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1.
Sensors (Basel) ; 21(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572727

RESUMO

In recent years, there has been an increasing demand for predictive and sensitive in vitro tools for drug discovery. Split complementation assays have the potential to enlarge the arsenal of in vitro tools for compound screening, with most of them relying on well-established reporter gene assays. In particular, ligand-induced complementation of split luciferases is emerging as a suitable approach for monitoring protein-protein interactions. We hereby report an intracellular nanosensor for the screening of compounds with androgenic activity based on a split NanoLuc reporter. We also confirm the suitability of using 3D spheroids of Human Embryonic Kidney (HEK-293) cells for upgrading the 2D cell-based assay. A limit of detection of 4 pM and a half maximal effective concentration (EC50) of 1.7 ± 0.3 nM were obtained for testosterone with HEK293 spheroids. This genetically encoded nanosensor also represents a new tool for real time imaging of the activation state of the androgen receptor, thus being suitable for analysing molecules with androgenic activity, including new drugs or endocrine disrupting molecules.


Assuntos
Androgênios , Medições Luminescentes , Nanotecnologia , Receptores Androgênicos , Genes Reporter , Células HEK293 , Humanos , Luciferases/genética , Receptores Androgênicos/genética
2.
Virology ; 556: 73-78, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33548599

RESUMO

The need to stem the current outbreak of SARS-CoV-2 responsible for COVID-19 is driving the search for inhibitors that will block coronavirus replication and pathogenesis. The coronavirus 3C-like protease (3CLpro) encoded in the replicase polyprotein is an attractive target for antiviral drug development because protease activity is required for generating a functional replication complex. Reagents that can be used to screen for protease inhibitors and for identifying the replicase products of SARS-CoV-2 are urgently needed. Here we describe a luminescence-based biosensor assay for evaluating small molecule inhibitors of SARS-CoV-2 3CLpro/main protease. We also document that a polyclonal rabbit antiserum developed against SARS-CoV 3CLpro cross reacts with the highly conserved 3CLpro of SARS-CoV-2. These reagents will facilitate the pre-clinical evaluation of SARS-CoV-2 protease inhibitors.


Assuntos
Técnicas Biossensoriais/métodos , Soros Imunes/imunologia , Luciferases/metabolismo , /metabolismo , Animais , Antivirais/farmacologia , /genética , Reações Cruzadas , Luciferases/genética , Inibidores de Proteases/farmacologia , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus da SARS/imunologia , Vírus da SARS/metabolismo , /genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
3.
Int J Mol Sci ; 22(2)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445497

RESUMO

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen-NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.


Assuntos
Técnicas Biossensoriais , Espaço Intracelular/metabolismo , Medições Luminescentes/métodos , Nanotecnologia , Concentração Osmolar , Genes Reporter , Células HEK293 , Humanos , Luciferases/química , Luciferases/genética , Luciferases/metabolismo , Proteínas Recombinantes , Relação Estrutura-Atividade
4.
Adv Exp Med Biol ; 1293: 281-293, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33398820

RESUMO

In this chapter, we introduce a relatively new, emerging method for molecular neuromodulation-bioluminescence-optogenetics. Bioluminescence-optogenetics is mediated by luminopsin fusion proteins-light-sensing opsins fused to light-emitting luciferases. We describe their structures and working mechanisms and discuss their unique benefits over conventional optogenetics and chemogenetics. We also summarize applications of bioluminescence-optogenetics in various neurological disease models in rodents.


Assuntos
Medições Luminescentes/métodos , Optogenética/métodos , Luciferases/genética , Medições Luminescentes/tendências , Optogenética/tendências
5.
Viruses ; 13(2)2021 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-33498923

RESUMO

The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.


