The activation of NLRP3 inflammasome contributes to the development of numerous chronic inflammatory diseases, including ulcerative colitis and stress-induced anxiety. Recent studies have revealed that NLRP3 K48-linked ubiquitination by several E3 ligases, including Trim31, leads to degradation. Neddylation is a process highly similar to ubiquitination by covalently conjugating NEDD8 to lysines in specific substrate proteins. Neddylation of substrate proteins alters their subcellular localization, stability, and activity. The role of neddylation in the NLRP3 inflammasome remains elusive. Here, we report that neddylation promotes the activation of the NLRP3 inflammasome in macrophages. Myeloid deficiency of UBA3, the catalytic subunit of the NEDD8-activating enzyme (NAE), renders mice resistant to dextran sodium sulfate-induced colitis. Inducible Uba3 deletion in microglia mitigates psychological stress-induced anxiety-like behavior. Neddylation blockade led to a reduced protein level of NLRP3 without affecting its mRNA level. Further exploration revealed that NLRP3 undergoes neddylation at K287 with Ube2M as the E2 and Smurf2 as an E3, respectively. NLRP3 neddylation hinders its interaction with Trim31 and thereby inhibits its K48-linked ubiquitination and subsequent degradation. MLN4924, a potent compound NAE inhibitor in phase 1/2/3 clinical trials for cancers, alleviates psychological stress-induced NLRP3 inflammasome activation, microglia inflammatory activation, and anxiety-like behavior, suggesting novel clinical activity of MLN4924.

Major depressive disorder (MDD) is a leading cause of disability worldwide. It remains elusive whether serum levels of cortisol, amino acids and their downstream neurotransmitters, and inflammatory cytokines can reflect the therapeutic response of MDD. Although music seems to be beneficial to MDD treatment, the optimal treatment strategy remains to be explored. Here, we report that the combination of pharmacotherapy and music therapy with both D major K. 448 and G minor K. 550, but not pharmacotherapy alone, significantly downregulated or presented numerical trends to downregulate serum levels of cortisol and norepinephrine (NE) in MDD patients. Furthermore, serum levels of L_Histidine, L_Glutamic acid, L_Aspartic acid, tumor necrosis factor-α (TNF-α), TNF-receptor I (TNFR I), and C-C motif ligand 11 (CCL11) were significantly downregulated or exhibited numerical trends to be downregulated by the combination of pharmacotherapy and music therapy, whereas those of cluster of differentiation 30 (CD30) exhibited opposite effects. Therefore, music therapy with both D major K. 448 and G minor K. 550 should be recommended as adjuvant therapy to MDD patients. Certain serum markers can reflect the therapeutic response of MDD. The therapeutic effects of music therapy may be attributed to augmented stress-coping ability and diminished systemic inflammation.

CD4+ T cells are central mediators of protective immunity to blood-stage malaria, particularly for their capacity in orchestrating germinal center reaction and generating parasite-specific high-affinity antibodies. T follicular helper (Tfh) cells are predominant CD4+ effector T cell subset implicated in these processes, yet the factors and detailed mechanisms that assist Tfh cell development and function during Plasmodium infection are largely undefined. Here we provide evidence that receptor for activated C kinase 1 (RACK1), an adaptor protein of various intracellular signals, is not only important for CD4+ T cell expansion as previously implied but also plays a prominent role in Tfh cell differentiation and function during blood-stage Plasmodium yoelii 17XNL infection. Consequently, RACK1 in CD4+ T cells contributes significantly to germinal center formation, parasite-specific IgG production, and host resistance to the infection. Mechanistic exploration detects specific interaction of RACK1 with STAT3 in P. yoelii 17XNL-responsive CD4+ T cells, ablation of RACK1 leads to defective STAT3 phosphorylation, accompanied by substantially lower amount of STAT3 protein in CD4+ T cells, whereas retroviral overexpression of RACK1 or STAT3 in RACK1-deficient CD4+ T cells greatly restores STAT3 activity and Bcl-6 expression under the Tfh polarization condition. Further analyses suggest RACK1 positively regulates STAT3 stability by inhibiting the ubiquitin-proteasomal degradation process, thus promoting optimal STAT3 activity and Bcl-6 induction during Tfh cell differentiation. These findings uncover a novel mechanism by which RACK1 participates in posttranslational regulation of STAT3, Tfh cell differentiation, and subsequent development of anti-Plasmodium humoral immunity.

