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DPP IV, otherwise known as CD26 lymphocyte T surface antigen, is a transmembrane glycoprotein also found in circulation in the blood. It plays an important role in several processes like glucose metabolism and T-cell stimulation. Moreover, it is overexpressed in renal, colon, prostate, and thyroid human carcinoma tissues. It can also serve as a diagnostic in patients with lysosomal storage diseases. The biological and clinical importance of having readouts for the activity of this enzyme, in physiological and disease conditions, has led us to design a near-infrared (NIR) fluorimetric probe that also has the characteristics of being ratiometric and excitable by two simultaneous NIR photons. The probe consists of assembling an enzyme recognition group (Gly-Pro) (Mentlein, 1999; Klemann et al., 2016) on the two-photon (TP) fluorophore (derivative of dicyanomethylene- 4H-pyran, DCM-NH2) disturbing its NIR characteristic internal charge transfer (ICT) emission spectrum. When the dipeptide group is released by the DPP IV-specific enzymatic action, the donor−acceptor DCM-NH2 is restored, forming a system that shows high ratiometric fluorescence output. With this new probe, we have been able to detect, quickly and efficiently, the enzymatic activity of DPP IV in living cells, human tissues, and whole organisms, using zebrafish. In addition, due to the possibility of being excited by two photons, we can avoid the autofluorescence and subsequent photobleaching that the raw plasma has when it is excited by visible light, achieving detection of the activity of DPP IV in that medium without interference., FEDER/Junta de Andaluc?a- Consejer?a de Transformaci?n Econ?mica, Industria, Con- ocimiento y Universidades/Proyecto A-FQM-230-UGR20 PID2020-114256RB-I00, Spanish Government FPU17/04749

Harmaline (1) and harmalol (2) represent two 3,4-dihydro-β-carboline (DHβCs) most frequently reported in a vast number of living systems. Fundamental aspects including the photosensitizing properties, cellular uptake, as well as the cyto- and phototoxicity of 1 and 2 were investigated herein. The molecular basis underlying the investigated processes are elucidated. Data reveal that both alkaloids show a distinctive pattern of extracellular DNA photodamage. Compound 1 induces a DNA photodamage profile dominated by oxidised purines and sites of base loss (AP sites), whereas 2 mostly induces single-strand breaks (SSBs) in addition to a small extent of purine oxidative damage. In both cases, DNA oxidative damage would occur through type I mechanism. In addition, a concerted hydrolytic attack is suggested as an extra mechanism accounting for the SSBs formation photoinduced by 2. Subcellular internalisation, cyto- and phototoxicity of 1 and 2 and the corresponding full-aromatic derivatives harmine (3) and harmol (4) also showed quite distinctive patterns in a structure-dependent manner. These results are discussed in the framework of the potential biological, biomedical and/or pharmacological roles reported for these alkaloids. Graphical abstract The subtle structural difference (i.e., the exchange of a methoxy group for a hydroxyl substituent at C(7)) between harmaline and harmalol, gives rise to distinctive photosensitizing and subcellular localisation patterns.

We thank the Ministerio de Economia y Competitividad (CTQ2017-85454-C2-1-P), Ministerio de Ciencia en Innovacion (PID2020-113059GB-C21) and Junta de Andalucia (P20_00162) (Spain) for funding and P. R. and A. O. G. also for FPU contracts. We also thank the UGR CSIRC for access to computational facilities. We also thank Big&Open Data Innovation Laboratory (BODaI-Lab), University of Brescia, granted by Fondazione Cariplo and Regione Lombardia, for access to resources of Computing Center CINECA (Bologna), Italy. Support from the Italian MIUR (grant N. 2017A4XRCA) is also acknowledged. VM acknowledges a Fellowship from Ikerbasque, the Basque Foundation for Science., A novel synthetic strategy based on a bifunctional stapled chiral nucleus from which segments of different lengths can be added to both ends of o-phenylene ethynylenes (o-OPEs) has been developed to obtain a new type of foldamer and a novel chiral Pd2L2 metallacycle. For the first time, an enantiopure fully conjugated helical foldamer having 14 phenyl rings and 13 alkynes is reported. The folded structure has four complete loops and is able to host three Ag(I) cations in their cavity with high binding constants. The complete photophysical and chiroptical (ECD, CPL and VCD) characterization of these foldamers has shown that these molecules show intense chiroptical responses with dissymmetry ratios in the range of 10−2. Theoretical modeling of these systems reveals the origin of these remarkable responses and points out a potential connection with the chiral induced spin selectivity (CISS) effect. The magnetic dipole moment is proposed as a key physical variable connecting the chiroptical properties and CISS-based spin filtering properties observed in chiral compounds., Ministerio de Economia y Competitividad (Spain) CTQ2017-85454-C2-1-P, Ministerio de Ciencia en Innovacion (Spain) PID2020-113059GB-C21, Junta de Andalucia European Commission P20_00162, Ministry of Education, Universities and Research (MIUR) 2017A4XRCA, Ikerbasque, the Basque Foundation for Science

