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1.
Chem Commun (Camb) ; 55(70): 10380-10383, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31397448

RESUMO

A strategy for the photoelectrochemical detection of miRNA with ultra-low background noise was developed using tungsten diselenide-cysteine-dopamine (WSe2/Cys/DA) as a nanoprobe coupled with mismatched catalytic hairpin assembly target recycling. A superior detection limit of 3.3 aM toward miRNA-221 was achieved.


Assuntos
Cisteína/química , Dopamina/química , Técnicas Eletroquímicas/métodos , MicroRNAs/análise , Sondas Moleculares/química , Nanoestruturas , Processos Fotoquímicos , Selênio/química , Tungstênio/química , Técnicas Biossensoriais , Catálise , Humanos , Limite de Detecção , MicroRNAs/sangue , Estudo de Prova de Conceito
2.
Chem Commun (Camb) ; 55(70): 10440-10443, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31410422

RESUMO

In this work, a unique dual-site controlled fluorescent probe was presented for the sensitive and concurrent detection of pH in the cytoplasm and lysosomes. With the probe, the simultaneous down-regulation of pH in the lysosomes and cytoplasm during autophagy has been successfully revealed for the first time.


Assuntos
Autofagia , Citoplasma/metabolismo , Regulação para Baixo , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Sondas Moleculares/química , Sobrevivência Celular , Células Hep G2 , Humanos , Espectrometria de Fluorescência
3.
Chem Commun (Camb) ; 55(62): 9080-9083, 2019 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-31287110

RESUMO

New strategies are required for the discovery of unknown bioactive molecules produced by gut microbiota in the human host. Herein, we utilize a chemoselective probe immobilized to magnetic beads for analysis of carbonyls in human fecal samples. We identified 112 metabolites due to femtomole analysis and an increased mass spectrometric sensitivity by up to six orders of magnitude.


Assuntos
Aldeídos/análise , Fezes/química , Microbioma Gastrointestinal/fisiologia , Cetonas/análise , Sondas Moleculares/análise , Sondas Moleculares/química , Aldeídos/metabolismo , Fezes/microbiologia , Feminino , Humanos , Cetonas/metabolismo , Masculino , Sondas Moleculares/síntese química , Estrutura Molecular , Tamanho da Partícula , Propriedades de Superfície
4.
Nat Commun ; 10(1): 3004, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285436

RESUMO

Identity determining transcription factors (TFs), or core regulatory (CR) TFs, are governed by cell-type specific super enhancers (SEs). Drugs to selectively inhibit CR circuitry are of high interest for cancer treatment. In alveolar rhabdomyosarcoma, PAX3-FOXO1 activates SEs to induce the expression of other CR TFs, providing a model system for studying cancer cell addiction to CR transcription. Using chemical genetics, the systematic screening of chemical matter for a biological outcome, here we report on a screen for epigenetic chemical probes able to distinguish between SE-driven transcription and constitutive transcription. We find that chemical probes along the acetylation-axis, and not the methylation-axis, selectively disrupt CR transcription. Additionally, we find that histone deacetylases (HDACs) are essential for CR TF transcription. We further dissect the contribution of HDAC isoforms using selective inhibitors, including the newly developed selective HDAC3 inhibitor LW3. We show HDAC1/2/3 are the co-essential isoforms that when co-inhibited halt CR transcription, making CR TF sites hyper-accessible and disrupting chromatin looping.


