Background and purpose:Intracellular deubiquitylating enzymes,such as ubiquitin-specific peptidase 2(USP2),play a pivotal role in regulating protein degradation and cellular homeostasis by modulating protein ubiquitin deconjugation,which have been implicated in the proliferation and survival of multiple myeloma(MM)cells.Targeting the inhibition of USP2 activity in MM cells might modulate their biological behavior.This study aimed to investigate regulatory effects of the leukotriene receptor antagonist montelukast sodium on USP2 in MM cells and its subsequent biological effects.Methods:An in vitro deubiquitination reaction system was established using purified USP2 protein and its substrate,the glutathione S-transferase(GST)tagged ubiquitin A-52 residue ribosomal protein fusion product(UbA52),known as GST-UbA52 protein.This system was used to characterize inhibitory effects of montelukast sodium on USP2 deubiquitinase activity.The MM cell lines MM1.S and H929 were used as in vitro models.Cellular thermal shift assay(CETSA)was subsequently employed to test interaction mode between montelukast sodium and USP2 in MM cells.Western blot assay was applied to detect expression levels of USP2 and its targeting regulators,including cell cycle supervisors cyclin D1(CCND1)and cyclin A1(CCNA1),classical signaling transducer KRAS and glucose regulated protein 78kD(GRP78),as well as apoptotic molecule C/EBP-homologous protein(CHOP)in MM1.S and H929 cells before and after the treatment with different concentrations of montelukast sodium.MM cells with either overexpression(H929-OE,MM1.S-OE)or knockdown(H929-LE,MM1.S-LE)of USP2 were generated using a lentiviral vector.Cell counting kit-8(CCK-8)and flow cytometry were utilized to detect the proliferation and apoptotic rates of H929-OE,MM1.S-OE,H929-LE and MM1.S-LE cells treated with montelukast sodium.Results:Montelukast sodium was found to inhibit USP2 mediated degradation of GST-UbA52 protein in a concentration-dependent manner,with a half inhibitory concentration(IC50)of 3.814 μmol/L.Additionally,montelukast sodium significantly enhanced the thermal stability of USP2 at temperatures of 49.1,53.2 and 56.4℃.It was also shown that montelukast sodium could down-regulate expressions of CCND1,CCNA1 and KRAS,while increase levels of GRP78 and CHOP in MM1.S and H929 cells.Furthermore,after treating with 40 μmol/L montelukast sodium for 24 h,the proliferation inhibition and apoptotic rate of H929-OE cells reached to(37.68±1.10)%and(18.99±0.26)%,while the proliferation inhibition and apoptotic rate of MM1.S-OE cells reached to(24.48±0.49)%and(33.29±0.75)%,which were significantly lower than those in H929 and MM1.S cells[H929:(57.19±1.93)%and(45.65±0.24)%;MM1.S:(50.04±0.53)%and(40.25±0.91)%;P＜0.05,n=3].Conversely,the proliferation inhibition and apoptotic rates of H929-LE and MM1.S-LE cells were significantly higher[H929-LE-1#:(80.70±1.60)%and(89.08±0.49)%;H929-LE-2#:(75.30±3.80)%and(82.41±1.07)%;MM1.S-LE-1#:(70.64±0.84)%and(67.63±0.21)%;MM1.S-LE-2#:(68.47±1.32)%and(85.90±0.18)%;P＜0.05,n=3].Conclusion:Montelukast sodium can target ubiquitin proteasome regulator USP2 and inhibit its deubiquitylating activity,which may modulate USP2 directing protein and trigger endoplasmic reticulum stress to induce cell cycle arrest and apoptosis in MM cells.