N-Methyl-β-carboline (βC) alkaloids, including normelinonine F (1b) and melinonine F (2b), have been found in a vast range of living species playing different biological, biomedical and/or pharmacological roles. Despite this, molecular bases of the mechanisms through which these alkaloids would exert their effect still remain unknown. Fundamental aspects including the photosensitizing properties and intracellular internalization of a selected group of N-methyl-βC alkaloids were investigated herein. Data reveal that methylation of the βC main ring enhances its photosensitizing properties either by increasing its binding affinity with DNA as a biomolecular target and/or by increasing its oxidation potential, in a structure- dependent manner. As a general rule, N(9)-substituted βCs showed the highest photosensitizing efficiency. With the exception of 2-methyl-harminium, all the N-methyl-βCs investigated herein induce a similar DNA photodamage profile, dominated largely by oxidized purines. This fact represents a distinctive behavior when comparing with N-unsubstituted-βCs. On the other hand, although all the investigated compounds might accumulate mainly into the mitochondria of HeLa cells, methylation provides a distinctive dynamic pattern for mitochondrial uptake. While rapid (passive) diffusion is most probably reponsible for the prompt uptake/release of neutral βCs, an active transport appears to mediate the (reatively slow) uptake of the quaternary cationic βCs. This might be a consequence of a distinctive subcellular localization (mitochondrial membrane and/or matrix) or interaction with intracellular components. Biomedical and biotechnological implications are also discussed herein., ANPCyT PICT-2015-0374 2016-0370 2018-3193, Spanish Ministry of Economy and Competitiveness CTQ201785658-R CTQ2014-55474-C2-2-R

Since the identification of human choline kinase as a protein target against cancer progression, many compounds have been designed to inhibit its function and reduce the biosynthesis of phosphatidylcholine. Herein, we propose a series of bioisosteric inhibitors that are based on the introduction of sulphur and feature improved activity and lipophilic/hydrophilic balance. The evaluation of the inhibitory and of the antiproliferative properties of the PL (dithioethane) and FP (disulphide) libraries led to the identification of PL 48, PL 55 and PL 69 as the most active compounds of the series. Docking analysis using FLAP suggests that for hits to leads, binding mostly involves an interaction with the Mg

This work was financially supported by CTQ2017-85685-R, CTQ2017-85454-C2-1-P, and CTQ2017-86125-P (MICIU/AEI/ERDF). J.M.P. and J.D.V. thank UEQ for funding. J.V.P. is supported by an FPU fellowship (FPU17/04749). D.A. was supported by the Italian Cystic Fibrosis Foundation (grant FFC#3/2019) and by Fondazione Cassa Rurale Trento Rovereto (ref 2018.256). The authors acknowledge the Universidad of Granada (Spain) cell culture and microscopy central facilities (CIC-UGR)., A new chloride-sensitive red fluorescent protein derived from Entacmaea quadricolor is described. We found that mBeRFP exhibited moderate sensitivity to chloride and, via sitedirected mutagenesis (S94V and R205Y), we increased the chloride affinity by more than an order of magnitude (kd = 106 ± 6 mM) at physiological pH. In addition, cis−trans isomerization of the chromophore produces a dual emission band with different chloride sensitivities, which allowed us to develop a ratiometric methodology to measure intracellular chloride concentrations., MICIU/AEI/ERDF CTQ2017-85685-R CTQ2017-85454-C2-1-P CTQ2017-86125-P, UEQ, Spanish Government FPU17/04749, Ministry of Health, Italy, Italian Cystic Fibrosis Research Foundation 3/2019, Fondazione Cassa Rurale Trento Rovereto 2018.256

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. ispartof: PHARMACEUTICS vol:13 issue:2 ispartof: location:Switzerland status: published

