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1.
Proc Natl Acad Sci U S A ; 117(26): 14694-14702, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554491

RESUMO

Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.


Assuntos
Encéfalo/enzimologia , DNA/química , Corantes Fluorescentes/química , Óxido Nítrico Sintase Tipo II , Animais , Encéfalo/metabolismo , Química Encefálica , DNA/metabolismo , Corantes Fluorescentes/metabolismo , Técnicas de Inativação de Genes , Camundongos , Microglia/química , Microglia/enzimologia , Microglia/metabolismo , Microscopia de Fluorescência , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Fagossomos/química , Fagossomos/metabolismo , Peixe-Zebra
2.
Nat Commun ; 11(1): 3114, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561744

RESUMO

Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immunoglobulin Gs (IgGs) at room temperature. The DNA origami epitopes (DOEs) allows programmed spatial distribution of epitope spikes, which enables direct imaging of functional complexes with atomic force microscopy (AFM). We establish the critical dependence of the IgG avidity on the lateral distance of epitopes within 3-20 nm at the single-molecule level. High-speed AFM imaging of transient conformations further provides structural and dynamic evidence for the IgG avidity from monovalent to bivalent in a single event, which sheds light on various applications including virus neutralization, diagnostic detection and cancer immunotherapy.


Assuntos
Afinidade de Anticorpos , Epitopos/ultraestrutura , Imunoglobulina G/ultraestrutura , Sondas Moleculares/ultraestrutura , Imagem Individual de Molécula/métodos , Complexo Antígeno-Anticorpo/ultraestrutura , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Epitopos/imunologia , Epitopos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Sondas Moleculares/imunologia , Sondas Moleculares/metabolismo , Nanotecnologia , Relação Estrutura-Atividade
3.
Nat Commun ; 11(1): 2828, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504003

RESUMO

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.


Assuntos
DNA de Cadeia Simples/metabolismo , RNA Polimerase III/metabolismo , Imagem Individual de Molécula/métodos , Fator de Transcrição TFIIIB/metabolismo , Transcrição Genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Transferência Ressonante de Energia de Fluorescência , Cinética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Sondas Moleculares/ultraestrutura , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Estabilidade Proteica , RNA Polimerase III/química , Proteínas Recombinantes/metabolismo , Proteína de Ligação a TATA-Box/metabolismo
4.
Br J Radiol ; 93(1111): 20200034, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32374626

RESUMO

Necrosis plays vital roles in living organisms which is related closely with various diseases. Non-invasively necrotic imaging can be of great values in clinical decision-making, evaluation of individualized treatment responses, and prediction of patient prognosis. This narrative review will demonstrate how the evolution of quinones for necrotic imaging has been promoted by searching for their active centers. In this review, we summarized the recent developments of various quinones with the continuous simplified π-conjugated cores in necrotic imaging and speculated their possible molecular mechanisms might be attributed to their intercalations with exposed DNA in necrotic tissues. We discussed their clinical challenges of necrotic imaging with quinones and their future translation studies deserved to be explored in personalized patient treatment.


Assuntos
Sondas Moleculares , Infarto do Miocárdio/patologia , Necrose/diagnóstico por imagem , Quinonas , Animais , Antraquinonas/química , Células/patologia , DNA/análise , Humanos , Sondas Moleculares/química , Infarto do Miocárdio/diagnóstico por imagem , Naftoquinonas/química , Quinonas/química , Quinonas/classificação , Ratos
5.
Nat Commun ; 11(1): 2399, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404879

RESUMO

The ability to monitor molecules volumetrically throughout the body could provide valuable biomarkers for studies of healthy function and disease, but noninvasive detection of molecular targets in living subjects often suffers from poor sensitivity or selectivity. Here we describe a family of potent imaging probes that can be activated by molecules of interest in deep tissue, providing a basis for mapping nanomolar-scale analytes without the radiation or heavy metal content associated with traditional molecular imaging agents. The probes are reversibly caged vasodilators that induce responses detectable by hemodynamic imaging; they are constructed by combining vasoactive peptides with synthetic chemical appendages and protein blocking domains. We use this architecture to create ultrasensitive biotin-responsive imaging agents, which we apply for wide-field mapping of targets in rat brains using functional magnetic resonance imaging. We also adapt the sensor design for detecting the neurotransmitter dopamine, illustrating versatility of this approach for addressing biologically important molecules.