Stress‐related illnesses are linked to the onset and progression of renal diseases and depressive disorders. To investigate stress‐induced changes in the renal transcriptome associated with the development of depressive behaviors, we generated here a chronic social defeat stress (CSDS) model of C57 BL/6 male mice and then performed RNA sequencing of the kidneys to obtain an inflammation‐related transcriptome. Administration of the antidepressant drug fluoxetine (10 mg·kg−1·day−1) during CSDS induction could partially alleviate renal inflammation and reverse CSDS‐induced depression‐like behaviors. Moreover, fluoxetine also modulated gene expression of stress‐related hormone receptors, including prolactin and melanin‐concentrating hormone. These results suggest that CSDS can induce gene expression changes associated with inflammation in the kidney of C57 BL/6 male mice, and this inflammation can be treated effectively by fluoxetine.

Major depressive disorder (MDD) is a highly prevalent psychiatric disorder. But the treatment of depression remains challenging. Anti-inflammatory treatments frequently produce antidepressant effects. EPO-derived helix-B peptide ARA290 has been reported to retain the anti-inflammatory and tissue-protective functions of EPO without erythropoiesis-stimulating effects. The effects of ARA290 on MDD remain elusive. This study established chronic unpredictable mild stress and chronic social defeat stress mouse models. Daily administration of ARA290 during chronic stress induction in two mouse models ameliorated depression-like behavior, similar to fluoxetine. With marginal effects on peripheral blood hemoglobin and red cells, ARA290 and fluoxetine reversed chronic stress-induced increased frequencies and/or numbers of CD11b+Ly6Ghi neutrophils and CD11b+Ly6Chi monocytes in the bone marrow and meninges. Furthermore, both drugs reversed chronic stress-induced microglia activation. Thus, ARA290 ameliorated chronic stress-induced depression-like behavior in mice through, at least partially, its anti-inflammatory effects.

Fulminant hepatitis (FH) is a life-threatening disease with partially understood pathogenesis.It has been demonstrated that myeloid-derived suppressor cells (MDSCs) are recruited into the liver during this process, and their augmented accumulation by various strategies protects against liver injury.However, the underlying mechanism(s) remain elusive.Receptor for activated C kinase 1 (RACK1), a multi-functional scaffold protein, is highly expressed in normal liver and has been implicated in liver physiology and diseases, but the in vivo role of hepatic RACK1 in FH remains unknown.Methods: Survival curves and liver damage were monitored to investigate the in vivo role of hepatic RACK1 in FH.The liver microenvironment was explored by microarray-based transcriptome analysis, flow cytometry, immunoblotting, and immunohistochemistry. MDSCs were identified with phenotypic and functional characteristics.Functional antibodies were used to target MDSCs.Co-culture techniques were used to study the underlying mechanism(s) of protection.The interaction of RACK1 with histone deacetylase 1 (HDAC1) and the consequent effects on HDAC1 ubiquitination were analyzed.Ectopic expression of HDAC1 with recombinant adeno-associated virus serotype 8 was conducted to confirm the role of HDAC1 in the protective effects of hepatic RACK1 deficiency against FH.Post-translational modifications of RACK1 were also investigated during the induction of FH.Results: Liver-specific RACK1 deficiency rendered mice resistant to FH. RACK1-deficient livers exhibited high basal levels of chemokine (C-X-C motif) ligand 1 (CXCL1) and S100 calcium-binding protein A9 (S100A9), associated with MDSC accumulation under steady-state conditions.Targeting MDSCs with an antibody against either Gr1 or DR5 abrogated the protective effects of liver-specific RACK1 deficiency.Accumulated MDSCs inhibited inflammatory cytokine production from macrophages and enhanced IκB kinase (IKK)/NF-κB pathway activation in hepatocytes.Further investigation revealed that RACK1 maintained HDAC1 protein level in hepatocytes by direct binding, thereby controlling histone H3K9 and H3K27 acetylation at the Cxcl1 and S100a9 promoters.Ectopic expression of HDAC1 in livers with RACK1 deficiency partially reversed the augmented Cxcl1/S100a9 → MDSCs → IKK/NF-κB axis.During FH induction, RACK1 was phosphorylated at serine 110, enhancing its binding to ubiquitin-conjugating enzyme E2T and promoting its ubiquitination and degradation.Conclusion: Liver-specific RACK1 deficiency protects against FH through accelerated HDAC1 degradation and the consequent CXCL1/S100A9 upregulation and MDSC accumulation.

