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1.
Invest Ophthalmol Vis Sci ; 61(3): 51, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32232350

RESUMO

Purpose: The lysozyme 2 (Lyz2 or LysM) cre mouse is extensively used to achieve genetic manipulation in myeloid cells and it has been widely employed in retinal research. However, LysM has been recently described to be expressed in brain neurons and there is a debate on whether it is also expressed by resident microglia in addition to infiltrating macrophages. Methods: We examined LysM-cre recombination in retinal tissue using a LysM-cre/tdTomato reporter mouse together with immunolabeling for several retinal cell markers. We further compared LysM-cre tdTomato recombination with that of Cdh5-cre driver, which is expressed in both endothelial and hematopoietic cells. Results: LysM-cre was strongly expressed in most microglia/resident macrophages in neonatal retinas (P8) and to a lesser extent in microglia of adult retinas. In addition, there was some neuronal recombination (8 %) of LysM-cre specifically in adult retinal ganglion cells and amacrine cells. After retinal ischemia-reperfusion injury, LysM-cre was strongly expressed in microglia/infiltrating macrophages. Cdh5-cre was expressed in endothelial and myeloid cells of P8 pups retinas. Unexpectedly, Cdh5 showed additional expression in adult mouse retinal ganglion cells and brain neurons. Conclusions: LysM-cre is expressed in macrophages and a subset of microglia together with a small but significant recombination of LysM-cre in the retinal neurons of adult mice. Cdh5 also showed some neuronal expression in both retina and brain of adult mice. These findings should be taken into consideration when interpreting results from central nervous system research using LysM-cre and Cdh5-cre mice.


Assuntos
Antígenos CD/metabolismo , Encéfalo/metabolismo , Caderinas/metabolismo , Integrases/metabolismo , Substâncias Luminescentes/metabolismo , Proteínas Luminescentes/metabolismo , Muramidase/metabolismo , Vasos Retinianos/metabolismo , Animais , Animais Recém-Nascidos , Pesquisa Biomédica , Diagnóstico por Imagem , Endotélio Vascular/metabolismo , Feminino , Genes Reporter , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Neurônios/metabolismo , Recombinação Genética , Traumatismo por Reperfusão/metabolismo , Células Ganglionares da Retina/metabolismo
2.
ACS Appl Mater Interfaces ; 12(11): 12383-12394, 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32091195

RESUMO

Drug-induced liver injury (DILI) is a widespread clinical problem. The pathophysiological mechanisms of DILI are complicated, and the traditional diagnostic methods for DILI have their limitations. Owing to its convenient operation, high sensitivity, and high specificity, luminescent sensing and imaging as an indispensable tool in biological research and clinical trials may provide an important means for DILI study. Herein, we report the rational design and preparation of a near-infrared dual-phosphorescent polymeric probe (P-ONOO) for exploring the DILI via specific imaging of peroxynitrite (ONOO-) elevation in vivo, which was one of early markers of DILI and very difficult to be detected due to its short half-life and high reactive activity. With the utilization of P-ONOO, the raised ONOO- was visualized successfully in the drug-treated hepatocytes with a high signal-to-noise ratio via ratiometric and time-resolved photoluminescence imaging. Importantly, the ONOO- boost in the acetaminophen-induced liver injury in real time was verified, and the direct observation of the elevated ONOO- production in ketoconazole-induced liver injury was achieved for the first time. Our findings may contribute to understanding the exact mechanism of ketoconazole-induced hepatotoxicity that is still ambiguous. Notably, this luminescent approach for revealing the liver injury works fast and conveniently.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Substâncias Luminescentes , Imagem Óptica/métodos , Ácido Peroxinitroso , Animais , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico por imagem , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Modelos Animais de Doenças , Irídio/química , Fígado/diagnóstico por imagem , Fígado/metabolismo , Substâncias Luminescentes/análise , Substâncias Luminescentes/química , Substâncias Luminescentes/metabolismo , Camundongos , Camundongos Nus , Ácido Peroxinitroso/análise , Ácido Peroxinitroso/metabolismo , Polímeros/química
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 229: 118014, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31923791