Assuntos
Antivirais/metabolismo , Luciferases/metabolismo , Inibidores de Proteases/metabolismo , /enzimologia , Antivirais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , /metabolismo , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Lentivirus/genética , Luciferases/genética , Inibidores de Proteases/farmacologia
6.
Methods Mol Biol ; 2130: 287-294, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33284452

RESUMO

Circadian rhythms in cellular function can be monitored in real time with bioluminescence imaging. In this approach, bioluminescence is produced by an enzymatic reaction, which can be used to report dynamic changes in gene or protein expression in living cells. Bioluminescence imaging in circadian experiments typically uses an ex vivo slice preparation, with the most commonly studied structure being the master clock in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Here we describe procedures for dissecting and collecting SCN slices for bioluminescence imaging experiments.


Assuntos
Relógios Circadianos , Conectoma/métodos , Núcleo Supraquiasmático/fisiologia , Animais , Genes Reporter , Luciferases/genética , Luciferases/metabolismo , Camundongos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Núcleo Supraquiasmático/metabolismo
7.
Methods Mol Biol ; 2130: 295-302, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33284453

RESUMO

Advances in imaging technology, combined with the development of bioluminescent reporters for core clock genes, has enabled the observation of spatiotemporal patterns of circadian rhythms in the suprachiasmatic nuclei (SCN). In particular, the PERIOD2::luciferase (PER2::LUC) knockin mouse has led to novel approaches for studying the heterogeneous circadian network in the SCN. This chapter describes how to automate the processing of PER2::LUC imaging data from SCN slices for generating spatiotemporal maps of circadian parameters like phase, period, and amplitude. These maps can be aligned and averaged to produce composite maps displaying common features across multiple slices. In addition, we describe a method for automated detection of cell-like regions of interest, to support the study of the neural network in the SCN.


Assuntos
Encéfalo/fisiologia , Ritmo Circadiano , Conectoma/métodos , Processamento de Imagem Assistida por Computador/métodos , Animais , Encéfalo/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
J Vis Exp ; (166)2020 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-33346188

RESUMO

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes , Genes Reporter , Luciferases/metabolismo , Mamíferos/metabolismo , Plasmídeos/genética , Animais , Sequência de Bases , Linhagem Celular , DNA/metabolismo , Reparo do DNA , Vetores Genéticos/metabolismo , Luciferases/genética , Oligonucleotídeos/metabolismo , RNA Guia/genética , Reprodutibilidade dos Testes , Ovinos , Transformação Genética
9.
Biochem Biophys Res Commun ; 533(4): 1477-1483, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33333713

RESUMO

Development of the mammalian central nervous system is an important process, which is accomplished through precise regulations of many different genes. Zinc finger protein 179 (Znf179) is one of the essential genes that plays a critical role in neuronal differentiation. In our previous study, Znf179 knockout mice displayed brain malformation and impaired brain functions. We have also previously shown that Znf179 involves in cell cycle regulation, but the regulatory mechanism of Znf179 expression is not yet fully characterized. Herein, we identified that Purα is an essential factor for the promotor activity of Znf179. We also showed concurrent expression of Znf179 and Purα during neuronal differentiation. We also found that overexpression of Purα increased Znf179 expression in neuronal differentiated P19 cells. Through its direct binding to Znf179, as shown using DAPA, Purα upregulates Znf179 expression, suggesting that Purα is important for the regulation of Znf179 expression during neuronal differentiation. Our data indicated that Purα is involved in the transcriptional regulation of Znf179 gene during neuronal differentiation, and is indispensable during the brain development.


Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Animais , Proteínas de Ligação a DNA/metabolismo , Luciferases/genética , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Regiões Promotoras Genéticas , Transcrição Genética
10.
PLoS One ; 15(12): e0244386, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33347482