BACKGROUND AND AIM:Previous studies have shown that the hepatitis C virus (HCV) core protein plays an important role in the metastasis of hepatocellular carcinoma (HCC) cells. This study aimed to identify the potential mechanism of HCV core protein in HCC. METHODS:A transcription factor microarray analysis was performed to identify the factors regulated by the HCV core protein. A comprehensive bioinformatics analysis approach was utilized to predict the functions, regulatory signaling pathways and downstream target genes of the differentially regulated transcription factors. Dual-luciferase assays, qPCR, Western blotting, ERK pathway inhibition experiments and siRNA knockdown experiments were performed to verify the effects of the HCV core protein on PEA3, SRF and c-Fos, as well asthe underlying mechanism. The migration/invasion assay and scratch assay served to confirm the metastasis-promoting mechanism of the HCV core protein. RESULTS:The results demonstrated that altered expression of PEA3, SRF and c-Fos mediated by the HCV core protein were associated with the MAPK/ERK pathway. c-Fos was a downstream target protein of PEA3 and SRF. Knockdown of PEA3-SRF/c-Fos expression and ERK pathway components suppressed the migration and invasion activity of hepatocytes by affecting MMP2 and MMP9 expression. CONCLUSION:We provided preliminary evidence that the role of the HCV core protein in promoting metastasis is at least partially dependent on the activation of the MAPK/ERK/PEA3-SRF/c-Fos/MMP2/MMP9 axis. These findings reveal a novel mechanism by which the HCV core protein promotes HCC metastasis and may provide new therapeutic targets for patients with metastatic HCC.

目的 获得小鼠干扰素调节因子7(IRF7)抗原,制备抗小鼠IRF7多克隆抗体并鉴定.方法 构建原核表达质粒pET?28a(+)?IRF7,在大肠杆菌中表达IRF7,用Ni柱纯化获得抗原后,分别采用MnJ胶体锰佐剂和弗氏佐剂制备免疫原,3次免疫后心脏取血分离血清,采用间接ELISA法测定血清效价、纯化试剂盒纯化抗体、Western印迹和免疫沉淀对抗体生物学活性进行鉴定.结果 在大肠杆菌中成功表达IRF7并获得纯蛋白.两种佐剂制备的多抗血清,效价均可达到810×103倍.血清纯化后获得抗IRF7抗体,经Western印迹鉴定,两个多克隆抗体均能够特异识别真核表达的IRF7蛋白,并在免疫沉淀外源表达IRF7时,其效果明显优于商业化抗体.结论 成功制备了抗IRF7多克隆抗体,为后续研究IRF7功能及作用机制奠定了基础.

