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1.
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
2.
Yakugaku Zasshi ; 139(12): 1531-1538, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31787640

RESUMO

Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are powerful molecular imaging methods for examining disease-related factors in the whole body using specific imaging probes. Recently, we tried to develop molecular imaging probes that specifically visualize pathological factors associated with cancers and infectious diseases. Although survivin is highly expressed in several cancers, its expression is undetectable in non-dividing tissues. Thus, we developed several small molecular imaging probes that target survivin. These ligands not only showed high affinity for survivin protein, but also showed consistent cellular accumulation with respect to survivin expression levels, thereby indicating the feasibility of their backbones as scaffolds for tumor-specific imaging agents that target survivin. Prion diseases are fatal neurodegenerative diseases characterized by the deposition of amyloid plaques containing abnormal prion protein aggregates (PrPSc). Thus, we developed flavonoids, acridines, and benzofurans as PrPSc-imaging probes. A styrylchromone derivative ([123I]SC-OMe) appears to be a particularly promising SPECT radioligand for monitoring prion deposit levels in living brains. Gallium-68 is a positron emitter in clinical PET applications that can be produced by a 68Ge/68Ga generator without a cyclotron. Notably, we developed new adsorbents for 68Ge by introducing N-methylglucamine groups into the Sephadex series to serve as a hydrophilic polymer matrix. We also demonstrated that generator-eluted 68Ga-citrate could be used for PET imaging of infectious mouse models. Our polysaccharide-based 68Ge/68Ga generators were shown to be prospectively cost-effective production systems for 68Ga radiopharmaceuticals. This Review describes the major findings of these three studies and the future prospect of these fields.


Assuntos
Doenças Transmissíveis/diagnóstico por imagem , Imagem Molecular/métodos , Técnicas de Sonda Molecular , Sondas Moleculares , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Acridinas , Animais , Benzofuranos , Flavonoides , Radioisótopos de Gálio , Humanos , Camundongos , Neoplasias/metabolismo , Compostos Radiofarmacêuticos , Survivina/metabolismo
3.
Integr Biol (Camb) ; 11(7): 315-329, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31712825

RESUMO

Commensal bacteria must colonize host mucosal surfaces to exert health-promoting properties, and bind to gastrointestinal tract (GIT) mucins via their cell surface adhesins. Considerable effort has been directed towards discovery of pathogen adhesins and their ligands to develop anti-infective strategies; however, little is known about the lectin-like adhesins and associated carbohydrate ligands in commensals. In this study, an in silico approach was used to detect surface exposed adhesins in the human commensal Lactobacillus paracasei subsp. paracasei, a promising probiotic commonly used in dairy product fermentation that presents anti-microbial activity. Of the 13 adhesin candidates, 3 sortase-dependent pili clusters were identified in this strain and expression of the adhesin candidate genes was confirmed in vitro. Mass spectrometry analysis confirmed the presence of surface adhesin elongation factor Tu and the chaperonin GroEL, but not pili expression. Whole cells were subsequently incubated on microarrays featuring a panel of GIT mucins from nine different mammalian species and two human-derived cell lines and a library of carbohydrate structures. Binding profiles were compared to those of two known pili-producing lactobacilli, L. johnsonii and L. rhamnosus and all Lactobacillus species displayed overlapping but distinct signatures, which may indicate different abilities for regiospecific GIT colonization. In addition, L. paracasei whole cells favoured binding to α-(2 â†’ 3)-linked sialic acid and α-(1 â†’ 2)-linked fucose-containing carbohydrate structures including blood groups A, B and O and Lewis antigens x, y and b. This study furthers our understanding of host-commensal cross-talk by identifying potential adhesins and specific GIT mucin and carbohydrate ligands and provides insight into the selection of colonization sites by commensals in the GIT.


Assuntos
Adesinas Bacterianas/química , Carboidratos/química , Microbioma Gastrointestinal , Glicômica/métodos , Lactobacillus paracasei , Animais , Aderência Bacteriana , Simulação por Computador , Fucose/química , Humanos , Lactobacillus , Lactobacillus johnsonii , Lactobacillus rhamnosus , Ligantes , Técnicas de Sonda Molecular , Probióticos , Ligação Proteica , RNA Bacteriano/isolamento & purificação , Propriedades de Superfície
4.
Proc Natl Acad Sci U S A ; 116(49): 24574-24582, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31744869

RESUMO

RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having deficiencies. Here we convert the common reagent dimethyl sulfate into a useful probe of all 4 RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base-pairing interactions in cells. PAIR-MaP has superior resolution compared to alternative experiments, can resolve multiple sets of pairing interactions for structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and 2 bacterial messenger RNA 5' untranslated regions reveals functionally important and complex structures undetected by prior analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells.