CDK5 kinase is activated through interactions with different partners including the p35/p25 regulators. Active CDK5 plays a key role in several neuronal functions and its hyperactivity contributes to a variety of neurodegenerative processes and several human cancers, in particular glioblastomas and neuroblastomas. In order to probe partners that interact with CDK5, we designed and synthesized fluorescent quinolimide-labelled peptides derived from the C-helix of CDK5, which were implemented to probe CDK5 partners in vitro and by in cellulo imaging in U87 glioblastoma and N2a neuroblastoma cells. Ectopic expression of a NIR fluorescent protein miRFP670 fusion with p25 (p25-miRFP670) in N2a cells induced FRET between a quinolimide-labelled peptide biosensor and p25-miRFP670, and fluorescence intensity was proportional to the expression level of p25, thereby demonstrating the potential of the quinolimide-labelled peptide biosensor to report on p25 relative abundance., This work was supported by the Ministerio de Ciencia e Innovación/ Agencia Estatal de Investigación grants FU2015-67284-R and PID2019- 104366RB-C22, the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación/European Regional Development Fund grant CTQ2017-85658-R, and the CSIC grant 201580E073, and the CNRS (Centre National de la Recherche Scientifique). We acknowledge the MRI imaging facility (Montpellier, France), a member of the national infrastructure France-BioImaging.

In recent years, the prevalence of amyloid neurodegenerative diseases such as Alzheimer&rsquo, s disease (AD) has significantly increased in developed countries due to increased life expectancy. This amyloid disease is characterized by the presence of accumulations and deposits of &beta, amyloid peptide (A&beta, ) in neuronal tissue, leading to the formation of oligomers, fibers, and plaques. First, oligomeric intermediates that arise during the aggregation process are currently thought to be primarily responsible for cytotoxicity in cells. This work aims to provide further insights into the mechanisms of cytotoxicity by studying the interaction of A&beta, aggregates with Neuro-2a (N2a) neuronal cells and the effects caused by this interaction. For this purpose, we have exploited the advantages of advanced, multidimensional fluorescence microscopy techniques to determine whether different types of A&beta, are involved in higher rates of cellular toxicity, and we measured the cellular stress caused by such aggregates by using a fluorogenic intracellular biothiol sensor. Stress provoked by the peptide is evident by N2a cells generating high levels of biothiols as a defense mechanism. In our study, we demonstrate that A&beta, aggregates act as seeds for aggregate growth upon interacting with the cellular membrane, which results in cell permeability and damage and induces lysis. In parallel, these damaged cells undergo a significant increase in intracellular biothiol levels.

In this work we evaluated by FLIM microscopy the preferential accumulation of long-lived acridone derivatives in mitochondria and lysosomes, based on a new concept of non-protonable and non-charged groups carriers. Thus, thiophene ring has been proved to act as mithocondria carrier whereas pyridine derivative is preferentially accumulated into lisosomes., Ministerio de Economía y Competitividad for funding (CTQ2017-85454-C2-1-P and CTQ2017-85658-R)., Junta de Andalucía, Ministerio de Economía y Competitividad

We have synthesized a new fluoride−containing xanthenic dye able to dynamically and quantitatively detect acetate anion, a biologically relevant analyte, in water. We studied deeply the photophysical properties of the compound and verified its use as an acetate probe in synthetic serum.

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., This work was funded by grants CTQ2017-85658-R, CTQ2014-55474-C2-2-R (Spanish Ministry of Economy and Competitiveness; Agencia Estatal de Investigacion, AEI; and European Regional Development Fund, ERDF), QM2012-790 (Junta de Andalucía), and a grant from the Fundación Botín.

International audience; Na+-H+ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser(46), Ser(162), Ser(181), Ser(269), Ser(280), Ser(291), Thr(293), Ser(299), and Ser(302) did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser(290) was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1 (PP1), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser(290). Mutating (VPF259)-V-257 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1-mediated Ser(290) dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that -sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser(290) rephosphorylation. Our results show that PP1 and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser(290) phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.

