Search

The development of new fluorescent probes for cellular imaging is currently a very active field because of the large potential in understanding cell physiology, especially targeting anomalous behaviours due to disease. In particular, red-emitting dyes are keenly sought, as the light in this spectral region presents lower interferences and a deeper depth of penetration in tissues. In this work, we have synthesized a red-emitting, dual probe for the multiplexed intracellular detection of biothiols and phosphate ions. We have prepared a fluorogenic construct involving a silicon-substituted fluorescein for red emission. The fluorogenic reaction is selectively started by the presence of biothiols. In addition, the released fluorescent moiety undergoes an excited-state proton transfer reaction promoted by the presence of phosphate ions, which modulates its fluorescence lifetime, τ, with the total phosphate concentration. Therefore, in a multidimensional approach, the intracellular levels of biothiols and phosphate can be detected simultaneously using a single fluorophore and with spectral clearing of cell autofluorescence interferences. We have applied this concept to different cell lines, including photoreceptor cells, whose levels of biothiols are importantly altered by light irradiation and other oxidants. [ABSTRACT FROM AUTHOR]

Recently, it was proposed that the thiophene ring is capable of promoting mitochondrial accumulation when linked to fluorescent markers. As a noncharged group, thiophene presents several advantages from a synthetic point of view, making it easier to incorporate such a side moiety into different molecules. Herein, we confirm the general applicability of the thiophene group as a mitochondrial carrier for drugs and fluorescent markers based on a new concept of nonprotonable, noncharged transporter. We implemented this concept in a medicinal chemistry application by developing an antitumor, metabolic chimeric drug based on the pyruvate dehydrogenase kinase (PDHK) inhibitor dichloroacetate (DCA). The promising features of the thiophene moiety as a noncharged carrier for targeting mitochondria may represent a starting point for the design of new metabolism-targeting drugs. [ABSTRACT FROM AUTHOR]

In this work the preferential accumulation of long-lived acridone derivatives in mitochondria were evaluated, based on a new concept of non-protonable and non-charged group carriers. In this sense, the thiophene ring has been proved to act, for the first time, as mitochondria targeting group. Moreover, the unique long lifetime of the dyes has allowed the determination of the polarity of the organelles by fluorescence lifetime imaging microscopy (FLIM), without causing remarkable cytotoxicity. The neutral nature of the vectoring group favors the synthetic process and prevents the modification of different organelle parameters as membrane potential, making these probes excellent candidates to organelle tracking. Image 1 • Thiophene ring acts as unreported non-charged mitochondrial targeting group. • Long-lived fluorescent probes allow polarity determination inside mitochondria and lysosomes. • Co-localization in organelles has been analyzed with FLIM microscopy taking advantage of time-gated technique. [ABSTRACT FROM AUTHOR]

• A new methodology for accurate microenvironment polarity maps is developed. • The method entails the orthogonal combination of lifetime and ratiometric imaging. • Long-lifetime acridone derivatives are excellent dyes for the new methodology. • Experimental and computational insights into the dyes' photophysics are reported. • Our new method can be extended to other environment targets, such as viscosity or pH. The cellular microenvironment is a complex medium due to high concentrations of proteins and an intertwined framework of cellular organelles. In particular, cellular micro-polarity controls several biological processes, since it modulates hydrophobic/hydrophilic interactions and, hence, recognition, signalling and binding events. In this work, we have developed an unprecedented methodology to construct accurate environment polarity images using multiparametric fluorescence microscopy, via a multi-linear calibration of orthogonal parameters: the fluorescence lifetime and the spectral shift of a series of solvatochromic dyes. For this approach, we have synthesized and fully characterized N-substituted 2-methoxy-9-acridone dyes as suitable bioimaging polarity probes. However, to fully comprehend the complex links between microenvironment polarity and the dyes' properties, we have endeavoured a multidisciplinary approach, in which we have studied the photophysics of our fluorophores using spectroscopic tools and state-of-the-art computational chemistry. This profound knowledge permitted to use these dyes as intracellular polarity probes, quantitatively and robustly probing the microenvironment of different cellular compartments. Our new methodology may pave the way to further developments in accurate sensing of cellular microenvironment parameters. [ABSTRACT FROM AUTHOR]