Neddylation, an important type of post-translational modification, has been implicated in innate and adapted immunity. But the role of neddylation in innate immune response against RNA viruses remains elusive. Here we report that neddylation promotes RNA virus-induced type I IFN production, especially IFN-α. More importantly, myeloid deficiency of UBA3 or NEDD8 renders mice less resistant to RNA virus infection. Neddylation is essential for RNA virus-triggered activation of Ifna gene promoters. Further exploration has revealed that mammalian IRF7undergoes neddylation, which is enhanced after RNA virus infection. Even though neddylation blockade does not hinder RNA virus-triggered IRF7 expression, IRF7 mutant defective in neddylation exhibits reduced ability to activate Ifna gene promoters. Neddylation blockade impedes RNA virus-induced IRF7 nuclear translocation without hindering its phosphorylation and dimerization with IRF3. By contrast, IRF7 mutant defective in neddylation shows enhanced dimerization with IRF5, an Ifna repressor when interacting with IRF7. In conclusion, our data demonstrate that myeloid neddylation contributes to host anti-viral innate immunity through targeting IRF7 and promoting its transcriptional activity.

The receptor for activated C kinase 1 (RACK1) adaptor protein has been implicated in viral infection. However, whether RACK1 promotes in vivo viral infection in mammals remains unknown. Moreover, it remains elusive how RACK1 is engaged in antiviral innate immune signaling. In this study, we report that myeloid RACK1 deficiency does not affect the development and survival of myeloid cells under resting conditions but renders mice less susceptible to viral infection. RACK1-deficient macrophages produce more IFN-α and IFN-β in response to both RNA and DNA virus infection. In line with this, RACK1 suppresses transcriptional activation of type 1 IFN gene promoters in response to virus infection. Analysis of virus-mediated signaling indicates that RACK1 inhibits the phosphorylation of IRF3/7. Indeed, RACK1 interacts with IRF3/7, which is enhanced after virus infection. Further exploration indicates that virus infection triggers AMPK activation, which in turn phosphorylates RACK1 at Thr50. RACK1 phosphorylation at Thr50 enhances its interaction with IRF3/7 and thereby limits IRF3/7 phosphorylation. Thus, our results confirm that myeloid RACK1 promotes in vivo viral infection and provide insight into the control of type 1 IFN production in response to virus infection.

目的 建立EV71抗原双抗体夹心检测方法,并初步探讨其在临床血清样品检测中的应用.方法 用抗EV71病毒单克隆抗体包被酶联反应板,加入待检标本反应后,用HRP标记的抗EV71病毒单克隆抗体进行检测,建立EV71抗原双抗体夹心检测方法;采用抗人IgM单克隆抗体包被96孔酶联板,辣根酶标记的EV71抗原,建立ELISA捕获方法检测抗EV71-IgM.2种方法同时检测230份体检人员血清,确定方法的cut-off(CO)值,并检测140份手足口病患者血清,采用GraraphPad Prism 4软件作图并绘制ROC曲线.结果 EV71抗原检测方法的CO值为0.168,抗EV71-IgMELISA检测方法的CO值为0.173.在140例手足口病患者血清中,EV71抗原检测方法的阳性检出率为45.00％,抗EV71-IgM ELISA检测方法的阳性检出率为42.86％,其中共同阳性患者45例,单独EV71抗原阳性患者18例,单独抗EV71-IgM抗体阳性患者15例,如果2种方法联合检测,其阳性检出率可提高到55.71％.结论 ELISA检测EV71抗原诊断HFMD具有很好的准确性,结合EV71-IgM抗体捕获检测技术,可进一步提高灵敏度,具有很好的临床应用前景.

Although targeted therapy has been extensively investigated for breast cancers, a molecular target with broad application is currently unavailable due to the high heterogeneity of these cancers. Mammaglobin-A (Mam-A), which is overexpressed in most breast carcinomas, has been proposed as a promising target. However, the lack of specific targeting moieties due to uncertain binding epitopes hampers further translational study. Here, seven potential epitopes of Mam-A were disclosed, and a unique epitope was then identified in most types of breast cancers, despite the genotypic heterogeneity. With phage display technology, the epitope was determined to be N-terminal amino acids 42-51 of Mam-A (N42-51). Then, the N42-51 epitope-specific monoclonal antibody, mAb785, was conjugated to poly lactic-co-glycolic acid (PLGA) nanoparticles loaded with therapeutic agents, thereby enhancing the drug uptake and therapeutic efficacy in different genotypes of breast cancers. The computer simulation of the N42-51 epitope and the mAb785 structures, as well as their interactions, further revealed the specific targeting mechanism of the mAb785-conjugated nanoparticles to breast cancers.