Ion homeostasis regulates critical physiological processes in the living cell. Intracellular chloride concentration not only contributes in setting the membrane potential of quiescent cells but it also plays a role in modulating the dynamic voltage changes during network activity. Dynamic chloride imaging demands new tools, allowing faster acquisition rates and correct accounting of concomitant pH changes. Joining a long-Stokes-shift red-fluorescent protein to a GFP variant with high sensitivity to pH and chloride, we obtained LSSmClopHensor, a genetically encoded fluorescent biosensor optimized for the simultaneous chloride and pH imaging and requiring only two excitation wavelengths (458 and 488 nm). LSSmClopHensor allowed us to monitor the dynamic changes of intracellular pH and chloride concentration during seizure like discharges in neocortical brain slices. Only cells with tightly controlled resting potential revealed a narrow distribution of chloride concentration peaking at about 5 and 8 mM, in neocortical neurons and SK-N-SH cells, respectively. We thus showed that LSSmClopHensor represents a new versatile tool for studying the dynamics of chloride and proton concentration in living systems.

Development of biomarkers of analytes with interest in clinic is an important field of study. In this work, we synthesized and analyzed the new fluorescent acetate-biomarker, Iso-PG. The mechanism of detection is the acetate buffer mediated proton transfer reaction. The rate constants involved were obtained, and we measured the change in the fluorescence lifetime produced as a consequence of the presence of acetate in the medium. Finally, we checked its potential use as acetate biomarker in synthetic serum

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., This work was funded by grants CTQ2014-56370-R, CTQ2014-53598, and CTQ2014-55474-C2-2-R from the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF), and grant FQM2012-790 from the Consejería de Innovación, Ciencia y Empresa (Junta de Andalucı́a), including costs to publish in open access.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Physical Chemistry A, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.acs.org/doi/10.1021/acs.jpca.5b07898., Dyes with near-red emission are of great interest because of their undoubted advantages for use as probes in living cells. In-depth knowledge of their photophysics is essential for employment of such dyes. In this article, the photophysical behavior of a new siliconsubstituted xanthene, 7-hydroxy-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-one (2-Me TM), was explored using absorption, steady-state, and time-resolved fluorescence. First, the near-neutral pH, ground state acidity constant of the dye, pK*N-A, was determined by both absorbance and steady-state fluorescence methods in the presence of very low buffer concentrations. Next, we determined whether the addition of phosphate buffer promoted the excited-state proton exchange reaction between the neutral and anion form of 2-Me TM in aqueous solutions at near-neutral pH. For this analysis, both the steady-state fluorescence method and time-resolved emission spectroscopy (TRES) were employed. The TRES experiments demonstrated a remarkably favored conversion of the neutral form to the anion form. Then, fluorescence decay traces recorded as a function of buffer concentration and pH were globally analyzed to determine the values of the excited-state rate constants. The revealed kinetic parameters were consistent with the TRES results, exhibiting a higher rate constant for deprotonation than for protonation, which implies an unusual low value of the excited-state acidity constant pK*N-A and therefore an enhanced photoacid behavior of the neutral form. Finally, we determined whether 2-Me TM could be used as a sensor inside live cells by measuring the intensity profile of the probe in different cellular compartments of HeLa 229 cells., This research was funded by MINECO (project CTQ2014-56370-R), and MINECO (project CTQ2014-53598).

A xanthene derivative, Granada Green dinitrobenzene sulfonate (GGDNBS), has been synthesized to assay cellular oxidative stress based on changes in the concentration of biothiols. The dye is able to react with biological thiols by a thiolysis reaction that promotes a change in fluorescence intensity. To demonstrate the usefulness of GGDNBS for in vivo oxidative stress measurements, 661 W photoreceptor-derived cells were exposed to light to induce ROS generation, and changes in GGDNBS fluorescence were measured. In these cells, GGDNBS fluorescence was correlated with the biothiol levels measured by an enzymatic method. Therefore, GGDNBS allows us to monitor changes in the levels of biothiols associated with ROS generation via single-cell bioimaging.

The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. However, the molecular basis and mechanism of amyloid aggregation are not completely understood. For many amyloidogenic proteins, certain oligomeric intermediates that form in the early aggregation phase appear to be the principal cause of cellular toxicity. Recent computational studies have suggested the importance of nonspecific interactions for the initiation of the oligomerization process prior to the structural conversion steps and template seeding, particularly at low protein concentrations. Here, using advanced single-molecule fluorescence spectroscopy and imaging of a model SH3 domain, we obtained direct evidence that nonspecific aggregates are required in a two-step nucleation mechanism of amyloid aggregation. We identified three different oligomeric types according to their sizes and compactness and performed a full mechanistic study that revealed a mandatory rate-limiting conformational conversion step. We also identified the most cytotoxic species, which may be possible targets for inhibiting and preventing amyloid aggregation.

