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1.
Methods Mol Biol ; 2807: 299-323, 2024.
Article in English | MEDLINE | ID: mdl-38743237

ABSTRACT

Ex vivo cervical tissue explant models offer a physiologically relevant approach for studying virus-host interactions that underlie mucosal HIV-1 transmission to women. However, the utility of cervical explant tissue (CET) models has been limited for both practical and technical reasons. These include assay variation, inadequate sensitivity for assessing HIV-1 infection and replication in tissue, and constraints imposed by the requirement for using multiple replica samples of CET to test each experimental variable and assay parameter. Here, we describe an experimental approach that employs secreted nanoluciferase (sNLuc) and current HIV-1 reporter virus technologies to overcome certain limitations of earlier ex vivo CET models. This method augments application of the CET model for investigating important questions involving mucosal HIV-1 transmission.


Subject(s)
Cervix Uteri , HIV Infections , HIV-1 , HIV-1/physiology , HIV-1/genetics , Humans , Cervix Uteri/virology , Cervix Uteri/metabolism , Female , HIV Infections/virology , Luciferases/genetics , Luciferases/metabolism , Genes, Reporter , Mucous Membrane/virology , Mucous Membrane/metabolism , Virus Replication
2.
ACS Chem Biol ; 19(5): 1035-1039, 2024 May 17.
Article in English | MEDLINE | ID: mdl-38717306

ABSTRACT

Red-shifted bioluminescence is highly desirable for diagnostic and imaging applications. Herein, we report a semisynthetic NanoLuc (sNLuc) based on complementation of a split NLuc (LgBiT) with a synthetic peptide (SmBiT) functionalized with a fluorophore for BRET emission. We observed exceptional BRET ratios with diverse fluorophores, notably in the red (I674/I450 > 14), with a brightness that is sufficient for naked eye detection in blood or through tissues. To exemplify its utility, LgBiT was fused to a miniprotein that binds HER2 (affibody, ZHER2), and the selective detection of HER2+ SK-BR-3 cells over HER2- HeLa cells was demonstrated.


Subject(s)
Luminescent Measurements , Humans , HeLa Cells , Luminescent Measurements/methods , Luciferases/genetics , Luciferases/metabolism , Receptor, ErbB-2/metabolism , Cell Line, Tumor , Fluorescent Dyes/chemistry
3.
Int J Mol Sci ; 25(9)2024 May 04.
Article in English | MEDLINE | ID: mdl-38732235

ABSTRACT

The formulation of novel delivery protocols for the targeted delivery of genes into hepatocytes by receptor mediation is important for the treatment of liver-specific disorders, including cancer. Non-viral delivery methods have been extensively studied for gene therapy. Gold nanoparticles (AuNPs) have gained attention in nanomedicine due to their biocompatibility. In this study, AuNPs were synthesized and coated with polymers: chitosan (CS), and polyethylene glycol (PEG). The targeting moiety, lactobionic acid (LA), was added for hepatocyte-specific delivery. Physicochemical characterization revealed that all nano-formulations were spherical and monodispersed, with hydrodynamic sizes between 70 and 250 nm. Nanocomplexes with pCMV-Luc DNA (pDNA) confirmed that the NPs could bind, compact, and protect the pDNA from nuclease degradation. Cytotoxicity studies revealed that the AuNPs were well tolerated (cell viabilities > 70%) in human hepatocellular carcinoma (HepG2), embryonic kidney (HEK293), and colorectal adenocarcinoma (Caco-2) cells, with enhanced transgene activity in all cells. The inclusion of LA in the NP formulation was notable in the HepG2 cells, which overexpress the asialoglycoprotein receptor on their cell surface. A five-fold increase in luciferase gene expression was evident for the LA-targeted AuNPs compared to the non-targeted AuNPs. These AuNPs have shown potential as safe and suitable targeted delivery vehicles for liver-directed gene therapy.