RESUMO

CpG-free pDNA was reported to facilitate sustained transgene expression with minimal inflammation in vivo as compared to CpG-containing pDNA. However, the expression potential and impact of CpG-free pDNA in in vitro model have never been described. Hence, in this study, we analyzed the transgene expression profiles of CpG-free pDNA in vitro to determine the influence of CpG depletion from the transgene. We found that in contrast to the published in vivo studies, CpG-free pDNA expressed a significantly lower level of luciferase than CpG-rich pDNA in several human cell lines. By comparing novel CpG-free pDNA carrying CpG-free GFP (pZGFP: 0 CpG) to CpG-rich GFP (pRGFP: 60 CpGs), we further showed that the discrepancy was not influenced by external factors such as gene transfer agent, cell species, cell type, and cytotoxicity. Moreover, pZGFP exhibited reduced expression despite having equal gene dosage as pRGFP. Analysis of mRNA distribution revealed that the mRNA export of pZGFP and pRGFP was similar; however, the steady state mRNA level of pZGFP was significantly lower. Upon further investigation, we found that the CpG-free transgene in non-integrating CpG-free pDNA backbone acquired increased nucleosome enrichment as compared with CpG-rich transgene, which may explain the observed reduced level of steady state mRNA. Our findings suggest that nucleosome enrichment could regulate non-integrating CpG-free pDNA expression and has implications on pDNA design.


Assuntos
Ilhas de CpG , Nucleossomos/genética , Plasmídeos/genética , Transgenes , Animais , Linhagem Celular , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Luciferases/genética , Células MCF-7 , Camundongos , Células NIH 3T3 , Especificidade da Espécie , Transfecção
11.
Int J Mol Sci ; 22(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374567

RESUMO

NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies.


Assuntos
Luciferases/química , Fenômenos Mecânicos , Sequência de Aminoácidos , Sequência de Bases , Engenharia Genética , Humanos , Luciferases/genética , Luciferases/isolamento & purificação , Luminescência , Medições Luminescentes , Microscopia de Força Atômica , Conformação Proteica , Dobramento de Proteína , Redobramento de Proteína , Estabilidade Proteica , Desdobramento de Proteína , Relação Estrutura-Atividade
12.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(10): 918-923, 2020 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-33148387

RESUMO

Objective To establish a triple negative breast cancer cell line stably expressing human epidermal growth factor receptor (EGFR) promoter and luciferase (Luc) reporter gene and to preliminarily verify its application. Methods Using genetic recombination technology, the lentiviral vector carrying Luc reporter and EGFR promoter sequence was designed and constructed to infect MDA-MB231 cells and obtain MDA-MB231-EGFR-Luc2 cell lines by the selection with puromycin. The Luc luminescence value after stimulating with EGFR activator EGF or inhibitor gefitinib regulating the EGFR promoter activities was detected. Results Gene sequencing and enzyme digestion verified that the lentiviral expression vector carrying Luc reporter vector recombined with EGFR promoter was successfully constructed. Lentivirus-infected MDA-MB231 cells were screened by puromycin, the MDA-MB231-EGFR-Luc2 cells stably expressing firefly Luc was obtained. EGF increased the Luc luminescence value of MDA-MB231-EGFR-Luc2 cells in a dose-dependent manner, while gefitinib did the opposite. Conclusion The cell line of MDA-MB231-EGFR-Luc2 containing EGFR promoter and Luc reporter gene has been successfully constructed, which provides a new cell model for high throughput screening of EGFR-targeting drugs.


Assuntos
Genes Reporter , Regiões Promotoras Genéticas , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Humanos , Luciferases/genética
13.
Nat Commun ; 11(1): 5214, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060595

RESUMO

A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC50 0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Ensaios de Triagem em Larga Escala/métodos , Testes de Neutralização/métodos , Pneumonia Viral/diagnóstico , Células A549 , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/genética , Betacoronavirus/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Humanos , Luciferases/genética , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
14.
Sci Rep ; 10(1): 16615, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024203

RESUMO

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.


