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1.
Int J Mol Sci ; 22(21)2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34769072

RESUMO

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Interferon-alfa/farmacologia , RNA Viral/metabolismo , SARS-CoV-2/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Exorribonucleases/genética , Vetores Genéticos , Células HeLa , Humanos , Interferon-alfa/administração & dosagem , Luciferases/genética , Luciferases/metabolismo , Naftiridinas/administração & dosagem , Naftiridinas/farmacologia , Oxidiazóis/administração & dosagem , Oxidiazóis/farmacologia , RNA Viral/efeitos dos fármacos , Replicon
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(5): 1429-1435, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34627421

RESUMO

OBJECTIVE: To establish the in vivo traceable acute myeloid leukemia mice model with Luciferase-Expressing KG1a Cells. METHODS: KG1a cells with stable luciferase gene expression (called as KG1a-Luc cells) were constructed by lentivirus transfection, then sifted out by puromycin. Eighteen male NOD-SCID-IL2rg-/-mice aged 8 to 12 weeks were randomly and equally divided into two groups: the control group and the KG1a-Luc group. The mice in KG1a-Luc group were injected with 200 µl PBS containing 5×106 KG1a-Luc cells through tail veins, and the mice in control group were injected with 200 µl PBS only. The bioluminescence imaging technology was used to monitor the tumor burden in vivo. The peripheral blood of the mice in both groups was analyzed by flow cytometry. After the mice were sacrificed, there were pathologic evaluations: bone marrow and spleens made into smears, and livers sliced to get paraffin sections. The survival time of the mice in the two groups was recorded and compared. RESULTS: KG1a cells expressing luciferase stably were successfully obtained. The tumor luminescence wildly spread at day 17 captured by in vivo imaging. The KG1a-Luc tumor cells could be detected in the peripheral blood of the mice, with the average percentage of (16.27±6.66)%. The morphology and pathology result showed that KG1a-Luc cells infiltrate was detected in bone marrow, spleens and livers. The survival time of the KG1a-Luc mice was notably shorter as compared with those in the control group, the median survival time was 30.5 days (95%CI: 0.008-0.260). CONCLUSION: The acute myeloid leukemia NOD-SCID-IL2rg-/-mouse model was successfully established by tail vein injection of 5×106 KG1a-Luc cells.


Assuntos
Leucemia Mieloide Aguda , Animais , Modelos Animais de Doenças , Subunidade gama Comum de Receptores de Interleucina , Luciferases/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
3.
Anal Chim Acta ; 1183: 338956, 2021 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-34627516

RESUMO

For the first time, a human cancer cell line was shown to grow and be functionally active on the particulate porous adsorbent surface of separated sample mixtures. This allowed the novel combination of chromatographic separations with human cells as biological detector. As exemplary screening for cancer treatment drugs, cytotoxic substances were directly discovered in Saussurea costus and ginseng samples using the Cytotox CALUX® osteosarcoma cells (with luciferase expressing reporter gene) as detector. In addition, rosiglitazone and pioglitazone were detected as luminescent zones upon binding to the PPARγ receptor expressed in the respective CALUX cell line that was grown on the surface of the adsorbent. This demonstrates the ability to address receptor-mediated signaling with this method, and opens the perspective to use our novel bioimaging method to identify bioactive molecules targeting a wide range of pathways with toxicological, pharmaceutical and nutraceutical relevance. The new bioimaging directly pointed to individual effective compounds in multi-component mixtures. Furthermore, discovered effective compounds were directly characterized by online elution to high-resolution mass spectrometry and fragmentation.