Subject(s)
Chitosan , Gene Transfer Techniques , Gold , Liver Neoplasms , Metal Nanoparticles , Humans , Gold/chemistry , Metal Nanoparticles/chemistry , Hep G2 Cells , Liver Neoplasms/therapy , Liver Neoplasms/genetics , Chitosan/chemistry , HEK293 Cells , Asialoglycoprotein Receptor/metabolism , Asialoglycoprotein Receptor/genetics , Caco-2 Cells , Luciferases/genetics , Luciferases/metabolism , Polyethylene Glycols/chemistry , Plasmids/genetics , Disaccharides/chemistry , Genetic Therapy/methods , Polymers/chemistry , Cell Survival/drug effects
4.
J Immunol Methods ; 529: 113682, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38705372

ABSTRACT

BACKGROUND: The measurement of antigen-specific serum IgE is common in clinical assessments of type I allergies. However, the interaction between antigens and IgE won't invariably trigger mast cell activation. We previously developed the IgE crosslinking-induced luciferase expression (EXiLE) method using the RS-ATL8 mast cell line; however, the method may not be sensitive enough in some cases. METHODS: In this study, we introduced an NF-AT-regulated luciferase reporter gene into the RBL-2H3 rat mast cell line and expressed a chimeric high-affinity IgE receptor (FcεRI) α chain gene, comprising an extracellular domain from humans and transmembrane/intracellular domains from rats. RESULTS: We generated multiple clones expressing the chimeric receptor. Based on their responsiveness and proliferation, we selected the HuRa-40 clone. This cell line exhibited significantly elevated human α chain expression compared to RS-ATL8 cells, demonstrating a 10-fold enhancement of antigen-specific reactivity. Reproducibility across different batches and operators was excellent. Moreover, we observed a detectable response inhibition by an anti-allergy drugs (omalizumab and cyclosporin A). CONCLUSIONS: HuRa-40 cells-which carry the human-rat chimeric IgE receptor-comprise a valuable reporter cell line for the EXiLE method. Their versatility extends to various applications and facilitates high-throughput screening of anti-allergy drugs.


Subject(s)
Immunoglobulin E , Luciferases , Mast Cells , Receptors, IgE , Receptors, IgE/metabolism , Receptors, IgE/genetics , Receptors, IgE/immunology , Animals , Humans , Mast Cells/immunology , Mast Cells/metabolism , Rats , Immunoglobulin E/immunology , Luciferases/genetics , Luciferases/metabolism , Cell Line , Genes, Reporter , Reproducibility of Results , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism
5.
Int J Mol Sci ; 25(10)2024 May 08.
Article in English | MEDLINE | ID: mdl-38791162

ABSTRACT

Early detection of drug-induced kidney injury is essential for drug development. In this study, multiple low-dose aristolochic acid (AA) and cisplatin (Cis) injections increased renal mRNA levels of inflammation, fibrosis, and renal tubule injury markers. We applied a serum amyloid A3 (Saa3) promoter-driven luciferase reporter (Saa3 promoter-luc mice) to these two tubulointerstitial nephritis models and performed in vivo bioluminescence imaging to monitor early renal pathologies. The bioluminescent signals from renal tissues with AA or CIS injections were stronger than those from normal kidney tissues obtained from normal mice. To verify whether the visualized bioluminescence signal was specifically generated by the injured kidney, we performed in vivo bioluminescence analysis after opening the stomachs of Saa3 promoter-luc mice, and the Saa3-mediated bioluminescent signal was specifically detected in the injured kidney. This study showed that Saa3 promoter activity is a potent non-invasive indicator for the early detection of drug-induced nephrotoxicity.


Subject(s)
Aristolochic Acids , Luciferases , Promoter Regions, Genetic , Serum Amyloid A Protein , Animals , Serum Amyloid A Protein/genetics , Serum Amyloid A Protein/metabolism , Mice , Luciferases/metabolism , Luciferases/genetics , Aristolochic Acids/toxicity , Genes, Reporter , Cisplatin/toxicity , Cisplatin/adverse effects , Luminescent Measurements/methods , Male , Kidney Diseases/chemically induced , Kidney Diseases/genetics , Kidney Diseases/metabolism , Kidney Diseases/pathology , Kidney/metabolism , Kidney/drug effects , Kidney/pathology , Disease Models, Animal , Mice, Inbred C57BL
6.
Int J Biol Macromol ; 269(Pt 1): 131864, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38692549