Assuntos
RNA/química , Ésteres do Ácido Sulfúrico/química , Regiões 5' não Traduzidas , Pareamento de Bases , Sobrevivência Celular , Endorribonucleases/genética , Escherichia coli/genética , Humanos , Células Jurkat , Modelos Moleculares , Imagem Molecular/métodos , Técnicas de Sonda Molecular , Sondas Moleculares/química , Conformação de Ácido Nucleico , Nucleotídeos/química , RNA/genética , RNA Longo não Codificante/química , RNA Mensageiro/química , Sequências Reguladoras de Ácido Ribonucleico
5.
Nat Commun ; 10(1): 4526, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586057

RESUMO

Genetically encoded probes monitoring H2O2 fluctuations in living organisms are key to decipher redox signaling events. Here we use a new probe, roGFP2-Tpx1.C169S, to monitor pre-toxic fluctuations of peroxides in fission yeast, where the concentrations linked to signaling or to toxicity have been established. This probe is able to detect nanomolar fluctuations of intracellular H2O2 caused by extracellular peroxides; expression of human aquaporin 8 channels H2O2 entry into fission yeast decreasing membrane gradients. The probe also detects H2O2 bursts from mitochondria after addition of electron transport chain inhibitors, the extent of probe oxidation being proportional to the mitochondrial activity. The oxidation of this probe is an indicator of steady-state levels of H2O2 in different genetic backgrounds. Metabolic reprogramming during growth in low-glucose media causes probe reduction due to the activation of antioxidant cascades. We demonstrate how peroxiredoxin-based probes can be used to monitor physiological H2O2 fluctuations.


Assuntos
Citosol/química , Peróxido de Hidrogênio/análise , Técnicas de Sonda Molecular , Peroxirredoxinas/química , Membrana Celular/química , Genes Reporter , Peróxido de Hidrogênio/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Mitocôndrias/química , Sondas Moleculares/química , Oxirredução , Engenharia de Proteínas , Schizosaccharomyces
6.
PLoS One ; 14(9): e0221714, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31479470

RESUMO

Imaging mass cytometry (IMC) is a technique allowing visualization and quantification of over 40 biological parameters in a single experiment with subcellular spatial resolution, however most IMC experiments are limited to endpoint analysis with antibodies and DNA stains. Small molecules containing tellurium are promising probes for IMC due to their cell permeability, synthetic versatility, and most importantly their application to sequential labelling with isotopologous probes (SLIP) experiments. SLIP experiments with tellurium-containing probes allow quantification of intracellular biology at multiple timepoints with IMC. Despite the promise of tellurium in IMC, there are unique challenges in image processing associated with tellurium IMC data. Here, we address some of these issues by demonstrating the removal of xenon background signal, combining channels to improve signal-to-noise ratio, and calculating isotope transmission efficiency biases. These developments add accuracy to the unique temporal resolution afforded by tellurium IMC probes.


Assuntos
Citometria por Imagem/métodos , Sondas Moleculares , Telúrio , Animais , Humanos , Processamento de Imagem Assistida por Computador , Isótopos/química , Isótopos/farmacocinética , Jejuno/anatomia & histologia , Jejuno/metabolismo , Camundongos , Técnicas de Sonda Molecular , Sondas Moleculares/química , Sondas Moleculares/farmacocinética , Razão Sinal-Ruído , Técnica de Subtração , Telúrio/química , Telúrio/farmacocinética , Xenônio/farmacocinética
7.
Forensic Sci Int Genet ; 42: 227-234, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31377480