Assuntos
Cromatografia , Linhagem Celular , Genes Reporter , Humanos , Luciferases , Espectrometria de Massas
4.
Mol Biol (Mosk) ; 55(5): 846-857, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34671006

RESUMO

DNA methylation is an essential epigenetic modification involved in numerous biological processes. Here, we present a cell-based system pLTR-Luc2P-EGFP for evaluation of DNA methylation in mammalian cells. In this system, the expression of reporter gene luciferase2P (Luc2P)-EGFP is under the control of HIV-1 promoter 5' long terminal repeat (LTR), which contains multiple CpG sites. Once these sites are methylated, the expression of Luc2P-EGFP is turned off, which may be visualized under fluorescence microscopy, with quantification performed in luciferase activity assay. As a proof of principle, pLTR-Luc2P-EGFP was methylated in vitro, and transfected into 293T cells, where the reduction of Luc2P-EGFP expression was confirmed. Premixed reporter DNA samples with the methylation levels varying from 0 to 100% were used for quantitative measurements of DNA methylation. The resulting standard curves indicated the accuracy of luciferase activity exceeding that of the Western blotting against EGFP. The Bland-Altman analysis showed that data from luciferase activity assay were in good agreement with the actual DNA methylation levels. In summary, we have established a reporter system coupled with reliable detection technique capable of efficient quantifying the changes in methylation in mammalian cells. This system may be utilized as a high throughput screening tool for identifying molecules that modulate DNA methylation.


Assuntos
Metilação de DNA , Epigênese Genética , Animais , Genes Reporter , Luciferases/genética , Regiões Promotoras Genéticas
5.
BMC Cancer ; 21(1): 1090, 2021 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-34627193

RESUMO

BACKGROUND: Glioblastoma (GBM) is characterized by progressive growth and metastasis. Numerous studies claim that the deregulation of circular RNAs (circRNAs) is associated with cancer progression. However, the role of circRNAs in GBM is largely limited. The purpose of this study was to investigate the functions of circCDC45 in GBM and provide a feasible functional mechanism to support its role. METHODS: The expression of circCDC45, miR-485-5p and colony-stimulating factor 1 (CSF-1) mRNA was examined using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed using cell counting kit - 8 (CCK-8) assay and colony formation assay. Cell migration and cell invasion were monitored using transwell assay. The protein levels of proliferation-related markers and CSF-1 were determined using western blot. The target relationship was predicted using bioinformatics tools and validated using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Animal models were constructed to verify the role of circCDC45 in vivo. RESULTS: The expression of circCDC45 and CSF-1 was elevated in GBM tissues and cells, while the expression of miR-485-5p was declined. Downregulation of circCDC45 or CSF-1 blocked GBM cell proliferation, invasion and migration as well as tumor growth in vivo. In mechanism, circCDC45 positively regulated the expression of CSF-1 by targeting miR-485-5p. Inhibition of miR-485-5p reversed the biological effects caused by circCDC45 downregulation in GBM cells. CONCLUSION: CircCDC45 promoted the progression of GBM by mediating the miR-485-5p/CSF-1 axis, and circCDC45 might be a promising plasmatic biomarker for GBM diagnosis and treatment.


Assuntos
Neoplasias Encefálicas/metabolismo , Proteínas de Ciclo Celular/fisiologia , Glioblastoma/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , MicroRNAs/metabolismo , RNA Circular/metabolismo , Animais , Neoplasias Encefálicas/patologia , Contagem de Células/métodos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação para Baixo , Inativação Gênica , Glioblastoma/patologia , Humanos , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Modelos Animais , Invasividade Neoplásica , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ensaio Tumoral de Célula-Tronco
6.
J Vis Exp ; (175)2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34633392

RESUMO

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient's breast cancer brain metastatic growth within the brain microenvironment.