Background and purpose:Intracellular deubiquitylating enzymes,such as ubiquitin-specific peptidase 2(USP2),play a pivotal role in regulating protein degradation and cellular homeostasis by modulating protein ubiquitin deconjugation,which have been implicated in the proliferation and survival of multiple myeloma(MM)cells.Targeting the inhibition of USP2 activity in MM cells might modulate their biological behavior.This study aimed to investigate regulatory effects of the leukotriene receptor antagonist montelukast sodium on USP2 in MM cells and its subsequent biological effects.Methods:An in vitro deubiquitination reaction system was established using purified USP2 protein and its substrate,the glutathione S-transferase(GST)tagged ubiquitin A-52 residue ribosomal protein fusion product(UbA52),known as GST-UbA52 protein.This system was used to characterize inhibitory effects of montelukast sodium on USP2 deubiquitinase activity.The MM cell lines MM1.S and H929 were used as in vitro models.Cellular thermal shift assay(CETSA)was subsequently employed to test interaction mode between montelukast sodium and USP2 in MM cells.Western blot assay was applied to detect expression levels of USP2 and its targeting regulators,including cell cycle supervisors cyclin D1(CCND1)and cyclin A1(CCNA1),classical signaling transducer KRAS and glucose regulated protein 78kD(GRP78),as well as apoptotic molecule C/EBP-homologous protein(CHOP)in MM1.S and H929 cells before and after the treatment with different concentrations of montelukast sodium.MM cells with either overexpression(H929-OE,MM1.S-OE)or knockdown(H929-LE,MM1.S-LE)of USP2 were generated using a lentiviral vector.Cell counting kit-8(CCK-8)and flow cytometry were utilized to detect the proliferation and apoptotic rates of H929-OE,MM1.S-OE,H929-LE and MM1.S-LE cells treated with montelukast sodium.Results:Montelukast sodium was found to inhibit USP2 mediated degradation of GST-UbA52 protein in a concentration-dependent manner,with a half inhibitory concentration(IC50)of 3.814 μmol/L.Additionally,montelukast sodium significantly enhanced the thermal stability of USP2 at temperatures of 49.1,53.2 and 56.4℃.It was also shown that montelukast sodium could down-regulate expressions of CCND1,CCNA1 and KRAS,while increase levels of GRP78 and CHOP in MM1.S and H929 cells.Furthermore,after treating with 40 μmol/L montelukast sodium for 24 h,the proliferation inhibition and apoptotic rate of H929-OE cells reached to(37.68±1.10)%and(18.99±0.26)%,while the proliferation inhibition and apoptotic rate of MM1.S-OE cells reached to(24.48±0.49)%and(33.29±0.75)%,which were significantly lower than those in H929 and MM1.S cells[H929:(57.19±1.93)%and(45.65±0.24)%;MM1.S:(50.04±0.53)%and(40.25±0.91)%;P＜0.05,n=3].Conversely,the proliferation inhibition and apoptotic rates of H929-LE and MM1.S-LE cells were significantly higher[H929-LE-1#:(80.70±1.60)%and(89.08±0.49)%;H929-LE-2#:(75.30±3.80)%and(82.41±1.07)%;MM1.S-LE-1#:(70.64±0.84)%and(67.63±0.21)%;MM1.S-LE-2#:(68.47±1.32)%and(85.90±0.18)%;P＜0.05,n=3].Conclusion:Montelukast sodium can target ubiquitin proteasome regulator USP2 and inhibit its deubiquitylating activity,which may modulate USP2 directing protein and trigger endoplasmic reticulum stress to induce cell cycle arrest and apoptosis in MM cells.

Abstract Social withdrawal is a kind of behavioral inhibition in social situations, which may increase the risk for maladjustment, internalizing and externalizing problems, interfering with psychological development and healthy growth. With the deepening understanding in sociology of development, child social withdrawal has gradually received extensive attention from scholars across the world. Understanding the phenomenon of child social withdrawal is important for in depth follow up research. Based on the literature review, the paper aims to summarize the types, mechanisms and influencing factors of social withdrawal in children, in order to provide scientific basis for formulating prevention strategies and early intervention programs in the future.