RESUMO

Eu(III) 2-{4-[(2-oxocyclopentyl)methyl]phenyl}propanoic acid complex (Eu-LPF), a novel low-toxic luminescent material based on energy transfer between the LPF ligand and Eu3+ ion, was synthesized and characterized by means of elemental analysis, thermogravimetric analyses, and FT-IR spectra. The spectroscopic properties of Eu-LPF were studied using UV-vis absorption spectroscopy and steady/transient state luminescence spectroscopy. Furthermore, the cytotoxicity of Eu-LPF on MCF-7 cells was investigated by MTT assay and flow cytometry. Its biocompatibility and utilization for cell imaging were studied as well. The results showed that Eu-LPF exhibited favorable luminescence properties, low toxicity and good biocompatibility, which endowed Eu-LPF with a potential capability for bioimaging and optical detection.


Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Neoplasias da Mama/patologia , Európio/metabolismo , Luminescência , Substâncias Luminescentes/metabolismo , Imagem Molecular/métodos , Fenilpropionatos/metabolismo , Anti-Inflamatórios não Esteroides/química , Apoptose , Neoplasias da Mama/metabolismo , Ciclo Celular , Proliferação de Células , Európio/química , Feminino , Humanos , Ligantes , Substâncias Luminescentes/química , Fenilpropionatos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Células Tumorais Cultivadas
4.
Biochem Biophys Res Commun ; 521(3): 674-680, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31685208

RESUMO

Galectin-3 (Gal-3) is a multifunctional glycan-binding protein that participates in many pathophysiological events and has been described as a biomarker and potential therapeutic target for severe disorders, such as cancer. Several probes for Gal-3 or its ligands have been developed, however both the pathophysiological mechanisms and potential biomedical applications of Gal-3 remain not fully assessed. Molecular imaging using bioluminescent probes provides great sensitivity for in vivo and in vitro analysis for both cellular and whole multicellular organism tracking and target detection. Here, we engineered a chimeric molecule consisting of Renilla luciferase fused with mouse Gal-3 (RLuc-mGal-3). RLuc-mGal-3 preparation was highly homogenous, soluble, active, and has molecular mass of 65,870.95 Da. This molecule was able to bind to MKN45 cell surface, property which was inhibited by the reduction of Gal-3 ligands on the cell surface by the overexpression of ST6GalNAc-I. In order to obtain an efficient and stable delivery system, RLuc-mGal-3 was adsorbed to poly-lactic acid nanoparticles, which increased binding to MKN45 cells in vitro. Furthermore, bioluminescence imaging showed that RLuc-mGal-3 was able to indicate the presence of implanted tumor in mice, event drastically inhibited by the presence of lactose. This novel bioluminescent chimeric molecule offers a safe and highly sensitive alternative to fluorescent and radiolabeled probes with potential application in biomedical research for a better understanding of the distribution and fate of Gal-3 and its ligands in vitro and in vivo.


Assuntos
Galectina 3/metabolismo , Luciferases de Renilla/metabolismo , Substâncias Luminescentes/metabolismo , Neoplasias/diagnóstico por imagem , Polissacarídeos/metabolismo , Animais , Linhagem Celular Tumoral , Galectina 3/análise , Galectina 3/genética , Humanos , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Substâncias Luminescentes/análise , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Imagem Óptica , Polissacarídeos/análise , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
5.
Adv Mater ; 31(39): e1902469, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31402525

RESUMO

Cells transport mass dynamically, crossing cell membranes to maintain metabolism and systemic homeostasis, through which biomolecules are also delivered to cells for gene editing, cell reprograming, therapy, and other purposes. Quantifying the translocation kinetics is fundamentally and clinically essential, but remains limited by fluorescence-based technologies, which are semi-quantitative and only provide kinetics information at cellular level or in discrete time. Herein, a real-time method of quantifying cell internalization kinetics is reported using functionalized firefly-luciferase nanocapsules as the probe. This quantitative assay will facilitate the rational design of delivery vectors and enable high-throughput screening of peptides and other functional molecules, constituting an effective tool for broad applications, including drug development and cancer therapy.