RESUMO

Next generation sequencing (NGS) technologies have enabled the possibility of analyzing a large number of SNPs simultaneously from multiple samples in a single experiment, for complementing the shortcomings of STR based methods. To efficiently genotype the desired SNPs, it is critical to optimize the library construction procedures. In this study, we formulated a strategy combining the molecular inversion probe (MIP) based target region capture method and NGS for genotyping 1245 SNPs. All the SNPs we selected exhibited high heterozygosity (minor allele frequency (MAF) > 0.3) according to 1000 genomes data. We applied the method to genotype a population of 210 unrelated individuals from the Hubei province of China and assessed the allele frequencies, Hardy-Weinberg equilibrium and linkage disequilibrium. The MAFs of more than 95% of the SNPs were ≥0.2, and no significant deviation or strong linkage was observed for 98% of the SNPs. The data indicated that, even within a relatively confined region, our SNP panel is suitable for individual identifications. Furthermore, we performed paternity test for 7 trio families using low quality DNA samples. The conclusions are in total agreement with these of STR-based analyses, with higher confidence indexes. Finally, we evaluated the performance of the MIP-NGS method with mock degraded DNA samples. We were able to genotype most of the SNPs even when the genomic DNA was sonicated to ˜100 bp range. In summary, we established a highly accurate and cost-effective method of SNP genotyping, which is potentially capable of solving complex issues encountered in forensic practices.


Assuntos
Genética Populacional , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Sonda Molecular , Polimorfismo de Nucleotídeo Único , China , Impressões Digitais de DNA , Grupos Étnicos/genética , Genética Forense/métodos , Frequência do Gene , Genótipo , Humanos
8.
Proc Natl Acad Sci U S A ; 116(37): 18285-18294, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31451653

RESUMO

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.


Assuntos
Cobre/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Glutationa/metabolismo , Técnicas de Sonda Molecular , Oncogenes/fisiologia , Transportador de Cobre 1/metabolismo , Fluoresceína , Células HEK293 , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Neoplasias/metabolismo , Oxirredução , Estresse Oxidativo , Rodaminas , Transdução de Sinais
9.
BMC Infect Dis ; 19(1): 738, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438880

RESUMO

BACKGROUND: The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5'UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results. METHODS: 100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5'UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank. RESULTS: All the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5'UTR and Core. Twenty-three samples were sequenced for two regions: 5' UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5'UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%. CONCLUSIONS: In this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular.


Assuntos
Técnicas de Genotipagem/métodos , Hepacivirus/genética , Hepatite C/diagnóstico , Técnicas de Sonda Molecular , Kit de Reagentes para Diagnóstico , Análise de Sequência de RNA/métodos , Regiões 5' não Traduzidas , Sequência de Bases , Comércio , Genômica/métodos , Genótipo , Técnicas de Genotipagem/economia , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Humanos , Técnicas de Sonda Molecular/economia , Filogenia , RNA Viral/análise , RNA Viral/isolamento & purificação , Kit de Reagentes para Diagnóstico/economia , Estudos Retrospectivos , Análise de Sequência de RNA/economia , Centros de Atenção Terciária
10.
J Dermatol ; 46(10): 917-921, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392741

RESUMO

While the etiology of sarcoidosis remains uncertain, mycobacteria have been suggested as a causative infectious agent. To investigate the causal relationship between mycobacteria and sarcoidosis, we performed a reverse blot hybridization assay (REBA) to identify mycobacteria from the skin samples of nine patients with sarcoidosis. Six of the nine samples were shown to be positive for mycobacteria by REBA, including Mycobacterium tuberculosis and non-tuberculous mycobacteria. This is the first study to identify mycobacteria from the skin samples of sarcoidosis patients using REBA, and our results could strengthen the etiologic association between mycobacteria and sarcoidosis.


Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Sarcoidose/microbiologia , Dermatopatias/microbiologia , Pele/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Sondas de DNA , DNA Bacteriano/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Sonda Molecular , Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas/genética , Reação em Cadeia da Polimerase , Sarcoidose/patologia , Pele/patologia , Dermatopatias/patologia
11.
PLoS One ; 14(7): e0220210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31344086

RESUMO

Photodynamic therapy (PDT) uses photosensitisers such as protoporphyrin IX (PpIX) to target tumours via the release of toxic singlet oxygen when irradiated. The effectivity of the treatment is limited by the innate properties of the photosensitizers; they typically exhibit inefficient accumulation in target tissue and high dark toxicity. Carbon dots (CDs) are biocompatible fluorescent nanoparticles which can improve PpIX cellular uptake and solubility. In this work, we present conjugates synthesised by host-guest encapsulation (PpIX@CD) and amide cross-linking (PpIX-CD). Characterization demonstrated conjugates have a loading efficiency of 34-48% and similar singlet oxygen production to PpIX. PpIX-containing CDs showed a 2.2 to 3.7-fold decrease in dark toxicity. PpIX-CD and PpIX@CD showed equivalent light-induced toxicity to PpIX in concentrations >1 µg/ml, leading to a 3.2 to 4.1-fold increase in photo-toxicity index (PI). The less soluble fraction of cross-linked conjugates (PpIX-CD)p did not show significant difference from PpIX. Confocal light scanning microscopy demonstrated rapid intracellular uptake and accumulation of conjugates. Our results demonstrate the variations between cross-linking strategies in CD-based conjugates, highlighting their potential as carriers in drug delivery and bioimaging applications.