Assuntos
Elementos Facilitadores Genéticos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Rabdomiossarcoma/genética , Acetilação/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Humanos , Simulação de Dinâmica Molecular , Sondas Moleculares/química , Sondas Moleculares/farmacologia , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Rabdomiossarcoma/patologia , Análise de Sequência de RNA , Transcrição Genética/efeitos dos fármacos
5.
Nat Commun ; 10(1): 2745, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227705

RESUMO

Small molecule probes are indispensable tools to explore diverse cellular events. However, finding a specific probe of a target remains a high challenge. Here we report the discovery of Fast-TRFS, a specific and superfast fluorogenic probe of mammalian thioredoxin reductase, a ubiquitous enzyme involved in regulation of diverse cellular redox signaling pathways. By systematically examining the processes of fluorophore release and reduction of cyclic disulfides/diselenides by the enzyme, structural factors that determine the response rate and specificity of the probe are disclosed. Mechanistic studies reveal that the fluorescence signal is switched on by a simple reduction of the disulfide bond within the probe, which is in stark contrast to the sensing mechanism of published probes. The favorable properties of Fast-TRFS enable development of a high-throughput screening assay to discover inhibitors of thioredoxin reductase by using crude tissue extracts as a source of the enzyme.


Assuntos
Descoberta de Drogas/métodos , Corantes Fluorescentes/química , Imagem Molecular/métodos , Sondas Moleculares/química , Tiorredoxina Redutase 1/metabolismo , Animais , Produtos Biológicos/farmacologia , Misturas Complexas , Dissulfetos/química , Corantes Fluorescentes/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Sondas Moleculares/metabolismo , Oxirredução , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxina Redutase 1/genética
6.
Int J Nanomedicine ; 14: 3723-3741, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31190821

RESUMO

Background: Inflammation and accumulation of macrophages are key features of unstable atherosclerotic plaques. The ability of macrophages to take up molecular probes can be exploited in new clinical imaging methods for the detection of unstable atherosclerotic lesions. We investigated whether modifications of human serum albumin (HSA) could be used to target macrophages efficiently in vitro. Materials and methods: Maleylated and aconitylated HSA were compared with unmodified HSA. Fluorescent or radiolabeled (89Zr) modified HSA was used in in vitro experiments to study cellular uptake by differentiated THP-1 cells and primary human macrophages. The time course of uptake was evaluated by flow cytometry, confocal microscopy, real-time microscopy and radioactivity measurements. The involvement of scavenger receptors (SR-A1, SR-B2, LOX-1) was assessed by knockdown experiments using RNA interference, by blocking experiments and by assays of competition by modified low-density lipoprotein. Results: Modified HSA was readily taken up by different macrophages. Uptake was mediated nonexclusively via the scavenger receptor SR-A1 (encoded by the MSR1 gene). Knockdown of CD36 and ORL1 had no influence on the uptake. Modified HSA was preferentially taken up by human macrophages compared with other vascular cell types such as endothelial cells and smooth muscle cells. Conclusions: Modified 89Zr-labeled HSA probes were recognized by different subsets of polarized macrophages, and maleylated HSA may be a promising radiotracer for radionuclide imaging of macrophage-rich inflammatory vascular diseases.


Assuntos
Macrófagos/metabolismo , Sondas Moleculares/química , Terapia de Alvo Molecular , Receptores Depuradores Classe B/metabolismo , Albumina Sérica Humana/química , Animais , Endocitose , Humanos , Maleatos/química , Fagocitose , Células THP-1 , Distribuição Tecidual
7.
BMC Bioinformatics ; 20(Suppl 8): 285, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182004

RESUMO

BACKGROUND: DNA is a promising candidate for the construction of biological devices due to its unique properties, including structural simplicity, convenient synthesis, high flexibility, and predictable behavior. And DNA has been widely used to construct the advanced logic devices. RESULTS: Herein, a molecular probe apparatus was constructed based on DNA molecular computing to perform fluorescent quenching and fluorescent signal recovery, with an ' ON/OFF' switching function. In this study, firstly, we program the streptavidin-mediated fluorescent quenching apparatus based on short-distance strand migration. The variation of fluorescent signal is acted as output. Then DNAzyme as a switching controller was involved to regulate the fluorescent signal increase. Finally, on this base, a cascade DNA logic gate consists of two logic AND operations was developed to enrich probe machine. CONCLUSION: The designed probe computing model can be implemented with readout of fluorescence intensity, and exhibits great potential applications in the field of bioimaging as well as disease diagnosis.


