By observing the buckled morphology of apertures, the structures can be classified into two groups: low (AR 1.6 and 2.0) and high (AR 4.0 and 5.3) AR groups, and crP decreases as AR increases in each group. To investigate the results theoretically, the analysis based on Euler’s buckling theory and slenderness ratio is conducted, indicating that the theory is only valid for the high-AR group herein. Besides, considering the correction factor, crP agrees well with the experimental results.A polymeric stencil with microdot apertures made by using polydimethylsiloxane (PDMS) molds with pillar patterns has many advantages, including conformal contact, easy processability, flexibility, and low cost compared to conventional silicon-based membranes. However, due to the inherent deformability of PDMS materials in response to external pressure, it is challenging to construct structurally stable stencils with high structural fidelity. Here, we propose a design rule on the buckling pressure for constructing polymeric stencils without process failure. To investigate the critical buckling pressure ( H/D) ratio) increases, the deformation of the structures is inevitable, which leads to the process failure of the membrane with apertures. To achieve reliability and reproducibility of the process and advance toward realization of large-scale fabrication and automatization, this issue should be addressed. Since previous studies did not address the processibility depending on the structural features and vertical pressure, process criteria considering the AR and vertical pressure should be developed. Here, we have established a design rule based on critical buckling pressure (crP) to prevent the buckling phenomenon. The experiments were conducted using different structures with various ARs of 1.6, 2.0, 4.0, and 5.33. Moreover, additional structures with different diameters and heights of the pillars with same ARs of 2.0 and 4.0 were prepared to verify whether the AR is the only determining factor for structural stability in the structural features.Membranes with microdot apertures are widely used in lithography masks, etching masks, microfluidic devices, membranes, and filters ,2,3. BasCommercially available UV curable polyurethane acrylate (PUA) resin  was used in the experiment. The PUA prepolymer resin consists of a photoinitiator, a UV curable releasing agent, and a prepolymer for crosslinking with acrylate functional groups.4F8 gas to detach the patterned PDMS polymer easily. Next, the mixture of PDMS  base and curing agent with a weight ratio of 10:1 was poured on the patterned silicon masters and cured for 2 h at 70 °C. The cured PDMS molds were carefully peeled from the silicon masters [To fabricate the patterned polydimethylsiloxane (PDMS) molds, PDMS was poured into the hole patterned silicon masters, which were made by photolithography and reactive ion etching. The surface of the silicon masters was treated with C−2  images were obtained using field emission SEM  with 10.0-kV acceleration voltage. Also, optical microscope (OM) images were obtained by optical microscopy . To confirm the shape of the apertures for each case, the constructed polymeric stencils were prepared by freeze-fracturing with liquid nitrogen and observed through SEM and OM images.crP depending on the geometrical features of the pillars to prevent buckling-induced process failure. This approach would provide concrete fundamentals for the fabrication criteria progressed from an empirical approach. To investigate the structural stability, six patterned PDMS molds with micropillars having different D and H were set. The detailed information on the fabrication process of PDMS molds and patterned silicon masters is provided in the Controlling the distance between the PDMS surfaces, which is adjusted by the externally applied pressure from the compressor in the system, is more important in the process of fabricating the polymeric stencil than applying pressure by hand to achieve reproducibility, reliability, uniformity, and accuracy. When the applied pressure is insufficient to secure the proper distance between the PDMS surfaces, the PUA layers, where the apertures should be formed, would be fully cured, resulting in the blocking of the apertures in the PUA stencil. However, if excessive pressure is applied, the pillars in the patterned PDMS in contact with the flat PDMS would be buckled or fractured, which is considered a process failure. The previous studies for fabricating a UV-cured membrane with apertures exhibit the need for investigation of properly applied pressure depending on the structural dimension as summarized in D = 50 μm, H = 80 μm, and AR = 1.6; (2) D = 40 μm, H = 80 μm, and AR = 2.0; (3) D = 50 μm, H = 100 μm, and AR = 2.0; (4) D = 30 μm, H = 120 μm, and AR = 4.0; (5) D = 40 μm, H = 160 μm, and AR = 4.0; (6) D = 30 μm, H = 160 μm, and AR = 5.3. The samples were mainly categorized into four kinds of AR . For the case of AR = 2.0 and 4.0, the samples with different D and H while fixing the AR were prepared to confirm if AR is the only factor in geometry, which determine the mechanical stability herein. With the prepared patterned PDMS mold, the fabrication of the polymeric stencil with microdot apertures was conducted with the sandwich-like assembly as mentioned above. The applied vertical pressure to the assembly was increased gradually and discretely through the imprint machine. For each case, the applied imprint force was converted into the pressure per single pillar. The pressure that induces the buckling phenomenon is denoted as the experimental buckling pressure.The scanning electron microscopy (SEM) images in D and H, affect the buckling phenomena in the same AR condition, the experiment was performed using two pillar structures with same AR = 2.0  were prepared, and the stencils were fabricated with each structure , 0.171 MPa (AR = 2.0), 0.229 MPa (AR = 4.0), and 0.140 MPa (AR = 5.3)  . Note thcrP, the end conditions of the pillar in the model should be set reflecting the behavior of the experimentally used PDMS pillar. In this analytical model, the bottom and the vertical line connected as one body represents the patterned PDMS, while the top cover represents the flat PDMS. The bottom-end condition of the pillar is fixed at the base, and the upper-end condition of the pillar is assumed to be rotation-fixed and translation-free. L without an applied load. crF) is applied to the pillar, which corresponds to the shape of the apertures in c′) is defined as the center point of the deflected pillar where the bending moment is zero. The theoretical crF can be obtained by solving the bending-moment equation considering the zero-moment at the point of injection (c′) in Equation (1), which is called Euler’s buckling equation . The crP in this equation corresponds to the pressure applied to a single pillar among numerous pillars in the patterned PDMS mold with an area of 9 cm2.I is the moment of inertia with respect to the principal axis of buckling, which is A is the top surface area of the pillar, which is L is the height of the pillar (H). E* = E/(1 − v2), where v is the Poisson’s ratio and E is the Young’s modulus. E* in Equations (1) and (2) is the combined elastic modulus, which is commonly used for large deformations considering the material’s Poisson’s ratio [*E is expressed as 4E/3 [’s ratio ,12,13. A as 4E/3 ,15.r. Based on the definition, the slenderness ratio can be expressed as follows:r) describes the cross-sectional area distribution in a pillar around its centroidal axis, which is L/r)c, which determines the applicability of Euler’s buckling equation, is obtained by assuming that the material’s critical stress (crP), and the relationship between L/r)c can be re-expressed by substituting Equation (2) into Equation (3) as follows:To elucidate the applicability of this equation to the cases in this study, the criteria for applying Euler’s buckling theory were investigated. Since Euler’s buckling equation is derived assuming that the pillars under the external load are extremely thin like a thread or string, it is not that suitable to assume such for the pillars used herein. In particular, the low-AR structures are likely to be excluded from the applicable range of Euler’s buckling equation. To set the criteria for the applicability of Euler’s buckling equation, the slenderness ratio was introduced and used, which is defined as the ratio of the length of the structure to its least radius of gyration it (σpl) . Euler’sL/r)c and assuming that After rearranging the equation for  [L/r)c was calculated as 9.906. By considering the relationship between the critical slenderness ratio and AR in Equation (3), the critical AR (cAR) as an indicator of applicability of Euler’s buckling equation was calculated as 2.4765. Thus, Euler’s buckling equation is only valid for the pillar structure with AR over ~2.5 in the case of using PDMS as molds. From the results, the calculated critical slenderness ratio, the theoretical crP, and the experimentally obtained buckling pressure are plotted in crP, the polymeric stencil was stably made without the buckling phenomenon. This result can be attributed to the inaccuracy in the following cases, which is not considered while analyzing the buckling phenomenon using Euler’s buckling equation [Inhomogeneity due to the inherent non-uniformity of the material and imperfect mixing process of PDMS base and curing agent of PDMS.The imperfect flatness of the UV-imprinting machine used in the experiment.Slightly different distances from the substrate to the top surface of each pillar due to the finely inclined PDMS mold.In Equation (5), ngth, σy ,17,18,19ngth, σy . E* is c.61 MPa) . Based oequation ,23:InhocAR due to the mismatch of the experimental result and Euler’s buckling theorem. Apparently, the resultant shapes of the low-AR structures were slanted without the curved wall, whereas the high-AR structures were bent with a curved wall. Also, the lateral shift of the pillar top surface