Assuntos
Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/metabolismo , Substâncias Luminescentes/química , Substâncias Luminescentes/metabolismo , Nanocápsulas/química , Animais , Linhagem Celular Tumoral , Cinética , Camundongos , Transporte Proteico
6.
Proc Natl Acad Sci U S A ; 116(38): 18911-18916, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31462497

RESUMO

Marine polychaetes Odontosyllis undecimdonta, commonly known as fireworms, emit bright blue-green bioluminescence. Until the recent identification of the Odontosyllis luciferase enzyme, little progress had been made toward characterizing the key components of this bioluminescence system. Here we present the biomolecular mechanisms of enzymatic (leading to light emission) and nonenzymatic (dark) oxidation pathways of newly described O. undecimdonta luciferin. Spectral studies, including 1D and 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction, of isolated substances allowed us to characterize the luciferin as an unusual tricyclic sulfur-containing heterocycle. Odontosyllis luciferin does not share structural similarity with any other known luciferins. The structures of the Odontosyllis bioluminescent system's low molecular weight components have enabled us to propose chemical transformation pathways for the enzymatic and nonspecific oxidation of luciferin.


Assuntos
Substâncias Luminescentes/química , Poliquetos/química , Animais , Vias Biossintéticas , Cor , Indóis/química , Indóis/metabolismo , Substâncias Luminescentes/metabolismo , Medições Luminescentes , Proteínas Luminescentes/metabolismo , Estrutura Molecular , Oxirredução , Poliquetos/metabolismo , Pirazinas/química , Pirazinas/metabolismo
7.
Biosens Bioelectron ; 141: 111403, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31176111

RESUMO

In the present work, peptide-functionalized upconversion@silica nanoparticle (termed as UCNP@SiO2@Cy5-pep)-based fluorescence resonance energy transfer (FRET) sensing platform has been fabricated for the detection of caspase-9 activity in vitro and in vivo. A Cy5 labeled peptide containing specific motif LEHD for caspase-9 cleavage was designed and conjugated with UCNP@SiO2 through covalent attachment. The red upconversion luminescence (UCL) emission of UCNP@SiO2 can be quenched by Cy5 while the green UCL emission of UCNP@SiO2 remains undisturbed. After the cleavage of LEHD by caspase-9, the Cy5 departed from UCNP@SiO2 surface, resulting in recovery of red UCL emission of UCNP@SiO2. The UCNP@SiO2@Cy5-pep has been successfully used to monitoring the changes of caspase-9 activity levels in apoptotic cancerous cells (MG-63 and SW480) by cisplatin-induction. Under same experimental conditions, it is found that the intracellular caspase-9 activity level of cisplatin-treated MG-63 cells is higher than that of cisplatin-treated SW480. This result is consistent with that of commercial caspase-9 activity kits. The UCL signal intensity ratio of red emission to green emission of UCNP (termed as R/G) is linearly dependent on the amount of MG-63 cells within the range of 5 × 103 to 1 × 106 cells (i.e., 0.5-100 U mL-1 caspase-9) with a limit of detection (LOD) of 675 cells (i.e., 0.068 U mL-1 caspase-9). Furthermore, the practicability of UCNP@SiO2@Cy5-pep is demonstrated by detection of caspase-9 activities in tumor tissues in vivo, and satisfactory results are obtained.