Assuntos
Neoplasias Encefálicas , Fenômenos Fisiológicos do Sistema Nervoso , Animais , Encéfalo , Feminino , Luciferases , Camundongos , Camundongos Nus , Microambiente Tumoral
7.
BMC Cancer ; 21(1): 1074, 2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34598688

RESUMO

BACKGROUND: The human miR-17-92 polycistron is the first reported and most well-studied onco-miRNA with a cluster of seven miRNAs. miR-17-5p, a member of the miR-17-92 family, plays an important role in tumor cell proliferation, apoptosis, migration and invasion. However, few studies have shown the role of miR-17-5p in the cell cycle of head and neck squamous cell carcinoma (HNSCC). METHODS: RT-qPCR was used to detect miR-17-5p expression levels in 64 HNSCC tissues and 5 cell lines. The relationship between the expression of miR-17-5p in the tissues and the clinical characteristics of the patients was analyzed. HNSCC cells were transfected with an miR-17-5p mimic or inhibitor to evaluate cell cycle distribution by flow cytometry. Cell cycle distribution of cells transfected with target gene was evaluated using flow cytometry. Dual-luciferase reporter assay was used to detect the regulatory effect of miR-17-5p on target gene expression. RESULTS: In the present study, we found that miR-17-5p expression in HNSCC tissues and cell lines was remarkably increased, and miR-17-5p is related to recurrence in HNSCC patients. Silencing miR-17-5p blocked HNSCC cells in G2/M phase, whereas its overexpression propelled cell cycle progression. More importantly, we verified that miR-17-5p negatively regulated CCNG2 mRNA and protein expression by directly targeting its 3'UTR. CONCLUSION: These findings suggest that miR-17-5p might act as a tumor promoter and prognostic factor for recurrence in HNSCC patients.


Assuntos
Ciclina G2/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Neoplasias de Cabeça e Pescoço/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , MicroRNAs/metabolismo , Recidiva Local de Neoplasia/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Regiões 3' não Traduzidas/genética , Apoptose/genética , Área Sob a Curva , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Ciclina G2/genética , Regulação para Baixo , Feminino , Inativação Gênica , Neoplasias de Cabeça e Pescoço/genética , Humanos , Luciferases/metabolismo , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , RNA Mensageiro/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Transfecção , Regulação para Cima
8.
Viruses ; 13(10)2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34696421

RESUMO

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.


Assuntos
Infecções por Coronavirus/veterinária , Deltacoronavirus/genética , Deltacoronavirus/metabolismo , Genes Reporter/genética , Luciferases/genética , Células A549 , Animais , Linhagem Celular , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/genética , Infecções por Coronavirus/patologia , Deltacoronavirus/crescimento & desenvolvimento , Cães , Genoma Viral/genética , Humanos , Luciferases/biossíntese , Células Madin Darby de Rim Canino , Nanoestruturas , Suínos , Doenças dos Suínos/virologia , Células Vero , Replicação Viral/genética
9.
Sci Rep ; 11(1): 18428, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531417

RESUMO

Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/análise , COVID-19/prevenção & controle , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Anticorpos Antivirais/análise , Brasil , COVID-19/imunologia , Humanos , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Mutação , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Estados Unidos , Vacinação
10.
Analyst ; 146(20): 6139-6144, 2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34486602

RESUMO

Serum copper levels are biomarkers for copper-related diseases. Quantification of levels of free copper (not bound to proteins) in serum is important for diagnosing Wilson's disease, in which the free copper concentration is elevated. Bioluminescence is commonly used in point-of-care diagnostics, but these assays require genetically engineered luciferase. Here, we developed a luciferase-independent copper detection platform. A luminogenic caged coelenterazine analogue (TPA-H1) was designed and synthesized to detect copper ions in human serum. TPA-H1 was developed by introducing a tris[(2-pyridyl)-methyl]amine (TPA) ligand, which is a Cu+ cleavable caging group, to the carbonyl group at the C-3 position of the imidazopyrazinone scaffold. The luciferin, named HuLumino1, is the product of the cleavage reaction of TPA-H1 with a copper ion and displays "turn-on" bioluminescence signals specifically with human serum albumin, which can be used to quantitatively analyse copper ions. TPA-H1 exhibited a fast cleavage of the protective group, high specificity, and high sensitivity for copper over other metal ions. This novel caged coelenterazine derivative, TPA-H1, can detect free copper ions in serum in a simple "mix-and-read" manner.


