Photosensitizing properties and subcellular localisation of 3,4-dihydro-ß-carbolines harmaline and harmalol.

Harmaline (1) and harmalol (2) represent two 3,4-dihydro-ß-carboline (DHßCs) 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. 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.

Virtual chromoendoscopy with iSCAN as an alternative method to dye-spray chromoendoscopy for dysplasia detection in long-standing colonic inflammatory bowel disease: a case-control study.

OBJECTIVE: Patients with long-standing colonic inflammatory bowel disease (cIBD) are at increased risk of developing colorectal cancer (CRC). Dye-spray chromoendoscopy (DCE) with targeted biopsies is the preferred technique for surveillance of dysplasia. Virtual chromoendoscopy (VCE) are arising to improve detection rates and adherence to surveillance guidelines, although its role is not yet well defined. The purpose of this study is to assess the effectiveness of VCE with iSCAN as an alternative method for dysplasia detection in cIBD. METHODS: Retrospective case-control study with 191 patients included, 98 in the DCE (Indigo carmine) group and 93 in the VCE (iSCAN, twin-mode 1-3) group. The dysplasia detection and the exploration time were analysed. A logistic regression analysis was performed to ascertain the factors related to colonic dysplasia. RESULTS: A total of 44 dysplastic lesions were detected in 21 (11%) patients. No differences were found in the per lesion and the per patient analysis (dysplastic versus non-dysplastic). Median withdrawal time was shorter in the VCE group than in the DCE group (9 min versus 14 min; p < .001). Location of lesions in the right colon was independently associated with an increased risk for colonic dysplasia (OR = 4.04, 95%CI 1.11-14.65; p = .034) after adjusting for age at inclusion, age at diagnosis, high risk for CRC and Kudo pit pattern. CONCLUSIONS: VCE with iSCAN presents a similar diagnostic performance to conventional DCE in the detection of colonic dysplasia in patients with long-standing cIBD. Furthermore, VCE with iSCAN is a less time-consuming surveillance alternative to DCE.

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport.

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 Ser46, Ser162, Ser181, Ser269, Ser280, Ser291, Thr293, Ser299, and Ser302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser290 Mutating 257VPF259 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 PP1α-mediated Ser290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that ß-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 Ser290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser290 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.

N-Methyl-ß-carboline alkaloids: structure-dependent photosensitizing properties and localization in subcellular domains.

N-Methyl-ß-carboline (ßC) 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-ßC alkaloids were investigated herein. Data reveal that methylation of the ßC 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 ßCs showed the highest photosensitizing efficiency. With the exception of 2-methyl-harminium, all the N-methyl-ßCs investigated herein induce a similar DNA photodamage profile, dominated largely by oxidized purines. This fact represents a distinctive behavior when comparing with N-unsubstituted-ßCs. 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 ßCs, an active transport appears to mediate the (reatively slow) uptake of the quaternary cationic ßCs. 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.

Seeding and Growth of ß-Amyloid Aggregates upon Interaction with Neuronal Cell Membranes.

In recent years, the prevalence of amyloid neurodegenerative diseases such as Alzheimer'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 ß-amyloid peptide (Aß) 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 Aß 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 Aß 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 Aß 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.

Inflammatory bowel disease in patients over the age of 70 y. Does the disease duration influence its behavior?

INTRODUCTION: The fastest growing segment of our population is that of people above 70 years of age. Elderly patients with IBD exhibit several specific problems. Our objective was to evaluate the clinical course, the side effects of the treatments and the need for surgery of elderly patients, regardless of the age of onset. MATERIALS AND METHODS: This was a cross-sectional study wherein retrospective data were collected from multiple centers from seven hospitals within the Valencia metropolitan area. Data were collected on patients older than 70 y with inflammatory bowel disease. RESULTS: We identified a total of 331 patients older than 70 years of age (5.3% of patients monitored at our centers). The mean age at the time of the study was 77.34 y (±5.39). Mesalamine were the most frequently used medications. Corticosteroids were used in 66% of the patients. However, the use of corticosteroids and biologics was less probable in older patients (OR 0.96, p = .06). The longer the disease progressed, the more immunosuppressive medications were used (OR 1.3, p = .052). Neoplasms appeared in 41 patients (13%). Of the 36 patients with tumors that appeared after the onset of the disease, 20 patients had not been treated with immunomodulators or biologics. CONCLUSIONS: Mesalamine was the most frequently used medication. There is no increased risk of tumors regarding the medications used. The use of immunosuppressive medications is more prevalent with longer disease progression times, although with a high rate of adverse events.

A Red-Emitting, Multidimensional Sensor for the Simultaneous Cellular Imaging of Biothiols and Phosphate Ions.

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.