of the low-AR structures was more alleviated than that of the high-AR structures under the comparable applied pressure. This trend implies that although the deformation of the low-AR structures occurs below the anticipated theoretical buckling pressure, the amount of deformation was restrained compared to the high-AR structures. It is well known that the large deformation for a short pillar is restrained, and since the structural stress is accumulated due to less deformation, it would be fractured rather than deformed. In this aspect, ideally, the low-AR structures under compression are likely to be expanded laterally , and a high-AR group (AR = 4.0 and 5.3). In each group, crP decreases as the AR increases, as expected. Also, the comparative analysis of crP based on Euler’s buckling theory was conducted to validate the buckling behavior. By considering the slenderness ratio, which is calculated as four times the AR in this study, we found the applicable range of Euler’s buckling theory. cAR was determined as 2.476; therefore, the analysis based on Euler’s buckling theory for the high-AR group is only valid herein. By considering a correction factor of 1.67, the experimentally obtained values and the calibrated theoretical buckling pressure agreed well for the high-AR group, but not for the low-AR group, in which the experimental approach rather than the theoretical approach should be considered. Such a design rule for preventing the buckling phenomenon can be used as an indicator to produce stable polymeric stencils with microdot apertures and can be advantageously applied to scalable processes, including the roll-to-roll process for manufacturing polymeric membranes for applications in biomedical, microfluidic, and flexible devices.We fabricated UV-cured polymeric stencils with microdot apertures having varied AR of the structures based on the scavenging effect of PDMS molds. In the fabrication process, the buckling phenomenon of the PDMS pillars was observed when the applied vertical pressure increased above a certain pressure, which is


N = 60). Lnc-ITSN1-2 expression was elevated in patients with AS compared to controls (P < 0.001). Baseline lnc-ITSN1-2 expression was positively associated with C-reaction protein (CRP) (P = 0.021), interleukin (IL)-1β (P = 0.020), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score (P = 0.040), and Ankylosing Spondylitis Disease Activity score with C-reactive protein (ASDASCRP) (P = 0.045) in patients with AS. Furthermore, lnc-ITSN1-2 expression declined during the treatment with adalimumab (P < 0.001). Notably, this reduction was more obvious in responders than non-responders. In conclusion, declined lnc-ITSN1-2 expression during the TNFα inhibitor treatment correlates with good treatment efficacy in patients with AS, suggesting its clinical value for AS management.Previous studies show that long noncoding RNA intersectin 1-2 (lnc-ITSN1-2) promotes the inflammation process and serves as a potential biomarker in autoimmune diseases, except for ankylosing spondylitis (AS). Therefore, this study aimed to explore the correlation of baseline lnc-ITSN1-2 expression with disease risk and activity of AS, and to investigate its longitudinal change with treatment response to a tumour necrosis factor alpha (TNFα) inhibitor in patients with AS. In total, 63 patients with AS receiving 12-week adalimumab treatment were included and their baseline clinical features were collected. Lnc-ITSN1-2 expression in peripheral blood mononuclear cells (PBMC) of patients with AS was detected by reverse transcription quantitative polymerase chain reaction. Meanwhile, Assessment in Spondyloarthritis International Society (ASAS) 40 response was evaluated at week 2 (W2), W4, W8, and W12. According to the ASAS40 response status at W12, patients with AS were classified into responders and non-responders. PBMC lnc-ITSN1-2 expression was also determined in healthy controls ( Ankylosing spondylitis (AS) is a chronic inflammatory disease that mainly affects the axial skeleton with unknown etiology Smith . Regardi+ T cells of inflammatory bowel disease (IBD) via regulating the microRNA (miR)-125a/interleukin (IL)-23R axis (Nie and Zhao Long noncoding RNAs (lncRNAs) are a collection of noncoding RNAs of relatively large size involved in numerous cellular and biological processes  > 2.1; (iv) willing to receive a 12-week adalimumab treatment. The patients were ineligible for inclusion if they met any of the following exclusion criteria: (i) known contraindications to adalimumab; (ii) dysfunction in liver, kidney, heart, or respiration; (iii) active infection; (iv) complicated with malignancies; (v) poor compliance to the study. Additionally, 60 healthy subjects who underwent physical examination in our hospital were recruited as healthy controls. In order to allow matching of the age and gender between healthy controls and the patients with AS, the age of healthy controls was limited to within 18–50 years, and the gender of healthy controls was limited at a ratio of 4:1 .This study was permitted by the institutional review board, and all subjects signed informed consent documents. This prospective cohort study enrolled 63 patients with AS between January 2019 and July 2020. The recruitment criteria were as follows: (i) diagnosis of AS; (ii) aged older than 18 years; (iii) had active disease which was defined as Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) > 4  and Ankylosing Spondylitis Disease Activity Score with C-reactive protein , total back pain, Patient’s Global Assessment of Disease Activity (PGADA), and ASDASg for 10 min and then used for determining the level of inflammatory cytokines by enzyme-linked immunosorbent assay (ELISA) using commercial human ELISA kits . For measurement of lnc-ITSN1-2 expression during the adalimumab treatment, PB samples of patients with AS were also collected at week 2 (W2), week 4 (W4), week 8 (W8), and week 12 (W12) after initiation of treatment, except for W0. Immediately after each sample collection, peripheral blood mononuclear cells (PBMC) were isolated by the density gradient separation method and then used to determine the lnc-ITSN1-2 expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR). As for healthy controls, PB samples were obtained during their physical examination and then were used for separation of PBMC to determine the lnc-ITSN1-2 expression by RT-qPCR as described below.Peripheral blood (PB) samples from patients with AS were collected prior to starting treatment (W0) to determine the level of inflammatory cytokines including TNFα, interleukin (IL)-1β, IL-6, and IL-17A. Serum was separated from the PB samples in a refrigerated centrifuge by centrifuging at 1000 to 2000×−ΔΔCt method with GAPDH as an internal reference. The primer sequence was designed according to a previous study as follows: lnc-ITSN1-2, forward primer (5′–3′) GCTTCACTCGCTTGCTTACA; reverse primer (5′–3′) GGTTCTGTCTTGCCTTCTGTT; GAPDH, forward primer (5′–3′) GACCACAGTCCATGCCATCAC; reverse primer (5′–3′) ACGCCTGCTTCACCACCTT  was used to isolate total RNA. After extraction, iScript™ cDNA Synthesis Kit (with random primer)  was used for cDNA synthesis. Then the PCR reaction was completed using QuantiNova SYBR Green PCR Kit . The lnc-ITSN1-2 relative expression was calculated by the 2All patients received a 12-week adalimumab treatment by receiving 40 mg adalimumab  through subcutaneous injection every 2 weeks. During the treatment, the treatment response was assessed at W2, W4, W8, and W12 using Assessment in Spondyloarthritis International Society (ASAS) 40 criteria and the detailed definition of the ASAS40 response was described in previous literature  curve analysis was used to determine the performance of lnc-ITSN1-2 expression in distinguishing different subjects. A P value less than 0.05 was considered as statistical significance. SPSS 21.0  was employed to execute data analysis.Comparison of lnc-ITSN1-2 expression between two groups was examined by Mann–Whitney The mean age of patients with AS was 34.0 ± 8.3 years Table . The numP < 0.001)                Fig. a. Additi01) Fig. b.Fig. 3LThe evaluation of ASAS40 response was conducted at each time point during treatment in patients with AS. There were 0 (0.0%), 11 (17.5%), 20 (31.7%), 28 (44.4%), and 35 (55.5%) patients who achieved ASAS40 response at W0, W2, W4, W8, and W12, respectively  , at W8 (P = 0.022) and at W12 (P = 0.010)  Fig. a. Furthe10) Fig. b.Fig. 5LP < 0.001)   Fig. a. A simiCRP) in patients with AS. Possible reasons might be that (a) Lnc-ITSN1-2 might promote the production of inflammatory cytokines (such as TNFα) and lead to increased inflammation and osteoclastogenesis (osteoclast formation), which further causes an imbalance between bone formation and bone destruction. Therefore, lnc-ITSN1-2 expression correlated with elevated AS risk  and increased disease activity reflected by DAS28 score in patients with RA (Gong et al. As commonly used biologics, TNFα inhibitors reduce inflammation rapidly through restraining TNFα binding to its receptor and further suppressing downstream inflammatory cytokine production (Corbett et al. Several limitations existed in this study. Firstly, the sample size was relatively small and therefore further study with a larger sample size is needed to validate these findings. Secondly, the underlying mechanism of lnc-ITSN1-2 expression in AS was not investigated; thus, it might be necessary to conduct relevant molecular studies. Finally, our study only investigated the correlation of longitudinal change of lnc-ITSN1-2 expression with treatment efficacy of adalimumab in patients with AS, while its association with treatment efficacy of other types of TNFα inhibitors, such as etanercept or infliximab, was not investigated. Hence, further study could be performed.In conclusion, reduced lnc-ITSN1-2 expression during the TNFα inhibitor treatment correlates with treatment efficacy in patients with AS, suggesting its clinical value for AS management.