Objective:We conducted a real-world multi-center clinical study with a large sample size to comprehensively evaluate the performance of three commercial hepatitis C virus (HCV) core antigen assays. The study aimed to evaluate the performance for their use in HCV infection screening, and to provide clues for further improving the sensitivity and specificity of the assays.Methods:Key performance indicators including the lower limit of detection (LOD), diagnostic sensitivity, and specificity of three HCV antigen assays (the Architect, Laibo, and ChemClin HCV core antigen assays) were evaluated using commercial seroconversion panels reflecting early HCV infection and clinical routine serum samples of outpatients and inpatients from 3 tertiary hospitals from January 2018 to April 2022. Factors that affect the performance indicators were further investigated.Results:The window period for detecting HCV infection with the three antigen assays was equal to or slightly longer than that of the RNA assay, but all are shorter than that of the anti-HCV assay. There was a good linear positive correlation between HCV core antigen and HCV RNA levels in treatment naive patients with hepatitis C ( r=0.90, P<0.01). For the most common genotype 1b strain in China, the LOD of the three HCV assays were equivalent to 531 IU/ml (Architect), 3,698 IU/mL (Laibo), and 4,624 IU/mL (ChemClin) HCV RNA, respectively. Due to the skewed distribution of HCV RNA levels in treatment-naive hepatitis C patients, more than 95% of the patients had viral loads higher than 6 166 IU/ml. Therefore, the three HCV antigens assays still maintained a satisfactory diagnostic sensitivity (94.33%-99.40%). Among 54 immunodeficient patients (leukemia patients) with HCV infection, 9% (5/54) had negative anti-HCV results, while the HCV antigen assays found all these infectors. Through further experiments, we revealed the amino acid polymorphism in the core region of genotype 3 strain impaired the sensitivity of all three HCV antigen assays. In addition, the sensitivity of the two domestic assays was impaired by anti-HCV antibodies in the serum. The specificity of HCV antigen assays for diagnosing hepatitis C is 99.94% to 99.98%. The rheumatoid factors, autoantibodies, and other unknown interference substances can lead to a small number of low level, "false positive" antigen results. Conclusions:HCV core antigen assay may be used as a satisfactory approach of infection screening, especially for the immunodeficient patents. However, the sensitivity and specificity of the assays are influenced by multiple factors, which should be further improved.

Objective To discuss the influence of microRNA(miR)-155/miR-21 on toll-like receptor 4 (TLR4) in children with sepsis.Methods Fifty children with sepsis who were hospita-lized in Pediatric Intensive Care Unit,Shenzhen Children's Hospital,were enrolled in the study,and 15 healthy children at the same age were selected as healthy control group.Expression levels of TLR4 protein and human leukocyte antigen(HLA)-DR in CD14 + monocytes (MC) were detected by using flow cytometry,and sepsis patients were divided into 2 groups according to whether they exceeded the value of HLA-DR by 30％ or not.Expression level of programmed cell death factor 4 (PDCDM) and inositol phosphatases 1 containing SH2 (SHIP1) were detected at the same time.MC were separated by CD14 + immune magnetic bead,and expression level of miR-155,miR-21 and tumor necrosis factor-α (TNF-α),interleukin-10 (IL-10) mRNA in CD14 + MC were detected by using real-time fluorescent quantitative PCR.Results Sepsis group consisted of 27 male and 23 female,and their ages were (2.34 ± 0.79) years old,among whom 9 patients died.There were 36 patients in the HLA-DR increase group and 14 patients in the HLA-DR decrease group.Expressions ofTLR4(2.33±0.90),miR-155[(7.19±3.75) ×10 3] and TNF-α[(21.98±14.15) ×10-2 pg/L] in CD14 + MC were higher in the HLA-DR increase group than those in the HLA-DR decrease group [1.24±0.60,(4.83 ±1.17) × 10-3,(14.18±5.45) ×10-2 μg/L] and healthy control group[1.57±0.55,(3.99 ± 1.29) × 10-3,(1.61 ± 0.84) × 10 2 pg/L],and the differences were statistically significant(F =11.943,7.583,18.538,all P ＜0.05),while the expressions of miR-21 (12.10 ±5.66),IL-10[(29.74 ± 12.55) × 10-4 μg/L] in CD14 + MC were lower in the HLA-DR increase group than those in the HLA-DR decrease group[4.68 ± 2.07,(12.50 ± 5.73) × 10-4 μg/L] and healthy control group [2.39 ± 0.86,(2.04 ± 0.92) × 10-4 μg/L],and the differences were statistically significant(F =41.673,54.991,all P ＜ 0.05).The levels of SH1P1 and PDCD4 decreased in sepsis compared with healthy control group[0.70 ±0.36)vs.(1.59 ±0.48);(1.55 ±0.56) vs.(3.01 ±0.70)],and the differences were statistically significant (t =7.682,8.339,all P ＜ 0.05),but SHIP1 decreased more significantly in the HLA-DR increase group than that in the HLA-DR decrease group [(0.60 ± 0.34) vs.(0.97 ± 0.26)],and the difference was statistically significant (F =39.214,P ＜ 0.05).PDCD4 decreased more significantly in the HLA-DR decrease group than that in the HLA-DR increase group (0.94 ±0.19 vs.1.79 ±0.47),the difference was statistically significant(F =65.367,P ＜ 0.05).Conclusions Regulation imbalance of miR-155/miR-21 may be one of the reasons for abnormal expression of TLR4 in children with sepsis,and it plays a role in enlarged or inhibited expression of TLR4 in the sepsis process which results in different immune status in sepsis patients.

