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1.
Heliyon ; 10(3): e25171, 2024 Feb 15.
Article in English | MEDLINE | ID: mdl-38352746

ABSTRACT

Depression is considered to be an "emotional disease" in ancient books of traditional Chinese medicine. Its clinical features are similar to those of "Lily disease" in the ancient Chinese medicine book Synopsis of the Golden Chamber written by Zhang Zhongjing in the Han Dynasty. Baihe Zhimu (Lilium lancifolium bulb and Anemarrhena asphodeloides rhizome) decoction (LBRAD) is the first prescription of "Lily Disease" in this book. It is also a special remedy for "Lily disease" after sweating. The classic recipe LBRAD consists of two herbs, fresh lily bulbs and dried Rhizoma Anemarrhena slice. It has the effect of supplementing nutrition and clearing heat, nourishing Yin and moistening. After more than two thousand years of clinical practice, it has been currently widely used in clinical treatment of depression. In this paper, the relationship between LBRAD and depression was systematically reviewed from both clinical and experimental studies, as well as the preparation, the clinical application, the pharmacological mechanism and the effective material basis for the treating depression of LBRAD. The core targets and biological processes of the depression treatment were explored through network pharmacological analysis, so as to speculate its potential mechanism. Finally, the association between LBRAD and post-COVID-19 depression was discussed. We concluded with a summary and future prospects. This review may provide a theoretical basis for the expansion of the clinical application of LBRAD and the development of new drugs for the treatment of depression, as well as new ideas for the secondary development of classical prescriptions.

2.
J Cosmet Dermatol ; 23(1): 271-283, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37464738

ABSTRACT

BACKGROUND: Adipose stem cell-derived exosomes (ADSC-EXO) and botulinum toxin type A (BTX-A) individually showed a therapeutic effect on skin wound repair. AIMS: This study investigated their synergistic effect on promoting skin wound healing in vitro and in vivo and the underlying molecular events. METHODS: ADSCs were isolated from Sprague-Dawley (SD) rats to obtain ADSC-EXO by ultrafiltration and ultracentrifugation and were confirmed using nanoparticle tracking analysis and transmission electron microscopy. Human skin fibroblasts (HSF) were cultured and treated with or without ADSC-EXO, BTX-A, or their combination. Changes in cell phenotypes and protein expression were analyzed using different in vitro assays, and a rat skin wound model was used to assess their in vivo effects. RESULTS: The isolated ADSC-EXO from primarily cultured ADSCs had a circular vesicle shape with a 30-180 nm diameter. Treatment of HSF with ADSC-EXO and/or BTX-A significantly accelerated HSF migration in vitro and skin wound healing in a rat model. Moreover, ADSC-EXO plus BTX-A treatment dramatically induced VEGFA expression but reduced COL III and COL I levels in vivo. ADSC-EXO and/or BTX-A treatment significantly upregulated TGF-ß3 expression on Day 16 after surgery but downregulated TGF-ß1 expression, suggesting that ADSC-EXO plus BTX-A promoted skin wound healing and reduced inflammatory cell infiltration. CONCLUSIONS: The ADSC-EXO plus BTX-A treatment demonstrated a synergistic effect on skin wound healing through upregulation of VEGF expression and the TGF-ß3/TGF-ß1 and COL III/COL I ratio.


Subject(s)
Botulinum Toxins, Type A , Exosomes , Rats , Humans , Animals , Botulinum Toxins, Type A/pharmacology , Exosomes/metabolism , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta3/metabolism , Rats, Sprague-Dawley , Stem Cells , Adipose Tissue
3.
Eur J Pharmacol ; 962: 176228, 2024 Jan 05.
Article in English | MEDLINE | ID: mdl-38042462