Assuntos
Cobre , Imidazóis , Humanos , Luciferases , Pirazinas
11.
PLoS Pathog ; 17(9): e1009840, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34499689

RESUMO

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, therapeutics that can help manage the disease are still required until immunity has been achieved globally. The identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2-ΔOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in-line with reported proteinuria and liver damage in patients with COVID-19. Using the nano-luciferase as a measure of virus replication we identified 35 drugs that reduced replication in Vero cells and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.


Assuntos
COVID-19/tratamento farmacológico , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Animais , Biomarcadores , Linhagem Celular , Chlorocebus aethiops , Hepatócitos/virologia , Humanos , Luciferases/farmacologia , Nanoestruturas , SARS-CoV-2/genética , Células Vero , Replicação Viral/efeitos dos fármacos
12.
Chem Commun (Camb) ; 57(77): 9906-9909, 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34494618

RESUMO

We report a method for detecting ligand-protein interactions occurring within cells using short peptide reactive tags appended to ligands and proteins, along with a split NanoLuc luciferase. This method can be applied to estimate the binding affinities of ligand-protein interactions and to detect the interactions of proteins with unstable synthetic ligands inside the cells.


Assuntos
Luciferases/química , Peptídeos/química , Linhagem Celular Tumoral , Humanos , Ligantes , Luciferases/metabolismo , Estrutura Molecular
13.
Acta Biomater ; 135: 225-233, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34496282

RESUMO

Over 30,000 protein-protein interactions with pathological implications have been identified; yet, discovering and investigating drugs that target these specific interactions is greatly limited by the inability to monitor native protein-protein interactions (PPIs) efficiently. The two most frequently used tools to monitor PPIs, resonance-energy transfer (RET) assays and protein complementation assays (PCA), face significant limitations. RET assays have a narrow working range of 10 to 50 Å, while PCA require permanent attachment of a reporter probe to a protein of interest by chemical conjugation or genetic engineering. We developed a non-invasive assay platform to measure PPIs without modifications to the proteins of interest and is functional at a greater working range than RET assays. We demonstrate our approach by monitoring the EGFR-HER2 heterodimerization on relevant cell surfaces, utilizing various EGFR- and HER2-specific binders (e.g., Fab, DARPin, and VHH) fused with small fragments of a tri-part split-luciferase derived from NanoLuc®. Following independent binding of the binder fusions to their respective targets, the dimerization of EGFR and HER2 induces complementation of the luciferase fragments into a functional native structure, producing glow-type luminescence. We have confirmed the functionality of the platform to monitor EGFR-HER2 dimerization induction and inhibition. STATEMENT OF SIGNIFICANCE: We describe a platform technology for rapid monitoring of protein-protein interactions (PPIs). Our approach is uses a luciferase split into three parts - two short peptide "tags" and a large third fragment. Each of the short peptides can be fused to antibodies which bind to domains of a target antigens which orients the two tags and facilitates refolding of an active enzyme. To our knowledge this is the first example of a split-enzyme used to monitor PPIs without requiring any modification of the target proteins. We demonstrate our approach on the important PPI of HER2 and EGFR. Significantly, we quantify stimulation and inhibition of these partners, opening the possibility of using our approach to assess potential drugs without engineering cells.


Assuntos
Anticorpos , Receptores ErbB , Dimerização , Receptores ErbB/metabolismo , Técnicas Imunoenzimáticas , Luciferases
14.
Int J Mol Sci ; 22(18)2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34576210