Assuntos
Caspase 9/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Substâncias Luminescentes/metabolismo , Peptídeos/metabolismo , Animais , Caspase 9/análise , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/metabolismo , Nanopartículas/ultraestrutura , Neoplasias/enzimologia
8.
Invest Ophthalmol Vis Sci ; 60(7): 2438-2448, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31158276

RESUMO

Purpose: Corneal endothelial dysfunction leads to corneal edema, pain, and vision loss. Adequate animal models are needed to study the safety and efficacy of novel cell therapies as an alternative to corneal transplantation. Methods: Primary human corneal endothelial cells (HCECs) were isolated from cadaveric donor corneas, expanded in vitro, transduced to express green fluorescent protein (GFP), loaded with superparamagnetic nanoparticles, and injected into the anterior chamber of adult rabbits immediately after endothelial cell or Descemet's membrane stripping. The same volume of balanced salt solution plus (BSS+) was injected in control eyes. We compared different models for inducing corneal edema in rabbits, and examined the ability of transplanted HCECs to reduce corneal edema over time by measuring central corneal thickness and tracking corneal clarity. GFP-positive donor cells were tracked in vivo using optical coherence tomography (OCT) fluorescence angiography module, and the transplanted cells were confirmed by human nuclei immunostaining. Results: Magnetic HCECs integrated onto the recipient corneas with intact Descemet's membrane, and donor identity was confirmed by GFP expression and immunostaining for human nuclei marker. Donor HCECs formed a monolayer on the posterior corneal surface and expressed HCEC functional markers of tight junction formation. No GFP-positive cells were observed in the trabecular meshwork or on the iris, and intraocular pressure remained stable through the length of the study. Conclusions: Our results demonstrate magnetic cell-based therapy efficiently delivers HCECs to restore corneal transparency without detectable toxicity or adverse effect on intraocular pressure. Magnetic delivery of HCECs may enhance corneal function and should be explored further for human therapies.


Assuntos
Transplante de Células/métodos , Doenças da Córnea/cirurgia , Sistemas de Liberação de Medicamentos , Epitélio Posterior/transplante , Terapia de Campo Magnético/métodos , Nanopartículas de Magnetita/química , Animais , Câmara Anterior/citologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Doenças da Córnea/patologia , Portadores de Fármacos , Epitélio Posterior/metabolismo , Epitélio Posterior/cirurgia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Pressão Intraocular , Substâncias Luminescentes/metabolismo , Modelos Animais , Coelhos , Doadores de Tecidos , Transfecção
9.
Mol Ther ; 27(6): 1074-1086, 2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31023523

RESUMO

Lentiviral vectors (LVs) are used for delivery of genes into hematopoietic stem and progenitor cells (HSPCs) in clinical trials worldwide. LVs, in contrast to retroviral vectors, are not associated with insertion site-associated malignant clonal expansions and, thus, are considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis affecting the erythroid, myeloid, and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. Nine insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPCs, which is concerning, and suggests that strong constitutive promoters should not be included in LVs.


Assuntos
Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/virologia , Lentivirus/genética , Transdução Genética , Animais , Antígenos CD34/metabolismo , Células Clonais , Terapia Genética/efeitos adversos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Substâncias Luminescentes/metabolismo , Macaca mulatta , Mutagênese Insercional/genética , Regiões Promotoras Genéticas , Processamento de Proteína/genética , Sequências Repetidas Terminais/genética , Transplante Autólogo
10.
ACS Sens ; 4(5): 1391-1398, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31002225

RESUMO

Oxygenation and tissue hypoxia play critical roles in mammalian biology and contribute to aggressive phenotypes in cancerous tumors, driving research to develop accurate and easy-to-implement methods for monitoring hypoxia in living cells and animal models. This study reports the chemiluminescent probe HyCL-4-AM, which contains a nitroaromatic sensing moiety and, importantly, an acetoxymethyl (AM) ester that dramatically improves operation in cells and animals. HyCL-4-AM provides a selective 60 000-fold increase in luminescence emission in the presence of rat liver microsomes (RLM). For cellular operation, the chemiluminescence response kinetics is sharply dependent on oxygen levels, enabling highly significant and reproducible measurement of hypoxia in living cells. Whole animal imaging experiments in muscle tissue and tumor xenografts show that HyCL-4-AM can differentiate between well oxygenated muscle tissue and hypoxic tumors, demonstrating potential for monitoring tumor reoxygenation via hyperoxic treatment.