Assuntos
Simulação por Computador , Sondas Moleculares/química , DNA/química , DNA Catalítico/metabolismo , Fluorescência , Lógica , Processamento de Sinais Assistido por Computador , Estreptavidina/química
8.
Talanta ; 202: 342-348, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171193

RESUMO

A molecular beacons (MBs) loaded on molybdenum disulfide (MoS2) nanosheets as fluorescence probes for sensitive and versatile detection of microRNAs (miRNAs) through hybridization chain reaction (HCR) has been designed. MoS2 was used as a adsorbent to capture the MBs and a selective fluorescence quencher to reduce the background signal. In the absence of miRNAs, HCR could not be triggered due to the stability of MB probes. The probes attached to the MoS2 surface, efficiently quenching fluorescence of the G-quadruplex/Thioflavin T. However, the presence of target miRNAs triggers the HCR process to generate large amount of HCR products. Meanwhile, the HCR products of long nanowires chain with abundant G-quadruplexes could not be adsorbed on the surface of MoS2, and therefore detach from the MoS2. Consequently, Thioflavin T could be embedded in G-quadruplexes and produced strong fluorescence signal. This fluorescence emission signal could achieve detection of miRNA as low as 4.2 pM and a wide linear ranges from 0.1 to 100 nM. In addition, a versatile fluorescence probe has been developed for detection of miRNA-21 by changing the miRNA-recognition domain of MB. Thus, the fluorescent probe would be a potential alternative tool for biomedical research and clinical molecular diagnostics.


Assuntos
Dissulfetos/química , Corantes Fluorescentes/química , Quadruplex G , MicroRNAs/análise , Sondas Moleculares/química , Molibdênio/química , Hibridização de Ácido Nucleico , Humanos
9.
Nat Chem ; 11(7): 629-637, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209299

RESUMO

In DNA, the loss of a nucleobase by hydrolysis generates an abasic site. Formed as a result of DNA damage, as well as a key intermediate during the base excision repair pathway, abasic sites are frequent DNA lesions that can lead to mutations and strand breaks. Here we present snAP-seq, a chemical approach that selectively exploits the reactive aldehyde moiety at abasic sites to reveal their location within DNA at single-nucleotide resolution. Importantly, the approach resolves abasic sites from other aldehyde functionalities known to exist in genomic DNA. snAP-seq was validated on synthetic DNA and then applied to two separate genomes. We studied the distribution of thymine modifications in the Leishmania major genome by enzymatically converting these modifications into abasic sites followed by abasic site mapping. We also applied snAP-seq directly to HeLa DNA to provide a map of endogenous abasic sites in the human genome.


Assuntos
DNA/genética , Genoma/genética , Análise de Sequência de DNA/métodos , Aldeídos/química , Sequência de Bases , DNA/química , Dano ao DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Leishmania major/genética , Sondas Moleculares/síntese química , Sondas Moleculares/química , Timina/química , Uracila-DNA Glicosidase/química
10.
Nat Commun ; 10(1): 2704, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221964

RESUMO

Attachment of lipid tails to oligonucleotides has emerged as a powerful technology in constructing cell membrane-anchorable nucleic acid-based probes. In practice, however, conventional lipid-conjugated oligonucleotides fail to distinguish among different cell membranes. Herein, a phosphorylated lipid-conjugated oligonucleotide (DNA-lipid-P) is reported for alkaline phosphatase (ALP)-dependent cell membrane adhesion. In the absence of ALP, DNA-lipid-P with its poor hydrophobicity shows only weak interaction with cell membrane. However, in the presence of the highly expressed plasma membrane-associated ALP, DNA-lipid-P is converted to lipid-conjugated oligonucleotide (DNA-lipid) by enzymatic dephosphorylation. As a result of such conversion, the generated DNA-lipid has greater hydrophobicity than DNA-lipid-P and is thus able to insert into cell membranes in situ. Accordingly, DNA-lipid-P enables selective anchoring on cell membranes with elevated ALP level. Since elevated ALP level is a critical index of some diseases and even cancers, DNA-lipid-P holds promise for cell membrane engineering and disease diagnostics at the molecular level.