RESUMO

G protein-coupled receptors (GPCRs) are targets of extracellular stimuli and hence occupy a key position in drug discovery. By specific and not yet fully elucidated coupling profiles with α subunits of distinct G protein families, they regulate cellular responses. The histamine H2 and H4 receptors (H2R and H4R) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the H2R and H4R, respectively, the molecular mechanism of which remained unclear. Using a combination of cellular experimental assays and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigated the coupling profiles of the H2R and H4R to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the H2R and H4R from the mini-G protein recruitment assays using HEK293T cells. Compared to H2R-mGs expressing cells, histamine responses were weaker (pEC50, Emax) for H2R-mGsi and -mGsq. By contrast, the H4R selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of H2R to mGs and H4R to mGsi revealed by the structural flexibility and free energy landscapes of the complexes. Although the mG α5 helices were consistently located within the HR binding cavity, alternative binding orientations were detected in the complexes. Due to the specific residue interactions, all mG α5 helices of the H2R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in H4R complexes, only mGsi was in the Gi-like orientation toward TM2, which was in agreement with Gs- and Gi-coupled GPCRs structures resolved by X-ray/cryo-EM. These cellular and molecular insights support (patho)physiological profiles of the histamine receptors, especially the hitherto little studied H2R function in the brain, as well as of the pharmacological potential of H4R selective drugs.


Assuntos
Proteínas de Ligação ao GTP/química , Ligantes , Simulação de Dinâmica Molecular , Engenharia de Proteínas/métodos , Receptores Histamínicos/química , Simulação por Computador , Microscopia Crioeletrônica , Sistemas de Liberação de Medicamentos , Células HEK293 , Histamina/química , Humanos , Luciferases/metabolismo , Distribuição Normal , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Receptores Histamínicos H2/metabolismo , Receptores Histamínicos H4/metabolismo , Transdução de Sinais , Raios X
15.
eNeuro ; 8(5)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34531280

RESUMO

The ability to measure changes in neuronal activity in a quantifiable and precise manner is of fundamental importance to understand neuron development and function. Repeated monitoring of neuronal activity of the same population of neurons over several days is challenging and, typically, low-throughput. Here, we describe a new biochemical reporter assay that allows for repeated measurements of neuronal activity in a cell type-specific manner. We coupled activity-dependent elements from the Arc/Arg3.1 gene with a secreted reporter, Gaussia luciferase (Gluc), to quantify neuronal activity without sacrificing the neurons. The reporter predominantly senses calcium and NMDA receptor (NMDAR)-dependent activity. By repeatedly measuring the accumulation of the reporter in cell media, we can profile the developmental dynamics of neuronal activity in cultured neurons from male and female mice. The assay also allows for longitudinal analysis of pharmacological treatments, thus distinguishing acute from delayed responses. Moreover, conditional expression of the reporter allows for monitoring cell type-specific changes. This simple, quantitative, cost-effective, automatable, and cell type-specific activity reporter is a valuable tool to study the development of neuronal activity in normal and disease-model conditions, and to identify small molecules or protein factors that selectively modulate the activity of a specific population of neurons.


Assuntos
Neurônios , Receptores de N-Metil-D-Aspartato , Animais , Feminino , Genes Reporter , Luciferases/genética , Masculino , Camundongos , Neurônios/metabolismo , Transdução de Sinais
16.
J Vis Exp ; (174)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34424228

RESUMO

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities. Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.


Assuntos
Medições Luminescentes , Optogenética , Luciferases/genética , Neurônios
17.
Immunity ; 54(9): 2143-2158.e15, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34453881

RESUMO

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Encéfalo/patologia , COVID-19/imunologia , Pulmão/patologia , SARS-CoV-2/fisiologia , Testículo/patologia , Enzima de Conversão de Angiotensina 2/genética , Animais , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Encéfalo/virologia , COVID-19/terapia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Luciferases/genética , Medições Luminescentes , Pulmão/virologia , Masculino , Camundongos , Camundongos Transgênicos , Testículo/virologia
18.
Cancer Sci ; 112(11): 4722-4735, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34431598