Assuntos
Ésteres/química , Substâncias Luminescentes/química , Substâncias Luminescentes/metabolismo , Células A549 , Animais , Hipóxia Celular , Sobrevivência Celular , Transformação Celular Neoplásica , Humanos , Cinética , Medições Luminescentes , Ratos
11.
Methods Mol Biol ; 1955: 147-163, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30868525

RESUMO

Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. Despite a global research effort, there have been no significant treatment advances for at least 40 years. Gaps in our knowledge of T. cruzi biology and pathogenesis have been major factors in limiting progress. In addition, the extremely low parasite burden during chronic infections has complicated the monitoring of both disease progression and drug efficacy, even in predictive animal models. To address these problems, we genetically modified T. cruzi to express a red-shifted luciferase. Mice infected with these highly bioluminescent parasites can be monitored by in vivo imaging, with exquisite sensitivity. However, a major drawback of bioluminescence imaging is that it does not allow visualization of host-parasite interactions at a cellular level. To facilitate this, we generated T. cruzi strains that express a chimeric protein that is both bioluminescent and fluorescent. Bioluminescence allows the tissue location of infection foci to be identified, and fluorescence can then be exploited to detect parasites in histological sections derived from excised tissue. In this article, we describe in detail the in vivo imaging and confocal microscopy protocols that we have developed for visualizing T. cruzi parasites expressing these dual-reporter fusion proteins. The approaches make it feasible to locate individual parasites within chronically infected murine tissues, to assess their replicative status, to resolve the nature of host cells, and to characterize their immunological context.


Assuntos
Doença de Chagas/patologia , Interações Hospedeiro-Parasita , Trypanosoma cruzi/fisiologia , Animais , Doença de Chagas/diagnóstico por imagem , Doença de Chagas/parasitologia , Modelos Animais de Doenças , Fluorescência , Humanos , Luciferases/análise , Luciferases/genética , Substâncias Luminescentes/análise , Substâncias Luminescentes/metabolismo , Medições Luminescentes/métodos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Confocal/métodos , Imagem Óptica/métodos , Trypanosoma cruzi/genética , Trypanosoma cruzi/isolamento & purificação , Imagem Corporal Total/métodos
12.
J Mol Biol ; 431(8): 1689-1699, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30878481

RESUMO

Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes and allows for continuous acquisition with a time resolution in the order of seconds, yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigor unattainable through classical methods.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Membranas Mitocondriais/metabolismo , Canais de Translocação SEC/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Luciferases/metabolismo , Substâncias Luminescentes/metabolismo , Medições Luminescentes/métodos , Transporte Proteico
13.
Biochemistry ; 58(12): 1689-1697, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30810040

RESUMO

In vivo bioluminescence imaging (BLI) has become a standard, non-invasive imaging modality for following gene expression or the fate of proteins and cells in living animals. Currently, bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Inconsistent results of experiments using different bioluminescent reporters, such as Akaluc and Antareas2, have been reported. Herein, we re-examined the inconsistency in several experimental settings and identified the factors, such as ATP dependency, stability in serum, and molecular sizes of luciferases, that contributed to the observed differences. We expect this study will make the research community aware of these factors and facilitate more accurate interpretation of BLI data by considering the nature of each bioluminescent reporter.