Assuntos
Carbono/química , Diagnóstico por Imagem/métodos , Portadores de Fármacos/síntese química , Técnicas de Sonda Molecular , Fotoquimioterapia/métodos , Protoporfirinas/química , Portadores de Fármacos/química , Portadores de Fármacos/uso terapêutico , Sistemas de Liberação de Medicamentos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Teste de Materiais , Nanoconjugados/química , Nanopartículas/química , Nanotubos de Carbono/química , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/química , Oxigênio Singlete/química , Testes de Toxicidade , Células Tumorais Cultivadas
12.
Am J Physiol Endocrinol Metab ; 317(1): E74-E84, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939051

RESUMO

Intrinsically labeled dietary proteins have been used to trace various aspects of digestion and absorption, including quantifying the contribution of dietary protein to observed postprandial amino acid and protein kinetics in human subjects. Quantification of the rate of appearance in peripheral blood of an unlabeled (tracee) amino acid originating from an intrinsically labeled protein (exogenous Ra) requires the assumption that there is no dilution of the isotope enrichment of the protein-bound amino acid in the gastrointestinal tract or across the splanchnic bed. It must also be assumed that the effective volume of distribution into which the tracer and tracee appear can be reasonably estimated by a single value and that any recycling of the tracer is minimal and thus does not affect calculated rates. We have assessed these assumptions quantitatively using values from published studies. We conclude that the use of intrinsically labeled proteins as currently described to quantify exogenous Ra systematically underestimates the true value. When used with the tracer-determined rates of amino acid kinetics, underestimation of exogenous Ra from the intrinsically labeled protein method likely translates to incorrect conclusions regarding protein breakdown, including the effect of a protein meal and the anabolic impact of the speed of digestion and absorption of amino acids. Estimation of exogenous Ra from the bioavailability of ingested protein has some advantages as compared with the intrinsically labeled protein method. We therefore conclude that the bioavailability method for estimating exogenous Ra is preferable to the intrinsically labeled protein method.


Assuntos
Proteínas na Dieta/farmacocinética , Marcação por Isótopo/métodos , Proteínas/metabolismo , Imagem Corporal Total/métodos , Aminoácidos/metabolismo , Aminoácidos/farmacocinética , Disponibilidade Biológica , Deutério , Proteínas na Dieta/metabolismo , Estudos de Avaliação como Assunto , Humanos , Íleo/metabolismo , Absorção Intestinal/fisiologia , Cinética , Técnicas de Sonda Molecular , Período Pós-Prandial
13.
Glycoconj J ; 36(2): 175-183, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30993518

RESUMO

Lectins, in combination with our established enzyme-linked lectin sorbent assay (ELLSA) and inhibition study, have been used as powerful tools in many glycoconjugate recognition studies. In this short review, we highlight the following: (i) The recognition profiles of Gal/GalNAc-specific lectins were updated and upgraded. (ii) Based on the cross-specificities of applied lectins, a new classification system was introduced. (iii) Applications of lectins for the detection and identification of N-glycan and/or Tn glycotope in glycoconjugates were intergraded. (iv) The polyvalency of the glycotopes in glycans was found to play a critical role in glycan-lectin recognition. This is an unexplored area of glycobiology and one of the most promising directions toward the coming glycoscience transformation.


Assuntos
Glicômica/métodos , Lectinas/metabolismo , Animais , Humanos , Lectinas/química , Técnicas de Sonda Molecular , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica
14.
Artigo em Inglês | MEDLINE | ID: mdl-30925315