Biological samples are a complex and heterogeneous matrix where different macromolecules with different physicochemical parameters cohabit in reduced spaces. The introduction of fluorophores into these samples, such as in the interior of cells, can produce changes in the fluorescence emission properties of these dyes, caused by the specific physicochemical properties of cells. This effect can be especially intense with solvatofluorochromic dyes, where changes in the polarity environment surrounding the dye can drastically change the fluorescence emission. In this article, we studied the photophysical behavior of a new dye and confirmed the aggregation-induced emission (AIE) phenomenon with different approaches, such as by using different solvent proportions, increasing the viscosity, forming micelles, and adding bovine serum albumin (BSA), through analysis of the absorption and steady-state and time-resolved fluorescence. Our results show the preferences of the dye for nonpolar media, exhibiting AIE under specific conditions through immobilization. Additionally, this approach offers the possibility of easily determining the critical micelle concentration (CMC). Finally, we studied the rate of spontaneous incorporation of the dye into cells by fluorescence lifetime imaging and observed the intracellular pattern produced by the AIE. Interestingly, different intracellular compartments present strong differences in fluorescence intensity and fluorescence lifetime. We used this difference to isolate different intracellular regions to selectively study these regions. Interestingly, the fluorescence lifetime shows a strong difference in different intracellular compartments, facilitating selective isolation for a detailed study of specific organelles., This research was funded by grants CTQ2017-85658-R and CTQ2014-55474-C2-2-R (Spanish Ministry of Economy and Competitiveness).

ortho-Oligo(phenylene)ethynylenes (o-OPEs) stapled with enantiopure 2,3-dihydroxybutane diethers have highly intense circular dichroism (CD) spectra and excellent circular polarized luminescence (CPL) responses (glum values up to 1.1 × 10-2), which are consistent with homochiral helically folded structures. In the presence of Ag(i), a change in the CPL emission is observed, representing the first example of CPL active small organic molecular emitters, which can be modulated by carbophilic interactions in a reversible manner, We thank Intramural CSIC project (201530E01) and MICINN (FEDER funded Grants CTQ2014-53598, CTQ2011-24783 and CTQ2014-53894-R) for financial support.

Six conformationally restricted BODIPY dyes with fused carbocycles were synthesized to study the effect of conformational mobility on their visible electronic absorption and fluorescence properties. The symmetrically disubstituted compounds (2, 6) have bathochromically shifted absorption and fluorescence spectral maxima compared to those of the respective asymmetrically monosubstituted dyes (1, 5). Fusion of conjugation extending rings to the α,β-positions of the BODIPY core is an especially effective method for the construction of boron dipyrromethene dyes absorbing and emitting at longer wavelengths. The fluorescence quantum yields Φ of dyes 1-6 are high (0.7 ≤ Φ ≤ 1.0). The experimental results are backed up by quantum chemical calculations of the lowest electronic excitations in 1, 2, 5, 6, and corresponding dyes of related chemical structure but without conformational restriction. The effect of the molecular structure on the visible absorption and fluorescence emission properties of 1-6 has been examined as a function of solvent by means of the recent, generalized treatment of the solvent effect, proposed by Catalán (J. Phys. Chem. B 2009, 113, 5951-5960). Solvent polarizability is the primary factor responsible for the small solvent-dependent shifts of the visible absorption and fluorescence emission bands of these dyes.

Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies., This work is funded by grant P10-FQM-6154 from the Consejeria de Innovacion, Ciencia y Empresa (Junta de Andalucia).

The size, diffusional properties, and dynamics of reverse water-in-oil nanoemulsions, or reverse micelles (RMs), have been widely investigated because of interest in this system as a model for biological compartmentalization. Here, we have employed fluorescence lifetime correlation spectroscopy (FLCS) to reveal the dynamics and sizes of aerosol-OT (AOT)/isooctane RMs using a fluorescent xanthene derivative called Tokyo Green II (TG-II). The dye undergoes a partition and a shift in its tautomeric equilibrium such that the TG-II anion remains in the inner micellar aqueous core, and the neutral quinoid form lies in the interfacial region. By applying FLCS, we specifically obtained the lifetime filtered autocorrelation curves of the anionic TG-II, which shows a characteristic lifetime of approximately 4 ns. Analysis of the FLCS curves provides the diffusion coefficient and hydrodynamic radius of the RMs as well as micelle dynamics in the same experiment. The FLCS curves show dynamics in the microsecond time range, which represents an interconversion rate that changes the distribution of the TG-II neutral and anionic forms in the hydrophobic interface and the water core.