This study determined that NRP1 was a gene that promotes gastric cancer. NRP1 induced EMT to enhance the migration and invasion ability of GC cells by activating PI3K/Akt signaling pathway. NRP1 was an independent prognostic marker for OS in GC patients and expected to be a therapeutic target for GC patients.We aimed to determine whether Neuropilin-1 (NRP1) promotes gastric cancer (GC) metastasis by inducing epithelial-mesenchymal transition (EMT), and to clarify its regulatory mechanism. Using the data of GC patients in The Cancer Genome Atlas (TCGA) and Gene Tissue Expression (GTEx) databases, combined with the data of GC patients in our medical center, the effect of NRP1 on the prognosis of GC patients were analyzed. Then, we investigated the role of NRP1 in GC metastasis and its potential mechanism. The level of NRP1 was up-regulated in GC tissues and associated with poor prognosis of GC patients. The expression of NRP1 was closely related to maximum tumor diameter, invasion depth, lymphnode metastasis, distant metastasis, and advanced TNM stage, and was an independent prognostic factor for overall survival (OS) in GC patients. Besides, the results of  Gastric cancer (GC) is one of the main causes of cancer-related deaths and poor 5-year survival of patients worldwide Several studies have found that epithelial-mesenchymal transition (EMT) is a sign of malignant tumor progression Neuropilin-1(NRP1) is a transmembrane glycoprotein that acts as a co-receptor for semaphorin (SEMA) and vascular endothelial growth factor and is a key regulator of nervous system development, angiogenesis, immunity, and tumorigenesis The study included 210 GC patients who underwent surgical resection in the General Surgery of Union Hospital  from June 2013 to June 2014. These patients did not receive preoperative chemotherapy or radiotherapy, and there were no severe heart, lung, or kidney comorbidities. All patients were diagnosed with a pathological confirmation. Tissue samples were obtained from excised specimens, including tumor tissues and peritumoral normal mucosal tissues (more than 5cm from the edge of the tumor), quickly frozen in liquid nitrogen, and stored at -80℃ until use. The clinicopathologic data including age, gender, tumor size (maximum diameter), tumor location, the degree of tumor differentiation, preoperative carcinoembryonic antigen (CEA), and TNM stage were collected from the patient's medical history. Tumor stage was determined according to the 8th edition of the American Joint Committee on Cancer (AJCC) TNM classification system. The median follow-up period for the surviving patients was 55.4 months (range 3-65 months). Overall survival (OS) was defined as the interval between surgery and death, or between surgery and the last observation date for surviving patients.This study was carried out according to the Code of Ethics of the World Medical Association (Declaration of Helsinki) and was approved by the research ethics committee of Union hospital, Huazhong University of Science and Technology, China (No. 2018LSZS085). Informed consent was obtained from all patients in this study.http://gepia.cancerpku.cn/index.html) All the work on GC's data from TCGA and GTEx database was carried out by using the online tool Gene Expression Profiling Interactive Analysis  staining was performed. All specimens were fixed in 4% formaldehyde solution, embedded into paraffin, and then cut into 4μm sheets. In the next step, the tissue slides were dewaxed with xylene, dehydrated by using gradient alcohol and retrieved antigen by microwave. NRP1 antibody  diluted to 1:200. The secondary antibody was labeled with peroxidase and developed with diaminobenzidine. The staining procedure was carried out according to the manufacturer's instructions. The result of immunohistochemistry (IHC) was judged by two experienced pathologists and quantified by using a semi-quantitative comprehensive scoring method based on the intensity of staining and the ratio of stained cells 2 at 37℃. SGC-7901 cells with lower endogenous NRP1 expression were selected for transfecting with NRP1 overexpression plasmids (pCDNA3.1 (1) -NRP1) or empty vectors (pCDNA3.1 (1)) . Lipofectamine 2000  was used as the transfection reagent according to the manufacturer's operating manual. Transfected SGC-7901 cells were selected with 600 mg/mL G418 . The transfection efficiency on cell lines with NRP1 over-expression plasmid or empty vector was evaluated by Western blotting and qRT-PCR.Five GC cell lines  and immortalized gastric mucosal cell line (GES-1) were obtained from the Cell Center of the Chinese Academy of Sciences . These cells were cultured in the recommended medium supplemented with 10% fetal bovine serum and incubated in 5% CO5) with NRP1 higher endogenous expression were cultured in 6-well plates until 50% confluence was reached, and then they were transfected with 100 nM of the indicated siRNA using riboFECT™ CP Reagent  according to the manufacturer's instructions. The efficiency of NRP1 knockdown was analyzed by Western blotting and qRT-PCR after 48 hours.Small interfering RNA (siRNA) oligonucleotides specific to NRP1 and siRNA control oligonucleotides were obtained from RiboBio Co. Ltd . MGC-803 cells . RNA was subsequently reverse transcribed to generate complementary DNA (cDNA) with a first-strand cDNA Synthesis Kit . Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to assess NRP1 and β-actin expression with LightCycler 480 SYBR Green I Master . The level of β-actin was used for normalization. The PCR primers used were as follows: NRP1  and β-actin .5 cells per well in a 24-well plate with a 12-mm round poly-lysine-coated glass coverslip. Cells after 4% paraformaldehyde fixation, coverslips were washed three times in PBS and then blocked with 5% BSA in PBS with 0.3% Triton X-100 for 1h at room temperature with gentle shaking, followed by incubation with anti-E-cadherin antibody  and anti-vimentin  overnight with gentle shaking at 4°C. Coverslips were then washed PBS and incubated for 1 h at room temperature with secondary anti-rabbit Alexa Fluor 594 (diluted 1:200 in 1% BSA). Coverslips were washed three times with PBS, incubated with a solution of DAPI (1:5000), and TO-PRO (1:1000) in water for 10 minutes to stain the nuclei, and imaged.Cells were seeded at 5×10https://imagej.net) and normalized to the quantified value of β-actin. The following antibodies were used in western blotting experiments, e.g. NRP1 , E-cadherin, N-cadherin, Vimentin, Snail, Twist, β-actin .Total protein was isolated from the cells and defined the concentration by using Coomassie Protein Assay . The protein sample from the previous step was separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking for 60 minutes in skim milk at room temperature, the membrane was incubated with specific primary antibody overnight at 4°C. The next day, the membrane was incubated with a secondary antibody  for 2 hours at room temperature. β-actin was served as a control. The signal on the membrane was detected and imaged by the Odyssey Infrared Imaging System . The expression of all proteins was quantified by Image J software (Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8) assay . Tested cells were seeded into 96-well plates  at a density of 1,000 cells/well. Cells could grow for 1, 2, 3, 4, 5, 6, and 7 days, and then 10μl of CCK-8 solution was added to each well and incubated at 37℃ for 4 h. Cell viability was detected by measurement of absorbance at 490nm.5) were seeded into the upper chambers in serum-free medium and fetal bovine serum as a chemoattractant. After 48h of incubation at 37℃ in 5%CO2, migrated and invaded cells were quantified by dissolving cell-bound crystal violet in 10% acetic acid, and absorbance was measured at 540nm.Transwell migration and invasion analysis were performed using a 24-well modified Boyden chamber  with or without Matrigel  coating, respectively. Cells .t test. Survival and univariate analysis were determined by Kaplan-Meier analysis. The Cox proportional hazards regression model was applied to perform a multivariate analysis. All statistical analyses were two-sided, and P value < 0.05 was considered statistically significant.Statistical analyses were performed with SPSS 25.0  and Prism software . Pearson's chi-squared test and Fisher's exact test were applied for categorical variables; continuous variables were analyzed by the Student's P<0.05, Fig. vs. 1.87±0.13, respectively; P<0.001, Fig. To reveal the expression of NRP1 in GC, the online tool GEPIA was performed to analyze the data of GC patients in the TCGA and GTEx databases. It was shown that the level of NRP1 mRNA in GC tissue was significantly higher than that in normal tissue (P<0.001), but was not significantly related to other clinicopathologic factors  were defined as high NRP1 protein expression, while the remaining 90 patients (42.9%) were classified as low NRP1 protein expression. We pooled the NPR1 level of GC tissue with patients' clinicopathologic factors  for statistical analysis. Statistical results showed that the level of NRP1 in GC was significantly related to tumor size, TNM stage, depth of tumor invasion, N stage, and distant metastasis . Furthermore, tumor size , TNM stage , tumor invasion depth , lymph node metastasis , and distant metastasis  were also risk factors for OS. Towards further identifying the independent prognostic factors for OS in GC patients, multivariate Cox regression analysis was performed on all OS risk factors determined by univariate analysis. As shown in Table P=0.001), TNM stage , and distant metastasis  were identified as an independent prognostic factor for OS in GC patients.In our study of 210 GC patients, 120 patients with high NRP1 expression had shorter OS than those with low NRP1 expression by using Kaplan-Meier analysis  and immortalized gastric mucosal cell line (GES-1) through qRT-PCR and Western-blotting. It was shown that NRP1 was significantly higher expressed in GC cell lines than in GES-1, and NRP1 expressed differently in our group of 5 GC cell lines , while NRP1-knockdown inhibited GC cell proliferation  To investigate how NRP1 activates the PI3K/AKT pathway (eg. ligands that were involved). The levels of several extracellular growth factors  in the cell culture supernatants were measured by ELISA. In 48-h cell culture, SGC-7901 transfected with NRP1 overexpression plasmid secreted more HGF into serum-free medium than other growth factors Fig. H. c-Met NRP1, a transmembrane glycoprotein, was originally involved in axon guidance and angiogenesis based on interaction with semaphorin and VEGF family EMT was the initial step of tumor cell invasion and metastasis. It manifested in the loss of cell polarity and cell adhesion to promote tumor cell invasion and metastasis P<0.05); (2) The expression of NRP1 was closely related to the TNM stage of GC patients; (3) The OS of GC patients with high NRP1 expression was significantly lower than that of patients with low NRP1 expression . Afterwards, we verified the above research results by using our GC cohort. It was indicated that the highly expression of NRP1 was related to tumor maximum diameter greater than 5cm, deeper invasion, severe lymph node metastasis, distant metastasis, later TNM stage, and shorter OS. Combined with Cox proportional hazards regression model analysis, NRP1 was identified as one of the independent prognostic factors for OS in GC patients.We used the online tool GEPIA to analyze GC patients' data from the TCGA and GTEx databases. The results were: (1) The level of NRP1 in normal mucosa tissues was significantly lower than that of GC tissues (In vitro, the results of CCK-8 and Transwell assay showed that NRP1 promoted the proliferation, migration, and invasion of GC cell, which was consistent with previous research In conclusion, our research demonstrated that NRP1 was upregulated in GC tissues and was associated with a poor prognosis. Moreover, NRP1 induced EMT to enhance the migration and invasion ability of GC cells by activating PI3K/Akt signaling pathway. NRP1 might be a novel prognostic factor and promising therapeutic target for GC patients.


