1β, and IL-6 in the serum, and IL-1β and IL-6 levels in the supernatant of HUVECs were measured using the specific ELISA kits according to the manufacturer’s instructions . Absorbance at 450 nm wavelength was measured, and the protein concentration was determined by interpolation on to absorbance curves generated by recombinant protein standards using iMark™ Microplate Absorbance Reader (Bio-Rad).TM Protein G-Agarose (Thermo Fisher) following the manufacturer’s instructions. After extensive washing, the electrophoresis loading buffer was added to the complexes and incubated for 5 min at 95 °C. Immunoprecipitated proteins were resolved by SDS-PAGE and analyzed by immunoblotting with indicated antibodies.HUVECs were transfected with Flag or Flag-TRIM47 plasmid using electroporation. The protease inhibitor MG132  was added for 1 h before harvesting. After 48 h of transfection, cells were lysed in CelLytic M Cell lysis buffer with protease inhibitors, phosphatase inhibitors, NEM, and ubiquitin aldehyde. HUVECs were harvested after 15 min-TNFα (10 ng/mL) exposure. The immune complexes were collected by incubation  with protein G-agarose (Sigma). Co-IP assays were performed by using Piercet test was used. For multiple comparisons, one-way ANOVA followed by Turkey’s post hoc analysis was used. The survival rate comparisons were made by the log-rank test. A value of P < 0.05 was considered significant.Statistical analysis was performed with GraphPad Prism software . Data were expressed as mean ± SD. For comparison between two groups, the unpaired Student’s Supplementary Materials


The glucocorticoid receptor (GR) at the blood–brain barrier (BBB) is involved in the pathogenesis of drug-resistant epilepsy with focal cortical dysplasia (FCD); however, the roles of GR isoforms GRα and GRβ in the dysplastic brain have not been revealed. We utilized dysplastic/epileptic and non-dysplastic brain tissue from patients who underwent resective epilepsy surgery to identify the GRα and GRβ levels, subcellular localization, and cellular specificity. BBB endothelial cells isolated from the dysplastic brain tissue (EPI-ECs) were used to decipher the key BBB proteins related to drug regulation and BBB integrity compared to control and transfected GRβ-overexpressed BBB endothelial cells. GRβ was upregulated in dysplastic compared to non-dysplastic tissues, and an imbalance of the GRα/GRβ ratio was significant in females vs. males and in patients > 45 years old. In EPI-ECs, the subcellular localization and expression patterns of GRβ, Hsp90, CYP3A4, and CYP2C9 were consistent with GRβ+ brain endothelial cells. Active matrix metalloproteinase levels and activity increased, whereas claudin-5 levels decreased in both EPI-ECs and GRβ+ endothelial cells. In conclusion, the GRβ has a major effect on dysplastic BBB functional proteins and is age and gender-dependent, suggesting a critical role of brain GRβ in dysplasia as a potential biomarker and therapeutic target in epilepsy. The glucocorticoid receptor (GR) has recently been uncovered as a critical molecular regulator of drug permeability and barrier integrity at the epileptic blood–brain barrier (BBB), where it is found to be overexpressed and to have accelerated maturation ,2,3. AftFocal cortical dysplasia (FCD) is a common epilepsy pathology that stems from focal malformations in the cerebral cortex , where nTo identify the involvement of the GRα and GRβ isoforms in FCD, we used cortical brain tissues from patients who underwent surgery for refractory epilepsy to determine: 1) the expression pattern of GRα and GRβ in dysplastic (epileptic) and non-dysplastic (relative control) tissues, 2) changes in GRα/GRβ ratio based on gender and age of these individuals, 3) the expression of neurovascular GR isoform localization in dysplastic vs. non-dysplastic brain tissue, and (4) the subcellular localization of GRα and GRβ based on the ASM combination taken by these patients before surgery as CYP dependent or a partially/independent metabolic pathway. To delineate the involvement of these two GR isoforms in BBB endothelial cells, where GR has been found to have a significant role ). All cell culture treatment experiments were performed in 100 mm Petri dishes at 70–80% confluency.We used primary microvascular endothelial cells derived from brain specimens resected from patients with drug-resistant epilepsy ,  earlier , obtainee flasks ,2. PrimaTo simulate the increase in GRβ expression observed in the dysplastic tissue compared to non-dysplastic, HBMECs were transfected with HA-tagged GRβ DNA (1.075 µg/µL). The custom HA-tagged GRβ plasmid was obtained from OriGene Technologies utilizing the open reading frame (ORF) from cat. RC220377  cloned in a pCMV6-AC-HA vector . To achieve this transfection, 5 µg of HA-GRβ DNA was mixed in serum-free Dulbecco’s modified eagle medium (DMEM/F12) and was later combined with a 30 µg mixture of lipofectamine  in serum-free DMEM to form the DNA+lipofectamine complex. This mixture was set aside for 25 min at room temperature. Once formed, this DNA+lipofectamine complex was combined with additional serum-free DMEM and added to the 100 mm Petri dish of 70% confluent HBMECs and left to incubate for 5 h at 37 °C. After the incubation was complete, the serum-free media containing the DNA+lipofectamine complex was aspirated, the plate was washed with 0.1 M phosphate-buffered saline (PBS), and the PBS was replaced with normal HBMEC media  until the following day when subsequent drug treatment experiments were performed. These transfected cells will be denoted throughout as “HBMEC+HA-GRβ”.To determine the effect of ASM (oxcarbazepine: OXC or levetiracetam: LEV) or GR agonist (dexamethasone: DEX) treatment on the subcellular localization of various protein targets crucial for drug metabolism/transport and BBB integrity, HBMECs, HBMEC+HA-GRβ, and EPI-ECs were divided into four treatment groups each: vehicle control, OXC (25 µg/mL), LEV (15 µg/mL), and DEX (10 µM) for 24 h. The cells were fractionated into cytoplasmic and nuclear fractions using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit as described above  at 6 h (HBMEC and HBMEC+ HA-GRβ) and 24 h  and analyzed by Western blot. Protein concentration was estimated by the Bradford method. Cell culture supernatant samples were also collected at each time point.MMP activity was determined by gelatin zymography using the samples obtained from the endothelial cell supernatant with and without drug treatment. A total of 20 µL of each sample was loaded into gelatin zymography gels  and run at 100 V for about 2 h . The gelt-test was used to compare dysplastic and non-dysplastic brain tissue regions of the same individual patient. One-way or two-way analysis of variance (ANOVA) was utilized to compare multiple groups, with a Tukey post hoc test. All data are presented as mean with standard deviation (SD), and p < 0.05 was considered to be statistically significant.Origin Pro 9.0 Software was used for data analysis and statistical interpretation of data. Paired