In this work, we have performed an in-depth study of the photophysics and solvatofluorochromism of a red-emitting Si-xanthene dye, an analog of Tokyo Magenta (TM) historically developed by Egawa et al. (Chem. Commun. 2011 , 47 , 4162–4164). The results show a strong dependency of the emission properties of 2-Me-4-OMe-TM on the polarity of the solvent. For instance, the dye exhibited an increase in its fluorescence lifetime with the decrease in solvent polarity. Therefore, in this work, this spectral behavior has been used as a new approach for determining the intracellular microenvironment polarity through the measurement of its fluorescence lifetime by Fluorescence Lifetime Imaging Microscopy (FLIM). Our experiments confirmed the ability of the dye to detect changes in polarity between different intracellular compartments. Image 1 • A silicon-substituted xanthene dye was synthesized and its photophysics studied. • The dye shows a strong solvatochromic behavior involving two emission bands. • A strong dependency on the solvent's acidity was observed in the red-shifted band. • The dye works as an intracellular polarity fluorescence lifetime sensor. [ABSTRACT FROM AUTHOR]

Background and Purpose: Pharmacological strategies aimed to facilitate neuronal renewal in the adult brain, by promoting endogenous neurogenesis, constitute promising therapeutic options for pathological or traumatic brain lesions. We have previously shown that non-tumour-promoting PKC-activating compounds (12-deoxyphorbols) promote adult neural progenitor cell (NPC) proliferation in vitro and in vivo, enhancing the endogenous neurogenic response of the brain to a traumatic injury. Here, we show for the first time that a diterpene with a lathyrane skeleton can also activate PKC and promote NPC proliferation.Experimental Approach: We isolated four lathyranes from the latex of Euphorbia plants and tested their effect on postnatal NPC proliferation, using neurosphere cultures. The bioactive lathyrane ELAC (3,12-di-O-acetyl-8-O-tigloilingol) was also injected into the ventricles of adult mice to analyse its effect on adult NPC proliferation in vivo.Key Results: The lathyrane ELAC activated PKC and significantly increased postnatal NPC proliferation in vitro, particularly in synergy with FGF2. In addition ELAC stimulated proliferation of NPC, specifically affecting undifferentiated transit amplifying cells. The proliferative effect of ELAC was reversed by either the classical/novel PKC inhibitor Gö6850 or the classical PKC inhibitor Gö6976, suggesting that NPC proliferation is promoted in response to activation of classical PKCs, particularly PKCß. ELAC slightly increased the proportion of NPC expressing Sox2. The effects of ELAC disappeared upon acetylation of its C7-hydroxyl group.Conclusions and Implications: We propose lathyranes like ELAC as new drug candidates to modulate adult neurogenesis through PKC activation. Functional and structural comparisons between ELAC and phorboids are included. [ABSTRACT FROM AUTHOR]

• A bacterial pepN activatable fluorescent biosensor based on ratiometric measurement. • The biosensor can be a tool to determine virulent bacteria in a few minutes. • NIR N-aminopeptidase fluorescent sensor useful in STED and excitable by two photons. • STED measurements show the hot spots where the enzymatic reaction begins. • We locate the sites where alanine amino peptidase is placed in bacteria. Bacterial proteases are relevant in pathological processes such as the survival, growth and development of microorganisms. In particular alanine amino peptidase (pepN), which is present in Gram (−) bacteria, is the only peptidase responsible for the ATP-independent degradation of cytosolic proteins in E. coli. These peptidases, including pepN, might be useful targets to fight bacterial infections, which are difficult to treat due to the increase in antibiotic resistance, as well as for diagnosis. In this work, we propose a new methodology for the identification of pepN-expressing bacteria by using a specific substrate, namely, DCM−NH−Ala. Substrate hydrolysis by pepN produces a sharp increase in the fluorescence band with peak at 662 nm when excited by a single photon at 480 nm or by two NIR photons (at approximately 800 nm). The emission kinetics are dependent on the intracellular pepN concentrations, which provide a powerful tool for detecting diverse virulent bacteria in a few minutes and with the inherent advantages of two-photon excitation. We resolved the enzymatic kinetics, obtained the Michaelis-Menten parameters (e.g., K M , v max , and k catalytic) and studied the photophysics of the released fluorophore DCM–NH 2. In addition, DCM–NH 2 meets the requirements for use in super-resolution microscopy. In bacteria with high pepN activity, the probe quickly initiates an enzymatic reaction at specific sites located on the bacterial membrane and some structures inside the bacterial body. [ABSTRACT FROM AUTHOR]