Assuntos
Luciferases/metabolismo , Medições Luminescentes/métodos , Trifosfato de Adenosina/metabolismo , Animais , Estabilidade Enzimática , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Imidazóis/metabolismo , Luciferases/sangue , Luciferases/genética , Substâncias Luminescentes/metabolismo , Camundongos Endogâmicos BALB C , Neoplasias/diagnóstico por imagem , Pirazinas/metabolismo , Distribuição Tecidual
14.
ACS Synth Biol ; 8(3): 474-481, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30721031

RESUMO

Enzymes are the ultimate entities responsible for chemical transformations in natural and engineered biosynthetic pathways. However, many natural enzymes suffer from suboptimal functional expression due to poor intrinsic protein stability. Further, stability enhancing mutations often come at the cost of impaired function. Here we demonstrate an automated protein engineering strategy for stabilizing enzymes while retaining catalytic function using deep mutational scanning coupled to multiple-filter based screening and combinatorial mutagenesis. We validated this strategy by improving the functional expression of a Type III polyketide synthase from the Atropa belladonna biosynthetic pathway for tropane alkaloids. The best variant had a total of 8 mutations with over 25-fold improved activity over wild-type in E. coli cell lysates, an improved melting temperature of 11.5 ± 0.6 °C, and only minimal reduction in catalytic efficiency. We show that the multiple-filter approach maintains acceptable sensitivity with homology modeling structures up to 4 Å RMS. Our results highlight an automated protein engineering tool for improving the stability and solubility of difficult to express enzymes, which has impact for biotechnological applications.


Assuntos
Aciltransferases/química , Aciltransferases/genética , Atropa belladonna/enzimologia , Biotecnologia/métodos , Ciência de Dados/métodos , Engenharia de Proteínas/métodos , Aciltransferases/metabolismo , Alcaloides de Belladona/metabolismo , Vias Biossintéticas , Códon sem Sentido , Estabilidade Enzimática/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Substâncias Luminescentes/química , Substâncias Luminescentes/metabolismo , Mutagênese , Mutação de Sentido Incorreto , Saccharomyces cerevisiae/metabolismo , Solubilidade , Temperatura de Transição
15.
Org Biomol Chem ; 17(9): 2413-2422, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30735222

RESUMO

Odorants constitute a small and chemically diverse group of molecules with ethanol functioning as a key odorant that induces reproductive toxicity and adverse chronic effects on the liver. Analytical tools designed so far for the detection of odorant molecules are relatively invasive. Therefore, a tool that can measure the corresponding rate changes of ethanol concentration in real-time is highly desirable. Here in this work, we report a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor for in vivo quantification of ethanol at the cellular level with high spatial and temporal resolution. A human odorant-binding protein (hOBPIIa) was flanked by fluorescent proteins ECFP (Enhanced Cyan Fluorescent Protein) and Venus at the N- and C-terminus respectively. The constructed FRET nanosensor was named the fluorescent indicator protein for odorants (FLIPO). FLIPO allows in vitro and in vivo determination of FRET changes in a concentration-dependent manner. The developed nanosensor is highly specific to ethanol, stable to pH changes and provides rapid detection rate response. FLIPO-42 is the most efficient nanosensor created that measures ethanol with an apparent affinity (Kd) of 4.16 µM and covers the physiological range of 500 nM to 12 µM ethanol measurement. FLIPO-42 can measure ethanol dynamics in bacterial, yeast and mammalian cells non-invasively in real time which proves its efficacy as a sensing device in both prokaryotic and eukaryotic systems. Taken together, a prototype for a set of nanosensors was established, potentially enabling the monitoring of dynamic changes of ethanol and investigate its uptake and metabolism with subcellular resolution in vivo and ex vivo. Furthermore, the advent of a set of novel nanosensors will provide us with the tools for numerous medical, scientific, industrial and environmental applications which would help to illuminate their role in biological systems.