Assuntos
Imagem Molecular/métodos , Sondas Moleculares/metabolismo , Peptídeos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Vasodilatadores/metabolismo , Animais , Biotina/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Dopamina/metabolismo , Células HEK293 , Humanos , Imagem por Ressonância Magnética/métodos , Sondas Moleculares/química , Neurotransmissores/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Ratos , Reprodutibilidade dos Testes , Vasodilatadores/química
6.
J Cancer Res Clin Oncol ; 146(8): 1941-1951, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32447486

RESUMO

PURPOSE: Currently, the routine screening program has insufficient capacity for the early diagnosis of lung cancer. Therefore, a type of chitosan-molecular beacon (CS-MB) probe was developed to recognize the miR-155-5p and image the lung cancer cells for the early diagnosis. METHODS: Based on the molecular beacon (MB) technology and nanotechnology, the CS-MB probe was synthesized self-assembly. There are four types of cells-three kinds of animal models and one type of histopathological sections of human lung cancer were utilized as models, including A549, SPC-A1, H446 lung cancer cells, tumor-initiating cells (TICs), subcutaneous and lung xenografts mice, and lox-stop-lox(LSL) K-ras G12D transgenic mice. The transgenic mice dynamically displayed the process from normal lung tissues to atypical hyperplasia, adenoma, carcinoma in situ, and adenocarcinoma. The different miR-155-5p expression levels in these cells and models were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The CS-MB probe was used to recognize the miR-155-5p and image the lung cancer cells by confocal microscopy in vitro and by living imaging system in vivo. RESULTS: The CS-MB probe could be used to recognize the miR-155-5p and image the lung cancer cells significantly in these cells and models. The fluorescence intensity trends detected by the CS-MB probe were similar to the expression levels trends of miR-155 tested by qRT-PCR. Moreover, the fluorescence intensity showed an increasing trend with the tumor progression in the transgenic mice model, and the occurrence and development of lung cancer were dynamically monitored by the differen fluorescence intensity. In addition, the miR-155-5p in human lung cancer tissues could be detected by the miR-155-5p MB. CONCLUSION: Both in vivo and in vitro experiments demonstrated that the CS-MB probe could be utilized to recognize the miR-155-5p and image the lung cancer cells. It provided a novel experimental and theoretical basis for the early diagnosis of the disease. Also, the histopathological sections of human lung cancer research laid the foundation for subsequent preclinical studies. In addition, different MBs could be designed to detect other miRNAs for the early diagnosis of other tumors.


Assuntos
Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/genética , MicroRNAs/análise , Células A549 , Animais , Quitosana/química , Detecção Precoce de Câncer/métodos , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Camundongos Transgênicos , MicroRNAs/biossíntese , MicroRNAs/genética , Imagem Molecular/métodos , Sondas Moleculares/química , Nanotecnologia
7.
Nat Commun ; 11(1): 1518, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251279

RESUMO

Size selectivity is an important mechanism for molecular recognition based on the size difference between targets and non-targets. However, rational design of an artificial size-selective molecular recognition system for biological targets in living cells remains challenging. Herein, we construct a DNA molecular sieve for size-selective molecular recognition to improve the biosensing selectivity in living cells. The system consists of functional nucleic acid probes (e.g., DNAzymes, aptamers and molecular beacons) encapsulated into the inner cavity of framework nucleic acid. Thus, small target molecules are able to enter the cavity for efficient molecular recognition, while large molecules are prohibited. The system not only effectively protect probes from nuclease degradation and nonspecific proteins binding, but also successfully realize size-selective discrimination between mature microRNA and precursor microRNA in living cells. Therefore, the DNA molecular sieve provides a simple, general, efficient and controllable approach for size-selective molecular recognition in biomedical studies and clinical diagnoses.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA Catalítico/química , Sondas Moleculares/química , Aptâmeros de Nucleotídeos/metabolismo , DNA Catalítico/metabolismo , MicroRNAs/metabolismo , Sondas Moleculares/metabolismo , Tamanho da Partícula , Precursores de RNA/metabolismo , Especificidade por Substrato
8.
Nat Chem Biol ; 16(5): 497-506, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32231343

RESUMO

We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.


Assuntos
Inibidores Enzimáticos/farmacologia , Nitrilos/química , Nitrilos/farmacologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Ferroptose/efeitos dos fármacos , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos SCID , Sondas Moleculares/química , Terapia de Alvo Molecular , Óxidos/química , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/química , Pró-Fármacos/química , Ratos Wistar , Selenocisteína/química , Selenocisteína/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
9.
Cancer Treat Res ; 180: 3-50, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32215865

RESUMO

Noninvasive imaging of functional and molecular changes in cancer has become an indispensable tool for studying cancer in vivo. Targeting the functional and molecular changes in cancer imaging provides a platform for the in vivo analysis of the mechanisms such as gene expression, signal transduction, biochemical reactions, regulatory pathways, cell trafficking, and drug action underlying cancer noninvasively. The main focus of imaging in cancer is the development of new contrast methods/molecular probes for the early diagnosis and the precise evaluation of therapy response. In clinical setup, imaging modalities facilitate screening, prediction, staging, biopsy and therapy guidance, therapy response, therapy planning, and prognosis of cancer. In this book chapter, we review different established and emerging in vivo imaging modalities and their applications in monitoring functional, molecular, and metabolic changes in cancer.