ABSTRACT

NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays, including gene expression analysis, protein-protein interaction, and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remain elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in living cells and in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in live cell and in vitro assays performed using lysates prepared from cells expressing the proteins and with the furimazine substrate. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity. SIGNIFICANCE: Bioluminescence has been extensively utilized in developing a variety of biological and biomedical assays. In this regard, engineering of brighter bioluminescent proteins, i.e. luciferases, has played a significant role. This is acutely exemplified by the engineering of the NLuc luciferase, which is small in size and displays much enhanced bioluminescence and thermal stability compared to previously available luciferases. While enhanced bioluminescent activity is desirable in a multitude of biological and biomedical assays, it would also be useful to develop variants of the protein that display a prolonged bioluminescence activity. This is specifically relevant in designing assays that require bioluminescence for extended periods, such as in the case of biosensors designed for monitoring slow enzymatic or cellular signaling reactions, without necessitating multiple rounds of luciferase substrate addition or any specialized reagents that result in increased assay costs. In the current manuscript, we report a mutant NLuc that possesses a stable and prolonged bioluminescence activity, albeit lower than the wild-type NLuc, and envisage a wider application of the mutant NLuc in designing biosensors for monitoring slower biological and biomedical events.


Subject(s)
Luciferases , Molecular Dynamics Simulation , Mutation , Luciferases/metabolism , Luciferases/genetics , Luciferases/chemistry , Humans , Hydrogen Bonding , Luminescent Measurements , Protein Conformation
7.
Methods Mol Biol ; 2808: 9-17, 2024.
Article in English | MEDLINE | ID: mdl-38743359

ABSTRACT

Protein-fragment complementation assays (PCAs) are powerful tools to investigate protein-protein interactions in a cellular context. These are especially useful to study unstable proteins and weak interactions that may not resist protein isolation or purification. The PCA based on the reconstitution of the Gaussia princeps luciferase (split-luc) is a sensitive approach allowing the mapping of protein-protein interactions and the semiquantitative measurement of binding affinity. Here, we describe the split-luc protocol we used to map the viral interactome of measles virus polymerase complex.


Subject(s)
Measles virus , Protein Binding , Protein Interaction Mapping , Protein Interaction Mapping/methods , Humans , Luciferases/metabolism , Luciferases/genetics , Viral Proteins/metabolism , RNA-Dependent RNA Polymerase/metabolism
8.
J Pharm Biomed Anal ; 245: 116185, 2024 Aug 01.
Article in English | MEDLINE | ID: mdl-38723556

ABSTRACT

Human epidermal growth factor receptor 2 (HER2) is a key player in the pathogenesis and progression of breast cancer and is currently a primary target for breast cancer immunotherapy. Bioactivity determination is necessary to guarantee the safety and efficacy of therapeutic antibodies targeting HER2. Nevertheless, currently available bioassays for measuring the bioactivity of anti-HER2 mAbs are either not representative or have high variability. Here, we established a reliable reporter gene assay (RGA) based on T47D-SRE-Luc cell line that expresses endogenous HER2 and luciferase controlled by serum response element (SRE) to measure the bioactivity of anti-HER2 antibodies. Neuregulin-1 (NRG-1) can lead to the heterodimerization of HER2 on the cell membrane and induce the expression of downstream SRE-controlled luciferase, while pertuzumab can dose-dependently reverse the reaction, resulting in a good dose-response curve reflecting the activity of the antibody. After optimizing the relevant assay parameters, the established RGA was fully validated based on ICH-Q2 (R1), which demonstrated that the method had excellent specificity, accuracy, precision, linearity, and stability. In summary, this robust and innovative bioactivity determination assay can be applied in the development and screening, release control, biosimilar assessment and stability studies of anti-HER2 mAbs.


Subject(s)
Antibodies, Monoclonal, Humanized , Biological Assay , Genes, Reporter , Luciferases , Neuregulin-1 , Receptor, ErbB-2 , Receptor, ErbB-2/genetics , Receptor, ErbB-2/immunology , Receptor, ErbB-2/antagonists & inhibitors , Humans , Cell Line, Tumor , Antibodies, Monoclonal, Humanized/pharmacology , Biological Assay/methods , Luciferases/genetics , Neuregulin-1/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/immunology , Breast Neoplasms/genetics , Female , Antineoplastic Agents, Immunological/pharmacology , Reproducibility of Results , Response Elements
9.
Nan Fang Yi Ke Da Xue Xue Bao ; 44(3): 578-584, 2024 Mar 20.
Article in Chinese | MEDLINE | ID: mdl-38597450