RESUMO

The Wnt/ß-catenin signaling pathway plays an important role in tissue homeostasis, and its malignant activation is closely related to the occurrence and development of many cancers, especially colorectal cancer with adenomatous polyposis coli (APC) and CTNNB1 mutations. By applying a TCF/lymphoid-enhancing factor (LEF) luciferase reporter system, the high-throughput screening of 18 840 small-molecule compounds was performed. A novel scaffold compound, C644-0303, was identified as a Wnt/ß-catenin signaling inhibitor and exhibited antitumor efficacy. It inhibited both constitutive and ligand activated Wnt signals and its downstream gene expression. Functional studies showed that C644-0303 causes cell cycle arrest, induces apoptosis, and inhibits cancer cell migration. Moreover, transcription factor array indicated that C644-0303 could suppress various tumor-promoting transcription factor activities in addition to Wnt/ß-catenin. Finally, C644-0303 suppressed tumor spheroidization in a 3-dimensional cell culture model and inhibited xenograft tumor growth in mice. In conclusion, we report a novel structural small molecular inhibitor targeting the Wnt/ß-catenin signaling pathway that has therapeutic potential for colorectal cancer treatment.


Assuntos
Polipose Adenomatosa do Colo/tratamento farmacológico , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Descoberta de Drogas , Feminino , Células HCT116 , Células HT29 , Humanos , Luciferases , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular/métodos , Esferoides Celulares/efeitos dos fármacos , Fatores de Transcrição TCF , Fatores de Transcrição/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
19.
PLoS One ; 16(8): e0254125, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34411144

RESUMO

Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cells to conduct studies in immune competent mice may lead to cell-extrinsic tdTomato/Luc-induced alterations in tumor growth and tumor immune microenvironment that need to be taken into consideration when evaluating the efficacy of anti-cancer drugs and vaccines in immunocompetent animal models.


Assuntos
Carcinoma Pulmonar de Lewis , Expressão Gênica , Genes Reporter/imunologia , Luciferases , Proteínas Luminescentes , Neoplasias Pulmonares , Microambiente Tumoral , Animais , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/imunologia , Linhagem Celular Tumoral , Luciferases/genética , Luciferases/imunologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Camundongos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
20.
Photochem Photobiol Sci ; 20(8): 1053-1067, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34347281

RESUMO

Firefly adult bioluminescence functions as signal communication between sexes. How sympatric sibling species with similar glow pattern recognize their conspecific mates remains largely unknown. To better understand the role of the luciferases of sympatric fireflies in recognizing mates, we cloned the luciferase genes of three sympatric forest dwelling fireflies (Diaphanes nubilus, Diaphanes pectinealis, and Diaphanes sp2) and evaluated their enzyme characteristics. Our data show that the amino acid (AA) sequences of all three luciferases are highly conserved, including the identities (D. nubilus vs D. pectinealis: 99%; D. nubilus vs Diaphanes sp2: 98.5%; D. pectinealis vs Diaphanes sp2: 99.4%) and the protein structures. Three recombinant luciferases produced in vitro all possess significant luminescence activity at pH 7.8, and similar maximum emission spectrum (D. nubilus: 562 nm; D. pectinealis and Diaphanes sp2: 564 nm). They show the highest activity at 10 °C (D. pectinealis, Diaphanes sp2) and 15 °C (D. nubilus), and completely inactivation at 45 °C. Their KM for D-luciferin and ATP were 2.7 µM and 92 µM (D. nubilus), 3.7 µM and 49 µM (D. pectinealis), 3.5 µM and 46 µM (Diaphanes sp2). Phylogenetic analyses support that D. nubilus is sister to D. pectinealis with Diaphanes sp2 at their base, which further cluster with Pyrocoelia. All combined data indicate that sympatric Diaphanes species have similar luciferase characteristics, suggesting that other strategies (e.g., pheromone, active time, etc.) may be adopted to recognize mates. Our data provide new insights into Diaphanes luciferases and their evolution.


Assuntos
Evolução Molecular , Vaga-Lumes/genética , Luciferases/genética , Simpatria , Animais , Clonagem Molecular
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