ABSTRACT
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293 T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Genetic Vectors , Promoter Regions, Genetic , Zebrafish , Zebrafish/genetics , Animals , Gene Editing/methods , Cell Line , Humans , Monophenol Monooxygenase/genetics , Monophenol Monooxygenase/metabolism , HEK293 Cells , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Connective tissue growth factor (Ctgf) is a matricellular protein with diverse biological function. It is regarded as a central regulator of tissue fibrosis and collagen synthesis in mammals. However, its roles in fish remain elusive. Here, a ctgf gene was cloned (NcCtgf), characterized and functionally studied in the chu's croaker (Nibea coibor). NcCtgf encoded a protein containing 346 amino acids, 38 conserved cysteine residues, 4 functional domains and a signal peptide. NcCtgf shared highest identity (99.4 %) to the Larimichthys crocea Ctgf protein. Phylogenetic tree revealed that NcCtgf clustered with the teleost Ctgfa and Ctgf of higher vertebrates, instead of teleost Ctgfb. NcCtgf was expressed with higher level in gonad, spleen, gill and swimming bladder than other tissues, and was up-regulated in swim bladder synchronously with collagen I genes by hydroxyproline and TGF-ß1 treatment. NcCtgf knockdown/overexpression inhibited/promoted collagen synthesis in swim bladder cell, respectively. Notably, NcCtgf protein could be secreted to cell culture medium and up-regulated collagen I expression in swim bladder cell. These findings indicate NcCtgf plays vital roles in collagen synthesis in swim bladder of Nibea coibor, and provide basis for further understanding of ctgf evolution and exploring new approach for enhancing collagen deposition in fish products during aquaculture.
Subject(s)
Connective Tissue Growth Factor , Perciformes , Animals , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Phylogeny , Urinary Bladder/metabolism , Collagen/genetics , Collagen/metabolism , Collagen Type I/genetics , Transforming Growth Factor beta1 , Perciformes/genetics , Perciformes/metabolism , Mammals/metabolismABSTRACT
The hemostatic effect of isinglass (dried swim bladder) in traditional Chinese medicine is well known. But its mechanism of action remains unclear. Here, mice were gavaged with the dried swim bladder of the chu's croaker (Nibea coibor). The hemostatic effect of swim bladder was investigated, tandem mass tag (TMT)-based quantitative proteomics analysis was performed to screen differentially abundant proteins associated with hemostasis in mouse serum. Results indicated that isinglass significantly shorten bleeding time and promoted coagulation after acute trauma (cut out mouse tail). In total, 57 differentially expressed proteins were identified in the sera between control and swim bladder group, of which 31 were up-regulated and 26 were down-regulated in swim bladder group. KEGG pathway enrichment analysis further demonstrated that the Neutrophil extracellular trap formation pathway was significantly affected. Combined with RT-qPCR verification, our findings further suggested that five candidate proteins in the pathway may be involved in the onset of hemostasis after swim bladder gavage, indicating their important role during the hemostasis process promoting by swim bladder. SIGNIFICANCE: Serum proteomics after swim bladder gavage described differentially enriched proteins related to hemostasis, and enriched pathways were validated. This study revealed the possible pathways involved in the hemostatic effect of swim bladder, which may provide a new effector target for the development of new hemostatic drugs.
Subject(s)
Hemostatics , Perciformes , Animals , Hemostasis , Hemostatics/metabolism , Mice , Perciformes/metabolism , Proteomics/methods , Urinary BladderABSTRACT
Iguratimod (IGU) is a novel disease modified anti-rheumatic drug, which has been found to act directly on B cells for inhibiting the production of antibodies in rheumatoid arthritis (RA) patients. Follicular helper T (Tfh) cells, a key T cell subsets in supporting B cell differentiation and antibody production, have been shown to play critical roles in RA. However, whether IGU can inhibit RA Tfh cells which further restrains B cell function remains unclear. Here, we aimed to explore the roles of IGU in regulating RA circulating Tfh (cTfh) cell function and investigate the potential mechanism associated with cell glucose metabolism. In our study, we found that IGU could act on RA-CD4+ T cells to reduce T cell-dependent antibody production. IGU decreased the percentage of RA cTfh cells and the expression of Tfh cell-related molecules and cytokines which were involved in B cell functions. Importantly, our data showed that IGU significantly restrained the cTfh cell function by inhibiting glucose metabolism, which relied on Hif1α-HK2 axis. In summary, we clarified a new target and mechanism of IGU by restraining RA cTfh cell function via inhibiting Hif1α-HK2-glucose metabolism axis. Our study demonstrates the potential application of IGU in the treatment of diseases related to abnormal metabolism and function of Tfh cells.
