RESUMEN
Human serum albumin exerts multifunctions, such as maintaining the oncotic pressure of plasma, carrying hydrophobic molecules, and acting as the most important antioxidant in the blood. Lower serum albumin levels are linked to several cardiovascular diseases, and dysfunction of albumin reabsorption in the kidney is linked to liver disease, renal disorder, and diabetes. Albumin is thus a powerful diagnostic and prognostic marker; however, its quantification in urine by readily affordable tools is challenging owing to its very low concentration. To address this issue, we developed a ratiometric fluorescent probe with multiple advantages through a systematic structure variation of a benzocoumarin fluorophore and, further, a prototype of a smartphone-based point-of-care device. We determined albumin levels in urine and observed that a smoking person has notably higher urine albumin than a nonsmoking person. The cheap device provides a promising tool for albumin-associated disease diagnosis in communities with limited resources.
Asunto(s)
Líquidos Corporales , Albúmina Sérica Humana , Humanos , Sistemas de Atención de Punto , Albúminas , UrinálisisRESUMEN
Elastase, a serine protease, plays important roles in our body in food digestion and defence against pathogens. Particularly, the elastase present in neutrophils is directly associated with inflammatory bowel disease (IBD). Through a rational approach, we have developed a fluorescent elastase probe that has multiple advantages for biological applications including two-photon ratiometric imaging capability. Using the probe, which is capable of detecting intracellular elastase activity associated with inflammation, we have investigated elastase level changes in various mouse organs under an IBD condition for the first time. The results reveal notably higher elastase levels in the liver and duodenum of the healthy mice than in the other investigated organs. Under the IBD condition, we observed significant elastase level changes in the liver, duodenum, colon, and lung. The downregulation of elastase in the liver under the IBD condition suggests migration of neutrophils into the upregulated organs. The notable upregulation of elastase in the duodenum is explained by self-production of elastase, in addition to the neutrophil migration from the liver. We have observed little elastase level changes in selected organs of immune-deficient mice raised under the normal and IBD conditions, which supports the neutrophil migration as the reason for perturbed elastase activity in the healthy mice. The results also suggest an important role of the liver in maintaining the immune response associated with the inflammation-induced elastase level changes. The probe offers an ideal tool for mapping neutrophil migration in body. Further understanding of the elastase-associated biology and diagnosis of IBD by monitoring affected organs are anticipated using the probe.
Asunto(s)
Colitis , Enfermedades Inflamatorias del Intestino , Animales , Inflamación , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/diagnóstico por imagen , Elastasa de Leucocito , Ratones , NeutrófilosRESUMEN
Impaired hepatic glucose and lipid metabolism are hallmarks of type 2 diabetes. Increased sulfide production or sulfide donor compounds may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver- enriched mitochondrial SOP enzyme thiosulfate sulfurtransferase (Tst-/- mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia, and fatty liver. Unexpectedly, hepatic sulfide levels are normal in Tst-/- mice because of exaggerated induction of sulfide disposal, with associated suppression of global protein persulfidation and nuclear respiratory factor 2 target protein levels. Hepatic proteomic and persulfidomic profiles converge on gluconeogenesis and lipid metabolism, revealing a selective deficit in medium-chain fatty acid oxidation in Tst-/- mice. We reveal a critical role of TST in hepatic metabolism that has implications for sulfide donor strategies in the context of metabolic disease.