The size was a key factor influencing the drug loading, release, and delivery efficacies of poly (lactic-co-glycolic) acid (PLGA) microspheres (MS). The purpose of the study was to explore a novel and simple method to prepare uniform-sized PLGA MS for drug delivery. Different from routine homogenizer, a vortex shaker was employed for emulsification during the preparation of PLGA MS. To enhance the homogeneity of resulted PLGA MS, a glass bead was used to facilitate the emulsification. The size of PLGA MS was controlled by adjusting the working frequency and time of vortex shaker. Then, selective centrifugation was used to further enrich PLGA MS of desired size. The resulted PLGA MS were systematically evaluated in anti-tubercular drug delivery. Compared with the routine stirring-based method, the novel method possessed a higher efficiency of emulsification and resulted in more homogenous MS under similar conditions. The produced PLGA particles were of a narrow size distribution, which could be 1 to 5 μm by adjusting the working frequency and time of vortex shaker. The narrow size distribution made it rather easy to enrich PLGA MS of specific size through selective centrifugation. Then, to evaluate the potential of the PLGA MS for drug delivery, 2–3-μm sized PLGA MS were selected to deliver anti-tubercular drug rifampicin (RFP). In vitro experiments demonstrated that the loaded RFP could be well-controlled release and delivered into macrophages. Anti-tubercular experiments showed that the uniform-sized MS as carriers of RFP significantly enhanced the therapeutic efficacy of the drug. Collectively, the study provided a novel and simple method for preparing uniform-sized PLGA MS, which may be of potentially wide applications.

目的:制备高活性EB病毒Zta优势表位抗原并初步评价抗原活性.方法:利用生物学软件分析Zta抗原的B细胞表位分布,选取含有优势表位的抗原区段,然后利用分子生物学技术进行克隆表达获得纯化抗原,采用ELISA方法初步评价优势表位抗原的检测性能.结果:筛选确定Zta优势表位抗原区段为1～185 aa,并获得了高效表达的Zta优势表位抗原,基于该抗原建立的Zta-IgA间接ELISA方法可以有效区分鼻咽癌患者和健康对照.结论:获得的优势表位抗原Zta可用于鼻咽癌患者的早期筛查和诊断.

目的:了解正常体检人群戊型肝炎病毒感染情况.方法:采用酶联免疫吸附法检测499例正常体检受试者血清HEV IgG和HEV IgM抗体水平.结果:在499例受试者中,HEV IgG总阳性率为13.63％ (68/499),HEV IgM总阳性率为3.21％ (16/499),共同阳性率为1.40％(9/499),共检出9例HEV IgM单阳性样本.男性人群中的抗体阳性率高于女性.体检人群中40岁～59岁HEV IgG阳性率显著高于20岁～39岁.结论:正常体检人群中存在HEV感染者,需要加强对正常体检人群的HEV抗体筛查,尤其是联合检测HEV IgG和HEV IgM可以提高检出率,避免漏检.

Hepatitis c virus (HCV) infection is one of major causes for chronic liver diseases worldwide and could lead to death. Development of effective HCV vaccines is a powerful auxiliary method of existing treatments. Adjuvants are necessary for modern vaccines to promote immune responses. Among the various nanomaterials that have been developed, multihydroxylated fullerene (C60(OH)22) has been proved as an efficient adjuvant for human immunodeficiency virus DNA vaccine. Here, we utilized three types of HCV recombinant proteins as antigens to investigate the activity of C60(OH)22 as a protein vaccine adjuvant. The proteins were carried by C60(OH)22 in a way of surface adsorption and self-assemble encapsulation. C60(OH)22 at a relatively low dose was sufficient to promote both humoral and cellular immune responses to HCV protein antigens and reduce the usage of antigen. These results demonstrated the positive adjuvant properties of C60(OH)22 when applied to protein vaccines.