Objective To investigate the expression of miR-146a in CD14+ monocytes (MC) of children with sepsis.Methods Forty children with sepsis in PICU were enrolled in the study.Fifteen children with systemic inflammatory response syndrome (SIRS) afteRsurgical treatment and fifteen healthy children were selected as control group.Expressions of miR-146a and TNF-α,IL-10 of CD14+ MC were detected by real-time quantitative PCR(qRT-PCR).HLA-DRlevels of CD14+ MC were detected by flow cytometry.Levels of high-sensitivity C-reactive protein (CRP) and procalcitonin (PCT) were detected.Results The HLA-DRlevels of CD14+ MC in all the children with sepsis were oveR30%.The miR-146a level of CD14+ MC were significantly higheRin sepsis group than SIRS group and healthy controls (P<0.05),and the level decreased when sepsis children were recovered (P<0.05).The TNF-α,IL-10 level of CD14+ MC in children with sepsis was significantly higheRthan healthy controls (P<0.05).Conclusion The expressions of CD14+ MC miR-146a in children with sepsis are significantly up-regulated.

Objective To investigate the expression of miR-146a in CD14+ monocytes (MC) of children with sepsis.Methods Forty children with sepsis in PICU were enrolled in the study.Fifteen children with systemic inflammatory response syndrome (SIRS) afteRsurgical treatment and fifteen healthy children were selected as control group.Expressions of miR-146a and TNF-α,IL-10 of CD14+ MC were detected by real-time quantitative PCR(qRT-PCR).HLA-DRlevels of CD14+ MC were detected by flow cytometry.Levels of high-sensitivity C-reactive protein (CRP) and procalcitonin (PCT) were detected.Results The HLA-DRlevels of CD14+ MC in all the children with sepsis were oveR30%.The miR-146a level of CD14+ MC were significantly higheRin sepsis group than SIRS group and healthy controls (P<0.05),and the level decreased when sepsis children were recovered (P<0.05).The TNF-α,IL-10 level of CD14+ MC in children with sepsis was significantly higheRthan healthy controls (P<0.05).Conclusion The expressions of CD14+ MC miR-146a in children with sepsis are significantly up-regulated.

Objective To explore the causes of persistent abnormal muscle response (AMR) after microvascular decompression (MVD) for hemifacial spasm (HFS) and the clinical outcomes of these patients.Methods MVD was performed under intraoperative electrophysiological monitoring of AMR in 372 HFS patients in 2014.Before MVD,the characteristic AMR of HFS was recorded in 326 patients.The patients were divided into two groups based on whether AMR disappeared or persisted following MVD;21 patients showed persistent AMR after successful MVD while AMR disappeared after decompression in the other 305 patients.The clinical features,treatment efficacy and postoperative complications were compared between these two groups.Results Gender,side of depression and mean age between the two groups showed no significant differences (P＞0.05).The immediate postoperative cure rate of the AMR disappeared group (88.9％) was significantly higher than that in the AMR persisted group (28.6％,P＜0.05).The follow-up cure rate showed no significant difference between the two groups (P＞0.05),and the postoperative and follow-up complications showed no significant differences (P＞0.05).Conclusion The long duration of HFS patients may be responsible for persistent AMR after successful decompression,and it is more likely for these patients to get delayed cured;their long-term outcomes showed no difference as compared with those in patients with disappeared AMR after MVD.