The 2,5-dioxopyrrolidin-1-yl-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)-3-methylbenzoate has been synthesized as an amine-reactive derivative able to yield stable covalently labeled biopolymers. The new derivative has been used to label polyribocytidilic acid (5'), poly(C), amine residues. TG-II-poly(C) exhibits monoexponential decay at the physiological pH range. In addition, both steady-state fluorescence intensity and fluorescence decay are also sensitive to solution pH. The large decrease in steady-state fluorescence upon hybridization allows it to be used as a nucleic acid probe in a homogeneous assay format. In summary, we report an efficient synthesis to obtain labeled RNA from commercially available materials in excellent yields.

The photophysical behavior of a new fluorescein derivative has been explored by using absorption and steady-state and time-resolved fluorescence measurements. The influence of ionic strength, as well as total buffer concentration, on both the absorbance and fluorescence has been investigated. The apparent acidity constant of the dye determined by absorbance is almost independent of the added buffer and salt concentrations. A semiempirical model is proposed to rationalize the variations in the apparent pKa values. The excited-state proton-exchange reaction around the physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the steady-state fluorescence and decay times. Fluorescence decay traces, collected as a function of total buffer concentration and pH, were analyzed by global compartmental analysis, yielding the following values of the rate constants describing excited-state dynamics: k01 = 1.29 x 10(10) s(-1), k02 = 4.21 x 10(8) s(-1), k21 approximately 3 x 10(6) M(-1) s(-1), k12B= 6.40 x 10(8) M(-1) s(-1), and k21B = 2.61 x 10(7) M(-1) s(-1). The decay rate constant values of k01, k21, k21B, along with the low molar absorption coefficient of the neutral form, mean that coupled decays are practically monoexponential at buffer concentrations higher than 0.02 M and any pH. Thus, the pH and buffer concentration can modulate the main lifetime of the dye.

The simultaneous detection of relevant metabolites in living organisms by using one molecule introduces an approach to understanding the relationships between these metabolites in healthy and deregulated cells. Fluorescent probes of low toxicity are remarkable tools for this type of analysis of biological systems in vivo. As a proof of concept, different naturally occurring compounds, such as biothiols and phosphate anions, were the focus for this work. The 2,4-dinitrobenzenesulfinate (DNBS) derivative of 9-[1-(4-tert-butyl-2-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (Granada Green; GG) were designed and synthesized. This new sulfinyl xanthene derivative can act as a dual sensor for the aforementioned analytes simultaneously. The mechanism of action of this derivative implies thiolysis of the sulfinyl group of the weakly fluorescent DNBS-GG by biological thiols at near-neutral pH values, thus releasing the fluorescent GG moiety, which simultaneously responds to phosphate anions through its fluorescence-decay time. The new dual probe was tested in solution by using steady-state and time-resolved fluorescence and intracellularly by using fluorescence-lifetime imaging microscopy (FLIM) in human epithelioid cervix carcinoma (HeLa) cells.

Since the very early years of microscopy development, scientists have pursued the ability to observe live cellular activity in order to probe the processes occurring inside cells in real time. Fluorescence microscopy has become an extraordinary tool for unraveling the myriad cellular processes and interactions that are relevant to understanding cell physiology. The intracellular sensing of certain analytes is of crucial importance to understanding some of these processes, such as the relation between cellular pH and metabolic states or the roles of specific ions in signaling pathways. However, the acquisition of quantitative information from the interiors of cells is not a trivial challenge. Ratiometric, intensity-based fluorescence microscopy approaches are commonly used, but they suffer from many systematic difficulties that make them unsuitable for quantitative sensing. Fluorescence lifetime imaging microscopy (FLIM) detects the time duration of fluorescence emission, taking advantage of the multidimensional nature of photon emission. FLIM-based intracellular sensing approaches, especially in the time domain in single-photon timing (SPT) mode, overcome many of the limitations of fluorescence-intensity methods. Herein, we review strategies for the FLIM-based intracellular sensing of local pH, ion concentration, and biomolecular interactions. In the first section, we demonstrate how in-depth knowledge of the photophysics of dyes can be useful in the development of FLIM sensors. Then, we review the growing field of nanoparticle-based FLIM sensors. Finally, the expanding detection capabilities of FRET and the use of FLIM for the larger-scale analysis of tissues are discussed.