Assuntos
Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Etanol/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/química , Substâncias Luminescentes/química , Proteínas Luminescentes/química , Receptores Odorantes/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Etanol/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Substâncias Luminescentes/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Imagem Óptica , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo
16.
Methods Mol Biol ; 1925: 1-14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30674012

RESUMO

Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10 µM, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.


Assuntos
Equorina/química , Cálcio/análise , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Equorina/metabolismo , Animais , Cálcio/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imidazóis/química , Imidazóis/metabolismo , Substâncias Luminescentes/metabolismo , Mitocôndrias/metabolismo , Modelos Moleculares , Pirazinas/química , Pirazinas/metabolismo , Cifozoários/química
17.
Methods Mol Biol ; 1925: 111-125, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30674021

RESUMO

Calcium (Ca2+) is a key player in cardiomyocyte homeostasis, and its roles span from excitation-contraction coupling to metabolic and structural signaling. Alterations in the function or expression of Ca2+-handling proteins are common findings in failing cardiomyocytes, which have been linked to impaired contractility and detrimental remodeling of the cellular structure. For these reasons, the study of intracellular Ca2+ handling in cardiomyocytes represents a central method in experimental molecular cardiology.


Assuntos
Sinalização do Cálcio , Cálcio/análise , Proteínas de Fluorescência Verde/análise , Microscopia de Fluorescência/métodos , Miócitos Cardíacos/metabolismo , Imagem Óptica/métodos , Animais , Cálcio/metabolismo , Células Cultivadas , Acoplamento Excitação-Contração , Proteínas de Fluorescência Verde/metabolismo , Substâncias Luminescentes/análise , Substâncias Luminescentes/metabolismo , Camundongos , Modelos Moleculares , Miócitos Cardíacos/citologia , Ratos
18.
Artigo em Inglês | MEDLINE | ID: mdl-30467537

RESUMO

Vibrio campbellii is a major pathogen in aquaculture. It is a causative agent of the so-called "luminescent vibriosis," a life-threatening condition caused by bioluminescent Vibrio spp. that often involves mass mortality of farmed shrimps. The emergence of multidrug resistant Vibrio strains raises a concern and poses a challenge for the treatment of this infection in the coming years. Inhibition of bacterial cell-to-cell communication or quorum sensing (QS) has been proposed as an alternative to antibiotic therapies. Aiming to identify novel QS disruptors, the 9H-fluroen-9yl vinyl ether derivative SAM461 was found to thwart V. campbellii bioluminescence, a QS-regulated phenotype. Phenotypic and gene expression analyses revealed, however, that the mode of action of SAM461 was unrelated to QS inhibition. Further evaluation with purified Vibrio fischeri and NanoLuc luciferases revealed enzymatic inhibition at micromolar concentrations. In silico analysis by molecular docking suggested binding of SAM461 in the active site cavities of both luciferase enzymes. Subsequent in vivo testing of SAM461 with gnotobiotic Artemia franciscana nauplii demonstrated naupliar protection against V. campbellii infection at low micromolar concentrations. Taken together, these findings suggest that suppression of luciferase activity could constitute a novel paradigm in the development of alternative anti-infective chemotherapies against luminescent vibriosis, and pave the ground for the chemical synthesis and biological characterization of derivatives with promising antimicrobial prospects.


Assuntos
Antibacterianos/administração & dosagem , Artemia/microbiologia , Luciferases Bacterianas/antagonistas & inibidores , Substâncias Luminescentes/metabolismo , Vibrioses/veterinária , Vibrio/efeitos dos fármacos , Animais , Fluorenos/administração & dosagem , Simulação de Acoplamento Molecular , Vibrioses/prevenção & controle , Compostos de Vinila/administração & dosagem
19.
Invest Ophthalmol Vis Sci ; 59(12): 4909-4920, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30347085