Assuntos
Fosfatase Alcalina/metabolismo , Membrana Celular/metabolismo , Sondas Moleculares/metabolismo , Oligonucleotídeos/metabolismo , Linhagem Celular Tumoral , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Sondas Moleculares/química , Oligonucleotídeos/química , Compostos Organofosforados/química , Fosforilação
11.
Talanta ; 200: 236-241, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036179

RESUMO

We report herein a rationally designed pyrene linked substrate for quantitative protease activity assay via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In this proof-of-concept study, a trypsin-specific peptide with the sequence of GGGGRG was selected to conjugate with pyrene forming a pyrene linked peptide probe, Py-GGGGRG. In the presence of trypsin, the Py-GGGGRG probe can be specifically hydrolyzed into Py-GGGGR. The introduction of pyrene greatly increased ionization efficiency of Py-peptides, and Py-peptides could be selectively captured from complex mixtures by a facially fabricated polystyrene coated MALDI plate through hydrophobic and π-π stacking interactions. As a result, trypsin activity can be directly quantified by relative intensity ratio of product and substrate via MALDI-TOF-MS without the use of external internal standard. A linear range of 0.1-10 µg/mL and a relatively low detection limit of 29 ng/mL were obtained. This method has also been successfully used for quantification of trypsin activity in urine and screening the inhibitors of trypsin. Besides, the proposed strategy was also validated for another protease, chymotrypsin, by using the probe Py-GGGGGGYG. Therefore, owing to simplicity, high-throughput capacity and quantificational accuracy, the proposed method shows great potential for activity assay of various proteases and screening their inhibitors via application of specific peptide sequences.


Assuntos
Sondas Moleculares/química , Peptídeo Hidrolases/análise , Peptídeos/química , Pirenos/química , Peptídeo Hidrolases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/análise , Tripsina/metabolismo
12.
Chem Commun (Camb) ; 55(45): 6417-6420, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31094364

RESUMO

An AIE dual-reactive supramolecular probe has been devised for the first time to simultaneously measure endogenous lipase and α-amylase activity in a homogeneous system. Fluorescence quantitative analysis of lipase and α-amylase in real biological samples enables rapid and accurate diagnosis of diseases.


Assuntos
Lipase/análise , Sondas Moleculares/química , alfa-Amilases/análise , Fluorescência , Humanos , Lipase/metabolismo , Substâncias Macromoleculares/química , alfa-Amilases/metabolismo
13.
Molecules ; 24(9)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31072045

RESUMO

A novel sensing system has been designed for the detection of cupric ions. It is based on the quenched fluorescence signal of carbon dots (CDs), which were carbonized from poly(vinylpyrrolidone) (PVP) and L-Cysteine (CYS). Cupric ions interact with the nitrogen and sulfur atoms on surface of the CDs to form an absorbed complex; this results in strong quenching of the fluorescence of the CDs via a fast metal-to-ligand binding affinity. The synthesized water-soluble CDs also exhibited a quantum yield of 7.6%, with favorable photoluminescent properties and good photostability. The fluorescence intensity of the CDs was very stable in high ionic strength (up to 1.0 M NaCl) and over a wide range of pH levels (2.0-12.0). This facile method can therefore develop a sensor that offers reliable, fast, and selective detection of cupric ions with a detection limit down to 0.15 µM and a linear range from 0.5 to 7.0 µM (R2 = 0.980). The CDs were used for cell imaging, observed that they were low toxicity to Tramp C1 cells and exhibited blue and green and red fluorescence under a fluorescence microscope. In summary, the CDs exhibited excellent fluorescence properties, and could be applied to the selective and sensitive detection of cupric ion and multicolor cell imaging.