RESUMO

An efficient and novel 2,5-bis(benzo[d]thiazol-2-yl)phenol scaffold-based ratiometric fluorescent probe BTP-Cys for the sensing of cysteine has been developed. The probe BTP-Cys with acrylates moiety, as recognition site, has been successfully constructed on account of the excited state intramolecular proton transfer (ESIPT) mechanism. Upon the treatment with Cys (0-250 µM), this probe BTP-Cys exhibits a dramatic fluorescent intensity ratios enhancement (from 0.03 to 18.3) and a large emission shift (113 nm). The detection limit of this probe is as low as 3.8 × 10-7 M. Importantly, the concentration and time dependent of Cys in bovine serum albumin (BSA) has also been measured, indicating that BTP-Cys could be a biocompatible and rapid probe for Cys in vitro.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Fenóis/química , Soroalbumina Bovina/análise , Animais , Bovinos , Cisteína/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Técnicas de Sonda Molecular , Soroalbumina Bovina/química , Espectrometria de Fluorescência
15.
Plant J ; 99(2): 270-285, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30900785

RESUMO

Compartmentation of photosynthetic reactions between mesophyll and bundle sheath cells is a key feature of C4 photosynthesis and depends on the cell-specific accumulation of major C4 enzymes, such as phosphoenolpyruvate carboxylase 1. The ZmPEPC1 upstream region, which drives light-inducible and mesophyll-specific gene expression in maize, has been shown to keep the same properties when introduced into rice (C3 plant), indicating that rice has the transcription factors (TFs) needed to confer C4 -like gene expression. Using a yeast one-hybrid approach, we identified OsbHLH112, a rice basic Helix-Loop-Helix (bHLH) TF that interacts with the maize ZmPEPC1 upstream region. Moreover, we found that maize OsbHLH112 homologues, ZmbHLH80, and ZmbHLH90, also interact with the ZmPEPC1 upstream region, suggesting that these C4 regulators were co-opted from C3 plants. A transactivation assay in maize mesophyll protoplasts revealed that ZmbHLH80 represses, whereas ZmbHLH90 activates, ZmPEPC1 expression. In addition, ZmbHLH80 was shown to impair the ZmPEPC1 promoter activation caused by ZmbHLH90. We showed that ZmbHLH80 and ZmbHLH90 bind to the same cis-element within the ZmPEPC1 upstream region either as homodimers or heterodimers. The formation of homo- and heterodimers with higher oligomeric forms promoted by ZmbHLH80 may explain its negative effect on gene transcription. Gene expression analysis revealed that ZmbHLH80 is preferentially expressed in bundle sheath cells, whereas ZmbHLH90 does not show a clear cell-specific expression pattern. Altogether, our results led us to propose a model in which ZmbHLH80 contributes to mesophyll-specific ZmPEPC1 gene expression by impairing ZmbHLH90-mediated ZmPEPC1 activation in the bundle sheath cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas de Plantas/fisiologia , Zea mays/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Sonda Molecular , Oryza/genética , Fotossíntese/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Zea mays/metabolismo
16.
Nat Commun ; 10(1): 1111, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30846702

RESUMO

Activated macrophages have the potential to be ideal targets for imaging inflammation. However, probe selectivity over non-activated macrophages and probe delivery to target tissue have been challenging. Here, we report a small molecule probe specific for activated macrophages, called CDg16, and demonstrate its application to visualizing inflammatory atherosclerotic plaques in vivo. Through a systematic transporter screen using a CRISPR activation library, we identify the orphan transporter Slc18b1/SLC18B1 as the gating target of CDg16.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Inflamação/diagnóstico por imagem , Inflamação/imunologia , Ativação de Macrófagos , Acridinas , Animais , Sistemas CRISPR-Cas , Células HeLa , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Knockout para ApoE , Técnicas de Sonda Molecular , Sondas Moleculares , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/metabolismo , Células RAW 264.7
17.
SLAS Technol ; 24(3): 298-307, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30707854

RESUMO

Multiplexing strategies, which greatly increase the number of simultaneously measured parameters in single experiments, are now being widely implemented by both the pharmaceutical industry and academic researchers. Color has long been used to identify biological signals and, when combined with molecular barcodes, has substantially enhanced the depth of multiplexed sample characterization. Moreover, the recent advent of DNA barcodes has led to an explosion of innovative cell sequencing approaches. Novel barcoding strategies also show great promise for encoding spatial information in transcriptomic studies, and for precise assessment of molecular abundance. Both color- and DNA-based barcodes can be conveniently analyzed with either a microscope or a cytometer, or via DNA sequencing. Here we review the basic principles of several technologies used to create barcodes and detail the type of samples that can be identified with such tags.