Asunto(s)
Diabetes Mellitus/patología , Dislipidemias/patología , Gluconeogénesis , Hígado/patología , Sulfuros/metabolismo , Tiosulfato Azufretransferasa/fisiología , Animales , Diabetes Mellitus/etiología , Diabetes Mellitus/metabolismo , Dislipidemias/etiología , Dislipidemias/metabolismo , Glucosa/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/metabolismo , Proteoma/metabolismoRESUMEN
Reactive oxygen species (ROS) produced by an inflammatory response in the brain are associated with various neurological disorders. To investigate ROS-associated neuroinflammatory diseases, fluorescent probes with practicality are in demand. We have investigated hypochlorous acid, an important ROS, in the brain tissues of neuroinflammation and maternal immune activation (MIA) model mice, using a new fluorescent probe. The probe has outstanding features over many known probes, such as providing two bright ratio signals in cells and tissues in deep-red/near-infrared wavelength regions with a large spectral separation, in addition to being strongly fluorescent, photo- and chemo-stable, highly selective and sensitive, fast responding, and biocompatible. We have found that the level of hypochlorous acid in the brain tissue of a neuroinflammatory mouse model was higher (2.7-4.0-fold) compared with that in normal brain tissue. Furthermore, the level of hypochlorous acid in the brain tissue of a MIA mouse model was higher (1.2-1.3-fold) compared with that in the normal brain tissue. The "robust" probe provides a practical tool for studying ROS-associated neurological disorders.
Asunto(s)
Encéfalo , Ácido Hipocloroso , Animales , RatonesRESUMEN
Human serum albumin (HSA) plays a pivotal role in drug release from its delivery vehicles such as cyclodextrins (CDs) by binding to the drugs. Here molecular recognition and binding of a drug mimic (CD1) to HSA have been explored in a microfluidic channel when CD1 is encapsulated in ß-cyclodextrin (ßCD) and heptakis(2,3,6-tri-O-methyl)-ß-cyclodextrin (TRIMEB), respectively, to investigate whether change of the host vehicle modulate the rate of drug binding to the serum protein. Molecular recognition of ßCD encapsulated CD1 by HSA occurs by the conformational selection fit mechanism leading to rapid binding of CD1 to HSA (k1 ~ 700 s-11) when the ßCD/CD1 complex interacts with HSA. In contrary, HSA recognizes CD1 encapsulated in TRIMEB by an induced fit mechanism leading to a significantly slower binding rate (k1 ~ 20.8 s-1) of the drug mimic to the protein. Thus molecular recognition controls the rate of HSA binding by CD1 which in turn modulates the rate of delivery of the drug mimic from its macrocyclic hosts. The remarkable change in the molecular recognition pathway of CD1 by HSA, upon change of the host from ßCD to TRIMEB, originates from significantly different conformational flexibility of the host/drug mimic complexes.
Asunto(s)
Albúmina Sérica Humana/química , Biomimética , Conformación de Carbohidratos , Ciclodextrinas/química , Liberación de Fármacos , Polarización de Fluorescencia , Humanos , Cinética , Microfluídica , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Albúmina Sérica Humana/metabolismo , Espectrometría de Fluorescencia , beta-Ciclodextrinas/químicaRESUMEN
Hypoxia, a condition of oxygen deficiency in tissues, features various diseases including solid tumor. Under hypoxia, several reductases such as nitroreductases are elevated. Based on this fact, we have investigated an indirect way to assess the hypoxia susceptibility of different organ tissues (mouse lung, heart, spleen, kidney, and liver) by detecting nitroreductase present within. Among the organs, the kidney showed a notable susceptibility to hypoxia, which was due to the renal medulla, not due to the renal cortex, as observed by ratiometric fluorescence imaging with a probe. The probe features ratiometric signaling, NIR-emitting, two-photon absorbing, and pH-insensitive emission properties, offering a practical tool for studying the nitroreductase activity and, furthermore, hypoxia-associated biological processes.
Asunto(s)
Colorantes Fluorescentes , Nitrorreductasas , Animales , Hipoxia , Ratones , Imagen Óptica , FotonesRESUMEN
The benzocoumarin dyes fluoresce negligibly in aqueous media but very strongly in cells, whereas representative conventional dyes display contrasting behaviour; the distinct emission behaviour of the fluorophores in organic solutions, in aqueous media, and in cell convinces the uniqueness of the cellular environment. The in cellulo superbright benzocoumarins also reveal an environment-insensitive emission behaviour, which is required for the reliable analysis via ratiometric imaging.