Assuntos
Anticorpos Antivirais/análise , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Infecções por Coronavirus/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/virologia , Genes Reporter , Células HEK293 , Humanos , Células Jurkat , Luciferases/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Fatores de Transcrição NFATC/genética , Receptores de IgG/genética , Receptores de IgG/imunologia , Elementos de Resposta , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção
15.
PLoS One ; 15(9): e0238153, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32998160

RESUMO

Bacillus cereus is a foodborne pathogen causing emesis and diarrhea in those affected. It is assumed that the non-hemolytic enterotoxin (Nhe) plays a key role in B. cereus induced diarrhea. The ability to trace Nhe activity is important for food safety. While assays such as PCR and ELISA exist to detect Nhe, those methods cannot differentiate between active and inactive forms of Nhe. The existing rabbit ileal loop bioassay used to detect Nhe activity is ethically disfavored because it uses live experimental animals. Here we present a custom built low-cost CCD based luminometer and applied it in conjunction with a cell-based assay using Vero cells transduced to express the luciferase enzyme. The activity of Nhe was measured as its ability to inhibit synthesis of luciferase as quantified by reduction of light emission by the luciferase reaction. Emitted light intensity was observed to be inversely proportional to Nhe concentration over a range of 7 ng/ml to 125 ng/ml, with a limit of detection of 7 ng/ml Nhe.


Assuntos
Enterotoxinas/metabolismo , Medições Luminescentes , Animais , Biocatálise , Chlorocebus aethiops , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Células Vero
16.
Int J Nanomedicine ; 15: 6689-6703, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32982227

RESUMO

Purpose: Nucleic acid-based therapies are a promising therapeutic tool. The major obstacle in their clinical translation is their efficient delivery to the desired tissue. We developed a novel nanosized delivery system composed of conjugates of α-tocopherol, polyethyleneimine, and polyethylene glycol (TPP) to deliver nucleic acids. Methods: We synthesized a panel of TPP molecules using different molecular weights of PEG and PEI and analyzed with various analytical approaches. The optimized version of TPP (TPP111 - the 1:1:1 molecular ratio) was self-assembled in water to produce nanostructures and then evaluated in diversified in vitro and in vivo studies. Results: Through a panel of synthesized molecules, TPP111 conjugate components self-assembled in water, forming globular shaped nanostructures of ~90 nm, with high nucleic acid entrapment efficiency. The polymer had low cytotoxicity in vitro and protected nucleic acids from nucleases. Using a luciferase-expressing plasmid, TPP111-plasmid nano-complexes were rapidly up-taken by cancer cells in vitro and induced strong transfection, comparable to PEI. Colocalization of the nano-complexes and endosomes/lysosomes suggested an endosome-mediated uptake. Using a subcutaneous tumor model, intravenously injected nano-complexes preferentially accumulated to the tumor area over 24 h. Conclusion: These results indicate that we successfully synthesized the TPP111 nanocarrier system, which can deliver nucleic acids in vitro and in vivo and merits further evaluation.


Assuntos
Nanoestruturas/administração & dosagem , Ácidos Nucleicos/administração & dosagem , Polietilenoglicóis/química , Polietilenoimina/química , alfa-Tocoferol/química , Células A549 , Animais , Sistemas de Liberação de Medicamentos/métodos , Endossomos/efeitos dos fármacos , Feminino , Técnicas de Transferência de Genes , Humanos , Luciferases/genética , Camundongos Nus , Peso Molecular , Nanoestruturas/química , Nanoestruturas/toxicidade , Plasmídeos/administração & dosagem , Plasmídeos/genética , Plasmídeos/farmacocinética , Polímeros/síntese química , Distribuição Tecidual , Transfecção/métodos , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Virology ; 548: 226-235, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32771769

RESUMO

Bovine leukemia virus (BLV) is a global problem that results in significant economic losses to the livestock industry. We developed three virus strains by inserting the HiBiT reporter tag from NanoLuc luciferase (NLuc) into limited sites within BLV molecular clones. Initial analysis for site selection of the tag insertion revealed a permissible site immediately downstream of the viral envelope gene. Therefore, NLuc activity could be used to measure virus copy numbers in the supernatant and the levels of cell infection. Productivity and growth kinetics of the reporter virus were similar to those of the wild-type strain; therefore, the reporter virus can be used to characterize the replication of chimeric viruses as well as responses to the antiviral drug, amprenavir. Collectively, our results suggest that the BLV reporter virus with a HiBiT tag insertion is a highly versatile system for various purposes such as evaluating virus replication and antiviral drugs.