Subject(s)
Arthritis, Rheumatoid , T Follicular Helper Cells , Chromones , Glucose/metabolism , Humans , Sulfonamides , T-Lymphocytes, Helper-InducerABSTRACT
Osteoarthritis (OA) is an age-related disease, which is mainly treated with oral, topical, and/or intra-articular options to relieve symptoms and lack of specific treatment measures. Fibroblasts (FLS) are crucial cells in joint inflammation and destruction. Cellular senescence plays an important role during OA pathogenesis and senescent cells exhibit cell-cycle arrest and senescence-associated secretory phenotype (SASP). SRY-related HMG-box 4 (SOX4) is a contributing factor during many developmental processes and is elevated in inflamed synovium than in noninflamed synovium from arthritis patients. This study was designed to investigate whether SOX4 participate in the pathogenesis of OA by affecting FLS senescence and explore the internal mechanism. Firstly, we found that FLS cells exhibited more cellular senescence in OA compared with control group. We also verified the role of reactive oxygen species (ROS)/TGF-ß signal in the induction of OA-FLS senescence. During the exploration of SOX4 in cell senescence, the results indicated that SOX4 activation promotes cell senescence and SASP of OA-FLS. Apart from that, we also confirmed that SOX4, regulated by ROS/TGF-ß signal, was critical transcription factor associated with OA-FLS senescence. Therefore, SOX4 is likely to be a novel therapeutic target and early diagnostic marker during OA pathogenesis.
Subject(s)
Cellular Senescence , Osteoarthritis , SOXC Transcription Factors , Transforming Growth Factor beta , Cells, Cultured , Fibroblasts/pathology , Humans , Reactive Oxygen Species , SOXC Transcription Factors/genetics , Signal TransductionABSTRACT
Leptin is over-secreted in many autoimmune diseases, which can promote dendritic cells (DCs) maturation and up-regulate the expression of inflammatory cytokines, but the underlying mechanisms are not fully elucidated. Considering the major role of leptin in maintaining energy balance and the significant role of glycolysis in DCs activation, our study aims to investigate whether leptin promotes the activation of DCs via glycolysis and its underlying mechanisms. We demonstrated that leptin promoted the activation of DCs, including up-regulating the expression of co-stimulatory molecules and inflammatory cytokines, enhancing the proliferation and T helper 17 (Th17) cell ratio in peripheral blood mononuclear cells (PBMC) co-cultured with leptin-stimulated DCs. Leptin also enhanced DCs glycolysis with increased glucose consumption, lactate production, and the expression of hexokinase 2 (HK2). In addition, the activation of DCs stimulated by leptin could be inhibited by the glycolysis inhibitor 2-deoxy-d-glucose (2-DG). To explore the signaling pathways involved in leptin-induced HK2 expression, we observed that the inhibitors of STAT3 (NSC74859) could repress the enhancement of HK2 triggered by leptin stimulation. Therefore, our results indicated that leptin promoted glycolytic metabolism to induce DCs activation via STAT3-HK2 pathway.