Glucocorticoids (GCs) act via the GC receptor (GR), a receptor ubiquitously expressed in the body where it drives a broad spectrum of responses within distinct cell types and tissues, which vary in strength and specificity. The variability of GR-mediated cell responses is further extended by the existence of GR isoforms, such as GRα and GRβ, generated through alternative splicing mechanisms. While GRα is the classic receptor responsible for GC actions, GRβ has been implicated in the impairment of GRα-mediated activities. Interestingly, in contrast to the popular belief that GRβ actions are restricted to its dominant-negative effects on GRα-mediated responses, GRβ has been shown to have intrinsic activities and “directly” regulates a plethora of genes related to inflammatory process, cell communication, migration, and malignancy, each in a GRα-independent manner. Furthermore, GRβ has been associated with increased cell migration, growth, and reduced sensitivity to GC-induced apoptosis. We will summarize the current knowledge of GRβ-mediated responses, with a focus on the GRα-independent/intrinsic effects of GRβ and the associated non-canonical signaling pathways. Where appropriate, potential links to airway inflammatory diseases will be highlighted. Glucocorticoids (GCs) are steroid hormones produced by the adrenal cortex in response to the hypothalamic–pituitary–adrenal axis activation . GCs conGCs act via the GC receptor (GR), a ubiquitously expressed receptor that drives a broad spectrum of responses within distinct cell types and tissues, which vary in strength and specificity ,4. The vWhile the GRα-dependent effects of GRβ gained a lot of attention, the GRα-independent effects of GRβ received less consideration. Indeed, in contrast to the popular belief that GRβ actions are restricted to its dominant-negative effects on GRα-mediated responses, GRβ has been shown to have intrinsic activities and “directly” regulates a plethora of genes related to inflammatory process, cell communication, migration, and malignancy, each in a GRα-independent manner ,13,14. FThe human GR (hGR) belongs to the nuclear hormone receptor family and acts as a ligand-inducible transcription factor. hGR is a modular protein consisting of an N-terminal transactivation domain (NTD), a DNA-binding domain (DBD), a hinge region, and a C-terminal ligand-binding domain (LBD) ,4,7. TheNR3C1 gene  located on chromosome 5 (5q31Y32) and is composed of nine exons. Alternative splicing near the 3′ UTR of the primary transcript generates GRα and GRβ isoforms; while the C-terminal end of GRα derives from the proximal portion of exon 9, the C-terminal end of GRβ derives from an alternative splice acceptor site in the distal portion of exon 9. Since exon 9 encodes the LBD, these isoforms differ significantly in their ligand-binding abilities. Indeed, while both GR isoforms are identical through amino acid 727, which includes NTD, DBD, and a part of the LBD, the rest of the LBD differs between the two isoforms. While GRα has an additional 50 amino acids that encode helices 11 and 12, necessary to form a hydrophobic pocket critical for the binding to GCs [The hGR is encoded by the  5 5q31Y3 and is cg to GCs . Similarg to GCs ,22,23,24g to GCs ,22,23,24While there is evidence showing that pro-inflammatory mediators such as TNFα and IFNγ selectively increase GRβ expression in airway cells , the molInterestingly, in human trabecular meshwork (TM) cells, Jain and colleagues showed that SRp20 favored the splicing of GRα, whereas SRp30c and SRp40 favored the splicing of GRβ  Figure . SimilarAltogether, these data indicate that SRps influence the alternative splicing of GR pre-mRNA and regulate the GRα/GRβ ratio in a cell-dependent manner. Moreover, other molecular factors, such as microRNAs (miRNAs), could also control the expression of GR isoforms ,38.miRNAs are small non-coding RNA molecules that have emerged as key post-transcriptional regulators of gene expression ,40. InteIn addition to humans, the GRβ isoform is expressed in several animal species ; however+ B cells and CD56+ natural killer cells at similar level than that of T cells [GRβ was originally described as an orphan receptor constitutively localized in the nucleus ,48,49. I T cells . A very  T cells .The effect of GCs on GRβ sub-cellular trafficking is also cell-specific. For example, dexamethasone treatment of COS-1 cells transfected with hGRβ cDNA showed that hGRβ primarily resided in the nucleus . In HeLaImportantly, several studies reported the ability of the GR antagonist, mifepristone (RU486), to bind GRβ . For exaSTAT1 gene [GRβ is frequently associated with GC insensitivity in a large number of inflammatory disorders, where its ability to regulate gene expression has mainly been accredited to its antagonism of GRα . HoweverAT1 gene .To further assess the GRα-independent effects of hGRβ, He and colleagues used knockout mice lacking GRα specifically in the liver (GR liver knockout (GRLKO) mice) . ExpressWhile the role of GRβ in physiological processes did not receive a lot of attention, recent evidence demonstrated its critical role in various physiological cell functions. Animal studies reveal that GRβ controls different metabolic functions, such as gluconeogenesis and lipid storage. For instance, the GRβ levels, but not GRα, were increased in mice and rats subjected to fasting–refeeding ,46. FurtPDK4) and G6Pase, was also inhibited by GRβ [The involvement of GRβ in hepatic functions has been further demonstrated in obese mice. For instance, in a high-fat diet (HFD)-induced obesity model, GRβ mRNA was elevated in adipose tissue and liver, but not in skeletal muscle, indicating that GRβ might control metabolic disorders . In agred by GRβ . AltogetThe involvement of GRβ in the modulation of inflammation has been demonstrated by different studies. For example, several reports demonstrated that GRβ was not only induced by a variety of inflammatory mediators ,58,59 buIL5 and IL13 genes in a histone deacetylases (HDACs)-dependent manner [Evidence from various cell types reported that GRβ differentially modulate GRα-mediated actions. For instance, a study performed in human neuroblastoma-derived BE(2)-C cells nicely demonstrated that GRβ exerts a selective dominant-negative effect on GRα-mediated transrepression activities but not on GRα-mediated transactivation activities. These findings may explain why the GC resistance seen in patients with inflammatory diseases is restricted to the immunosuppressive/anti-inflammatory effects of GC, which are driven by transrepression mechanisms, but not to the metabolic effects of GC, which are driven by transactivation mechanisms . Howevert manner . TogetheGRβ has been also incriminated in the regulation of cell migration ,63. For LAMB2, RAP1B, ITGA3, or ITGB1 [TNC) and laminin A4 (LAMA4) [Additional studies in HeLa and U-2 OS cells showed that GRβ through its intrinsic/GRα-independent transcriptional activities modulates migration by differentially regulating the expression of genes associated with extracellular matrix (ECM)-receptor interactions, as well as regulating those involved in the actin cytoskeleton, focal adhesion, and cell communication, such as or ITGB1 . Moreoveor ITGB1  while do (LAMA4) . These dIn order to examine whether targeting GRβ has any anti-migratory effects, several studies sought to identify and target the factors involved in the transcriptional regulation of GRβ. For instance, Hinds’ group showed that the overexpression of miR-144 in T24 bladder cancer cells significantly increased GRβ, but not GRα . InteresAltogether, these studies clearly highlight the modulatory role of GRβ in regulating cancer cell migration, albeit in a cell-specific manner. Further studies are needed to examine whether GRβ modulate the migration of other cell types, such as airway smooth muscle, as the increased migration of these cells constitutes a key feature of asthma pathogenesis.While the anti-proliferative properties of GCs are mediated through GRα, GRβ has been linked to promoting cell growth ,68. InteThe PI3K/Akt pathway has long been known to promote cell growth and survival signals . PTEN isThe Wnt intracellular signaling pathway promotes cell growth, migration, and differentiation in several cell types and its dysregulation was shown to facilitate tumor progression. β-catenin and TCF-4 are the main signaling molecules involved in the canonical Wnt pathway ,75. InteTogether, these findings clearly indicate the pro-proliferative potential of GRβ in different cell types involving different signaling pathways, including the PTEN/Akt/PI3K and/or Wnt/β-catenin/TCF signaling pathways. These findings further suggest that targeting GRβ could be a promising strategy in the treatment of diseases associated with aberrant cell growth.BCL2), and this independently of GRα [GCs are frequently used in cancer treatment because of their ability to induce cell death; however, some cancer cells are resistant to the apoptotic effects of GCs ,78. Study of GRα ,14.In non-cancer cell lines, such as PBMCs derived from patients with severe asthma, the increase in GRβ expression induced by cytokines such as IL-17 and IL-23 was associated with a reduction in the pro-apoptotic effects of dexamethasone . SimilarAltogether, these studies suggest that the GRβ effects on apoptosis is cell-specific. However, whether such an effect of GRβ is due to its intrinsic or extrinsic activities remain to be further investigated.Traditional views describe GRβ as a lowly expression GR isoform, mainly located in the nucleus, not expressed in mice, and unable to bind any ligand, modulating GC actions only through a dominant-negative effect on GRα. New evidence, however, challenged these concepts and now demonstrated, albeit in cell-specific manner, that (i) the ability of GRβ to bind ligands, such as RU486; (ii) the localization of GRβ both in the cytoplasm and the nucleus; (iii) the capability of GRβ to modulate gene expression and different physiological functions through its intrinsic activities; and (iv) GRβ expression in mice. These opposing views could be due, at least partially, to the fact that earlier studies used (i) cells lacking endogenous expression of GRβ, relying mostly on ectopic expression of GRβ , transformed cells (rat hepatoma cells or leukemia cell lines), or yeast system, making physiological and clinical interpretation/extrapolation very challenging; and (ii) cell-based transient assays, assessing the synthetic reporter gene activities, rather than the endogenous, more relevant GC-target genes. In addition, the cell-, tissue-, and species-specific expression of regulatory molecules, e.g., miRNA and SRps regulating GRβ transcriptional induction, further contributed to such controversy. Finally, the fact that an earlier report failed to demonstrate the expression of GRβ in mice, dramatically limited the studies examining the role of GRβ in vivo in murine models. This emerging concept regarding the intrinsic activities of GRβ will not only uncover new mechanisms regulating cellular and physiological functions but may also provide a promising strategy in the treatment of diseases where GRβ plays a pathogenic role.