Phosphate ions play important roles in signal transduction and energy storage in biological systems. However, robust chemical sensors capable of real-time quantification of phosphate anions in live cells have not been developed. The fluorescein derivative dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivatives at approximately physiological pH resulting in the dependence of the dye's nanosecond fluorescence decay time on the phosphate buffer concentration. This allows the 2-Me-4-OMe TG dye to be used with fluorescence lifetime imaging microscopy (FLIM) as a real-time phosphate intracellular sensor in cultured cells. This methodology has allowed the time course of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe TG. These changes were consistent with increased alkaline phosphatase activity in the extracellular medium as a marker of the differentiation process.

Ficoll has been widely used as a crowding agent to mimic intracellular media because it is believed to be noninteracting and is composed of mixed sizes such that smaller and larger diffusing solutes can be studied. Due to the interest that the fluorescent dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (TG-II) as a fluorometric probe of phosphate ions in intracellular media could generate, we describe the spectral characteristics of the system TG-II-Ficoll in aqueous solution by means of absorption spectroscopy, steady-state fluorescence, time-resolved fluorescence, time-resolved emission spectroscopy, and fluorescence lifetime correlation spectroscopy. The spectral characteristics found are consistent with the formation of an adsorption complex on the surface of Ficoll, probably due to hydrogen bonding between TG-II and Ficoll. In addition, the diffusion coefficient calculated for the association was similar to the diffusion coefficient previously recovered for Ficoll in the same experimental conditions. Therefore, our overall data clearly demonstrate that Ficoll is not an inert crowding agent when in the presence of fluorescein derivative dyes.

Fluorescence Lifetime Correlation Spectroscopy (FLCS) allows to extract autocorrelation functions from different emitters by using Time-Correlated Single Photon Counting information, extending the applicability of conventional Fluorescence Correlation Spectroscopy (FCS) [1].Here, we have used a xanthene derivative, so-called Tokyo Green II (TG-II) [2,3], to study the size, diffusional properties, and dynamics of reverse water-in-oil micelles of Aerosol-OT (AOT) in isooctane (iC8). The dye undergoes a partition between the inner micellar aqueous core, in which the TG-II anion is the main form, and the AOT lipophilic side chains, where the TG-II neutral species is predominant. By applying FLCS we obtained the lifetime filtered autocorrelation (AC) curves of the anionic TG-II, which shows a characteristic lifetime of 3.70 ns, under different conditions. Fitting the TG-II anion AC curves to the appropriate model provided the diffusion coefficient and hydrodynamic radius of micelles at different ratios [water] / [AOT]. Interestingly, the AC functions also showed a strong anticorrelation time in the microsecond time range. This time represents micelle dynamics that changes the distribution of the TG-II neutral and anionic forms in the hydrophobic interface and the water core.[1] Kapusta et al., J. Fluoresc.17 (2007), 43.[2] Paredes et al., Phys. Chem. Chem. Phys.11 (2009), 5400.[3] Paredes et al., Phys. Chem. Chem. Phys.12 (2010), 323.

The buffer-mediated proton-transfer reactions of the fluorescent xanthenic derivative 9-[1-(2-Methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (TG-II) have been studied in different aqueous media. We have employed various buffers to investigate the influence of donor/acceptor systems with different anion and/or cation chemical constituents on the kinetic parameters of proton-transfer. The kinetic parameters were recovered both in the ground-state by means of Fluorescence Lifetime Correlation Spectroscopy (FLCS) and in the excited-state by means of Time Correlated Single Photon Counting (TCSPC) and Global Compartmental Analysis (GCA). Both ground- and excited- deprotonation and protonation recovered rate constants in the presence of either phosphate or acetate buffer as donor/acceptor systems were similar. The presence of Tris-HCl buffer does not promote the excited-state proton-transfer (ESPT) reaction. The results indicate the influence of the ions on the ground-state proton-transfer (GSPT) rates and concomitantly on the ESPT reaction. The proton-transfer rate constants recovered here show a trend correlated with the Hofmeister series or the Marcus classification of ions.

