RESUMO
We report the first diselenide-based probe for the selective detection of thioredoxin reductase (TrxR), an enzyme commonly overexpressed in melanomas. The probe design involves conjugation of a seminaphthorhodafluor dye with a diselenide moiety. TrxR reduces the diselenide bond, triggering a fluorescence turn-on response of the probe. Kinetic studies reveal favorable binding of the probe with TrxR with a Michaelis-Menten constant (Km ) of 15.89â µm. Computational docking simulations predict a greater binding affinity to the TrxR active site in comparison to its disulfide analogue. Inâ vitro imaging studies further confirmed the diselenide probe exhibited improved signaling of TrxR activity compared to the disulfide analogue.
Assuntos
Corantes Fluorescentes/uso terapêutico , Tiorredoxina Dissulfeto Redutase/metabolismo , HumanosRESUMO
The current five-year survival rate of <5% for pancreatic ductal adenocarcinoma (PDAC) is compounded by late diagnosis, a lack of PDAC-specific intraoperative guidance to ensure complete resection, and the ineffectiveness of current therapies. Previously, utilizing compound 1, a fluorophore with inherent PDAC selectivity, PDAC was visualized both in vivo and ex vivo in a murine model. In the current study, human PDAC tissue is targeted. Compound 1 selectively stains ducts of the adenocarcinoma versus the surrounding stroma, enabling the imaging of PDAC in frozen tissue sections with high contrast. To enhance the potential of 1 for intraoperative applications, the ex vivo staining protocol was optimized for rapid margin assessment, with a final staining time ofâ¯~15â¯min. To measure diagnostic performance, the area under a receiver operating characteristic (ROC) curve was measured for the identification of ductal adenocarcinoma vs. stroma. The bright fluorescence contrast enabled quantitative determination of PDAC (or precancerous PanIN lesions) versus healthy pancreas tissue in human tissue array samples.
Assuntos
Carcinoma Ductal Pancreático/diagnóstico por imagem , Imagem Óptica/métodos , Neoplasias Pancreáticas/diagnóstico por imagem , Animais , Humanos , CamundongosRESUMO
Fluorescent small molecules enable researchers and clinicians to visualize biological events in living cells, tissues, and organs in real time. Herein, the focus is on the structure and properties of the relatively rare benzo[ a]xanthenes that exhibit enhanced steric and electronic interactions due to their annulated structures. Three types of fluorophores were synthesized: (i) pH- and solvent-dependent seminaphthorhodafluors, (ii) pH- and solvent-independent seminaphthorhodafluors, and (iii) pH-independent but solvent-sensitive seminaphthorhodamines. The probes exhibited promising far-red to near-infrared (NIR) emission, large Stoke shifts, broad full width at half-maximum (fwhm), relatively high quantum yields, and utility in immunofluorescence staining. Deviation of the π-system from planarity due to changes in the fluorophore ionization state resulted in fluorescence properties that are atypical of common xanthene dyes.
Assuntos
Corantes Fluorescentes/química , Xantenos/química , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Imunofluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacocinética , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Molecular , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Rodaminas/síntese química , Rodaminas/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Eletricidade Estática , Relação Estrutura-Atividade , Xantenos/síntese química , Xantenos/farmacocinéticaRESUMO
E-cigarette aerosol emission studies typically focus on benchmarking toxicant levels versus those of cigarettes. However, such studies do not fully account for the distinct chemical makeup of e-liquids and their unique properties. These approaches often conclude that there are fewer and lower levels of toxins produced by e-cigarettes than by cigarettes. In 2015, we reported the discovery of new hemiacetals derived from the reaction of formaldehyde and the e-liquid solvents. The main finding was that they constituted a significant proportion of potentially undetected formaldehyde. Moreover, unlike gaseous formaldehyde, the hemiacetals reside in the aerosol particulate phase, and thus are capable of delivering formaldehyde more deeply into the lungs. However, the findings were criticized by those claiming that some of the results were obtained under conditions that are averse to vapers. A "reinvestigation" of our study was recently published addressing this latter issue. However, this reinvestigation ignored major details, including no mention of the formaldehyde hemiacetals. Herein, we isolated both gaseous formaldehyde and formaldehyde hemiacetals at an intermediate power level claimed, in the "reinvestigation", to be relevant to "non-averse," "normal" usage. The results were that both gaseous formaldehyde and formaldehyde from hemiacetals were produced at levels above OSHA workplace limits.