The psychosocial rehabilitation of an individual with mental illness is an evidence-based approach to reducing the burden of the illness and the associated stigma globally. Specifically, in Africa, it has promising scope for African life and the African economy. Psychosocial rehabilitation is described as a set of approaches that aim to assist an individual in achieving restoration from a state of dependency caused by schizophrenia to a state of being an independent decision-maker. However, there seems to be a dearth of literature and implementation of psychosocial rehabilitation in Africa. Therefore, it is necessary to map studies on how psychosocial rehabilitation is conducted for people living in Africa with the most chronic form of mental illness, schizophrenia.This study will adopt the Arksey and O’Malley scoping review framework to search and compile relevant studies. This process will involve three steps: title screening, to be performed solely by the principal investigator, followed by abstract and full-text screening, to be performed independently by two reviewers . Rayyan QCRI, a systematic reviews web app, will be used for tracking the screening records, and data charting form will be used to extract basic data of included studies. The risk of bias in the articles identified for screening will be assessed by the Mixed Method Appraisal Tool (MMAT). Finally, the content analysis of the screened studies will be performed with NVivo.This study has the likelihood of revealing a research gap in psychosocial rehabilitation approaches and methods. The review results will constitute part of the available evidence that the researchers aim to adopt in the broader part of the project, which aims to develop implementation strategies for the psychosocial rehabilitation of chronic mental illnesses, specifically schizophrenia, in Sub-Sahara Africa. The implementation process also encompasses disseminating the findings of this review to stakeholders, which will enhance their knowledge of the current state of Sub-Saharan Africa and may stimulate support for the implementation of rehabilitation strategies. Schizophrenia has been declared the most chronic mental illness and the mental illness that attracts the most self-blame in Sub-Saharan Africa, as opposed to alcoholism and drug addiction, which attract the most self-blame in Western countries . The belThe disabling effect of schizophrenia not only affects individuals but also affects their families and health practitioners. The burden on families stems from monitoring the patient’s hygiene, the use of medication, comportment at social gatherings, follow-up with health care providers, and the need to deal with mood, cognitive and behavioural changes –13. The Additionally, community members refuse to enter into marital relationships with the siblings of individuals with schizophrenia even when those siblings do not have the ailment . This imThe burden of schizophrenia on health practitioners is related to the scarcity of human resources, which the World Health Organization  has declEvidence from global research shows that over 200 251–261) mental illness disability-adjusted life years (DALYs p.a./1 million pop.) may be averted through psychosocial rehabilitation interventions compared with 149–160 DALYs averted through medication alone , 31. Spe1 mental PSR implementation in high-income countries has led to status disclosure, reduced institutionalization, self-awareness, increased productivity and reduced stigma. This result was contrary to experiences of Sub-Saharan African countries of labelling and intense stigmatization , 35. ThiTo explore the existing evidence on psychosocial rehabilitation of schizophrenia and its approaches in Sub-Saharan AfricaTo examine the opportunities available to individuals with schizophrenia and the outcomes of studies or exposure to the available opportunities in Sub-Saharan AfricaTo refine future PSR research questions, to fill gaps in evidence on rehabilitation research in Sub-Saharan AfricaThe objectives of the scoping review are as follows:Setting the research questionIdentifying the relevant studiesSelection of studiesData chartingCollation, summary generation and report writingThe protocol is for a scoping review of studies on the PSR of individuals with schizophrenia in Sub-Saharan Africa. The review will be guided by the Arksey and O’Malley framework . This frThe review is underpinned by the following question: what work has been done in the psychosocial rehabilitation of people living with schizophrenia in Sub-Saharan Africa?What is the existing evidence on the psychosocial rehabilitation of schizophrenia and its approaches in Sub-Saharan Africa?What was the expressed goal of the individuals with schizophrenia in Sub-Saharan Africa, and what difference did participating in rehabilitation make in their life generally and specifically in the achievement of their life goal?What are the existing gaps in psychosocial rehabilitation research in South Sahara, Africa?The sub-questions are as follows:Primary data (from published and unpublished studies) that address the research question will be identified for possible review. The searches for published literature will be performed using the LibGuides online bibliography and ten databases  and selected databases under the listed platforms, ProQuest , Embase, Medline) and EbscoHost  will be consulted. The following sites will also be consulted for unpublished literature: Google, Google Scholar, African Journals Online, and HINARI. In addition to the electronic search of databases, the search approach will include checking the references of included studies and related reviews and contacting experts in searching grey literature sources such as conference proceedings. The search terms will include psychosocial rehabilitation AND rehabilitation AND schizophrenia AND Sub-Saharan Africa. Three library experts from three different countries were consulted. One was a medical school librarian, and two were librarians from schools of public health to guide search terms for “schizophrenia”, “rehabilitation” and “Sub-Saharan Africa” (the full list of variations on the search strategy is available in the search sample below). According to reports of library experts, schizophrenia can also be conceptualized as a chronic mental illness, while rehabilitation is conceptualized as productivity. In alternate words, Sub-Saharan Africa is also regarded as Black Africa. The variant terms will also be considered in the study. Duplicates will be removed, and the eligibility criteria will be applied to select studies that will be reviewed in the study.The 2015 study population concept and context (PCC) scoping review framework by Arksey and O’Malley, shown in Table A specific duration will guide the search strategy for this review, and studies before 2000 will not be considered for review because the global advocacy of the mental health burden and rehabilitation started in 2000 . SpecifiThis stage comprises three phases. The first phase is the search stage which consists of 1) developing and refining a search in one database, 2) translating the search to all the other databases and 3) importing/gathering the results in software (Endnote) to remove duplicates. The second and the third phases are the screening phases which involve a title, abstract and full article screening using Rayyan QCRI, a systematic reviews web app, for screening and tracking the screening records.Phase 1: This phase is operationalized in this protocol as pilot testing. Existing reviews on schizophrenia and psychosocial rehabilitation were consulted to elaborate the searches for each concept, and the findings are presented in Table Phase 2: This involves the title screening of the studies from each database. The keywords with the variant search terms as displayed in Table The eligible titles will be exported into a storage file . The records of the titles screened will be documented as shown in Table The records of the titles screened will be kept in three columns. The first column consists of the keywords inserted into the search space. The second column will record each database where the search was conducted. The last column consists of the number of studies retrieved from each database.Phase 3: The abstract and full-text screening will be performed by two screeners  using Rayyan QCRI software. Tables The data charting form indicated in Table The studies screened for inclusion in this review will be collated in NVivo. The content analysis approach will be used to analyze each of the studies in NVivo. Themes will be derived from the categories of meaning units of each of the studies. The theme derivation will evolve from the meaning units in each category. The meaning units are information derived from the study based on the review objectives, and it will form the basis of the report on rehabilitation. The report will be written based on the themes generated, which will inform the conclusion and recommendations of the review.The risk of bias in each of the identified articles will be assessed using the Mixed Method Appraisal Tool (MMAT) version 2011. The tool will serve as a scientific assessment instrument for checking the rigour of qualitative methodologies, the validity of quantitative instruments, the suitability of methodologies and the appropriateness of mixed-method approaches . The priBecause of the identified need to concentrate effort on the rehabilitation of individuals with schizophrenia, the investigators are interested in knowing the scientific work that has been performed in terms of the psychosocial rehabilitation of people living with schizophrenia in Sub-Sahara Africa. The scoping review can reveal the gap in rehabilitation in Sub-Saharan Africa and provide information on available strategies. This scoping review will form part of the research questions of the main study that the investigators plan to conduct in the most populous country in Sub-Sahara Africa, Nigeria.