ABSTRACT

OBJECTIVE: G protein-coupled receptor 124 (GPR124) regulates central nervous system angiogenesis and blood-brain barrier (BBB) integrity, and its deficiency aggravates BBB breakdown and hemorrhagic transformation in ischemic mice. However, excessive GPR124 expression promotes inflammation in atherosclerotic mice. In this study, we aimed to elucidate the role of GPR124 in hypoxia/ischemia-induced cerebrovascular endothelial cell injury. METHODS: bEnd.3 cells were exposed to oxygen-glucose deprivation (OGD), and time-dependent changes in GPR124 mRNA and protein expression were evaluated using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The effects of GPR124 overexpression or knockdown on the expression of pyroptosis-related genes were assessed at the mRNA and protein levels. Tadehaginoside (TA) was screened as a potential small molecule targeting GPR124, and its effects on pyroptosis-related signaling pathways were investigated. Finally, the therapeutic efficacy of TA was evaluated using a rat model of transient middle cerebral artery occlusion/reperfusion (tMCAO/R). RESULTS: During OGD, the expression of GPR124 initially increased and then decreased over time, with the highest levels observed 1 h after OGD. The overexpression of GPR124 enhanced the OGD-induced expression of NLRP3, Caspase-1, and Gasdermin D (GSDMD) in bEnd.3 cells, whereas GPR124 knockdown reduced pyroptosis. Additionally, TA exhibited a high targeting ability to GPR124, significantly inhibiting its function and expression and suppressing the expression of pyroptosis-related proteins during OGD. Furthermore, TA treatment significantly reduced the cerebral infarct volume and pyroptotic signaling in tMCAO/R rats. CONCLUSIONS: Our findings suggest that GPR124 mediates pyroptotic signaling in endothelial cells during the early stages of hypoxia/ischemia, thereby exacerbating ischemic injury.


Subject(s)
Inflammasomes , Reperfusion Injury , Animals , Mice , Rats , Endothelial Cells/metabolism , Glucose/metabolism , Hypoxia/metabolism , Infarction, Middle Cerebral Artery/metabolism , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Oxygen/metabolism , Pyroptosis , Receptors, G-Protein-Coupled/metabolism , Reperfusion Injury/metabolism , RNA, Messenger/metabolism
4.
Sci Rep ; 13(1): 9771, 2023 06 16.
Article in English | MEDLINE | ID: mdl-37328486

ABSTRACT

T-box transcription factor 15 (TBX15) is upregulated in a variety of tumors and has been reported to promote uncontrolled proliferation of tumor cells and induce tumor cells to avoid apoptosis, thus accelerating the malignant transformation of malignant tumors. However, the prognostic value of TBX15 in glioma and its relationship with immune infiltration remain unknown. In this study, we intended to explore the prognostic value of TBX15 and its link to glioma immune infiltration and examine TBX15 expression in pan-cancer using RNAseq data in TPM format from TCGA and GTEx. TBX15 mRNA and protein expressions in glioma cells and adjacent normal tissue were detected and compared by RT-qPCR and Western blot. The effect of TBX15 on survival was assessed by Kaplan-Meier Method. The correlation between TBX15 upregulation and the clinicopathological characteristics of glioma patients was assessed by using TCGA databases, and the relationship between TBX15 and other genes in glioma was evaluated by using TCGA data. The top 300 genes most significantly associated with TBX15 were selected to establish a PPI network through the STRING database. The relationship between TBX15 mRNA expression and immune cell infiltration was explored by using ssGSEA and the TIMER Database. It was found that TBX15 mRNA expression in glioma tissues was significantly higher than that in the adjacent normal tissues, and this difference was most obvious in high-grade gliomas. TBX15 expression was increased in human gliomas and associated with worse clinicopathological characteristics and poorer survival prognosis in glioma patients. In addition, elevated TBX15 expression was linked to a collection of genes involved in immunosuppression. In conclusion, TBX15 played an important role in immune cell infiltration in glioma and may prove to be a predictor of the prognosis in glioma patients.


Subject(s)
Brain Neoplasms , Glioma , Humans , Brain Neoplasms/pathology , Clinical Relevance , Glioma/pathology , Prognosis , RNA, Messenger/genetics , T-Box Domain Proteins/genetics
5.
Front Biosci (Landmark Ed) ; 28(5): 94, 2023 05 22.
Article in English | MEDLINE | ID: mdl-37258471