ABSTRACT

OBJECTIVE: To investigate the regulatory role of miR-26b-3p in proliferation, migration and invasion of glioma. METHODS: The expressions of miR-26b-3p and cAMP-responsive element binding protein 1 (CREB1) in gliomas of different pathological grades were detected with RT-qPCR and Western blotting. Bioinformatic methods were used to analyze the target sequence of miRNA-26b-3p binding to CREB1, and dual luciferase gene reporter experiment was performed to explore the mechanism for targeted regulation of CREB1 by miR-26b-3p. Glioma U251 cells were treated with miR-26b-3p mimic or inhibitor, and the changes in CREB1 expression and cell proliferation, migration, invasion and apoptosis were determined with Western blotting, CCK-8 assay, wound healing assay, Transwell assay, and flow cytometry. RESULTS: The expression of miR-26b-3p decreased while CREB1 expression increased significantly as the pathological grade of gliomas increased (P < 0.05). Dual luciferase gene reporter experiment confirmed that CREB1 was a downstream target of miR-26b-3p. Inhibition of miR-26b-3p significantly upregulated the expression of CERB1, suppressed apoptosis and promoted proliferation and invasion of glioma cells, and overexpression of miR-26b-3p produced the opposite effects (P < 0.05). CONCLUSION: MiR-26b-3p regulates CREB1 expression to modulate apoptosis, proliferation, migration and invasion of glioma cells, thereby participating in tumorigenesis and progression of glioma.


Subject(s)
Glioma , MicroRNAs , Humans , Apoptosis/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Gene Expression Regulation, Neoplastic , Glioma/genetics , Glioma/pathology , Luciferases/genetics , MicroRNAs/metabolism
10.
Commun Biol ; 7(1): 442, 2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38600349

ABSTRACT

Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1. To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase (Fluc) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo.


Subject(s)
Cytochrome P-450 CYP1A1 , Receptors, Aryl Hydrocarbon , Mice , Animals , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A1/metabolism , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Luciferases/genetics , Liver/metabolism , Lung/metabolism
11.
Methods Mol Biol ; 2795: 43-53, 2024.
Article in English | MEDLINE | ID: mdl-38594526

ABSTRACT

The pace of circadian rhythms remains relatively unchanged across a physiologically relevant range of temperatures, a phenomenon known as temperature compensation. Temperature compensation is a defining characteristic of circadian rhythms, ensuring that clock-regulated processes occur at approximately the same time of day across a wide range of conditions. Despite the identification of several genes involved in the regulation of temperature compensation, the molecular mechanisms underlying this process are still not well understood. High-throughput assays of circadian period are essential for the investigation of temperature compensation. In this chapter, we present a luciferase imaging-based method that enables robust and accurate examination of temperature compensation in the plant circadian clock.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Circadian Clocks , Temperature , Circadian Clocks/genetics , Arabidopsis/genetics , Circadian Rhythm/genetics , Luciferases/genetics , Arabidopsis Proteins/genetics
12.
Endocrinology ; 165(6)2024 Apr 29.
Article in English | MEDLINE | ID: mdl-38679471

ABSTRACT

The glycoprotein receptors, members of the large G protein-coupled receptor family, are characterized by a large extracellular domains responsible for binding their glycoprotein hormones. Hormone-receptor interactions are traditionally analyzed by ligand-binding assays, most often using radiolabeling but also by thermal shift assays. Despite their high sensitivity, these assays require appropriate laboratory conditions and, often, purified plasma cell membranes, which do not provide information on receptor localization or activity because the assays typically focus on measuring binding only. Here, we apply bioluminescence resonance energy transfer in living cells to determine hormone-receptor interactions between a Gaussia luciferase (Gluc)-luteinizing hormone/chorionic gonadotropin receptor (LHCGR) fusion and its ligands (human chorionic gonadotropin or LH) fused to the enhanced green fluorescent protein. The Gluc-LHCGR, as well as other Gluc-G protein-coupled receptors such as the somatostatin and the C-X-C motif chemokine receptors, is expressed on the plasma membrane, where luminescence activity is equal to membrane receptor expression, and is fully functional. The chimeric enhanced green fluorescent protein-ligands are properly secreted from cells and able to bind and activate the wild-type LHCGR as well as the Gluc-LHCGR. Finally, bioluminescence resonance energy transfer was used to determine the interactions between clinically relevant mutations of the hormones and the LHCGR that show that this bioassay provides a fast and effective, safe, and cost-efficient tool to assist the molecular characterization of mutations in either the receptor or ligand and that it is compatible with downstream cellular assays to determine receptor activation/function.