Long noncoding RNA PCAT1 (lnc‐PCAT1) involves in the proliferation and drug sensitivity of multiple myeloma (MM), while its prognostic role in MM patients is still obscure. This study aimed to explore the association of lnc‐PCAT1 with MM risk, clinical characteristics, treatment response, progression‐free survival (PFS), and overall survival (OS).A total of 83 symptomatic MM patients were enrolled in this study. Additionally, 30 healthy bone marrow donors as health controls were also recruited. Bone marrow plasma cell samples of MM patients and health donors were collected. Lnc‐PCAT1 in bone marrow plasma cells was detected by reverse transcription‐quantitative polymerase chain reaction.p < 0.001), and receiver operating characteristic (ROC) curve showed that lnc‐PCAT1 had excellent capability of discriminating MM patients from health donors . In MM patients, lnc‐PCAT1 was correlated with bone lesion (p = 0.024), higher β2‐MG (p = 0.005), LDH (p = 0.037), and presence of Del (17p) (p = 0.029). Lnc‐PCAT1 was also associated with poor ISS stage (p = 0.013) and R‐ISS stage (p = 0.005). Besides, lnc‐PCAT1 was reduced after treatment (p < 0.001); meanwhile, lnc‐PCAT1 before treatment was correlated with lower CR (p = 0.046) but not ORR (p = 0.185). Additionally, lnc‐PCAT1 after treatment was associated with lower CR (p = 0.003) and ORR (p = 0.010). Furthermore, baseline Inc‐PCAT1 high and Inc‐PCAT1 increase after treatment were correlated with worse PFS and OS .Lnc‐PCAT1 was increased in MM patients than in health donors (Lnc‐PCAT1 dysregulation serves as a biomarker for diagnosis and prognosis for MM. p ＜ 0.001), and receiver operating characteristic (ROC) curve showed that lnc‐PCAT1 had excellent capability of discriminating MM patients from health donors . In MM patients, lnc‐PCAT1 was correlated with bone lesion (p = 0.024), higher β2‐MG (p = 0.005), LDH (p = 0.037), and presence of Del (17p) (p = 0.029). Lnc‐PCAT1 was also associated with poor ISS stage (p = 0.013) and R‐ISS stage (p = 0.005). Besides, lnc‐PCAT1 was reduced after treatment (P ＜ 0.001); meanwhile, lnc‐PCAT1 before treatment was correlated with lower CR (p = 0.046) but not ORR (p = 0.185). Additionally, lnc‐PCAT1 after treatment was associated with lower CR (p = 0.003) and ORR (p = 0.010). Furthermore, baseline Inc‐PCAT1 high and Inc‐PCAT1 increase after treatment were correlated with worse PFS and OS . Lnc‐PCAT1 dysregulation serves as a biomarker for diagnosis and prognosis for MM.Long noncoding RNA PCAT1 (lnc‐PCAT1) involves in the proliferation and drug sensitivity of multiple myeloma (MM), while its prognostic role in MM patients is still obscure. This study aimed to explore the association of lnc‐PCAT1 with MM risk, clinical characteristics, treatment response, progression‐free survival (PFS), and overall survival (OS). A total of 83 symptomatic MM patients were enrolled in this study. Additionally, 30 healthy bone marrow donors as health controls were also recruited. Bone marrow plasma cell samples of MM patients and health donors were collected. Lnc‐PCAT1 in bone marrow plasma cells was detected by reverse transcription‐quantitative polymerase chain reaction. Lnc‐PCAT1 was increased in MM patients than in health donors ( Besides, this study also aimed to explore the correlation of lnc‐PCAT1 with MM prognosis, including treatment response, progression‐free survival (PFS), and overall survival (OS).22.1In this prospective study, 83 symptomatic MM patients admitted to the hospital between March 2017 and September 2020 were consecutively enrolled. All eligible patients were newly diagnosed as symptomatic MM according to the International Myeloma Working Group (IMWG) diagnostic criteria2.2−ΔΔCt method. Primers were designed referring to a previous study.For the investigation of lnc‐PCAT1 expression, bone marrow samples of MM patients were collected at diagnosis and at the completion of 3 to 4 cycles of induction therapy, respectively. The bone marrow samples of health donors were collected on their donation. Immediately after collection, CD138‐immunomagnetic beads (Miltenyi Biotec) were used to sort out plasma cells from the bone marrow samples. Subsequently, reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) was carried out to determine the expression of lnc‐PCAT1 in the plasma cells. The plasma cells were treated by TRIzol™ Reagent (Thermo Fisher Scientific) to extract total RNA, which were then submitted to perform reverse transcription using iScript™ cDNA Synthesis Kit (with random primer) (Bio‐Rad). After that, qPCR was carried out with QuantiNova SYBR Green PCR Kit (Qiagen). GAPDH was served as reference gene. The relative quantitative analysis of lnc‐PCAT1 expression was conducted with the use of 22.3Baseline clinical features and staging information  stage, International Staging System (ISS) stage, and revised ISS (R‐ISS) stage2.4p value < 0.05 in the corresponding analysis.SPSS 24.0 (IBM Corp.) and GraphPad Prism 7.01 (GraphPad Software Inc.) were used for data analysis and graph plotting, respectively. Categorical data were described as count with percentage. Continuous data distribution was analyzed using Kolmogorov–Smirnov (K–S) test, and median with interquartile range (IQR) or mean with standard deviation (SD) was calculated for descriptive analysis. Wilcoxon rank sum test or Kruskal–Wallis test was used for comparison of lnc‐PCAT1 expression in independent samples. Wilcoxon signed‐rank test was used for comparison of lnc‐PCAT1 expression in paired samples. The correlation of lnc‐PCAT1 with disease stage was analyzed by Spearman's rank correlation test. The receiver operating characteristic (ROC) curve and the area under the curve (AUC) were used to estimate the ability of lnc‐PCAT1 expression in identifying different subjects. Kaplan–Meier curve was plotted to display survival profiles of different subjects. Log‐rank test was applied for the determination of PFS and OS difference between subjects. In the survival analysis, “lnc‐PCAT1 decline” was defined as lnc‐PCAT1 expression declined after treatment compared with that before treatment; “lnc‐PCAT1 increase” was defined as lnc‐PCAT1 expression increased after treatment compared with that before treatment. Statistical significance was concluded if there was a 33.12‐MG, and LDH were 1.8 (1.2–2.2) mg/dl, 34.0 (28.0–38.0) g/L, 5.6 (3.0–9.6) mg/L, and 206.3 (172.9–253.7) U/L, respectively. As for the chromosomal abnormality, there were 8 (9.6%) patients having t , 4 (4.8%) patients having t , and 6 (7.2%) patients having Del (17p). Additionally, 8 (9.6%) patients were at DS stage II and 75 (90.4%) patients were at DS stage III, while no patients were at DS stage I; 12 (14.5%) patients were at ISS stage I, 28 (33.7%) patients were at ISS stage II and 43 (51.8%) patients were at ISS stage III; 7 (8.4%) patients were at R‐ISS stage I, 38 (45.8%) patients were at R‐ISS stage II and 38 (45.8%) were at R‐ISS stage III.A total of 83 symptomatic MM patients were enrolled in this study. The baseline characteristics of MM patients were summarized in Table 3.2N = 83) than in the health donors (N = 30) (p < 0.001)  Figure .3.3p = 0.024), >5.5 mg/L β2‐MG (p = 0.005), >220 U/L LDH (p = 0.037), and presence of Del (17p) (p = 0.029) (Table p = 0.013)  .As shown in Table ) Figure  and R‐IS) Figure . However) Figure , Table 23.4p < 0.001). Besides, lnc‐PCAT1 expression was declined in 61 (73.5%) patients and increased in 22 (26.5%) patients after treatment    and BLD regimens (p < 0.001), lnc‐PCAT1 expression was decreased after treatment ) compared with before treatment ) (.302) com) Figure  and B. B) Figure . Howevert Figure  and E.3.5n = 23) was lower than that in the non‐CR patients (n = 60) (p = 0.046)  and the non‐ORR patients (n = 24) (p = 0.185)  than the non‐CR patients (n = 60) (p = 0.003)  compared with that in the non‐ORR patients (n = 24) (p = 0.010)     patients and non‐ORR patients (p = 0.013) after treatment compared with before treatment .Lnc‐PCAT1 expression before treatment in the CR patients  Figure . Otherwi) Figure . In addi) Figure . Moreove) Figure . Additio) Figure  and non‐) Figure  after tr) Figure . In addit Figure  and E. Nt Figure  (p > 0.03.6p = 0.009)     = 2.383 (1.218–4.660)), t  = 4.502 (1.003–20.201)), and R‐ISS stage  = 2.395 (1.321–4.343)) were all independent predictive factors for shorter PFS; meanwhile, lnc‐PCAT1 decline (vs. increase) (after treatment) was an independent predictive factor for longer PFS  = 0.353 (0.189–0.659))  = 6.753 (1.972–23.121)) and t  = 4.528 (1.008–20.336)) were independent predictive factors for worse OS  was correlated with shorter accumulating PFS  Figure  and accu) Figure . What is) Figure  and accu) Figure . In addi) Figure . MoreoveS Figure .42‐MG, LDH, and presence of Del (17p) in MM; meanwhile, lnc‐PCAT1 expression was associated with poor prognostic risk stratification, including ISS stage and R‐ISS stage; and (3) baseline lnc‐PCAT1 high expression and its longitude increase during treatment were correlated with worse CR, PFS and OS.In this study, we aimed to explore the correlation of lnc‐PCAT1 with MM risk, clinical characteristics, and prognosis, which observed that (1) lnc‐PCAT1 expression was increased in the MM patients; (2) lnc‐PCAT1 expression was correlated with bone lesion, higher βAs to lnc‐PCAT1 expression in the hematological malignancies, lnc‐PCAT1 is enhanced in acute myeloid leukemia patients.2‐MG concentration of MM.2‐MG, LDH, and presence of Del (17p) in MM; moreover, it was associated with poor risk stratification in ISS stage and R‐ISS stage. The possible reasons could be that (1) lnc‐PCAT1 promotes plasma cell growth, resulting in MM progression,2‐MG and LDH concentration2‐MG (above‐mentioned), which was a feature of the poor ISS stage; thus, lnc‐PCAT1 was related to the poor ISS stage; meanwhile, lnc‐PCAT1 was positively associated with higher LDH, which exceeded the normal level of LDH constituting the most advanced subtype of R‐ISS stage, thus lnc‐PCAT1 was correlated with poor R‐ISS stage.In terms of the association of lncRNA expression with clinical characteristics of MM, an interesting study shows that lnc‐PCAT1 is positively correlated with βRegarding the correlation of lnc‐PCAT1 expression with prognosis in patients with MM, previous studies elucidate that high expression of NIMA‐related kinase 2, a target gene of lnc‐PCAT1, is related to poor prognosis and inferior survival in MM.Although a lot of findings were identified in this study, there were still some limitations. First, with a small sample size, this study might have low statistical power. Further validation in the more and same number of patients and health controls might be needed. Second, our study evaluated the correlation of lnc‐PCAT1 expression with clinical characteristics and prognosis of patients having symptomatic MM, while the correlation of lnc‐PCAT1 expression with asymptomatic MM, might be further evaluated. Third, this study did not investigate the molecular mechanism of lnc‐PCAT1 involved in MM progression; thus, in vivo and in vitro experiments might be necessary to be further conducted.Conclusively, lnc‐PCAT1 associates with elevated disease risk and unfavorable ISS stage, R‐ISS stage, treatment response, and survival of MM. It may potentially serve as a biomarker to predict MM prognosis, further improving the management of MM patients.The authors declare that they have no conflicts of interest.