ABSTRACT

BACKGROUND: Huang Qi (HQ, Astragalus) and Hong Hua (HH, Safflower), two Chinese herbal remedies, are widely used to treat coronary heart disease (CHD). However, the underlying mechanisms of this herb pair remain unclear. The aim of this study was to determine the potential synergistic effects and mechanisms of Astragalus-Safflower in the treatment of CHD. METHODS: Network pharamcology was performed to identify the core components, targets, and key genes of Astragalus-Safflower herbal pair (ASHP) for the treatment of CHD. Enrichment analysis was performed to identify overlapping genes. Ultrahigh-performance liquid chromatography coupled with Q-Exactive MS/MS (UHPLC-QE-MS) was used to detect the blood component of rat ASHP drug-containing serum, which is also considered to be the core components of the ASHP. Molecular docking of ASHP core compounds with core proteins of the pyroptosis pathway mediated by the NLR family pyrin domain containing 3 (NLRP3) inflammasomes. In vivo experiments were conducted to verify the effect and mechanism of ASHP in the CHD mice model. RESULTS: 54 active compounds and 404 target genes were identified from ASHP, and 1576 targets for CHD with 90 overlapping genes for both. IL6, AKT1, IL1B, TP53, VEGFA, PTGS2, MMP9, CCL2, CXCL8 and EGF were the key hub target genes. Enrichment analysis of Kyoto Encyclopedia of Gene and Genome (KEGG) revealed that the NLRP3 inflammasome-mediated signaling pathway was one of the more critical signaling pathways. The UHPLC-QE-MS was used to identify the rat ASHP containing serum enrollment compound as calycosin and isorhamnetin. Molecular docking showed that quercetin, kaempferol, apigenin, calycosin and isorhamnetin possessed good binding sites with NLRP3 and Caspase-1. Animal experiments showed that the expression of NLRP3, Caspase-1, GSDMD, IL-1ß mRNA and protein levels were elevated in mouse models of CHD, and decreased after intervention with ASHP. CONCLUSIONS: ASHP can effectively treat CHD, and the mechanism may be related to the inhibition of the NLRP3 inflammasome-mediated pathway.


Subject(s)
Carthamus tinctorius , Mice , Animals , Rats , Network Pharmacology , Inflammasomes/genetics , Molecular Docking Simulation , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Tandem Mass Spectrometry , Caspases
6.
BMC Cardiovasc Disord ; 22(1): 546, 2022 12 13.
Article in English | MEDLINE | ID: mdl-36513971

ABSTRACT

BACKGROUND: Recent study has shown that the transient receptor potential vanilloid 2 (TRPV2) channel was exclusively upregulated in patients with atrial fibrillation (AF), and that this overexpression might be detrimental for occurrence and maintenance of AF. We aimed to characterize the expression levels of TRPV2 mRNA in peripheral blood mononuclear cells (PBMCs) with/without early recurrence of atrial fibrillation (ERAF) after radiofrequency catheter ablation (RFCA), and to find a reliable predictor for ERAF. METHODS: 65 patients of AF, who underwent RFCA successfully, then divided into two groups according to ERAF during following 3 months. PBMCs were isolated from whole blood by Ficoll gradient centrifugation before and after RFCA. Gene set enrichment analysis was performed to evaluate TRPV channels expression levels and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping was used for pathway enrichment analysis. RESULTS: There was no significant difference in the TRPV2 mRNA expression level between the two groups before RFCA, while without ERAF group of TRPV2 expression was markedly reduced compared to ERAF group after RFCA. Moreover, the number of TRPV2 expression was confirmed as an independent predictor for the first time through receiver operating characteristic and Kaplan-Meier survival curve analysis. It should be pointed out that the above results were only used to predict ERAF, and have no predictive significance for late recurrence of atrial fibrillation according to the current data. Additionally, ERAF was inversely correlated with P wave dispersion. KEGG mapping further clustered 41 pathways, revealing that ''cyclic guanosine monophosphate-protein kinase G signaling pathway'' was significantly enriched. CONCLUSIONS: We firstly assume that downregulated expression of peripheral TRPV2 appear in patients without ERAF after RFCA. TRPV2 may thus represent a novel predictor of early phase after successful radiofrequency ablation.


Subject(s)
Atrial Fibrillation , Catheter Ablation , Humans , Atrial Fibrillation/diagnosis , Atrial Fibrillation/genetics , Atrial Fibrillation/surgery , Leukocytes, Mononuclear , Recurrence , Treatment Outcome , Catheter Ablation/adverse effects , Catheter Ablation/methods , RNA, Messenger/genetics , TRPV Cation Channels/genetics
7.
BMC Cardiovasc Disord ; 22(1): 339, 2022 07 29.
Article in English | MEDLINE | ID: mdl-35906548