Assuntos
Vírus da Leucemia Bovina/genética , Animais , Antivirais/farmacologia , Genes Reporter , Vírus da Leucemia Bovina/efeitos dos fármacos , Vírus da Leucemia Bovina/crescimento & desenvolvimento , Vírus da Leucemia Bovina/fisiologia , Luciferases/análise , Luciferases/genética , Luciferases/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos
18.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(9): 987-990, 2020 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-32820513

RESUMO

OBJECTIVE: To analyze the action of miRNA-326 on its target gene BCL-XL and the molecular mechanism of platelet apoptosis regulated by miRNAs. METHODS: Dual-luciferase vectors containing respectively the wild-type and mutant 3'-untranslated region (3'UTR) fragments of the BCL-XL gene were constructed with firefly and renilla luciferases and transfected into 293T cells. Relative fluorescence intensities of the transfected cells were measured. RESULTS: Dual-luciferase reporter gene vectors for PsiCHECK- BCL-XL -3'UTR-WT (wild-type) and PsiCHECK- BCL-XL -3' UTR-MT (variant) were respectively constructed. Relative fluorescence intensities of the 293T cells co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3'UTR-WT plasmid were significantly lower compared with the control group (co-transfected by a miRNA-326 negative sequence and PsiCHECK- BCL-XL -3' UTR-WT plasmid) ( P = 0.034). The relative fluorescence intensity was also significantly reduced in cells co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3' UTR-WT plasmid compared with the mutant control group co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3'UTR-MT plasmid (P = 0.022). CONCLUSION: miRNA-326 may participate in the regulation of platelet apoptosis by acting on the 3'-UTR of the BCL-XL gene.


Assuntos
Regiões 3' não Traduzidas , Apoptose , Plaquetas/citologia , MicroRNAs/genética , Proteína bcl-X/genética , Genes Reporter , Células HEK293 , Humanos , Luciferases/genética
19.
Nucleic Acids Res ; 48(17): e100, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32797168

RESUMO

Tracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR (R2 = 0.9722) and NHEJ (R2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo. Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1-10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.


Assuntos
Genes Reporter , Luciferases/genética , Reparo de DNA por Recombinação , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Copépodes/enzimologia , Reparo do DNA por Junção de Extremidades , Feminino , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Luciferases/metabolismo , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase Multiplex/métodos , Imagem Óptica/métodos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Análise de Sequência de DNA/métodos
20.
PLoS Biol ; 18(8): e3000792, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745129

RESUMO

A ubiquitous feature of the circadian clock across life forms is its organization as a network of cellular oscillators, with individual cellular oscillators within the network often exhibiting considerable heterogeneity in their intrinsic periods. The interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clock's entrainability, but our knowledge of mechanisms governing period heterogeneity within circadian clock networks remains largely elusive. In this study, we aimed to explore the principles that underlie intercellular period variation in circadian clock networks (clonal period heterogeneity). To this end, we employed a laboratory selection approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging from 22 to 28 h. We report that a single parent clone can produce progeny clones with a wide distribution of circadian periods, and this heterogeneity, in addition to being stochastically driven, has a heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our clone panel, we found that inheritance of expression patterns in at least three clock genes might govern clonal period heterogeneity in circadian clock networks. Furthermore, we provide evidence suggesting that heritable epigenetic variation in gene expression regulation might underlie period heterogeneity.


Assuntos
Proteínas CLOCK/genética , Relógios Circadianos/genética , Ritmo Circadiano/genética , Epigênese Genética , Redes Reguladoras de Genes , Animais , Proteínas CLOCK/metabolismo , Linhagem Celular Tumoral , Células Clonais , Perfilação da Expressão Gênica , Genes Reporter , Heterogeneidade Genética , Humanos , Padrões de Herança , Luciferases/genética , Luciferases/metabolismo , Camundongos , Células NIH 3T3 , Osteoblastos/citologia , Osteoblastos/metabolismo , Processos Estocásticos
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