Asunto(s)
Cumarinas/química , Fluorescencia , Colorantes Fluorescentes/química , Línea Celular Tumoral , Humanos , SolucionesRESUMEN
Organic fluorophores emitting in the far-red/near-infrared (NIR) wavelength region are in great demand for minimal autofluorescence and reduced light scattering in deep tissue or whole body imaging. Currently, only a few classes of far-red/NIR fluorophores are available including widely used cyanine dyes, which are susceptible to photobleaching and form nonfluorescent aggregates. Even rare are those far-red/NIR emitting dyes that have two-photon imaging capability. Here we report a new class of far-red/NIR-emitting dyes that are photo-stable, very bright, biocompatible, and also two-photon absorbing. The introduction of an electron-withdrawing group such as N-acyl or N-alkoxycarbonyl groups on the C-10-amino substituent of the new julolidine-derived amino-Si-pyronin dyes (ASiPj), which emit in the far-red region, causes large bathochromic shifts, leading to NIR-emitting amino-Si-pyronin dyes (NIR-ASiPj) having high cellular stability. Furthermore, the ASiPj-NIR-ASiPj couple offers a novel ratiometric bioimaging platform with a large spectral gap, as demonstrated here with a boronate-containing NIR-ASiPj derivative that is converted to the corresponding ASiPj dye upon reaction with hydrogen peroxide.
RESUMEN
The promising features of fluorescence spectroscopy have inspired a quest for fluorescent probes for analysis and monitoring of molecular interactions in biochemical, medical, and environmental sciences. To overcome the competitive supramolecular interactions in aqueous media encountered with conventional molecular-recognition-based probes, the use of reaction-based probes that involve making or breaking of covalent bonds has emerged as a complementary sensing strategy to realize higher selectivity and sensitivity with larger spectroscopic changes. In spite of the enormous efforts, the development of reaction-based fluorescent probes meets with certain challenges in terms of their practical applications, demanding "intelligent design" of probes with an appropriate fluorophore attached to an efficient reactive moiety at the right place. This Account summarizes the results of our efforts made in the development and fine-tuning of reaction-based fluorescent probes toward those goals, classified by the type of analyte (anions, metal cations, and biomolecules) with notes on the challenges and achievements. The reaction-based approach was demonstrated to be powerful for the selective sensing of anions (cyanide and (amino)carboxylates) for the first time, and later it was extended to develop two-photon probes for bisulfite and fluoride ions. The reaction-based approach also enabled selective sensing of noble metal ions such as silver, gold, and palladium along with toxic (methyl)mercury species and paramagnetic copper ions. Furthermore, microscopic imaging and monitoring of biologically relevant species with reaction-based two-photon probes were explored for hydrogen sulfide, hypochlorous acid, formaldehyde, monoamine oxidase enzyme, and ATP.