ABSTRACT

BACKGROUND: Coronary atherosclerosis (CA) is the most common type of atherosclerosis. However, the inherent pathogenesis and mechanisms of CA are unclear, and the relationship with ferroptosis-related genes (FRGs) has not been reported. The purpose of this study was to use bioinformatics techniques to evaluate potential therapeutic targets for CA.Please provide the given name for author "Dingshun".Please provide the given name for author "Dingshun". METHODS: First, the GSE132651 dataset was acquired from the Gene Expression Omnibus database. Gene Ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and Protein-Protein interaction network were successively conducted. Next, overlapping genes between hub genes and CA genes were found. FRGs were found when comparing the CA group with the normal group. The correlation between overlapping genes and FRGs was further analyzed. At last, we performed Elisa to validate the expression of these genes in human blood specimens. Mice aortic tissues were used for western blot to detect the expression of proteins. RESULTS: Based on the GSE132651 dataset, 102 differentially expressed genes were identified. Five overlapping genes between hub genes and CA genes were found (CCNA2, RRM2, PBK, PCNA, CDK1). TFRC and GPX4 were found to be FRGs. TFRC was positively correlated with CCNA2, PBK, PCNA, CDK1, RRM2, with CDK1 being the strongest correlation. GPX4 was negatively correlated with these genes, among which CCNA2 was the strongest correlation. The ELISA results showed that CCNA2, CDK1, and TFRC expression were markedly increased in serum of the CA samples compared with controls, while GPX4 expression was markedly decreased in the CA samples. The western blot results show that GPX4 expression was lower in the model group, TFRC, CDK1, and CCNA2 protein expression were high in the model group. CONCLUSIONS: Ferroptosis-related genes GPX4 and TFRC were closely correlated with the identified overlapping genes CCNA2 and CDK1, which may serve as targeted therapies for the treatment of CA.


Subject(s)
Coronary Artery Disease , Ferroptosis , Animals , Computational Biology/methods , Coronary Artery Disease/genetics , Ferroptosis/genetics , Humans , Mice , Proliferating Cell Nuclear Antigen
8.
Article in English | MEDLINE | ID: mdl-35607519

ABSTRACT

Safflower has long been used to treat coronary heart disease (CHD). However, the underlying mechanism remains unclear. The goal of this study was to predict the therapeutic effect of safflower against CHD using a network pharmacology and to explore the underlying pharmacological mechanisms. Firstly, we obtained relative compounds of safflower based on the TCMSP database. The TCMSP and PubChem databases were used to predict targets of these active compounds. Then, we built CHD-related targets by the DisGeNET database. The protein-protein interaction (PPI) network graph of overlapping genes was obtained after supplying the common targets of safflower and CHD into the STRING database. The PPI network was then used to determine the top ten most significant hub genes. Furthermore, the DAVID database was utilized for the enrichment analysis on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). To validate these results, a cell model of CHD was established in EAhy926 cells using oxidized low-density lipoprotein (ox-LDL). Safflower was determined to have 189 active compounds. The TCMSP and PubChem databases were used to predict 573 targets of these active compounds. The DisGeNET database was used to identify 1576 genes involved in the progression of CHD. The top ten hub genes were ALB, IL6, IL1B, VEGFA, STAT3, MMP9, TLR4, CCL2, CXCL8, and IL10. GO functional enrichment analysis yielded 92 entries for biological process (BP), 47 entries for cellular component (CC), 31 entries for molecular function (MF), and 20 signaling pathways, which were obtained from KEGG pathway enrichment screening. Based on these findings, the FoxO signaling pathway is critical in the treatment of CHD by safflower. The in vitro results showed that safflower had an ameliorating effect on ox-LDL-induced apoptosis and mitochondrial membrane potential. The western blot results showed that safflower decreased Bax expression and acetylation of FoxO1 proteins while increasing the expression of Bcl-2 and SIRT1 proteins. Safflower can be used in multiple pathways during CHD treatment and can exert anti-apoptotic effects by regulating the expression of Bax, Bcl-2, and SIRT1/FoxO1 signaling pathway-related proteins.