Assuntos
Neoplasias/diagnóstico por imagem , Meios de Contraste , Detecção Precoce de Câncer , Humanos , Sondas Moleculares
10.
Nat Commun ; 11(1): 1250, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144257

RESUMO

Currently, there are no non-invasive tools to accurately diagnose wound and surgical site infections before they become systemic or cause significant anatomical damage. Fluorescence and photoacoustic imaging are cost-effective imaging modalities that can be used to noninvasively diagnose bacterial infections when paired with a molecularly targeted infection imaging agent. Here, we develop a fluorescent derivative of maltotriose (Cy7-1-maltotriose), which is shown to be taken up in a variety of gram-positive and gram-negative bacterial strains in vitro. In vivo fluorescence and photoacoustic imaging studies highlight the ability of this probe to detect infection, assess infection burden, and visualize the effectiveness of antibiotic treatment in E. coli-induced myositis and a clinically relevant S. aureus wound infection murine model. In addition, we show that maltotriose is an ideal scaffold for infection imaging agents encompassing better pharmacokinetic properties and in vivo stability than other maltodextrins (e.g. maltohexose).


Assuntos
Corantes Fluorescentes/administração & dosagem , Imagem Molecular/métodos , Miosite/diagnóstico por imagem , Infecção da Ferida Cirúrgica/diagnóstico por imagem , Trissacarídeos/administração & dosagem , Animais , Carbocianinas/administração & dosagem , Carbocianinas/química , Modelos Animais de Doenças , Estabilidade de Medicamentos , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Injeções Intravenosas , Medições Luminescentes/métodos , Camundongos , Microscopia de Fluorescência/métodos , Sondas Moleculares/administração & dosagem , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Miosite/microbiologia , Técnicas Fotoacústicas/métodos , Ratos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo , Infecção da Ferida Cirúrgica/microbiologia , Trissacarídeos/química , Trissacarídeos/metabolismo
11.
Chem Commun (Camb) ; 56(17): 2610-2613, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32016272

RESUMO

We have synthesized a turn-on fluorescent probe, termed NB4OH, to detect cellular hypochlorite. NB4OH is mainly localized in the endoplasmic reticulum and detects ClO- in foam cells. The fluorescence change of the probe was explained by theoretical calculation as a PET process. The probe holds great promise for application in biomedical research, including atherosclerosis research.


Assuntos
Aterosclerose/metabolismo , Retículo Endoplasmático/metabolismo , Células Espumosas/metabolismo , Ácido Hipocloroso/metabolismo , Sondas Moleculares/metabolismo , Humanos , Espectrometria de Fluorescência
12.
Food Chem ; 317: 126433, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32092613

RESUMO

Highly catalytic and stable N-doped carbon dots (N-CDs) were prepared rapidly by microwave procedure using glucose as precursor and ammonium sulfite as N-dopant. The reduction of AgNO3 by trisodium citrate (TCA) was slow to form nanosilver (AgNP), and the N-CDs exhibited strong catalysis of the AgNP reaction. The formed AgNPs were used as indicator in the presence of Vitoria blue B (VBB) molecule probe with a SERS peak at 1615 cm-1. With the increase of nancatalyst N-CDs concentration, the AgNP reaction speed up, and the SERS peak of VBB enhanced linearly due to formation of more AgNPs as substrate. In the presence of avidin (Ad), the SERS peak weakened. Upon addition of biotin, the SERS peak enhanced due to turn on the indicator nanoreaction. The enhanced SERS signal had a good linear relationship with the biotin concentration in range of 0.0006-0.021 ng/mL, with a detection limit of 0.3 pg/mL.