Assuntos
Carbono/química , Cobre/análise , Imagem Tridimensional/métodos , Sondas Moleculares/síntese química , Pontos Quânticos/química , Animais , Carbono/toxicidade , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fluorescência , Íons , Camundongos Transgênicos , Sondas Moleculares/química , Espectroscopia Fotoeletrônica , Pontos Quânticos/toxicidade , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Poluentes Químicos da Água/análise
14.
J Chromatogr A ; 1601: 385-387, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31122730

RESUMO

Although the inverse gas chromatography technique has been used to measure the surface acid-base properties for almost 50 years, several basic problems still hinder its further development and application. One of the problems is that for the polar probe molecules of dichloromethane, acetone, ethyl acetate, tetrahydrofuran, and ethanol, the molecular area of each probe has different values in literature. In this paper, we point out the incorrect molecular area values presented in the literature by comparing these values with those indicated in Smallwood's Handbook of Organic Solvent Properties. The correct molecular area values of 11 polar probes are determined by plotting the molecular areas of the polar probes versus the van der Waals surface areas reported in the handbook. The correct molecular areas for six common polar probes, namely, dichloromethane, trichloromethane, acetone, ethyl acetate, diethyl ether, and tetrahydrofuran, are 38.0, 44.0, 42.5, 48.0, 47.0, and 45.0 Å2, respectively. Benzene is a specific molecule with a conjugated π-bond structure, and its molecular area is estimated to be larger than that of other molecules. Therefore, benzene is unsuitable for use as a probe molecule.


Assuntos
Cromatografia Gasosa/métodos , Cromatografia Gasosa/normas , Sondas Moleculares/química , Acetatos/química , Acetona/química , Ácidos/química , Etanol/química , Furanos/química , Cloreto de Metileno/química , Solventes/química , Propriedades de Superfície , Termodinâmica
15.
Inorg Chem ; 58(11): 7295-7302, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31091081

RESUMO

Cerium oxide (CeO x) with a reversible surface Ce3+/Ce4+ redox pair has played an important role in catalytic reactions, whereas catalase mimetics of CeO x have attracted little attention in the field of biotherapy. Herein, a smart photosensitizer-cerium oxide nanoprobe was developed to represent a promising paradigm in high-performance photodynamic therapy. The photosensitizer was linked to CeO x nanoparticles through a substrate peptide (EGPLGVRGK) of matrix metalloproteinase-2 (MMP-2). The smart nanoprobe could be converted from the "silent state" before arriving at the cancer cells to the "activated state" within the cells to turn on the fluorescence and 1O2 generation when the peptide linker (EGPLGVRGK) was cut by the cancer biomarker MMP-2. Moreover, CeO x played the role of an excellent catalase-like compound to decompose endogenous hydrogen peroxide to relieve tumor hypoxia. Via the conventional application of CeO x, our study showed innovatively how a smart nanoprobe could relieve tumor hypoxia and achieve a therapeutic effect for highly selective and efficient personalized treatment.


Assuntos
Cério/química , Cério/metabolismo , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Nanopartículas/química , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/química , Sequência de Aminoácidos , Transporte Biológico , Clorofila/análogos & derivados , Clorofila/química , Clorofila/farmacologia , Células Hep G2 , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Modelos Moleculares , Conformação Molecular , Peptídeos/química , Fármacos Fotossensibilizantes/farmacologia
16.
Chem Commun (Camb) ; 55(42): 5934-5937, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-31049519

RESUMO

A small-molecule photoacoustic probe based on cyanine dyes was developed by taking advantage of the nucleophilic substitution reaction of H2S with chlorine. The probe demonstrated specific response to H2S with ratiometric photoacoustic signals in the NIR region, which enabled real-time, accurate, high-resolution imaging of endogenous H2S in vivo.