9.
Entropy (Basel) ; 24(1)2022 Jan 12.
Article in English | MEDLINE | ID: mdl-35052143

ABSTRACT

The maximum correntropy Kalman filter (MCKF) is an effective algorithm that was proposed to solve the non-Gaussian filtering problem for linear systems. Compared with the original Kalman filter (KF), the MCKF is a sub-optimal filter with Gaussian correntropy objective function, which has been demonstrated to have excellent robustness to non-Gaussian noise. However, the performance of MCKF is affected by its kernel bandwidth parameter, and a constant kernel bandwidth may lead to severe accuracy degradation in non-stationary noises. In order to solve this problem, the mixture correntropy method is further explored in this work, and an improved maximum mixture correntropy KF (IMMCKF) is proposed. By derivation, the random variables that obey Beta-Bernoulli distribution are taken as intermediate parameters, and a new hierarchical Gaussian state-space model was established. Finally, the unknown mixing probability and state estimation vector at each moment are inferred via a variational Bayesian approach, which provides an effective solution to improve the applicability of MCKFs in non-stationary noises. Performance evaluations demonstrate that the proposed filter significantly improves the existing MCKFs in non-stationary noises.

10.
Front Pharmacol ; 13: 1079185, 2022.
Article in English | MEDLINE | ID: mdl-36703734

ABSTRACT

Cardiovascular disease (CVD) is one of the leading causes of death worldwide. Atherosclerosis is the pathological basis of atherosclerotic cardiovascular disease (ASCVD). Atherosclerosis is now understood to be a long-term immune-mediated inflammatory condition brought on by a complicated chain of factors, including endothelial dysfunction, lipid deposits in the artery wall, and monocyte-derived macrophage infiltration, in which both innate immunity and adaptive immunity play an indispensable role. Recent studies have shown that atherosclerosis can be alleviated by inducing a protective immune response through certain auto-antigens or exogenous antigens. Some clinical trials have also demonstrated that atherosclerotic is associated with the presence of immune cells and immune factors in the body. Therefore, immunotherapy is expected to be a new preventive and curative measure for atherosclerosis. In this review, we provide a summary overview of recent progress in the research of immune mechanisms of atherosclerosis and targeted therapeutic pathways.

11.
Cell Rep ; 32(7): 108044, 2020 08 18.
Article in English | MEDLINE | ID: mdl-32814047

ABSTRACT

Type I interferon (IFN) plays an essential role in the host innate immune responses. Several ubiquitin-conjugating enzyme (E2) family members were reported to regulate type I IFN production and host antiviral immune responses. However, the molecular mechanisms are still not fully understood. Here, we report that UBE2S acts as a negative regulator in the type I IFN signaling pathway. Ectopic expression of UBE2S inhibits host antiviral immune responses and enhances viral replications, whereas deficiency of UBE2S enhances host antiviral immune responses and suppresses viral replications both in vitro and in vivo. Inhibition of type І IFN production by UBE2S is independent on its E2 and E3 enzymic activity. Mechanistically, UBE2S interacts with TBK1 and recruits ubiquitin-specific protease 15 (USP15) to remove Lys63 (K63)-linked polyubiquitin chains of TBK1. Our findings reveal a role of the UBE2S-USP15-TBK1 axis in the regulation of host antiviral innate immune responses.


Subject(s)
Interferon Type I/antagonists & inhibitors , Protein Serine-Threonine Kinases/immunology , Ubiquitin-Conjugating Enzymes/immunology , Virus Replication/immunology , Animals , Cattle , HEK293 Cells , Humans , Interferon Type I/biosynthesis , Interferon Type I/immunology , Mice , Mice, Knockout , Mice, Transgenic , Ubiquitination
12.
Mamm Genome ; 31(3-4): 110-116, 2020 04.
Article in English | MEDLINE | ID: mdl-32318815

ABSTRACT

RNA polymerase III is an essential enzyme in eukaryotes for synthesis of tRNA, 5S rRNA, and other small nuclear and cytoplasmic RNAs. Thus, RNA polymerase III promoters are often used in small hairpin RNA (shRNA) expression. In this study, the porcine H1, U6, and 7SK RNA polymerase III type promoters were cloned into a pcDNA3.1( +) expression vector containing a shRNA sequence targeting enhanced green fluorescent protein (EGFP). PK and DF-1 cells were cotransfected with the construction of recombinant interference expression vector and the EGFP expression vector, pEGFP-N1. The average fluorescence intensity of EGFP in transfected cells was measured by fluorescence microscopy and flow cytometry. Real-time PCR was used to detect expressed shRNAs and the relative expression of EGFP, to confirm the activity of the promoters. The results showed that the activity of porcine 7SK promoter is stronger than the U6 promoter, which is in turn stronger than porcine H1. While the high levels of expression of the U6 and 7SK promoters saturate the shRNAs level in the host cell, which can cause cytotoxicity and tissue damage. Therefore, porcine H1 promoter is effective for expression of shRNA, and may be an excellent tool to knockdown gene expression in pigs for functional genomics studies. The results also lay a foundation for the development of porcine RNAi technology and genetically modified porcine research.