Subject(s)
Green Fluorescent Proteins , Protein Binding , Humans , Green Fluorescent Proteins/metabolism , Green Fluorescent Proteins/genetics , Receptors, LH/metabolism , Receptors, LH/genetics , Luciferases/metabolism , Luciferases/genetics , Animals , Bioluminescence Resonance Energy Transfer Techniques/methods , Chorionic Gonadotropin/metabolism , HEK293 Cells , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/genetics , Energy Transfer , Glycoproteins/metabolism , Luminescent Measurements/methods
13.
Emerg Microbes Infect ; 13(1): 2348525, 2024 Dec.
Article in English | MEDLINE | ID: mdl-38661428

ABSTRACT

To assess the clinical applicability of a semi-quantitative luciferase immunosorbent assay (LISA) for detecting antibodies against Treponema pallidum antigens TP0171 (TP15), TP0435 (TP17), and TP0574 (TP47) in diagnosing and monitoring syphilis. LISA for detection of anti-TP15, TP17, and TP47 antibodies were developed and evaluated for syphilis diagnosis using 261 serum samples (161 syphilis, 100 non-syphilis). Ninety serial serum samples from 6 syphilis rabbit models (3 treated, 3 untreated) and 110 paired serum samples from 55 syphilis patients were used to assess treatment effects by utilizing TRUST as a reference. Compared to TPPA, LISA-TP15, LISA-TP17, and LISA-TP47 showed a sensitivity of 91.9%, 96.9%, and 98.8%, specificity of 99%, 99%, and 98%, and AUC of 0.971, 0.992, and 0.995, respectively, in diagnosing syphilis. Strong correlations (rs = 0.89-0.93) with TPPA were observed. In serial serum samples from rabbit models, significant differences in the relative light unit (RLU) were observed between the treatment and control group for LISA-TP17 (days 31-51) and LISA-TP47 (day 41). In paired serum samples from syphilis patients, TRUST titres and the RLU of LISA-TP15, LISA-TP17, and LISA-TP47 decreased post-treatment (P < .001). When TRUST titres decreased by 0, 2, 4, or ≥8-folds, the RLU decreased by 17.53%, 31.34%, 48.62%, and 72.79% for LISA-TP15; 8.84%, 17.00%, 28.37%, and 50.57% for LISA-TP17; 22.25%, 29.79%, 51.75%, and 70.28% for LISA-TP47, respectively. Semi-quantitative LISA performs well for syphilis diagnosis while LISA-TP17 is more effective for monitoring syphilis treatment in rabbit models and clinical patients.


Subject(s)
Antibodies, Bacterial , Antigens, Bacterial , Sensitivity and Specificity , Syphilis , Treponema pallidum , Syphilis/diagnosis , Syphilis/microbiology , Syphilis/blood , Treponema pallidum/immunology , Animals , Humans , Rabbits , Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Male , Female , Adult , Luciferases/genetics , Syphilis Serodiagnosis/methods , Middle Aged , Disease Models, Animal , Young Adult
14.
Sci Rep ; 14(1): 9710, 2024 04 27.
Article in English | MEDLINE | ID: mdl-38678103

ABSTRACT

Among the several animal models of α-synucleinopathies, the well-known viral vector-mediated delivery of wild-type or mutated (A53T) α-synuclein requires new tools to increase the lesion in mice and follow up in vivo expression. To this end, we developed a bioluminescent expression reporter of the human A53T-α-synuclein gene using the NanoLuc system into an AAV2/9, embedded or not in a fibroin solution to stabilise its expression in space and time. We first verified the expression of the fused protein in vitro on transfected cells by bioluminescence and Western blotting. Next, two groups of C57Bl6Jr mice were unilaterally injected with the AAV-NanoLuc-human-A53T-α-synuclein above the substantia nigra combined (or not) with fibroin. We first show that the in vivo cerebral bioluminescence signal was more intense in the presence of fibroin. Using immunohistochemistry, we find that the human-A53T-α-synuclein protein is more restricted to the ipsilateral side with an overall greater magnitude of the lesion when fibroin was added. However, we also detected a bioluminescence signal in peripheral organs in both conditions, confirmed by the presence of viral DNA corresponding to the injected AAV in the liver using qPCR.