Assuntos
Acetais/isolamento & purificação , Aerossóis/isolamento & purificação , Formaldeído/isolamento & purificação , Acetais/toxicidade , Aerossóis/toxicidade , Cromatografia Líquida de Alta Pressão , Sistemas Eletrônicos de Liberação de Nicotina , Formaldeído/toxicidade , Espectroscopia de Ressonância MagnéticaRESUMO
Herein we utilize the similar though divergent nucleophilic properties of cysteine, homocysteine, and glutathione to achieve the selective detection of cysteine under mildly acidic conditions. This enables the specific in situ detection of lysosomal cysteine. Employing time-dependent fluorescent imaging of probe-labeled A549 cells, we demonstrate that dexamethasone-induced apoptosis is not dependent on lysosomal cysteine. This methodology can thus produce useful information about pathogenesis associated with cysteine and lysosomes.
Assuntos
Apoptose/efeitos dos fármacos , Cisteína/análise , Dexametasona/farmacologia , Lisossomos/química , Imagem Óptica , Células A549 , Animais , Humanos , Microscopia Confocal , Estrutura Molecular , Células Tumorais Cultivadas , Peixe-ZebraRESUMO
Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for highly multiplexed fluorescence microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four to five. Herein, we synthesized a library of 95 novel boron-dipyrromethene (BODIPY)-based fluorophores and screened their photophysical, optical and spectral properties for their utility in multicolor microscopy. A subset of our BODIPY-based fluorophores yielded varied length Stokes shifts probes, which were used to create a five-color image using a single excitation with confocal laser scanning microscopy for the first time. Combining these novel fluorophores with conventional fluorophores could facilitate imaging in up to nine to ten colors using linear unmixing based microscopy approaches.
RESUMO
Electronic cigarettes enabling enhanced airflow have grown in popularity in recent years. The objective of this study is to show that flow rates modulate the levels of specific aerosol toxicants produced in electronic cigarettes. Flow rates used in various laboratory investigations involving e-cigarettes have varied widely to date, and can thus promote interlaboratory variability in aerosol product profiles. The thermal decomposition of hydroxyacetone and glycolaldehyde is less favorable at lower temperatures, supporting the observations of these products at higher flow rates/lower heating coil temperatures. Higher temperatures promote the formation of acetaldehyde from hydroxyacetone and formaldehyde from both hydroxyacetone and glycolaldehyde. A separate finding is that greater airflow can also expose users to concerning levels of e-liquid solvents. Under the modest conditions studied, propylene glycol aerosol levels are found at above the acceptable inhalation levels defined by NASA, and in range of the generally recognized as safe levels for daily ingestion.
RESUMO
The electronic cigarette solvents propylene glycol and glycerol are known to produce toxic byproducts such as formaldehyde, acetaldehyde and acrolein. However, the aerosol toxin yield depends upon a variety of chemical and physical variables. The formaldehyde hemiacetals derived from these solvents were reported as major electronic cigarette aerosol components by us in 2015. In the study described herein, the formaldehyde hemiacetals were found at higher levels than those of free formaldehyde via an orthogonal sample collection protocol. In addition, the common aldehyde collection methods for electronic cigarettes, such as impingers and sorbent tubes containing DNPH, significantly underestimate the levels of formaldehyde. The reason for this is that formaldehyde hemiacetals follow other reaction pathways, such as the formation of a less reactive full cyclic acetal catalyzed by the acidity of the DNPH solution and the silica. We found that formaldehyde hemiacetals are a considerable fraction of the total formaldehyde produced in electronic cigarette that cannot be determined accurately by DNPH derivatization methods. Although the health effects of the hemiacetals are not yet known, they warrant further investigation.
Assuntos
Aerossóis/química , Sistemas Eletrônicos de Liberação de Nicotina , Formaldeído/análogos & derivados , Formaldeído/análise , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância MagnéticaRESUMO
BACKGROUND/OBJECTIVE: The heating of the fluids used in electronic cigarettes ("e-cigarettes") used to create "vaping" aerosols is capable of causing a wide range of degradation reaction products. We investigated formation of benzene (an important human carcinogen) from e-cigarette fluids containing propylene glycol (PG), glycerol (GL), benzoic acid, the flavor chemical benzaldehyde, and nicotine. METHODS/MAIN RESULTS: Three e-cigarette devices were used: the JUULTM "pod" system (provides no user accessible settings other than flavor cartridge choice), and two refill tank systems that allowed a range of user accessible power settings. Benzene in the e-cigarette aerosols was determined by gas chromatography/mass spectrometry. Benzene formation was ND (not detected) in the JUUL system. In the two tank systems benzene was found to form from propylene glycol (PG) and glycerol (GL), and from the additives benzoic acid and benzaldehyde, especially at high power settings. With 50:50 PG+GL, for tank device 1 at 6W and 13W, the formed benzene concentrations were 1.9 and 750 µg/m3. For tank device 2, at 6W and 25W, the formed concentrations were ND and 1.8 µg/m3. With benzoic acid and benzaldehyde at ~10 mg/mL, for tank device 1, values at 13W were as high as 5000 µg/m3. For tank device 2 at 25W, all values were ≤~100 µg/m3. These values may be compared with what can be expected in a conventional (tobacco) cigarette, namely 200,000 µg/m3. Thus, the risks from benzene will be lower from e-cigarettes than from conventional cigarettes. However, ambient benzene air concentrations in the U.S. have typically been 1 µg/m3, so that benzene has been named the largest single known cancer-risk air toxic in the U.S. For non-smokers, chronically repeated exposure to benzene from e-cigarettes at levels such as 100 or higher µg/m3 will not be of negligible risk.