Subject(s)
Gene Expression/genetics , Promoter Regions, Genetic/genetics , RNA Polymerase III/genetics , RNA, Small Interfering/genetics , Animals , Base Sequence , Cell Line , Genetic Therapy/methods , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , RNA Interference/physiology , Swine , Transfection/methods
13.
Cell Rep ; 26(5): 1258-1272.e4, 2019 01 29.
Article in English | MEDLINE | ID: mdl-30699353

ABSTRACT

DExD/H-box helicase members are key receptors for recognizing viral nucleic acids, and they regulate retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-mediated type I interferon (IFN) production. Here, we report that the DExD/H-box helicase family member DExD/H-box RNA helicase 19 (DDX19) is a negative regulator of type I IFN production. Ectopic expression of DDX19 suppressed poly(I:C) (polyinosinic-polycytidylic acid)- and Sendai-virus-induced type I IFN production, whereas knockdown of DDX19 expression enhanced type I IFN production. Mechanistically, DDX19 inhibited TANK-binds kinase 1 (TBK1)- and inhibitor-κb kinase ε (IKKε)-mediated phosphorylation of interferon regulatory factor 3 (IRF3) by disrupting the interaction between TBK1 or IKKε and IRF3. Additionally, DDX19 recruited Lamtor2 and then formed the TBK1-IKKε-Lamtor2-DDX19-IRF3 complex to suppress IFN production by promoting TBK1 and IKKε degradation. We generated Ddx19 knockout mice using transcription activator-like effector nucleases (TALENs) and found that Ddx19 deficiency in vivo augmented type I IFN production, resulting in suppression of encephalomyocarditis virus replication. These data show that DDX19 is an important negative regulator of RLR-mediated type I IFN production.


Subject(s)
I-kappa B Kinase/metabolism , Interferon Regulatory Factor-3/metabolism , Interferon Type I/biosynthesis , Nucleocytoplasmic Transport Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteolysis , Animals , HEK293 Cells , Humans , Mice , Phosphorylation , Protein Binding , Proteins , RAW 264.7 Cells , THP-1 Cells
14.
Poult Sci ; 98(1): 430-439, 2019 Jan 01.
Article in English | MEDLINE | ID: mdl-30085302

ABSTRACT

The generation of transgenic chickens is of both biomedical and agricultural significance, and recently chicken transgenesis technology has been greatly advanced. However, major issues still exist in the efficient production of transgenic chickens. This study was designed to optimize the production of enhanced green fluorescence protein (EGFP)-transgenic broilers, including egg windowing at the blunt end (air cell) of egg, and the direct transfection of circulating primordial germ cells by microinjection of the Tol2 plasmid-liposome complex into the early embryonic dorsal aorta. For egg windowing, we discovered that proper manipulation of the inner shell membrane at the blunt end could improve the rate of producing G0 transgenic roosters. From 27 G0 roosters, we successfully collected semen with EGFP-positive sperms from 16 and 19 roosters after direct fluorescence observation and fluorescence-activated cell sorting analyses (13 detected by both methods), respectively. After artificial insemination using the G0 rooster with the highest number of EGFP fluorescent sperm, one G1 EGFP transgenic broiler (1/81, 1.23%) was generated. Our results indicate that appropriate egg windowing and screening of potentially transgene-positive roosters can improve the production of germline-transmitted transgenic birds.


Subject(s)
Animals, Genetically Modified , Chickens/genetics , Gene Transfer Techniques/veterinary , Transfection/veterinary , Animals , Embryo, Nonmammalian , Female , Genetic Vectors , Green Fluorescent Proteins/genetics , Insemination, Artificial/veterinary , Male , Transfection/methods , Transgenes
15.
Vaccine ; 33(31): 3758-65, 2015 Jul 17.
Article in English | MEDLINE | ID: mdl-26036948

ABSTRACT

To prevent a future influenza A virus subtype pandemic outbreak, developing a broad-spectrum vaccine would be highly beneficial. The ion channel protein M2 is highly conserved in a diverse number of influenza A virus subtypes. This distinguishing characteristic makes M2 an attractive vaccine target for a broadly protective vaccine. We expressed a full-length M2 protein which was C-terminally fused to a small peptide in Escherichia coli. Because this recombinant M2 (rM2) protein forms multimeric complexes with high molecular weight, it serves as a potential immunogen. Antibodies induced by the rM2 protein prevented the replication of different subtypes of influenza A virus both in vitro and in vivo. Animal study demonstrated that rM2 immunization protected mice against influenza A virus infection via limiting replication of virus progeny in vivo and attenuating lung damage. As such, the M2 protein is a highly potential candidate for next generation vaccine development with the capability of protecting against various influenza A virus subtypes.