Subject(s)
Dependovirus , Fibroins , Genetic Vectors , Luminescent Measurements , Mice, Inbred C57BL , alpha-Synuclein , Animals , alpha-Synuclein/metabolism , alpha-Synuclein/genetics , Dependovirus/genetics , Humans , Mice , Luminescent Measurements/methods , Genetic Vectors/genetics , Fibroins/metabolism , Central Nervous System/metabolism , Male , Luciferases/metabolism , Luciferases/genetics
15.
J Virol Methods ; 327: 114932, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38582378

ABSTRACT

Senecavirus A (SVA) is a newly identified picornavirus associated with swine vesicular disease and neonatal mortality. The development of an SVA incorporating an exogenous reporter gene provides a powerful tool for viral research. In this study, we successfully constructed a recombinant SVA expressing Gaussia Luciferase (Gluc), termed rSVA-Gluc. The growth kinetics of rSVA-Gluc in BHK-21 cells were found to be comparable to those of the parental virus, and Gluc activity paralleled the virus growth curve. Genetic analysis revealed stable inheritance of the inserted reporter protein genes for at least six generations. We evaluated the utility of rSVA-Gluc in antiviral drug screening, and the results highlighted its potential as an effective tool for such purposes against SVA. DATA AVAILABILITY STATEMENT: The data that support the findings of this study are available on request from the corresponding author.


Subject(s)
Antiviral Agents , Genes, Reporter , Luciferases , Picornaviridae , Picornaviridae/genetics , Picornaviridae/drug effects , Animals , Antiviral Agents/pharmacology , Cell Line , Luciferases/genetics , Luciferases/metabolism , Cricetinae , Drug Evaluation, Preclinical/methods
16.
J Biol Chem ; 300(5): 107277, 2024 May.
Article in English | MEDLINE | ID: mdl-38588804

ABSTRACT

Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase, and its dysfunction is involved in the onset of cancer and neurodegenerative disorders. PP2A functions as a trimeric holoenzyme whose composition is regulated by the methyl-esterification (methylation) of the PP2A catalytic subunit (PP2Ac). Protein phosphatase methylesterase-1 (PME-1) is the sole PP2Ac methylesterase, and the higher PME-1 expression is observed in various cancer and neurodegenerative diseases. Apart from serving as a methylesterase, PME-1 acts as a PP2A inhibitory protein, binding directly to PP2Ac and suppressing its activity. The intricate function of PME-1 hinders drug development by targeting the PME-1/PP2Ac axis. This study applied the NanoBiT system, a bioluminescence-based protein interaction assay, to elucidate the molecular mechanism that modulates unknown PME-1/PP2Ac protein-protein interaction (PPI). Compound screening identified that the CHK1 inhibitors inhibited PME-1/PP2Ac association without affecting PP2Ac methylation levels. CHK1 directly phosphorylates PP2Ac to promote PME-1 association. Phospho-mass spectrometry identified multiple phospho-sites on PP2Ac, including the Thr219, that affect PME-1 interaction. An anti-phospho-Thr219 PP2Ac antibody was generated and showed that CHK1 regulates the phosphorylation levels of this site in cells. On the contrary, in vitro phosphatase assay showed that CHK1 is the substrate of PP2A, and PME-1 hindered PP2A-mediated dephosphorylation of CHK1. Our data provides novel insights into the molecular mechanisms governing the PME-1/PP2Ac PPI and the triad relationship between PP2A, PME-1, and CHK1.