Human vasorin (VASN) as a type I transmembrane protein, is a potential biomarker of hepatocellular carcinoma, which could expedite HepG2 cell proliferation and migration significantly in vitro. The ectodomain of VASN was proteolytically released to generate soluble VASN (sVASN), which was validated to be the active form. Among several monoclonal antibodies produced against sVASN, the clone V21 was found to bind with the recombinant human sVASN (rhsVASN) with the highest affinity and specificity, and also have inhibitory effects on proliferation and migration of HepG2 cells. Hence the phage-displayed peptide library was screened against the antibody V21. The positive phage clones were isolated and sequenced, and one unique consensus motifs was obtained. The result of sequence alignment showed that the conserved motif had similarity to VASN(Cys432-Cys441), embedded in the epidermal growth factor (EGF)-like domain. The synthetic mimotope peptide V21P1 and V21P2 were confirmed to bind with V21 and could compete with rhsVASN in ELISA assay. And they could also almost completely reverse the inhibitory effect of V21 on HepG2 migration and proliferation. Furthermore, the antibodies produced against V21P1 were able to bind not only with the peptide V21P1, but also with rhsVASN and the natural VASN from HepG2 cell. Our results showed that V21 seemed to be a functional antibody. The mimotopes toward V21 might mimic the functional domain of VASN, which would be helpful to exploit VASN functions and act as a candidate target for developing therapeutic antibodies against VASN.

Objective: We aimed to determine whether IP-10 and RANTES plasma levels can be used in diagnosis and monitoring of pulmonary tuberculosis (PTB). Methods: Plasma levels of cytokines/chemokines were measured using a Bio-Plex' multiplex cytokine assay system in a cohort containing 457 clinically suspected PTB patients including a training set (n = 41)and two independent WA sets A (n = 242) and B (n = 174). Results: Plasma levels of IP-10 and RANTES were significantly higher in PTB patients than healthy controls' in both training and independent test sets (P < 0.05). Compared with other combinations, the combination of IP10 and RANTES had the best performance with an AUC of 1.0 in training set. The performance characteristic of this model was successfully validated in independent WA set A although this combination only resulted in a slightly improvement of AUC value in independent test set B. Plasma IP-10 and RANTES levels were weakly and positively correlated with blood glucose concentrations. Moreover, IP-10 levels were positively correlated with CRP and ESR in PTB patients. Furthermore, in response to therapy, both IP-10 and RANTES levels significantly decreased over the period of 6 months (P < 0.001). Conclusions: Taken together, combination of IP-10 and RANTES could be potentially used as diagnostic and monitoring biomarker in PTB management.

Objective To explore the significance of the combined determination of glutamic acid decarboxylase antibody(GA-DA),insulinoma-associated 2 molecule antibody(IA-2A) and Zinc transporter 8 antibody and Islet-specific glucose-6-phosphatase catalytic subunit-related protein autoantibody (IGRPA) in the diagnosis of type 1 diabetes.Methods GADA,IA-2A,ZnT8A and IGRPA autoantibody in serum were conducted for detection by enzyme-linked immunosorbent assay (ELISA) in 45 patients with type 1 diabetes mellitus,50 type 2 diabetic patients and 50 normal controls.Results The detection rates of IA-2A,GADA,ZnT8A and IGRPA in patients with type 1 diabetes were 28.89％,53.33％,71.11％ and 77.78％.The positive rate of four autoantibodies in type 1 diabetes mellitus was significantly higher than those in type 2 diabetes group and control group,with the differences of statistical significance(P ＜0.05).The sensitivity of the combined autoantibodies detection was higher than that of the single detection.The sensitivity and the area under the curve of GADA/IA-2A/ZnT8A/1G-RPA were 95.56％ and 0.988,respectively.Conclusion The combined detection of GADA,IA-2A,ZnT8A and IGRPA can significantly improve the detection rate of type 1 diabetes mellitus,and has important significance for the diagnosis of type 1 diabetes mellitus.