Subject(s)
Cross Protection , Orthomyxoviridae Infections/prevention & control , Viral Matrix Proteins/immunology , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Disease Models, Animal , Lung/pathology , Lung/virology , Mice, Inbred BALB C , Orthomyxoviridae Infections/pathology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Viral Matrix Proteins/genetics
16.
Retrovirology ; 12: 36, 2015 Apr 28.
Article in English | MEDLINE | ID: mdl-25928027

ABSTRACT

BACKGROUND: As a member of the tumor necrosis factor receptor (TNFR) protein superfamily, equine lentivirus receptor 1 (ELR1) has been shown to be expressed in various equine cells that are permissive for equine infectious anemia virus (EIAV) replication. The EIAV Tat protein (eTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter through a unique mechanism that requires the recruitment of the equine cyclin T1 (eCT1) cofactor into the viral TAR RNA target element. In vitro studies have demonstrated that mouse fibroblast cell lines (e.g., NIH 3T3 cells) that express the EIAV receptor ELR1 and eCT1 support the productive replication of EIAV. Therefore, we constructed transgenic eCT1- and ELR1-expressing mice to examine whether they support in vivo EIAV replication. FINDINGS: For the first time, we constructed mice transgenic for ELR1 and eCT1. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis confirmed that ELR1 and eCT1 were expressed in the transgenic mouse tissues, particularly in the intestines, spleen and lymph nodes. Consistent with the results of EIAV infection in NIH 3T3 cells expressing ELR1 and eCT1, mouse embryonic fibroblasts (MEFs) from the transgenic mice could support EIAV replication. More importantly, this virus could infect and replicate in mouse blood monocyte-derived macrophages (mMDMs). Macrophages are the principle target cell of EIAV in its natural hosts. Furthermore, after the transgenic mice were inoculated with EIAV, the virus could be detected not only in the plasma of the circulating blood but also in multiple organs, among which, the spleen and lymph nodes were the predominant sites of EIAV replication. Finally, we found that consistent with high viral replication levels, the relevant pathological changes occurred in the spleen and lymph nodes. CONCLUSIONS: Our results show that mice transgenic for ELR1 and eCT1 are susceptible to EIAV infection and replication. Further, EIAV infection can cause lesions on the spleen and lymph nodes, similar to those frequently observed in horses, the natural hosts. Therefore, ELR1 and eCT1 are essential in vivo for EIAV invasion and replication.


Subject(s)
Cyclin T/biosynthesis , Equine Infectious Anemia/virology , Gene Expression , Infectious Anemia Virus, Equine/growth & development , Receptors, Virus/biosynthesis , Animal Structures/virology , Animals , Blotting, Western , Cyclin T/genetics , Disease Models, Animal , Equine Infectious Anemia/pathology , Gene Expression Profiling , Horses , Lymph Nodes/pathology , Mice , Mice, Transgenic , Real-Time Polymerase Chain Reaction , Receptors, Virus/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Spleen/pathology , Virus Replication
18.
Med Oncol ; 31(3): 843, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24442426

ABSTRACT

We aimed to elucidate whether serum VEGFR2 concentration before and after transarterial chemoembolization (TACE) can predict survival in patients with unresectable hepatocellular carcinoma (HCC). Serum VEGFR2 concentrations were serially measured in 169 patients with advanced HCC before and after TACE. We defined a decrease in the serum VEGFR2 level>10% from the pretreatment level as response. Serum VEGFR2 concentrations decreased in 44 (26.0%) patients at week 4. Patients who had a VEGFR2 response at week 4 had a longer median survival than those who did not have a VEGFR2 decrease (19.0 vs. 9.8 months, p<0.001). Clinical variables associated with OS in addition to VEGFR2 response also included extrahepatic metastases (p=0.005) and vascular invasion (p=0.035). VEGFR2 decrease after TACE (p=0.012) and presence of extrahepatic metastases (p=0.02) were independently associated with OS by multivariate analysis. A serum VEGFR2 concentration decrease at 4 weeks after TACE may predict favorable overall survival in patients with advanced HCC.