Assuntos
Biotina/análise , Análise de Alimentos/métodos , Prata/química , Análise Espectral Raman/métodos , Animais , Avidina/química , Carbono/química , Catálise , Citratos/química , Análise de Alimentos/instrumentação , Limite de Detecção , Nanopartículas Metálicas/química , Leite/química , Técnicas de Sonda Molecular/instrumentação , Sondas Moleculares/química , Compostos Orgânicos/química , Pontos Quânticos/química , Nitrato de Prata/química , Análise Espectral Raman/instrumentação
13.
Nat Chem Biol ; 16(2): 170-178, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932721

RESUMO

C1 domains are lipid-binding modules that regulate membrane activation of kinases, nucleotide exchange factors and other C1-containing proteins to trigger signal transduction. Despite annotation of typical C1 domains as diacylglycerol (DAG) and phorbol ester sensors, the function of atypical counterparts remains ill-defined. Here, we assign a key role for atypical C1 domains in mediating DAG fatty acyl specificity of diacylglycerol kinases (DGKs) in live cells. Activity-based proteomics mapped C1 probe binding as a principal differentiator of type 1 DGK active sites that combined with global metabolomics revealed a role for C1s in lipid substrate recognition. Protein engineering by C1 domain swapping demonstrated that exchange of typical and atypical C1s is functionally tolerated and can directly program DAG fatty acyl specificity of type 1 DGKs. Collectively, we describe a protein engineering strategy for studying metabolic specificity of lipid kinases to assign a role for atypical C1 domains in cell metabolism.


Assuntos
Diacilglicerol Quinase/química , Diacilglicerol Quinase/metabolismo , Engenharia de Proteínas/métodos , Animais , Domínio Catalítico , Cromatografia Líquida , Diacilglicerol Quinase/genética , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Metabolômica/métodos , Sondas Moleculares/química , Ácidos Fosfatídicos/metabolismo , Domínios Proteicos , Proteômica/métodos , Ratos , Especificidade por Substrato , Espectrometria de Massas em Tandem
14.
Anal Chim Acta ; 1098: 66-74, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31948588

RESUMO

A streamlined analytical workflow was developed for the analysis of infant drink samples using a miniature mass spectrometry system preceded by solid-phase microextraction (SPME) and extraction nano-electrospray ionization. Potential chemical contaminants in infant drinks (milk, lactic acid bacteria beverage, and fruit juice) were extracted and enriched using a custom-made stainless-steel SPME probe, which was coated with a thin layer of polyaniline and multi-walled carbon nanotubes nanocomposites (PANI/MWCNTs) by electrochemical deposition. The resulting porous microstructure has a larger surface area and enhanced microextraction efficiency, with enrichment factors ranging from 3055 to 8695 for exemplary analytes of antibiotics, bisphenol A, and perfluorinated compounds. The enriched analytes on the electrochemically fabricated SPME probe were simultaneously desorbed and ionized within a pulled glass capillary by extraction nano-electrospray ionization. The ionized species were subjected to instrumental analysis on a miniature ion trap mass spectrometer with adequate tandem mass spectrometry capability. The developed method was optimized and validated in terms of sensitivity, linearity, repeatability, and recovery. The integrated experimental protocol combining SPME, ambient ionization, and miniature mass spectrometry is promising for rapid, on-site screening of hazardous substances in food to ensure consumer health.


Assuntos
Laticínios/análise , Técnicas Eletroquímicas , Contaminação de Alimentos/análise , Alimentos Infantis/análise , Sondas Moleculares/química , Microextração em Fase Sólida , Humanos , Lactente , Espectrometria de Massas
15.
World Neurosurg ; 138: 608-618, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31953096

RESUMO

This paper used magnetic resonance diffusion kurtosis imaging to observe the acute cerebral infarction model of mice, and studied the imaging changes of ischemic penumbra after perfusion of model for rat middle cerebral artery occlusion experiment, and combined with the physiologic changes of mice. The damage of neurons was evaluated by the evolution of N-methyl-D-aspartate receptors to provide a corresponding imaging basis for the diagnosis and treatment of ischemic penumbra. The research shows that the diffusivity value decreases with time, and the diffusion kurtosis increases with time. The difference in diffusivity between different parts of the same time point and the same part of the same point (except the edge relative to the normal area) is statistically different. Learning significance was set at P < 0.05. The expression of N-methyl-D-aspartate receptor 2A in tissue homogenate increased overall, and expression in synaptic membrane, synaptic membrane, and light membrane decreased. The expression of N-methyl-D-aspartate acid receptor 2B in tissue homogenate, synaptic membrane, and light cell membrane decreased, and it increased first and then decreased in the synaptic membrane. The studies confirmed that magnetic resonance imaging has a certain clinical diagnostic value for the penumbra evolution mechanism and neuronal injury of acute cerebral infarction, which deserves further study.