Objective To investigate the effects of miR-155/miR-21 on the expression of TLR4 in patients with Kawasaki disease (KD).Methods Forty-five children with acute KD were enrolled into the study.Children were divided into the group with coronary artery lesions (KD-CAL+) and the group without coronary artery lesions (KD-CAL-).The age-matched twenty healthy children were included as the control group.The levels of TLR4 expression were analyzed by flow cytometry.The CD14+ monocytes in the peripheral blood were separated by CD14+ immunomagnetic beads.Real-time polymerase chain reaction (PCR) were performed to evaluate the expression of miR-155,miR-21 and interleukin (IL)-10,tumor necrosis factor (TNF)-α,IL-6,IL-1β in CD14+ monocytes at mRNA level.The concentration of matixmetalloproteinase (MMP)-9,HSP60,high mobility group box-1 protein (HMGB1) were measured by enzyme-linked immunosorbentassay (ELISA).T test was used to compare the differences between groups and P＜0.05 was considered to be statistically significant.Results The levels of Toll-like receptor 4 (TLR4) expression in children with acute KD were significantly higher than those in the control group [(12.4±5.1)％ vs (4.6±1.6)％,t=5.55,P＜0.05],and the levels of TLR4 expression in the KD-CAL+ group were remarkably elevated compared with the KD-CAL+group [(17.1±4.6)％ vs (9.6±2.7)％,t=5.89,P＜0.05].The plasma concentrations of HMGB11 [(10.2±7.6 vs 3.2±1.0) ng/ml,t=3.55,P＜0.05] and HSP60 [(56±23 vs 18±7) ng/ml,t=4.90,P＜0.05] in the KD group were increased(P＜0.05),but significantly decreased aftcr treatment with IVIG (P＜0.05).The levels of miR-155 [(21.0±27.0)×10 vs (4.6±3.1)×10-3,t=2.57,P＜0.05] and miR-21 (11.6±4.0 vs 8.7±2.2,t=2.78,P＜0.05)expression in the KD group were significantly higher than those in the controls (P＜0.05).The level of miR-155 expression in the KD-CAL+ group was apparently higher than that in the KD-CAL-group [(4.2±3.0)×10 vs (11.0±1.5)×104,t=4.07,P＜0.05] and the expression of miR-21 was lower than that in the KD-CAL-group (8.8±2.5 vs 12.8±4.0,t=3.46,P＜0.05).Compared with the controls,the levels of IL-10 mRNA expression in the KD group were significantly increased [(34.9 ±17.5)×10 vs (1.0±9.4)×10 4,t =5.62,P＜0.05),and IL-10 expression in the KD-CAL-group was higher than that in the KD-CAL+ group [(26.6±13.4)×104 vs (39.5±1.8)×10,t=2.36,P＜0.05].The plasma concentrations of MMP-9 in the KD group were remarkably higher than those in the controls [(1 141±541) pg/ml vs (304±94) pg/ml,t=4.83,P＜0.05],the concentration of MMP-9 in the KD-CAL+ group was higher than that in the KD-CAL-group [(1 350±625) pg/ml vs (945±370) pg/ml,t=2.21,P＜0.05].Conclusion The dysregulation of miR-155 and miR-21 may be one of the reasons that result in over-expression of TLR4 in patients with acute KD,and the persistently increased HSP60 and HMGB1 may trigger or amplify TLR4 expression.

OBJECTIVETo investigate the expressions of midkine (MK) and Ki67 in gastric carcinoma (GC) and their relation with the clinicopathologic characteristics.

METHODSThe expressions of MK and Ki67 in 71 GC and 20 adjacent normal tissues were evaluated by immunohistochemistry.

RESULTSThe expressions of MK and Ki67 were significantly higher in GC than in adjacent normal tissues (76.1% vs 0, and 73.2% vs 5%, respectively, P<0.05). MK and Ki67 expressions were correlated with the infiltration depth, lymph node metastasis and TNM stage (P<0.05), and a significant correlation was found between MK and Ki67 expressions (P<0.05).

CONCLUSIONGC tissues have high expressions of MK and Ki67, indicating that MK and Ki67 play important roles in promoting the tumorigenesis and progression of GC.

Cytokines ; metabolism ; Disease Progression ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; metabolism ; Lymphatic Metastasis ; Nerve Growth Factors ; Stomach Neoplasms ; metabolism