Subject(s)
Carboxylic Ester Hydrolases , Checkpoint Kinase 1 , Protein Phosphatase 2 , Protein Phosphatase 2/metabolism , Protein Phosphatase 2/genetics , Humans , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/genetics , Carboxylic Ester Hydrolases/metabolism , Carboxylic Ester Hydrolases/genetics , Phosphorylation , Luciferases/metabolism , Luciferases/genetics , Protein Binding , HEK293 Cells
17.
BMC Genomics ; 25(1): 325, 2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38561670

ABSTRACT

BACKGROUND: Non-coding RNA is a key epigenetic regulation factor during skeletal muscle development and postnatal growth, and miR-542-3p was reported to be conserved and highly expressed in the skeletal muscle among different species. However, its exact functions in the proliferation of muscle stem cells and myogenesis remain to be determined. METHODS: Transfection of proliferative and differentiated C2C12 cells used miR-542-3p mimic and inhibitor. RT-qPCR, EdU staining, immunofluorescence staining, cell counting kit 8 (CCK-8), and Western blot were used to evaluate the proliferation and myogenic differentiation caused by miR-542-3p. The dual luciferase reporter analysis and rescued experiment of the target gene were used to reveal the molecular mechanism. RESULTS: The data shows overexpression of miR-542-3p downregulation of mRNA and protein levels of proliferation marker genes, reduction of EdU+ cells, and cellular vitality. Additionally, knocking it down promoted the aforementioned phenotypes. For differentiation, the miR-542-3p gain-of-function reduced both mRNA and protein levels of myogenic genes, including MYOG, MYOD1, et al. Furthermore, immunofluorescence staining immunized by MYHC antibody showed that the myotube number, fluorescence intensity, differentiation index, and myotube fusion index all decreased in the miR-542-3p mimic group, compared with the control group. Conversely, these phenotypes exhibited an increased trend in the miR-542-3p inhibitor group. Mechanistically, phosphatase and tensin homolog (Pten) was identified as the bona fide target gene of miR-542-3p by dual luciferase reporter gene assay, si-Pten combined with miR-542-3p inhibitor treatments totally rescued the promotion of proliferation by loss-function of miR-542-3p. CONCLUSIONS: This study indicates that miR-542-3p inhibits the proliferation and differentiation of myoblast and Pten is a dependent target gene of miR-542-3p in myoblast proliferation, but not in differentiation.


Subject(s)
MicroRNAs , MicroRNAs/genetics , MicroRNAs/metabolism , Epigenesis, Genetic , Cell Proliferation/genetics , Cell Differentiation/genetics , RNA, Messenger/metabolism , Muscle Development/genetics , Myoblasts , Luciferases/genetics , Luciferases/metabolism
18.
Methods Mol Biol ; 2788: 273-285, 2024.
Article in English | MEDLINE | ID: mdl-38656520

ABSTRACT

Epigenetic editing, also known as EpiEdit, offers an exciting way to control gene expression without altering the DNA sequence. In this study, we evaluate the application of EpiEdit to plant promoters, specifically the MLO (mildew locus o) gene promoter. We use a modified CRISPR-(d)Cas9 system, in which the nuclease-deficient Cas9 (dCas9) is fused to an epigenetic modifier, to experimentally demonstrate the utility of this tool for optimizing epigenetic engineering of a plant promoter prior to in vivo plant epigenome editing. Guide RNAs are used to deliver the dCas9-epigenetic modifier fusion protein to the target gene sequence, where it induces modification of MLO gene expression. We perform preliminary experiments using a plant promoter cloned into the luciferase reporter system, which is transfected into a human system and analyzed using the dual-luciferase reporter assay. The results suggest that this approach may be useful in the early stages of plant epigenome editing, as it can aid in the selection of appropriate modifications to the plant promoter prior to conducting in vivo experiments under plant system conditions. Overall, the results demonstrate the potential of CRISPR (d)Cas9-based EpiEdit for precise and controlled regulation of gene expression.