Assuntos
Infarto Cerebral/terapia , Imagem por Ressonância Magnética/métodos , Técnicas de Sonda Molecular , Células-Tronco Neurais/transplante , Paralisia/terapia , Células-Tronco Pluripotentes/transplante , Animais , Infarto Cerebral/diagnóstico por imagem , Infarto Cerebral/patologia , Membro Posterior/diagnóstico por imagem , Membro Posterior/patologia , Sondas Moleculares , Paralisia/diagnóstico por imagem , Ratos
16.
Chem Commun (Camb) ; 56(9): 1317-1324, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31904034

RESUMO

G-quadruplexes are nucleic acids secondary structures that can be formed in guanine-rich sequences. More than 30 years ago, their formation was first observed in telomeric DNA. Since then, a number of other sequences capable of forming G-quadruplex structures have been described and increasing evidence supporting their formation in the context of living cells has been accumulated. To fully underpin the biological significance of G-quadruplexes and their potential as therapeutic targets, several chemical-biology tools and methods have been developed to map and visualise these nucleic acids secondary structures in human cells. In this review, we critically present the most relevant methods developed to investigate G-quadruplex prevalence in human cells and to study their biological functions, presenting the next key chemical-biology challenges that need to be addressed to fully unravel G-quadruplex mediated biology and their therapeutic potential.


Assuntos
DNA/química , Quadruplex G , Genoma Humano , Sondas Moleculares/química , RNA/química , DNA/genética , Humanos , Ligação de Hidrogênio , RNA/genética
17.
Chem Commun (Camb) ; 56(10): 1464-1480, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31967621

RESUMO

This review discusses the advantages of using luminescent d6-transition-metal complexes as cell probes for optical microscopy. In particular it focusses on the Thomas group's use of specific complexes as "building blocks" toward the construction of biomolecular binding substrates, with DNA being a particular target. Using this approach, a range of new imaging probes for conventional optical microscopy, nanoscopy and transmission electron microscopy have been identified. Through selection of specific metal centres and by substitution of coordinated ligands we illustrate how new chemotherapeutics, photo-therapeutics, and theranostics have been identified and developed from the original architectures.


Assuntos
Complexos de Coordenação/química , DNA/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Apoptose/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Meios de Contraste/química , Meios de Contraste/metabolismo , Complexos de Coordenação/metabolismo , Complexos de Coordenação/toxicidade , DNA/metabolismo , Humanos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Nanomedicina Teranóstica
18.
Nat Commun ; 11(1): 81, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900403

RESUMO

Live-cell Raman imaging based on bioorthogonal Raman probes with distinct signals in the cellular Raman-silent region (1800-2800 cm-1) has attracted great interest in recent years. We report here a class of water-soluble and biocompatible polydiacetylenes with intrinsic ultrastrong alkyne Raman signals that locate in this region for organelle-targeting live-cell Raman imaging. Using a host-guest topochemical polymerization strategy, we have synthesized a water-soluble and functionalizable master polydiacetylene, namely poly(deca-4,6-diynedioic acid) (PDDA), which possesses significantly enhanced (up to ~104 fold) alkyne vibration compared to conventional alkyne Raman probes. In addition, PDDA can be used as a general platform for multi-functional ultrastrong Raman probes. We achieve high quality live-cell stimulated Raman scattering imaging on the basis of modified PDDA. The polydiacetylene-based Raman probes represent ultrastrong intrinsic Raman imaging agents in the Raman-silent region (without any Raman enhancer), and the flexible functionalization of this material holds great promise for its potential diverse applications.


Assuntos
Células/citologia , Imagem Molecular/métodos , Sondas Moleculares/química , Polímero Poliacetilênico/química , Análise Espectral Raman/métodos , Alquinos/química , Células/química , Células HeLa , Humanos , Imagem Molecular/instrumentação , Sondas Moleculares/síntese química , Polímero Poliacetilênico/síntese química , Análise Espectral Raman/instrumentação
19.
PLoS One ; 15(1): e0227341, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923258

RESUMO

Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability.


Assuntos
Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Malária/enzimologia , Animais , Antimaláricos/química , Permeabilidade da Membrana Celular , Humanos , Malária/diagnóstico por imagem , Malária/tratamento farmacológico , Sondas Moleculares/química , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Sulfonas , Triptofano
20.
Nat Rev Genet ; 21(3): 151-170, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31780816

RESUMO

Cell-free biology is the activation of biological processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent technical renaissance has facilitated high-yielding (grams of protein per litre), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biological design. These advances provide exciting opportunities to profoundly transform synthetic biology by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compounds, on-demand biomanufacturing, building cells from the bottom up, and next-generation educational kits.


Assuntos
Sistema Livre de Células , Expressão Gênica , Sondas Moleculares
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