Assuntos
Benzeno/química , Sistemas Eletrônicos de Liberação de Nicotina , Ciclização , Descarboxilação , Desidratação , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Espectroscopia de Ressonância Magnética , Nicotina/análise , OxirreduçãoRESUMO
Molecular probes that selectively highlight pancreatic cancer (PC) tissue have the potential to improve pancreatic ductal adenocarcinoma (PDAC) margin assessment through the selective highlighting of individual PC cells. Herein, we report a simple and unique family of systematically modified red and near-infrared fluorescent probes that exhibit a field-effect-derived redshift. Two of thirteen probes distributed to the normal mouse pancreas following systemic administration. One selectively accumulated in genetically modified mouse models of PDAC. The probe exhibited intracellular accumulation and enabled visualization of four levels of the structure, including the whole organ, resected tissue, individual cells, and subcellular organelles. In contrast to the small-molecule probes reported previously, it possesses an inherent affinity toward PDAC cells and thus does not require conjugation to any targeting agent. The fluorescent probe can thus promote new strategies not only for precision image-guided surgery, but also for PC detection, monitoring of therapeutic outcomes, and basic research.
RESUMO
The very close structural similarities between cysteine and homocysteine present a great challenge to achieve their selective detection using regular fluorescent probes, limiting the biological and pathological studies of these two amino thiols. A coumarin-based fluorescent probe was designed featuring pH-promoted distinct turn-on followed by ratiometric fluorescence responses for Cys and turn-on fluorescence response for Hcy through two different reaction paths. These specific responses demonstrate the activity differences between Cys and Hcy qualitatively for the first time. The probe could also be used for Cys and Hcy imaging in living cells.
Assuntos
Cumarínicos/química , Cisteína/química , Corantes Fluorescentes/química , Homocisteína/química , Técnicas Biossensoriais , Rastreamento de Células , Ciclização , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Cinética , Soluções , Espectrometria de Fluorescência/métodos , Compostos de Sulfidrila/químicaRESUMO
Cisplatin is widely used to treat a variety of cancers. However, ototoxicity and nephrotoxicity remain serious side effects of cisplatin-based chemotherapy. In order to inform the study of cisplatin's off-target effects, a new drug-fluorophore conjugate was synthesized that exhibited utility as a tracer to determine the cellular uptake and in vivo distribution of cisplatin. This probe will serve as a useful tool to facilitate investigations into the kinetics and biodistribution of cisplatin and its associated side effects in preclinical models after systemic administration.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Cisplatino/administração & dosagem , Cisplatino/uso terapêutico , Animais , Relação Dose-Resposta a Droga , Humanos , Neoplasias/tratamento farmacológicoRESUMO
Due to the biological and industrial importance of hypochlorous acid, the development of optical probes for HOCl has been an active research area. Hypochlorous acid and hypochlorite can oxidize electron-rich analytes with accompanying changes in molecular sensor spectroscopic profiles. Probes for such processes may monitor HOCl levels in the environment or in an organism and via bio-labeling or bioimaging techniques. This review summarizes recent developments in the area of chromogenic and fluorogenic chemosensors for HOCl.
Assuntos
Técnicas de Química Analítica/instrumentação , Corantes Fluorescentes/química , Ácido Hipocloroso/análise , Ácido Hipocloroso/química , Animais , Cor , Humanos , OxirreduçãoRESUMO
A series of geometrically well-defined cationic fluorophores were designed based on molecular mechanics. They contain biaryl linkers to impart rigidity preventing intramolecular folding between a conjugated biomolecule and fluorophore. All probes have absorption and emission maxima within 20 nm from Texas Red, as predicted by TDDFT calculations and validated experimentally.