Assuntos
Técnicas Citológicas/métodos , Técnicas de Sonda Molecular , Coloração e Rotulagem/métodos , Automação Laboratorial/métodos , Citometria de Fluxo , Ensaios de Triagem em Larga Escala/métodos , Microscopia
18.
Biotechnol J ; 14(7): e1800645, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30791223

RESUMO

Herein, the ribonuclease H (RNase H) activity assay based on the target-activated DNA polymerase activity is described. In this method, a detection probe composed of two functional sequences, a binding site for DNA polymerase and a catalytic substrate for RNase H, serves as a key component. The detection probe, at its initial state, suppresses the DNA polymerase activity, but it becomes destabilized by RNase H, which specifically hydrolyzes RNA in RNA/DNA hybrid duplexes. As a result, DNA polymerase recovers its activity and initiates multiple primer extension reactions in a separate TaqMan probe-based signal transduction module, leading to a significantly enhanced fluorescence "turn-on" signal. This assay can detect RNase H activity as low as 0.016 U mL-1 under optimized conditions. Furthermore, its potential use for evaluating RNase H inhibitors, which have been considered potential therapeutic agents against acquired immune deficiency syndrome (AIDS), is successfully explored. In summary, this approach is quite promising for the sensitive and accurate determination of enzyme activity and inhibitor screening.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Descoberta de Drogas/métodos , Ensaios Enzimáticos/métodos , Técnicas de Sonda Molecular , Ribonuclease H , Estabilidade Enzimática , Ribonuclease H/análise , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/metabolismo
19.
Biotechnol J ; 14(5): e1800647, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30810268

RESUMO

Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody-binding protein as an adapter can simplify antibody functionalization by forming a specific antibody-bound complex and introducing site-specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody-binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein ("lasso"), which binds human immunoglobulin G1 (IgG1) with K D = 0.53 n m and a dissociation rate that is 55- to 84-fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme-linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12-fold. The small size of the lasso and a long half-life of dissociation make the peptide a useful tool in antibody detection and immobilization.


Assuntos
Afinidade de Anticorpos/imunologia , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Peptídeos/química , Domínios Proteicos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Sítios de Ligação , Sítios de Ligação de Anticorpos , Biotina , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Cisteína/química , Ensaio de Imunoadsorção Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/imunologia , Humanos , Imobilização , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Cinética , Modelos Moleculares , Técnicas de Sonda Molecular , Peptídeo Hidrolases , Ligação Proteica , Especificidade por Substrato , Leveduras
20.
RNA ; 25(1): 82-89, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30309880

RESUMO

Many approaches exist to detect RNA using complementary oligonucleotides. DNA ligation-based techniques can improve discrimination of subtle sequence variations, but they have been difficult to implement for direct RNA analysis due to the infidelity and inefficiency of most DNA ligases on RNA. In this report, we have systematically studied if ribonucleotide substitutions in padlock probes can provide higher catalytic efficiencies for Chlorella virus DNA ligase (PBCV-1 DNA ligase) and T4 RNA ligase 2 (T4Rnl2) on RNA. We provide broad characterization of end-joining fidelity for both enzymes in RNA-templated 3'-OH RNA/5'-pDNA chimeric probe ligation. Both ligases showed increased ligation efficiency toward chimeric substrates on RNA. However, end-joining fidelity of PBCV-1 DNA ligase remained poor, while T4Rnl2 showed a somewhat better end-joining fidelity compared to PBCV-1 DNA ligase. The recently presented invader padlock (iLock) probes overcome the poor end-joining fidelity of PBCV-1 DNA ligase by the requirement of target-dependent 5' flap removal prior to ligation. Here we show that two particular ribonucleotide substitutions greatly improve the activation and ligation rate of chimeric iLock probes on RNA. We characterized the end-joining efficiency and fidelity of PBCV-1 DNA ligase and T4Rnl2 with chimeric iLock probes on RNA and found that both enzymes exhibit high ligation fidelities for single nucleotide polymorphisms on RNA. Finally, we applied the chimeric probe concept to directly differentiate between human and mouse ACTB mRNA in situ, demonstrating chimeric padlock and iLock probes as superior to their DNA equivalents.


Assuntos
Técnicas de Sonda Molecular , Sondas de Oligonucleotídeos/genética , RNA/análise , RNA/genética , Actinas/genética , Animais , Sequência de Bases , DNA Ligases/metabolismo , Humanos , Camundongos , RNA Ligase (ATP)/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Especificidade da Espécie , Especificidade por Substrato , Proteínas Virais/metabolismo
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