Subject(s)
Biomarkers, Tumor/blood , Carcinoma, Hepatocellular/blood , Chemoembolization, Therapeutic , Liver Neoplasms/blood , Vascular Endothelial Growth Factor Receptor-2/blood , Carcinoma, Hepatocellular/mortality , Carcinoma, Hepatocellular/secondary , Carcinoma, Hepatocellular/therapy , Enzyme-Linked Immunosorbent Assay , Female , Follow-Up Studies , Humans , Liver Neoplasms/mortality , Liver Neoplasms/pathology , Liver Neoplasms/therapy , Male , Middle Aged , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Staging , Prognosis , Prospective Studies , Survival Rate
19.
Zhonghua Yi Xue Za Zhi ; 93(23): 1830-2, 2013 Jun 18.
Article in Chinese | MEDLINE | ID: mdl-24124720

ABSTRACT

OBJECTIVE: To investigate the abnormal expression of microRNAs (miR-216, miR-222, miR-181) in the serum of patients with hepatocellular carcinoma (HCC) and its clinical significance. METHODS: Serum miRNAs expression was investigated in 49 patients with HCC and 25 healthy normal controls by using real-time PCR technique, and then correlations between miRNAs expression and the clinicopathological features and prognosis of HCC patients were evaluated. RESULTS: No differences were observed between the HCC patients and healthy controls with respect to the expressions of serum miR-181 and miR-216 (P > 0.05), while miR-222 was found to be significantly overexpressed in HCC serum samples (6.1 ± 6.6 vs 1.2 ± 0.6, P < 0.01). According to the median fold change of miR-222 (3-fold) in all HCC serum samples, we divided the 49 patients into two groups:a low expression group (25 patients) and a high expression group (24 patients).High level of miR-222 expression was correlated with cirrhosis (P < 0.01), tumor number (P = 0.013), portal vein tumor thrombosis (P = 0.002) and TNM stage (P = 0.020), while not correlated with serum alpha-fetoprotein level, tumor size and HbsAg. Kaplan-Meier survival analysis showed that the median overall survival in the high miR-222 expression group was significantly shorter than that in the low expression group (8.7 months vs 16.5 months, P = 0.036). In multivariate Cox analysis, TNM stage and serum miR-222 expression were two independent prognostic factors. CONCLUSION: Serum miR-222, upregulated in HCC, maybe helpful in prognosis of HCC patients.


Subject(s)
Carcinoma, Hepatocellular/blood , Liver Neoplasms/blood , MicroRNAs/blood , Adult , Aged , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Case-Control Studies , Female , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Male , Middle Aged , Neoplasm Staging , Prognosis , Up-Regulation
20.
Res Vet Sci ; 95(3): 1006-11, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24074690

ABSTRACT

To extend the use of RNAi in chicken, we have developed a RNA interference (RNAi) system using a shortened chicken 7SK (ch7SK) promoter. The results stated that the cloned ch7SK promoter includes multiple Oct-1 motifs, SPH domain, PSE and TATA box, without CACCC box. All RNAi groups driven by ch7SK promoter showed significant mean fluorescence intensity (MFI) reduction. In the pch7SK-shEGFP transfected DF-EGFP cell culture, the MFI reduction ratio was smaller than the pmU6-shEGFP did. In the pmU6-shEGFP transfected Vero-EGFP cell culture, the MFI was reduced significantly than the pch7SK-shEGFP did. In summary, the essential part of ch7SK promoter was capable of efficiently expressing shRNAs with relatively different interfering degrees in avian and mammalian cells, respectively. Our results suggest that ch7SK promoter is an efficient alternative to commercially mouse U6 promoter in shRNA expression with chicken cells, and provide references for furthering functional genome analysis and disease resistant breeding in chicken.


Subject(s)
Promoter Regions, Genetic/genetics , RNA Interference , RNA, Small Interfering/genetics , Transcription, Genetic/genetics , Animals , Chickens/genetics , Chlorocebus aethiops , Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Gene Knockdown Techniques/veterinary , Mice , Promoter Regions, Genetic/physiology , RNA Interference/physiology , RNA, Small Interfering/physiology , Species Specificity , Transcription, Genetic/physiology , Vero Cells
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