Assuntos
Sulfeto de Hidrogênio/química , Sondas Moleculares/química , Técnicas Fotoacústicas/métodos , Animais , Cloro/química , Camundongos , Espectroscopia de Luz Próxima ao Infravermelho
17.
Expert Rev Med Devices ; 16(5): 341-350, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30990129

RESUMO

INTRODUCTION: Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose (FDG) presents some limitations for imaging of hepatocellular carcinoma (HCC), the most common primary hepatic malignancy. AREAS COVERED: The authors discuss the accuracy and limitations of FDG for HCC detection. Then, authors examine the recent advances in PET tracers other than FDG for the biological and prognostic characterization of HCC such as 11C-acetate, 11C-choline, and its 18F-labeled derivatives. EXPERT COMMENTARY: FDG PET can be helpful for the identification of the more aggressive and poorly differentiated HCC. 11C-acetate is readily incorporated into intracellular phosphatidylcholine membranes and proved useful for the in vivo biological characterization of the more differentiated and less aggressive HCC. Nevertheless, the short half-life of 11C- radionuclide limits the clinical application of this compound. 11C-choline, another surrogate biomarker of cell membrane biosynthesis, has been demonstrated effective for HCC imaging. The availability of choline derivatives labeled with 18F-radionuclide (i.e. 18F-fluoroethylcholine, 18F-fluorocholine) has overcome the drawbacks due to 11C, thus triggering the clinical applications of choline PET for HCC diagnosis and management. Further research needs to be conducted to better define the alternative or complementary role of these PET probes for the characterization of HCC, with particular regard to the dual-tracer PET-CT modality.


Assuntos
Carcinoma Hepatocelular/diagnóstico por imagem , Neoplasias Hepáticas/diagnóstico por imagem , Sondas Moleculares/química , Tomografia por Emissão de Pósitrons , Humanos , Imagem Molecular
18.
Chemistry ; 25(35): 8236-8239, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-30990914

RESUMO

Many smart magnetic resonance imaging (MRI) probes provide response to a biomarker based on modulation of their rotational correlation time. The magnitude of such MRI signal changes is highly dependent on the magnetic field and the response decreases dramatically at high fields (>2 T). To overcome the loss of efficiency of responsive probes at high field, with fast-field cycling magnetic resonance imaging (FFC-MRI) we exploit field-dependent information rather than the absolute difference in the relaxation rate measured in the absence and in the presence of the biomarker at a given imaging field. We report here the application of fast field-cycling techniques combined with the use of a molecular probe for the detection of Zn2+ to achieve 166 % MRI signal enhancement at 3 T, whereas the same agent provides no detectable response using conventional MRI. This approach can be generalized to any biomarker provided the detection is based on variation of the rotational motion of the probe.


Assuntos
Complexos de Coordenação/química , Gadolínio/química , Zinco/análise , Biomarcadores/análise , Técnicas Biossensoriais/métodos , Complexos de Coordenação/síntese química , Campos Eletromagnéticos , Ligantes , Limite de Detecção , Imagem por Ressonância Magnética/métodos , Sondas Moleculares/química , Albumina Sérica Humana/química , Termodinâmica
19.
Nat Chem ; 11(6): 552-561, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30936521

RESUMO

Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.


Assuntos
Alquinos/química , Sondas Moleculares/química , Prenilação de Proteína , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Espectrometria de Massas , Camundongos Knockout , Prenilação de Proteína/efeitos dos fármacos , Proteínas/química , Proteoma/química
20.
Nat Protoc ; 14(5): 1401-1424, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988508

RESUMO

The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically encoded glutamate indicators (GEGIs) allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed accelerated versions of GEGIs that allow investigation of synaptic release during 100-Hz trains. Here, we describe the detailed procedures for design and characterization of fast iGluSnFR variants in vitro, transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser-scanning microscopy. As the released glutamate spreads from a point source-the fusing vesicle-it is possible to localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By using a spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak amplitude of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.


Assuntos
Técnicas Biossensoriais/métodos , Ácido Glutâmico/análise , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia , Região CA3 Hipocampal/citologia , Células Cultivadas , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Humanos , Microscopia Confocal , Sondas Moleculares/análise , Sondas Moleculares/química , Sondas Moleculares/genética , Sondas Moleculares/metabolismo , Imagem Óptica , Transfecção
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