Asunto(s)
Colorantes Fluorescentes/química , Adenosina Trifosfato/análisis , Ácidos Carboxílicos/análisis , Cianuros/análisis , Formaldehído/análisis , Sulfuro de Hidrógeno/análisis , Ácido Hipocloroso/análisis , Metales Pesados/análisis , Monoaminooxidasa/análisis , Monoaminooxidasa/metabolismo , Espectrometría de FluorescenciaRESUMEN
Enzyme-mediated catalysis is attributed to enzyme-substrate interactions, with models such as "induced fit" and "conformational selection" emphasizing the role of protein conformational transitions. The dynamic nature of the protein structure, thus, plays a crucial role in molecular recognition and substrate binding. As large-scale protein motions are coupled to water motions, hydration dynamics play a key role in protein dynamics, and hence, in enzyme catalysis. Here, microfluidic techniques and time-dependent fluorescence Stokes shift (TDFSS) measurements are employed to elucidate the role of nanoscopic water dynamics in the interaction of an enzyme, α-Chymotrypsin (CHT), with a substrate, Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) in the cationic reverse micelles of benzylhexadecyldimethylammonium chloride (BHDC/benzene) and anionic reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT/benzene). The kinetic pathways unraveled from the microfluidic setup are consistent with the "conformational selection" fit for the interaction of CHT with AMC in the cationic reverse micelles, whereas an "induced fit" mechanism is indicated for the anionic reverse micelles. In the cationic reverse micelles of BHDC, faster hydration dynamics (≈550â ps) aid the pathway of "conformational selection", whereas in the anionic reverse micelles of AOT, the significantly slower dynamics of hydration (≈1600â ps) facilitate an "induced fit" mechanism for the formation of the final enzyme-substrate complex. The role of water dynamics in dictating the mechanism of enzyme-substrate interaction becomes further manifest in the neutral reverse micelles of Brij-30 and Triton X-100. In the former, the faster water dynamics aid the "conformational selection" pathway, whereas the significantly slower dynamics of water molecules in the latter are conducive to the "induced fit" mechanism in the enzyme-substrate interaction. Thus, nanoscopic water dynamics act as a switch in modulating the pathway of recognition of an enzyme (CHT) by the substrate (AMC) in reverse micelles.
Asunto(s)
Quimotripsina/metabolismo , Cumarinas/metabolismo , Dispositivos Laboratorio en un Chip , Micelas , Oligopéptidos/metabolismo , Agua/metabolismo , Aniones/metabolismo , Cationes/metabolismo , Diseño de Equipo , Fluorescencia , Cinética , Especificidad por Sustrato , Tensoactivos/metabolismoRESUMEN
The maintenance of cell membrane fluidity is of critical importance for various cellular functions. At lower temperatures when membrane fluidity decreases, plants and cyanobacteria react by introducing unsaturation in the lipids, so that the membranes return to a more fluidic state. To probe how introduction of unsaturation leads to reduced membrane fluidity, a model cationic lipid dioctadecyldimethylammonium bromide (DODAB) has been chosen, and the effects of an unsaturated lipid monoolein (MO) on the structural dynamics and phase behavior of DODAB have been monitored by quasielastic neutron scattering and time-resolved fluorescence measurements. In the coagel phase, fluidity of the lipid bilayer increases significantly in the presence of MO relative to pure DODAB vesicles and becomes manifest in significantly enhanced dynamics of the constituent lipids along with faster hydration and orientational relaxation dynamics of a fluorophore. On the contrary, MO restricts both lateral and internal motions of the lipid molecules in the fluid phase (>330 K), which is consistent with relatively slow hydration and orientational relaxation dynamics of the fluorophore embedded in the mixed lipid bilayer. The present study illustrates how incorporation of an unsaturated lipid at lower temperatures (below the phase transition) assists the model lipid (DODAB) in regulating fluidity via enhancement of dynamics of the constituent lipids.
Asunto(s)
Cationes/química , Glicéridos/química , Membrana Dobles de Lípidos/química , Rastreo Diferencial de Calorimetría , Compuestos de Amonio Cuaternario/químicaRESUMEN
Lipid vesicles composed of cationic dioctadecyldimethylammonium bromide (DODAB) and neutral 1-monooleoyl-rac-glycerol (MO) are promising non-viral carriers of nucleic acids for delivery into cells. Among them, higher cell transfection efficiency was displayed by DODAB-rich vesicles than those enriched with MO. Structural relaxation of these mixed lipid vesicles plays a key role in their cell transfection efficiency because structural organization of the DODAB-rich vesicles are different from that of the MO-rich vesicles. Polarization-gated anisotropy in conjunction with picosecond resolved emission transients of a novel fluorophore 6-acetyl-(2-((4-hydroxycyclohexyl)(methyl)amino)naphthalene) (ACYMAN) has been employed to probe relaxation dynamics in pure DODAB vesicles, and in mixed vesicles of DODAB with varying content of MO. Both orientational relaxation of ACYMAN and relaxation dynamics of its local environment are retarded significantly in mixed lipid vesicles with increasing MO content, from a mole fraction (χ MO) of 0.2 to that of 0.8 which is consistent with increased rigidity of the MO-rich (χ MO > 0.5) vesicles relative to the DODAB-rich (χ MO < 0.5) vesicles. Therefore, higher structural rigidity of the MO-rich vesicles (χ MO > 0.5) gives rise to their lower cell transfection efficiency than the more flexible DODAB-rich (χ MO < 0.5) vesicles as observed in previous in vivo studies (Biochim. Biophys. Acta, Biomembr., 2014, 1838, 2555-2567).