The interactions between trimethine cyanine homodimer dye, BOBO-3 (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-pyridinium tetraiodide), and single-stranded homo-oligonucleotides, double-stranded complementary homo-oligonucleotides, and high-molecular-weight double-stranded polyhomonucleotides have been investigated in detail using absorption and both steady-state and time-resolved fluorescence spectroscopy. In this work, we describe the differences in the binding behavior of BOBO-3 with double-stranded DNA (dsDNA) having different base contents. The fluorescence intensity of BOBO-3 interacting with deoxyadenosine-deoxythymidine (dAdT) dsDNA was higher than with the deoxyguanosine-deoxycytidine (dGdC) double helix. However, the BOBO-3 lifetime was longer in dGdC-rich dsDNA than in dsDNA with many dAdT sites. This result was detected at both the ensemble level and the single-molecule level. This behavior is a consequence of the dye's interacting with dsDNA on two kinds of binding sites. This phenomenon also occurs in natural dsDNA (Ruedas-Rama, M. J.; Orte, A.; Crovetto, L.; Talavera, E. M.; Alvarez-Pez, J. M. J. Phys. Chem. B 2010, 114, 1094-1103). By using a time-resolved fluorescence methodology and the McGhee-von Hippel theory for two overlapping, noncooperative binding modes, we obtained the equilibrium binding constants and the number of occupied sites for each binding mode of BOBO-3 in dAdT and dGdC binding sites. BOBO-3 has a higher affinity for dAdT sites and occupies 4.0 +/- 1.0 sites in its primary binding mode, whereas in dGdC-rich double strands, BOBO-3 covers 6.2 +/- 1.1 sites and has a lower affinity. These differences in the binding features and spectral properties of BOBO-3 may be used to develop approaches to identify GC- or AT-rich regions within large strands of dsDNA.

Buffer-mediated proton exchange reactions of a xanthenic dye were studied in the ground and the excited state by single molecule and bulk fluorescence techniques, respectively. The rate constant obtained supported the uniformity of the process in the ground and the excited state, and the need of adequate character of the buffer species to be able to promote excited-state reactions.

The photophysical behaviour of the new fluorescein derivative 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one has been explored by using absorption, and steady-state, time-resolved and single-molecule fluorescence measurements. The apparent ground-state acidity constant of the dye determined by both the absorbance and steady-state fluorescence is almost independent of the added buffer and salt concentrations. The excited-state proton exchange reaction around the physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the steady-state fluorescence and decay times. Fluorescence decay traces, collected as a function of total buffer concentration and pH, were analyzed by global compartmental analysis (GCA) to elucidate the values of the excited-state rate constants. The features of this system make the fluorescence decays monoexponential at pH values and phosphate buffer concentrations higher than 6.10 and 0.2 M respectively, with the possibility of tuning the fluorescence lifetime value by changing pH or buffer concentrations. The tuned lifetimes obtained by means of phosphate concentration at constant pH have also been recovered at the single-molecule level.

The proton-transfer ground-state rate constants of the xanthenic dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (TG-II), recovered by Fluorescence Lifetime Correlation Spectroscopy (FLCS), have proven to be useful to quantitatively reflect specific cation effects in aqueous solutions (J. M. Paredes, L. Crovetto, A. Orte, J. M. Alvarez-Pez and E. M. Talavera, Phys. Chem. Chem. Phys., 2011, 13, 1685-1694). Since these phenomena are more sensitive to anions than to cations, in this paper we have accounted for the influence of salts with the sodium cation in common, and the anion classified according to the empirical Hofmeister series, on the proton transfer rate constants of TG-II. We demonstrate that the presence of ions accelerates the rate of the ground-state proton-exchange reaction in the same order than ions that affect ion solvation in water. The combination of FLCS with a fluorophore undergoing proton transfer reactions in the ground state, along with the desirable feature of a pseudo-dark state when the dye is protonated, allows one unique direct determination of kinetic rate constants of the proton exchange chemical reaction.

The Danish study of positron emission tomography (PET)–CT versus conventional staging (CS) in non-small cell lung cancer has been reported twice now1 2 and corrected once.3 However, there are discrepancies in numbers between the manuscripts,1 2 which is surprising given the …

The basic pathologic process in multiple myeloma is the neoplastic proliferation of a single clone of plasma cells. Although the events which trigger autonomous cell growth are not well understood, the secretion of an M component, a serum or urinary immunoglobulin molecule or a light chain fragment by the vast majority of myeloma cells has provided a biologic marker which has greatly facilitated the study of this disease Some of the more recent clinical concepts which have evolved from studies on the plasma cell and the immunoglobulin molecule are discussed.