Accuracy of magnetic resonance enterography in assessing response to therapy and mucosal healing in patients with Crohn's disease.

BACKGROUND & AIMS: We assessed the accuracy of magnetic resonance enterography (MRE) in monitoring response to therapy in patients with Crohn's disease (CD) using ileocolonoscopy as a reference standard. METHODS: We performed a prospective multicenter study of 48 patients with active CD and ulcers in at least one ileocolonic segment. All patients underwent ileocolonoscopy and MRE at baseline and 12 weeks after completing treatment with corticosteroids (CS) or anti-tumor necrosis factor agents. Disease activity was quantified using Crohn's Disease Endoscopic Index of Severity (CDEIS) and Magnetic Resonance Index of Activity (MaRIA). The primary analysis was to determine the accuracy of MRE in identification of healing, defined as the disappearance of ulcers in endoscopy examination. Additional analyses established the accuracy of MRE in determining endoscopic remission (a CDEIS score <3.5) and change in severity based on consideration of all segments. RESULTS: MRE determined ulcer healing with 90% accuracy and endoscopic remission with 83% accuracy. The mean CDEIS and MaRIA scores significantly changed at week 12 in segments with ulcer healing, based on endoscopic examination (CDEIS: 21.28 ± 9.10 at baseline vs 2.73 ± 4.12 at 12 weeks; P < .001 and MaRIA: 18.86 ± 9.50 at baseline vs 8.73 ± 5.88 at 12 weeks; P < .001). The MaRIA score accurately detected changes in lesion severity (Guyatt score: 1.2 and standardized effect size: 1.07). MRE was as reliable as endoscopy in assessing healing; no significant changes in CDEIS or MaRIA scores were observed in segments with persistent ulcers, based on endoscopic examination (CDEIS: 26.43 ± 9.06 at baseline vs 20.77 ± 9.13 at 12 weeks; P = .18 and MaRIA: 22.13 ± 8.42 at baseline vs 20.77 ± 9.17 at 12 weeks; P = .42). The magnitude of change in CDEIS scores correlated with those in MaRIA scores (r = 0.51; P < .001). CONCLUSIONS: MRE evaluates ulcer healing with a high level of accuracy when ileocolonoscopy is used as the reference standard. The MaRIA is a valid, responsive, and reliable index assessing response to therapy in patients with CD.

New Dual Fluorescent Probe for Simultaneous Biothiol and Phosphate Bioimaging.

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.

Photophysics of a Live-Cell-Marker, Red Silicon-Substituted Xanthene Dye.

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 silicon-substituted xanthene, 7-hydroxy-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-one (2-Me TM), was explored by means absorption, steady-state, and time-resolved fluorescence. First, the near-neutral pH, ground-state acidity constant of the dye, pKN-A, was determined by absorbance and steady-state fluorescence at very low buffer concentrations. Next, we determined whether the addition of phosphate buffer promoted the excited-state proton-transfer (ESPT) reaction among 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, the values of the excited-state rate constants were determined by global analysis of the fluorescence decay traces recorded as a function of pH, and buffer concentration. 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.

Ratiometric Two-Photon Near-Infrared Probe to Detect DPP IV in Human Plasma, Living Cells, Human Tissues, and Whole Organisms Using Zebrafish.

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.

New bioisosteric sulphur-containing choline kinase inhibitors with a tracked mode of action.

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 Mg2+ cofactor, or its destabilization. The most active compounds of the two series are capable of inducing apoptosis following the mitochondrial pathway and to significantly reduce the expression of anti-apoptotic proteins such as the Mcl-1. The fluorescence properties of the compounds of the PL library allowed the tracking of their mode of action, while PAINS (Pan Assays Interference Structures) filtration databases suggest the lack of any unspecific biological response.

Effects of the anion salt nature on the rate constants of the aqueous proton exchange reactions.

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.

Visible absorption and fluorescence spectroscopy of conformationally constrained, annulated BODIPY dyes.

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,ß-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.

Early amyloidogenic oligomerization studied through fluorescence lifetime correlation spectroscopy.

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.

Dynamics of water-in-oil nanoemulsions revealed by fluorescence lifetime correlation spectroscopy.

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.

Influence of the solvent on the ground- and excited-state buffer-mediated proton-transfer reactions of a xanthenic dye.

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.

Photophysics of the interaction between a fluorescein derivative and Ficoll.

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.

New Red-Emitting Chloride-Sensitive Fluorescent Protein with Biological Uses.

A new chloride-sensitive red fluorescent protein derived from Entacmaea quadricolor is described. We found that mBeRFP exhibited moderate sensitivity to chloride and, via site-directed 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.

Chimeric Drug Design with a Noncharged Carrier for Mitochondrial Delivery.

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.