RESUMEN
Fluorescent compounds with distinct photophysical properties are essential for the development of optical probes for chemical, biological, and environmental species, in addition to optoelectronic devices. In this context, we synthesized a series of 3-substituted-8-hydroxybenzo[g]coumarin derivatives and characterized their photophysical and cellular imaging properties. Being dipolar π-extended coumarin analogues, they have intramolecular charge-transfer character and good two-photon imaging capability, as shown for two selected dyes. Most of the dyes emit in a wavelength range of 530-580 nm in aqueous media and show large Stokes shifts as high as 197 nm. In spite of its dipolar nature, the 3-pyridinium-substituted derivative 5h notably shows insignificant solvatochromism as well as viscosity- and polarity-insensitive emission intensity, offering an ideal dye platform for probing biological targets. As a demonstration, we prepared an esterase probe based on it, which shows ratiometric sensing behavior.
RESUMEN
A fluorescent probe that enables ratiometric imaging of endogenous hypochlorous acid (HOCl) in cells and tissues by two-photon microscopy is developed based on a red-emitting acetyl-benzocoumarin (AcBC) dye. An oxathiolane group in the probe reacts with HOCl to generate the AcBC dye, which involves a ratiometric fluorescence change only toward HOCl along with high sensitivity.
Asunto(s)
Colorantes Fluorescentes/química , Ácido Hipocloroso/análisis , Animales , Encéfalo/metabolismo , Encéfalo/patología , Cumarinas/química , Células HeLa , Humanos , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica , Fotones , Rodaminas/química , Espectrometría de FluorescenciaRESUMEN
Decreased growth hormone (GH) and thyroid hormone (TH) signaling are associated with longevity and metabolic fitness. The mechanisms underlying these benefits are poorly understood, but may overlap with those of dietary restriction (DR), which imparts similar benefits. Recently we discovered that hydrogen sulfide (H2S) is increased upon DR and plays an essential role in mediating DR benefits across evolutionary boundaries. Here we found increased hepatic H2S production in long-lived mouse strains of reduced GH and/or TH action, and in a cell-autonomous manner upon serum withdrawal in vitro. Negative regulation of hepatic H2S production by GH and TH was additive and occurred via distinct mechanisms, namely direct transcriptional repression of the H2S-producing enzyme cystathionine γ-lyase (CGL) by TH, and substrate-level control of H2S production by GH. Mice lacking CGL failed to downregulate systemic T4 metabolism and circulating IGF-1, revealing an essential role for H2S in the regulation of key longevity-associated hormones.