Subject(s)
Dendritic Cells/immunology , Glycolysis/immunology , Leptin/metabolism , Aminosalicylic Acids/pharmacology , Benzenesulfonates/pharmacology , Cell Communication/immunology , Cell Line , Cell Proliferation/drug effects , Coculture Techniques , Dendritic Cells/metabolism , Healthy Volunteers , Hexokinase/metabolism , Humans , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Primary Cell Culture , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Signal Transduction/immunology , Th17 Cells/immunology , Th17 Cells/metabolism , Up-Regulation/immunologyABSTRACT
Huaier, a traditional Chinese medicine, is currently used to treat certain types of cancer in the clinic and is also regarded as an immune-modulating and immune-enhancing agent that regulates immune cells. Emerging evidence indicates that an imbalance of immune cells, such as CD4+ T helper (Th) lymphocytes, contributes to the progression of immune thrombocytopenia (ITP), but the effects of Huaier on the regulation of CD4+ T cells are not yet fully elucidated. In the present study, Jurkat cells and peripheral blood mononuclear cells (PBMCs) from patients with ITP and healthy volunteers were treated with Huaier aqueous extract (HR). The CCK-8 assay revealed that HR suppressed the proliferation of Jurkat cells in a dose-dependent manner, whereas 3 mg/mL could decrease cell viability by 50%. At the latter concentration, the activation of CD4+ T cells from patients with ITP was partially attenuated. In addition, HR could correct the unbalanced Th1/Th2 polarization and inhibit the secretion of pro-inflammatory factors interleukin (IL)-2, tumor necrosis factor-α, and interferon-γ. It also suppressed Treg and facilitated Th17 differentiation, but did not change the levels of IL-10 and transforming growth factor-ß. Thus, this study provides more information on how Huaier regulates cellular immunity and improves our understanding of the use of Huaier in ITP.
Subject(s)
CD4-Positive T-Lymphocytes/drug effects , Immunomodulating Agents/pharmacology , Purpura, Thrombocytopenic, Idiopathic/drug therapy , Adolescent , Adult , Aged , Aged, 80 and over , CD4-Positive T-Lymphocytes/immunology , Cells, Cultured , Complex Mixtures/chemistry , Humans , Immunomodulating Agents/chemistry , Jurkat Cells , Male , Medicine, Chinese Traditional , Middle Aged , Purpura, Thrombocytopenic, Idiopathic/immunology , Trametes/chemistry , Young AdultABSTRACT
Leptin, an adipokine sharing structural characteristics of the long-chain helical cytokine family with the crucial role as a regulator in energy homeostasis, has been paid more and more attention to its immunoregulatory function. Emerging evidence has indicated the roles of leptin on autoimmune diseases such as systemic lupus erythematous (SLE), multiple sclerosis (MS), rheumatoid arthritis (RA) and psoriasis, implying that leptin may be involved in autoimmune disorders. It is very definite that there exists immunocyte dysfunction in RA patients. Growing data has manifested that leptin is increased in both serum and synovial fluid of RA patients compared to healthy controls, suggesting leptin probably takes part in the pathogenesis of RA. The aim of this review is to discuss about what we currently know with regard to the role of leptin in immune system and its effects on RA crucial cells. To clarify the role of leptin in the pathogenesis of RA is beneficial to both the treatment and medical study.
Subject(s)
Arthritis, Rheumatoid/etiology , Arthritis, Rheumatoid/metabolism , Disease Susceptibility , Leptin/metabolism , Animals , Arthritis, Rheumatoid/pathology , Biomarkers , Disease Susceptibility/immunology , Gene Expression Regulation , Humans , Leptin/blood , Leptin/genetics , Lymphocytes/immunology , Lymphocytes/metabolism , Macrophages/immunology , Macrophages/metabolismABSTRACT
Time difference of arrival (TDoA) technology is widely utilized for source localization, which stimulates many studies on performance-evaluation approaches for TDoA localization systems. Some approaches using simulations are designed merely for a simple Line-of-Sight (LoS) scenario while some other ones using experiments show high cost and inefficiency. This paper proposes an integrated approach to evaluate a TDoA localization system in an area with a complicated environment. Radio propagation graph is applied through a simulation to obtain channel impulse responses (CIRs) between a source to be located and the TDoA sensors for the area. Realistic signals received by the sensors in baseband are emulated combining the source transmitted signal and the CIRs. A hardware unit takes charge of sending the radio emulated received signals to the system under test, which is consistent with real experimental measurements. Statistical analysis of the system is allowed based on localization errors obtained comparing the system's estimates with the ground truth of the source location. Verified results for LoS and non-LoS scenarios with variable transmitted signal bandwidths and signal-to-noise ratios, as well as for three variations of the sensor locations in an automobile circuit, show the usability of the proposed experiment-free performance-evaluation approach.