Subject(s)
CRISPR-Cas Systems , Epigenesis, Genetic , Gene Editing , Genes, Reporter , Luciferases , Promoter Regions, Genetic , Humans , Gene Editing/methods , Luciferases/genetics , Luciferases/metabolism , RNA, Guide, CRISPR-Cas Systems/genetics , HEK293 Cells
19.
Front Biosci (Elite Ed) ; 16(1): 6, 2024 Feb 27.
Article in English | MEDLINE | ID: mdl-38538524

ABSTRACT

BACKGROUND: This research explores the significance of miR-215-5p and vasculogenic mimicry (VM) in forecasting the prognosis for hepatocellular carcinoma (HCC). METHODS: We analyzed HCC-associated miRNA expression profiles using data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). Samples included tissue and blood from 80 early-stage HCC patients and serum from 120 healthy individuals. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed to measure miR-215-5p and zinc finger E-box binding homeobox 2 (ZEB2) gene expressions. Hematoxylin and eosin (H&E) and CD34/Periodic Acid-Schiff (PAS) double staining assessed VM presence in HCC tissue sections. Bioinformatics tools predicted interactions between miR-215-5p and ZEB2, confirmed through luciferase reporter assays. We also examined the impact of miR-215-5p or ZEB2 overexpression on HCC cell invasion, migration, and VM formation using scratch, Transwell invasion assays, and Matrigel 3D cultures. RESULTS: Bioinformatics analysis indicated that miR-215-5p was under-expressed in HCC, particularly in cases with vascular invasion, which correlated with worse patient outcomes. In contrast, ZEB2, targeted by miR-215-5p, was overexpressed in HCC. RT-qPCR validated these expression patterns in HCC tissues. Among the HCC patients, 38 were VM positive and 42 VM negative. Logistic regression highlighted a negative correlation between miR-215-5p levels and VM positivity in HCC tissues and a positive correlation for ZEB2 with VM positivity and tumor vascular invasion. Lower miR-215-5p levels were linked to increased HCC recurrence and metastasis. Both bioinformatics analysis and luciferase assays demonstrated a direct interaction between miR-215-5p and ZEB2. Enhancing miR-215-5p levels reduced ZEB2 expression, consequently diminishing invasion, migration, and VM formation of the HCC cells in vitro. CONCLUSIONS: miR-215-5p expression inversely correlates with VM occurrence in HCC tissues, while ZEB2 expression shows a direct correlation. By targeting ZEB2, miR-215-5p may hinder VM in HCC tissues, helping to prevent vascular invasion and HCC recurrence. Thus, miR-215-5p emerges as a vital prognostic indicator for predicting vascular invasion and recurrence in HCC.


Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , MicroRNAs , Humans , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Cell Line, Tumor , Luciferases/genetics , Luciferases/metabolism , Cell Proliferation , Gene Expression Regulation, Neoplastic , Cell Movement/genetics
20.
Plant Physiol Biochem ; 209: 108544, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38520965

ABSTRACT

Flower abscission is an important developmental process that can significantly reduce the yield of horticultural plants. We previously reported that SmMYB113 is a key transcription factor promoting anthocyanin biosynthesis and improve fruit quality. However, the overexpression of SmMYB113 in eggplant increased flower drop rate and reduced fruit yield. Here, we elucidate the regulatory mechanisms of SmMYB113 on flower abscission in eggplant. RNA-seq analysis indicated that the regulation of flower abscission by SmMYB113 was associated with altered expression of genes related to ethylene biosynthesis and signal transduction, including ethylene biosynthetic genes SmACS1, SmACS8 and SmACO4. Then, the ethylene content in flowers and the function of ethephon (ETH, which promotes fruit ripening) and 1-Methylcyclopropene (1-MCP, which acts as an ethylene perception inhibitor) were analyzed, which revealed that SmMYB113 directly regulates ethylene-dependent flower abscission. Yeast one-hybrid and dual-luciferase assays revealed that SmMYB113 could directly bind to the promoters of SmACS1, SmACS8, and SmACO4 to activate their expression. Through construction of a yeast two-hybrid (Y2H) screening library, the protein SmERF38 was found to interact with SmMYB113, and verified by Y2H, bimolecular fluorescence complementation (BiFC), and luciferase complementation assay. Furthermore, dual-luciferase assays showed that SmERF38 enhanced the role of SmMYB113 on the promoters of SmACS1. Our results provided new insight into the molecular mechanism of flower abscission in eggplant.


Subject(s)
Solanum melongena , Transcription Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Solanum melongena/genetics , Solanum melongena/metabolism , Saccharomyces cerevisiae/metabolism , Gene Expression Regulation, Plant , Ethylenes/metabolism , Flowers/metabolism , Luciferases/genetics , Luciferases/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism
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