Asunto(s)
Sulfuro de Hidrógeno/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Hígado/metabolismo , Animales , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Dextrotiroxina/metabolismo , Femenino , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Relaxation dynamics at the surface of biologically important macromolecules is important taking into account their functionality in molecular recognition. Over the years it has been shown that the solvation dynamics of a fluorescent probe at biomolecular surfaces and interfaces account for the relaxation dynamics of polar residues and associated water molecules. However, the sensitivity of the dynamics depends largely on the localization and exposure of the probe. For noncovalent fluorescent probes, localization at the region of interest in addition to surface exposure is an added challenge compared to the covalently attached probes at the biological interfaces. Here we have used a synthesized donor-acceptor type dipolar fluorophore, 6-acetyl-(2-((4-hydroxycyclohexyl)(methyl)amino)naphthalene) (ACYMAN), for the investigation of the solvation dynamics of a model protein-surfactant interface. A significant structural rearrangement of a model histone protein (H1) upon interaction with anionic surfactant sodium dodecyl sulphate (SDS) as revealed from the circular dichroism (CD) studies is nicely corroborated in the solvation dynamics of the probe at the interface. The polarization gated fluorescence anisotropy of the probe compared to that at the SDS micellar surface clearly reveals the localization of the probe at the protein-surfactant interface. We have also compared the sensitivity of ACYMAN with other solvation probes including coumarin 500 (C500) and 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran (DCM). In comparison to ACYMAN, both C500 and DCM fail to probe the interfacial solvation dynamics of a model protein-surfactant interface. While C500 is found to be delocalized from the protein-surfactant interface, DCM becomes destabilized upon the formation of the interface (protein-surfactant complex). The timescales obtained from this novel probe have also been compared with other femtosecond resolved studies and molecular dynamics simulations.
Asunto(s)
Colorantes Fluorescentes/química , Proteínas/química , Colorantes Fluorescentes/metabolismo , Histonas/química , Simulación de Dinámica Molecular , Reproducibilidad de los Resultados , Dodecil Sulfato de Sodio/química , Tensoactivos/químicaRESUMEN
Ratiometric imaging by two-photon microscopy can offer a viable tool for the relative quantification of biological analytes inside tissue with minimal influence from environmental factors that affect fluorescence signal. We demonstrate the ratiometric imaging of formaldehyde at the suborgan level using a two-photon fluorescent probe, which involves pixel-to-pixel ratiometric data transformation. This study reveals for the first time a high level of formaldehyde around the crypts of mouse small intestine, implicating its possible protective role along with the released antimicrobials from the Paneth cells.
Asunto(s)
Colorantes Fluorescentes/química , Formaldehído/análisis , Intestino Delgado/química , Imagen Óptica , Fotones , Animales , Ratones , Microscopía Fluorescente , Estructura MolecularRESUMEN
Ciprofloxacin, a fluoroquinolone antibiotic, is widely used for the treatment of bacterial infection in humans due to its broad antibacterial spectrum. An excessive use or overdose of ciprofloxacin on the other hand can cause several adverse effects not only to humans but also to microorganisms. Unabsorbed ciprofloxacin in the body is mostly excreted through urine and finally goes to the environment, providing a drug resistance pressure on bacteria. Hence a simple and efficient detection method of ciprofloxacin is necessary, which, for example, can be used to analyze ciprofloxacin content in urine. Although ciprofloxacin itself shows inherent fluorescence, direct fluorescent detection of ciprofloxacin in raw urine sample is difficult due to autofluorescence of urine by other components. Herein we report that a Tb(III) complex of DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) can be efficiently sensitized by ciprofloxacin to emit luminescence separately from the urine autofluorescence wavelength region. Tb-DO3A shows excellent sensitivity with a detection limit of three parts per billion in aqueous buffer solution. Further, Tb-DO3A is used to detect ciprofloxacin with high sensitivity and selectivity in a raw urine sample without any purification or separation procedures in the concentrations ranging from 1 µg·mL-1 to 50 µg·mL-1. The direct measurement of ciprofloxacin excreted in urine may be used to control overdose of the drug.
Asunto(s)
Ciprofloxacina/orina , Elementos de la Serie de los Lantanoides/química , Luminiscencia , Mediciones Luminiscentes/métodos , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Espectrometría de FluorescenciaRESUMEN
The selective detection of zinc ions in lysosomes over that in cytosol is achieved with a fluorescent probe, which enabled the fluorescence imaging of endogenous zinc ions in lysosomes of NIH 3T3 cells as well as mouse hippocampal tissues by two-photon microscopy under excitation at 900 nm.