The androgen receptor (AR) is a member of the ligand-activated nuclear receptor family of transcription factors. AR’s transactivation activity is turned on by the binding of androgens, the male sex steroid hormones. AR is critical for the development and maintenance of the male phenotype but has been recognized to also play an important role in human diseases. Most notably, AR is a major driver of prostate cancer (CaP) progression, which remains the second leading cause of cancer deaths in American men. Androgen deprivation therapies (ADTs) that interfere with interactions between AR and its activating androgen ligands have been the mainstay for treatment of metastatic CaP. Although ADTs are effective and induce remissions, eventually they fail, while the growth of the majority of ADT-resistant CaPs remains under AR’s control. Alternative approaches to inhibit AR activity and bypass resistance to ADT are being sought, such as preventing the interaction between AR and its cofactors and coregulators that is needed to execute AR-dependent transcription. For such strategies to be efficient, the 3D conformation of AR complexes needs to be well-understood and AR-regulator interaction sites resolved. Here, we review current insights into these 3D structures and the protein interaction sites in AR transcriptional complexes. We focus on methods and technological approaches used to identify AR interactors and discuss challenges and limitations that need to be overcome for efficient therapeutic AR complex disruption. N-terminal domain (NTD) which contains a ligand-independent transcriptional function (AF) AF-1, a central DNA binding domain (DBD) and a C-terminal ligand-binding domain (LBD) which harbors the ligand-activated AF-2. DBD and LBD are connected through a short hinge domain (AR is a ligand-activated transcription factor that belongs to the nuclear receptor (NR) family ,2. Its me domain . The manner in which AR is activated has been reviewed extensively in the past. To summarize, androgen binding to the AR LBD induces a conformational change, cytoplasmic to nuclear re-localization, and, following dimerization via intra- and intermolecular NTD-LBD interactions, binding of AR through its DBDs to Androgen Response Elements (AREs), the canonical AR binding site in target genes. ARE-bound AR then recruits transcriptional regulators to modulate the expression of target genes ,4,5,6 F.Until recently, the structure of full-length DNA-bound AR has remained elusive. Information on AR’s putative overall 3D conformation, and the contribution of AR’s domain interactions, homodimerization, and DNA-binding to the overall conformation of AR transcriptional complexes was derived from in vitro X-ray crystallography or computational modeling of individual LBD or DBD domains, either associated with small DNA fragments or small peptides from AR-associated proteins in the presence of a few ligands e.g., ,7). The . The 7])AR is widely expressed . BecauseIn view of AR’s central role in bypassing CaP’s response to the selective pressure of ADT, therapeutic strategies to inhibit AR’s activity other than impeding its ligand activation are being sought. Some of these approaches, which have not (yet) transitioned into the clinic, are directly targeting AR, such as AR degraders  in the cell nucleus that may either not be expressed endogenously or not expressed at the same level or in the same cell compartment in human prostate cancer cells, it is prone to false-positive results. The absence of post-translational modifications in yeast that do occur in mammalian cells adds to this problem, as does the observation that traditional yeast 2 hybrid assays do not perform optimally when the bait protein possesses intrinsic transactivation activity, such as the AR NTD. Some of these issues can be overcome, for instance, by co-expression of a kinase in yeast cells to ensure the presence of relevant phosphorylation marks  or the uStructural and/or conformational information on the AR transcriptional complex that can be derived from 2 hybrid assays, however, appears very limited. The focus on different AR domains, regions, and even short AR amino acid sequences that are conserved in AR among species  and short AR motifs . Prey fPhage display assays do suffer for some of the limitations mentioned above for 2 hybrid assays; they also rely on (over)expression of protein domains or peptides that may not fully represent endogenous post-translational modifications and localization. Limitation in terms of size of library or type of protein that can be expressed is mentioned already above. On the other hand, phage displays tend to be easy to perform, relatively cheap, adaptable and amenable to high throughput approaches. They are able to select peptides and proteins with high affinity and specificity that may be developed further and moved forward as preclinical AR-inhibitory therapeutics. As proof-of-principle, an FxxLF-containing AR-interacting peptide functioned as peptide antagonist of AR-dependent transcription . Phage dFrom a conformational and structural perspective, important novel information ensued from phage display assay findings. Apart from fine-tuning the protein/peptide sequences that most stringently interact with AR, deeper insights in AR structure-function relationships and its allosteric regulation have been obtained. First, AR-binding peptides that reflect variations on the LxxLL, FxxLF, and WxxLF motif theme have been combined with the AR LBD in X-ray crystallography studies . These eOther approaches that have the potential to screen for novel AR interactors have, to our knowledge, not yet been used (extensively) for this purpose and are mentioned here for completeness. For instance, protein arrays have a similar set up as DNA (oligo) arrays but involve spotting proteins instead of DNA on a slide to screen for interactors from cell or tissue lysates. The commercial Marconi peptide array uses NR-coregulator derived peptides to screen for NR interactors and showed interaction between an LxxLL motif in the coregulator TRIM24 and the AR LBD . It shouThe assays discussed above represent screens for novel AR interactors that were performed in non-CaP model systems. Alternative strategies to identify AR-associated cofactors and coregulators have focused on better defining the composition of the endogenous AR transcriptional complexes in CaP cells. A number of approaches that vary in bait protein purification steps, stabilization and/or labelling of protein-protein interactions and are coupled to mass spectrometry (MS)-based identification of AR interactors have been applied to this end.In this first approach, various antibody- or tag-based methods are used to affinity-purify (AP) the bait protein (here: AR) and its interactors from CaP cells or tissues. The purified bait and its associated complex are then washed extensively to eliminate non-specific interactions and subjected to a MS approach for characterization The whole protein eluate can be subjected to MS, which allows for a more unbiased characterization of AR associated proteins. Alternatively, a more selective approach, which narrows down the search for interactors based on size and concentrates samples, involves running the pulled-down proteins on an SDS-PAGE gel, excising the gel fragment of interest and enzymatic digestion of this fragment prior to MS data acquisition. Further refinements to AP-MS are possible. For instance, IPs can be done from a specific cell compartment, such as the nucleus to enrich for proteins with a transcriptionally relevant function. Enrichment for activators or repressors of AR action can be achieved by first treating cells with AR agonists or antagonists.N- and C-terminal domains purified Ku70 and Ku80 as AR coactivators [To better understand AR complex composition, several studies used GST-tagged AR that was immobilized on beads or columns as bait and isolated interactors from CaP cell lysates were identified via MS analysis. A study that screened for interactors of GST-labelled AR tivators . In othetivators ). An advtivators ,84. The N- or C-terminal) may influence the normal bait expression levels of folding patterns and thereby its function and interactome. None-the-less, using tagged versions of AR in AP-MS has expanded the AR interactome, for instance ChREBP [An important limitation to these assays, however, is that they rely on the availability of a specific antibody that is suitable for efficient immunoprecipitation (IP). This problem can be overcome by using tagged versions of the bait protein, in which for instance a FLAG, His, GFP, Myc or HA tag is fused in frame that can be readily IP’ed with a commercial antibody. The tagged bait is then overexpressed in cells prior to IP with a tag-targeting antibody, more effectively IP’ed and its interactome defined. Since this method does lead to artificially high cellular levels of proteins, it may skew the ratios with interactors, protein interfaces and access to epitopes. This may be overcome by careful titrating overexpressed proteins or combining their ectopic expression with depletion of the endogenously expressed bait protein. The addition of a tag and the site to which it is added , formaldehyde is used to crosslink endogenous protein-protein interactions ,87. FormAnother way to overcome the problems with isolating transient or labile protein-protein interactions and to focus on proteins in the immediate vicinity of the bait is evidenced by biotin-based proximity labeling assays. In such assays, a promiscuous biotin-labeling enzyme is targeted by genetic fusion to the bait protein and to a subcellular compartment. The enzymes used in these assays are constantly being refined. Currently, TurboID has substantially higher activity than the previously described BioID . A recenIn combination, these MS-based characterization efforts on AR transcriptional complexes have considerably expanded the spectrum of its coregulators and cofactors. Alternatives are possible, for instance (MS) analyses of complexes bound to ARE-containing regions ,94. A liAR’s role in CaP biology is tightly regulated by its interaction with other cellular proteins which affect its binding to ligand, nuclear translocation, folding and transcriptional activity. Over the past few decades, hundreds of AR-interacting proteins have been identified using different unbiased approaches ,37. TogeThat disrupting AR complexes may have therapeutic potential when AR-driven CaP has lost responsiveness to ADT is supported by a multitude of studies. In these reports, expression of AR-associated coregulators or cofactors is silenced by siRNA, shRNA or CRISPR approaches, or dominant-negative or catalytic-dead versions of diverse coregulators are overexpressed. Moreover, coregulator-derived decoy proteins or peptides that compete with coregulators for AR binding have shown promise in model systems after short term treatments ,73. PeptThe structure-function basis for the majority of these AR-regulator interactions is yet to be defined, which will likely be critical to explore their full potential for therapeutic intervention . EffectiThe first cryoEM study on full length DNA-bound AR has recently been published . As discIn anticipation of the renewed insights in AR complex composition, conformation and stoichiometry that the above-mentioned technological advances and others that may emerge can deliver, it is important to consider a few questions will need to be addressed and prioritized to deliver the most useful information that can be move forward clinically. For instance, to which of the hundreds of AR-associated transcriptional regulators should these efforts be directed to ensure clinical applicability? It seems reasonable that an AR interactor pursued for therapeutic intervention contributes to AR activity that drives ADT-recurrent CaP progression. Its AR-dependent activity or expression may increase also during CaP progression. Ideally, its function would not be impacted by CaP genomic heterogeneity . On the With respect to the proposed therapeutic targeting, several other questions remain. From a molecular perspective, as protein–protein interactions in transcriptional complexes display a tiered hierarchy , what leConsidering and prioritizing these relevant issues will be important to ultimately employ the anticipated novel molecular insights in AR complex formation in an evidence–based manner for therapeutic intervention in treatment-resistant CaP.