RESUMO

Purpose: Temporal and reversible control of protein expression in vivo is a central goal for many gene therapies, especially for strategies involving proteins that are detrimental to physiology if constitutively expressed. Accordingly, we explored whether protein abundance in the mouse retina could be effectively controlled using a destabilizing Escherichia coli dihydrofolate reductase (DHFR) domain whose stability is dependent on the small molecule, trimethoprim (TMP). Methods: We intravitreally injected wild-type C57BL6/J mice with an adeno-associated vector (rAAV2/2[MAX]) constitutively expressing separate fluorescent reporters: DHFR fused to yellow fluorescent protein (DHFR.YFP) and mCherry. TMP or vehicle was administered to mice via oral gavage, drinking water, or eye drops. Ocular TMP levels post treatment were quantified by LC-MS/MS. Protein abundance was measured by fundus fluorescence imaging and western blotting. Visual acuity, response to light stimulus, retinal structure, and gene expression were evaluated after long-term (3 months) TMP treatment. Results: Without TMP, DHFR.YFP was efficiently degraded in the retina. TMP achieved ocular concentrations of ∼13.6 µM (oral gavage), ∼331 nM (drinking water), and ∼636 nM (eye drops). Oral gavage and TMP eye drops stabilized DHFR.YFP as quickly as 6 hours, whereas continuous TMP drinking water could stabilize DHFR.YFP for ≥3 months. Stabilization was completely and repeatedly reversible following removal/addition of TMP in all regimens. Long-term TMP treatment had no impact on retina function/structure and had no effect on >99.9% of tested genes. Conclusions: This DHFR-based conditional system is a rapid, efficient, and reversible tool to effectively control protein expression in the retina.


Assuntos
Antagonistas do Ácido Fólico/uso terapêutico , Terapia Genética , Vetores Genéticos , Substâncias Luminescentes/metabolismo , Parvovirinae/genética , Retina/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/uso terapêutico , Administração Oral , Animais , Proteínas de Bactérias/metabolismo , Western Blotting , Cromatografia Líquida , Eletrorretinografia , Escherichia coli/enzimologia , Injeções Intravítreas , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos , Reação em Cadeia da Polimerase em Tempo Real , Retina/metabolismo , Espectrometria de Massas em Tandem , Tetra-Hidrofolato Desidrogenase/metabolismo , Acuidade Visual/fisiologia
20.
Talanta ; 189: 92-99, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30086981

RESUMO

Trypsin, as one of important proteases, is specific for catalyzing the hydrolysis of peptide and ester bonds containing lysine and arginine residues at the C-terminus. The level of trypsin in biological fluids can serve as a reliable and specific diagnostic biomarker for pancreatic function and its pathological changes. Herein, we demonstrate the application of phosphorescent Cu NCs for trypsin detection for the first time depending on the electron transfer between Cu NCs and cyt c. Cyt c and Cu NCs were selected as the quencher and the fluorophore, respectively. Cu NCs could bind to the positively charged cyt c through electrostatic and hydrophobic interactions, and the phosphorescence of Cu NCs was efficiently quenched by the metal-containing heme of cyt c. In the presence of trypsin, cyt c was digested, thus phosphorescence of Cu NCs remained. Therefore, a new and continuous phosphorescence assay for the detection of trypsin activity and its inhibitor screening was established. The plot of relative fluorescence versus trypsin concentration obtains a good linear detection range from 0 to 20 ng/mL (R2 = 0.9657), and a detection limit of 2 ng/mL, which is much lower than 20 ng/mL of the sensor in buffer solution because of urine amplifying the phosphorescence signal of Cu NCs based on the FRET strategy. This assay still has been successfully applied to trypsin inhibitor screening, demonstrating its potential application in drug discovery.


Assuntos
Cobre/química , Citocromos c/metabolismo , Ensaios Enzimáticos/métodos , Limite de Detecção , Nanoestruturas/química , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Animais , Cobre/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Transporte de Elétrons , Humanos , Substâncias Luminescentes/química , Substâncias Luminescentes/metabolismo , Tripsina/urina
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