RESUMO
Elevated homocysteine levels are a well-known independent risk factor for cardiovascular disease. To date, relatively few selective fluorescent probes for homocysteine detection have been reported. The lack of sensing reagents and remaining challenges largely derive from issues of sensitivity and/or selectivity. For example, homocysteine is a structural homologue of the more abundant (ca, 20-25 fold) aminothiol cysteine, differing only by an additional methylene group side chain. Fluorescein tri-aldehyde, described herein, has been designed and synthesized as a sensitive and selective fluorophore for the detection of homocysteine in human plasma samples. It responds to analytes selectively via a photoinduced electron transfer (PET) inhibition process that is modulated by predictable analyte-dye product hybridization and ionization states. Mulliken population analysis of fluorescein tri-aldehyde and its reaction products reveals that the characteristic formation of multiple cationic of homocysteine-derived heterocycles leads to enhanced relative negative charge build up on the proximal phenolate oxygen of the fluorophore as a contributing factor to selective emission enhancement.
Assuntos
Aldeídos/química , Fluoresceína/química , Corantes Fluorescentes/química , Homocisteína/sangue , Humanos , Estrutura Molecular , Espectrometria de FluorescênciaRESUMO
The first solid phase extraction materials for selective lysophosphatidic acid (LPA) enrichment from human plasma are described. Molecularly imprinted polymers were designed, synthesized and evaluated as cartridge fillings. They enabled a relatively rapid and simple extraction protocol for LPA without any need for multiple liquid-liquid extraction steps. The five major subspecies of lysophosphatidic acid are readily separated from all other native plasma phospholipids, including those well-known to interfere with LPA quantitation, such as phosphatidylcholine and lysophosphatidylcholine. Outstanding LPA purity is obtained via these solid phase materials in a tandem extraction setup.
Assuntos
Lisofosfolipídeos/sangue , Lisofosfolipídeos/isolamento & purificação , Impressão Molecular/métodos , Polímeros/química , Extração em Fase Sólida/métodos , Cromatografia Líquida de Alta Pressão , Humanos , Extração Líquido-Líquido , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A method for detecting glutathione selectively in whole blood deposited on filter paper is described. GSH is fractionated from proteins, hemoglobin and other potentially interfering components and determined using a resorufin-acrylate fluorescent probe. The relative standard deviation is lower than 5% (n = 5). Recoveries of GSH from whole blood are between 94% and 108.6%.
Assuntos
Análise Química do Sangue/métodos , Glutationa/sangue , Corantes Fluorescentes/química , Glutationa/química , Humanos , Concentração de Íons de Hidrogênio , Oxazinas/químicaRESUMO
Direct determination of total lysophosphatidic acid (LPA) was accomplished using newly developed spiroguanidines derived from rhodamine B as universal fluorogenic probes. Optimum conditions for the quantitative analysis of total LPA were investigated. The linear range for the determination of total LPA is up to 5 µM with a limit of detection of 0.512 µM.
Assuntos
Corantes Fluorescentes/química , Guanidinas/química , Lisofosfolipídeos/química , Rodaminas/química , Estrutura MolecularRESUMO
Many studies have shown that glutathione (GSH) and cysteine (Cys) / homocysteine (Hcy) levels are interrelated in biological systems. To unravel the complicated biomedical mechanisms by which GSH and Cys/Hcy are involved in various disease states, probes that display distinct signals in response to GSH and Cys/Hcy are highly desirable. In this work, we report a rhodol thioester (1) that responds to GSH and Cys/Hcy with distinct fluorescence emissions in neutral media. Probe 1 reacts with Cys/Hcy to form the corresponding deconjugated spirolactam via a tandem native chemical ligation (NCL) reaction. This intramolecular spirocyclization leads to the "quinone - phenol" transduction of rhodol dyes, and an excited-state intramolecular proton transfer (ESIPT) process between the phenolic hydroxyl proton and the aromatic nitrogen in the benzothiazole unit occurs upon photoexcitation, thus affording 2-(2'-hydroxyphenyl) benzothiazole (HBT) emission (454 nm). In the case of the tripeptide GSH, only transthioesterification takes place removing the intramolecular photo-induced electron transfer (PET) process caused by the electron deficient 4-nitrobenzene moiety giving rise to a large fluorescence enhancement at the rhodol emission band (587 nm). The simultaneous detection of GSH and Cys/Hcy is attributed to the significantly different rates of intramolecular S,N-acyl shift of their corresponding thioester adducts derived from 1. The utility of probe 1 has been demonstrated in various biological systems including serum and cells.
RESUMO
We report the detection of homocysteine over cysteine based upon characteristic differences between 5- and 6-membered heterocyclic amines formed upon reaction with aldehyde-bearing compounds. Homocysteine-derived thiazinane-4-carboxylic acids are more basic than cysteine-derived thiazolidines-4-carboxylic acids. Fluorescence enhancement in response to homocysteine is achieved by tuning pH and excitation wavelength.