This indicates that the P300 BCI paradigm is relatively reliable and may work well in various situations.Since the emergence of head-mounted displays (HMDs), researchers have attempted to introduce virtual and augmented reality  in brain–computer interface (BCI) studies. However, there is a lack of studies that incorporate both AR and VR to compare the performance in the two environments. Therefore, it is necessary to develop a BCI application that can be used in both VR and AR to allow BCI performance to be compared in the two environments. In this study, we developed an opensource-based drone control application using P300-based BCI, which can be used in both VR and AR. Twenty healthy subjects participated in the experiment with this application. They were asked to control the drone in two environments and filled out questionnaires before and after the experiment. We found no significant ( The brain–computer interface (BCI) is a technology that allows for direct communication between humans and a computer through the user’s brain activity and mental states . A BCI cP300 BCI is one of the popular paradigms used in the BCI field. P300 is a positive event-related potential (ERP) that appears approximately 300 ms after the stimulus . The P30Many BCI studies in the past were based upon 2D visual stimulation but the recent development of head-mounted displays (HMDs) has paved the way to the commercialization of combined BCI+VR  or BCI+AR  . Indeed,With the emergence of HMDs, some BCI studies have been conducted that combine VR or AR using a HMD. However, the development of BCI content in VR and AR environments has not been carried out actively because of the limited performance of BCI-based control, the two technologies’ inherent complexity, as well as the difficulty in designing effective UIs based upon BCI . FurtherTo do so, we developed a BCI drone control application in this study that can be used in both VR and AR. In VR, a virtual drone was controlled with virtual maps, while in AR, a real drone was controlled and real-time video could be viewed with a drone camera. Using this application, we conducted an experiment with 20 subjects and compared the differences in the VR and AR environments.In general, AR using head-mounted displays (HMD) includes optical see-through (OST) and video see-through (VST) . The OSTTo compare the performance in VR and AR effectively, we focused on one BCI paradigm (P300), two environments (VR and AR), and one task (mobile drone control).We selected the P300 paradigm for our drone control system for the following reasons. First, drone control systems require rapid interaction between users and a drone, as the direction in which a user wants to go must be controlled with a rapid response with higher accuracy. Second, compared to the motor imagery BCI paradigm, P300-based BCIs require less training time and achieve a higher accuracy and information transfer rate (amount of information sent per unit of time). Third, P300 allows for a larger number of possible commands ,25, whicThe first objective of this paper was to introduce an opensource-based drone control application using P300 BCI in the AR and VR environments. This application can allow people who are physically challenged to travel outside their normal environment and increase their autonomy by controlling a drone with their brainwaves alone. In addition, it also allows for more immersive experiences by combining BCI with VR and AR environments using a HMD.The second objective of this paper was to study the feasibility of combining BCI and VR or AR technologies and compare the VR and AR environments. We evaluated the two environments from two perspectives: online performance and user experience. We compared the classification accuracy and analyzed the differences in event-related potential (ERP) signals in each environment. We also compared user satisfaction in the two environments and their effect according to preference.In this project, we used OpenViBE, Python, Unity 3D, and Tello for Unity, which are competitive with respect to portability, scalability, and online performance.OpenViBE for integration overall: This is an open-source software platform that is specialized in integrating various components of the brain–computer interface . OpenViBPython for signal processing: Signal processing algorithms were implemented in Python. Brainwaves were processed with Python to detect the P300 signal and the target button that a user saw was predicted by a pre-constructed classifier.Unity 3D for VR and AR application: Unity 3D is a cross-platform game engine that can be used to create three-dimensional, VR, and AR games, as well as simulations and other experiences . In thisTello for Unity to control the drone: Unity API was used to control a DJI Tello drone in the AR mode . This APAll the source codes for the drone control system are available through the Github repository .The drone control system’s environment and composition are shown in Re-referencing: seven channels  were re-referenced using a channel on the left ear.Bandpass filtering: data were filtered to the frequency band of 0.1–30 Hz using the SciPy package with the 5th order Butterworth filter .Epoching: data were segmented to 0–1000 ms epochs from each stimulus onset.Baseline Correction: Each epoch’s mean value was subtracted from the epoch. EEG signals are prone to amplitude shifts attributable to such factors including changes in impedance or noise, which can be fatal in the data analysis. Baseline correction compensates for this random amplitude shift.Consecutive Trial Averaging: to improve the signal-to-noise ratio (SNR), segmented epochs were averaged continuously by 20 epochs.Resampling: Brain signals were digitized originally at a sampling rate of 300 Hz. To reduce the data size, signals were down-sampled to 100 Hz.Basically, the signal acquired was processed through a pre-defined pipeline, as shown in The EEG data acquired during the training session were processed through the steps in The characteristics of the P300 component in an ERP may differ in appearance, latency, and amplitude depending upon users’ individual differences, age effects, and ultradian rhythms . Thus, iIn the training session, the user looks at the target button according to the instructions on the top. In one selection of a training session, seven buttons blink 30 times with a fixed ISI in random sequence. “Blink” in this system refers to visual stimulation using a color that toggles between gray and yellow. A button toggles to yellow for 100 ms and re-toggles to gray for 100 ms to give one visual stimulation.After the training session, online sessions are run to control the drone. The button interface for online sessions is shown in The drone control application is divided into VR and AR contents depending upon the environment. To compare the performance in VR and AR, 20 subjects  participated in this experiment with the same equipment in the same place and performed one task (mobile drone control) in VR and AR sequentially. The Public Institutional Bioethics Committee designated by the Ministry of Health and Welfare approved the study (P01-201812-11-004) and all subjects were informed about the experiment and their right to stop the experiment if/when they felt uncomfortable or experienced 3D motion sickness before the experiment. All subjects signed a written consent form prior to this experiment and were paid for their participation. None of them had participated in a BCI experiment before this study.Each experiment took a total of 50 min. The experimental procedure is shown in The EEG data were recorded with DSI-VR300, which has a HMD (HTC-Vive) and a headset to measure the EEG a 40]. T. T40]. TTwo questionnaires were generated pre and post-task . Before Primarily, we investigated the online performance, peak latency, and amplitude of the ERP component to compare the VR and AR environments. The scores and answers to the questionnaires were also examined to assess any differences between the two. We used the Wilcoxon signed-rank tests to analyze the data, as the Kolmogorov–Smirnov test indicated that most of the data did not follow a normal distribution. We set the significance level to 0.05.Before the analysis of the experimental results, we looked at all subjects’ full P300 waves in 30 selections (15 each in VR and AR) one by one. In one selection, there was 20 epochs (or blinks) per button. In total, 140 epochs (20 epochs × 7 buttons) were required to predict one directional button. If the maximum amplitude of the ERP in each epoch exceeded 80 μV, which is over the conventional range of P300 amplitude ,43, the Performance in each environment, 3D motion sickness, and environment preference are shown in p > 0.05).The classification accuracy was calculated to evaluate the two-drone control system. To compare the difference in ERP signals in the two environments, we analyzed the latency and amplitude of the peak in the P300. In this research study, AR for comparison with VR is VR-assisted and video see through-based. Three-hundred epochs (15 selection × 20 blinks) in one environment were averaged per subject; we identified the peak of the P300 component in the averaged ERP manually and calculated the peak value’s amplitude and latency.As shown in p > 0.05).The Wilcoxon signed-rank tests were used to test the statistical significance of the ERP because the Kolmogorov–Smirnov test showed that the latency and amplitude dataset also did not follow a normal distribution. The Wilcoxon signed-rank test showed that the latency and amplitude of the P300 peak point did not differ significantly in the VR and AR environments  and analyzed the ERP signal depending upon the directions.p > 0.05, FDR corrected [As observed, prominent peaks presented at approximately 0.4 s for both VR and AR. To examine the difference in peak amplitude between the two environments, we collected the mean amplitude from 0.3 to 0.5 s in each direction and conducted a statistical test (Wilcoxon rank-sum test). However, we found no statistically significant difference between the AR and VR peak amplitudes in all directions .p > 0.05).Furthermore, we collected all trials from all subjects and attempted to calculate all subjects’ direction-based accuracy. We also evaluated the users’ experience using the answers from the two questionnaires in p > 0.05).To compare the users’ satisfaction in VR and AR, seven items  were evaluated. Each item was scored from one to five points on VR and AR and three points indicates moderate or neutral. The mean score and standard deviation for the seven items evaluated are shown in n = 7) was 93.1% in VR and 89.33% in AR, which indicates that VR accuracy was slightly higher than that in AR. In addition, the group that preferred AR (n = 10) showed greater accuracy in AR (90.67%) than VR (85.48%). However, the statistical test showed no significant difference between the two .The mean accuracy between VR and AR did not differ significantly in r = 0.68, p < 0.05).BCI users’ self-prediction of their accuracy is useful information for understanding BCI performance variation ,47,48,49In this study, we demonstrated a P300 BCI drone control application for VR and AR environments and showed that the two environments did not differ significantly in performance (classification accuracy and ERP patterns) and user experience .We extended the conventional 2D-based BCI experiment to a 3D-based BCI experiment in both virtual reality (VR) and augmented reality (AR) using a HMD. A wearable HMD-based BCI is promising as it provides a more user-friendly BCI system for hands-free interaction with real and virtual objects in AR and VR environments . These 3In BCI studies, the user’s experience is one of the most important aspects of the system’s feasibility. We compared the two environments using not only performance but also users’ experience in a real-world environment and all participants showed a positive response in both VR and AR. This confirms the feasibility of applications available in both VR and AR with respect to users’ satisfaction as well as performance.This work encourages more exciting and promising new research topics to further develop the association between BCI and mixed reality, which uses both VR and AR. In addition to the P300 paradigm, other paradigms, such as motor imagery and SSVEP, can also be tested in a mobile drone control system in VR and AR for more practical applications.Both VR and AR offer immersive and interactive effects but even when the same HMD is used, some elements such as UI and contents can lead to different user satisfactions. Thus, we investigated the participants’ preferred environment as well as their satisfaction.Although the subjects expressed the same positive opinion about the two environments, they had their own preferred environment and corresponding reasons. The reasons why some preferred VR were “interest in a virtual environment” and “feeling of playing the game”, while the reasons why others preferred AR were “strong immersion due to the AR”, “controlling of real drone”, and “liveliness of real time video”.n = 7) or AR (n = 10). In general, a small sample size can influence statistical tests’ significance [ificance  and a laificance . Thus, wNow we discuss the potential limitations and issues with respect to user-friendly BCI applications. As in traditional ERP-based systems, this drone control application requires a training session before use. During the session, a classifier for the online session is constructed by applying a supervised algorithm to adjust the user’s ERP signal . HoweverVarious studies have been conducted to minimize or reduce the calibration process. Lotte et al. proposed regularized canonical correlation analysis combined with a regularized linear discriminant analysis in 2009 , and in Another aspect to improve in our experiment is to include an evaluation of the drone’s free flight through user’s intention. In this experiment, the subjects had to move the drone according to designated instructions to test their accuracy. If the result of a user’s P300 classification matched the specified instruction, the drone moved in that direction; otherwise, it did not. In fact, controlling the drone in the direction that the user wants it to go is one of the important points in this system. Therefore, in the future, we need to test the drone’s free flight performance through users’ intention.The last issues are related to the basic concept of this study. Many P300 BCI applications have been implemented in a desktop environment (2D monitor screen). Hence, a desktop-based BCI application may provide a different user experience. In this study, we only compared VR with AR. Thus, investigating any differences in user feedback in the three environments would provide insightful information that may be helpful in designing a new BCI application. Furthermore, different control paradigms  may provide different experiences as well. Considering the methods used to command vary somewhat across control paradigms, VR and AR should be tested in different BCI types as well. Finally, we recruited 20 subjects and used data from only 17. Although this is not a small sample, more data will help strengthen the results, particularly for group analysis . In the future, we will consider these issues in further studies.We developed a P300 BCI drone control application that worked with VR and AR. To check the difference between and influence of the different environments, an online experiment was conducted with 20 subjects and their opinions were collected using a questionnaire. We found that all subjects controlled a drone in the VR and AR environments well and expressed the same positive opinions about both environments. These results demonstrate that VR and AR do not differ in performance (including ERP patterns) and user experience. Thus, we conclude that the P300 BCI paradigm is relatively stable and works well in various situations.