Search

Controlling the nano- and micropatterning of metal structures is an important requirement for various technological applications in photonics and biosensing. This work presents a method for controllably creating silver micropatterns by laser-induced photosculpting. Photosculpting is driven by plasmonic interactions between pulsed laser radiation and silver nanorods (AgNRs) in aqueous suspension; this process leads to optical binding forces transporting the AgNRs in the surroundings, while electronic thermalization results in photooxidation, melting, and ripening of the AgNRs into well-defined 3D structures. This work call these structures Airy castles due to their structural similarity with a diffraction-limited Airy disk. The photosculpted Airy castles contain emissive Ag nanoclusters, allowing for the visualization and examination of the aggregation process using luminescence microscopy. This work comprehensively examines the factors that define the photosculpting process, namely, the concentration and shape of the AgNRs, as well as the energy, power, and repetition rate of the laser. Finally, this work investigates the potential applications by measuring the metal-enhanced luminescence of a europium-based luminophore using Airy castles., MCIN/AEI/10.13039/501100011033 and the European Regional Development Fund (ERDF). Grant Number: CTQ2017-85658-R MCIN/AEI/10.13039/501100011033. Grant Number: PID2020-114256RB-I00 European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie. Grant Number: 101007934 Fundação para a Ciência e a Tecnologia, I.P. Grant Numbers: UIDB/00100/2020, 2022.04076.PTDC

In this work, we describe the application of the self-assembled europium complex 1:Eu(III) (where 1: diethyl 8-methoxy-2-oxo-1,2,4,5-tetrahydro-cyclopenta[de]quinolin-3-yl)phosphonate) for the analysis of water content of wheat flour as well as from a variety of other food raw materials. The methodology herein described represents a robust and accurate way for the detection of small amounts of water in food, providing comparing results with well-established methods such as thermogravimetry and Karl-Fischer titration. Interestingly, as an advantage over other methods, our luminescent-based approach can be implemented in lab-on-a-chip microfluidic devices for real-time and on-line detection of water content of food samples. Additionally, the remarkably long luminescence lifetime of Eu(III) allows the use of state-of-the-art imaging strategies based on PLIM microscopy for the direct visualization of unique water content maps of flour grains.

We report on the preparation and characterization of two dimensional (2D) films of (S,S,P)-1 and (R,R,M)-1 ortho-oligophenylene ethylene (o-OPE) enantiomers presenting high values of circularly polarized luminescence (CPL). The amphiphilic character of these two molecules allows a precise 2D self-assembly at the air/water interface and an efficient transfer onto a glass solid support. The morphological and chiroptical characterization of the solid supports after the transfer of 1, 8, 16 and 32 Langmuir films of (S,S,P)-1 and (R,R,M)-1 has been carried out. The strong chiroptical values of these monomers allow reliable ECD measurements to be obtained after a single transfer, with ECD values increasing as the number of transferred films increases. The semi-liquid behavior of the monomers on the solid substrate allows CPL measurements free of photoselection artifacts that show values similar to those obtained in solution and independent of monomer concentration. All these properties have allowed us to develop the first simple organic molecule (SOM)-based chiroptical barcoding presenting positive and negative regions as a proof of concept., Spanish Ministry of Science and Innovation (AEI, Spain) PID2020-112744GB-I00 Ministry of Science and Innovation, Spain (MICINN) Spanish Government MCIN/AIE, FSE "El FSE invierte en tu futuro" in Spain, FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento/Proyecto PID2020-113059GB-C21/AEI PRE2021-097546 FPU16/02597 P20_00162

We report a platform combining multicomponent reaction synthesis and automated cell-based screening to develop biocompatible NIR-BODIPY fluorophores. From a library of over 60 fluorophores, we optimised compound NIRBD-62c as a multimodal probe with suitable properties for STED superresolution and fluorescence lifetime imaging. Furthermore, we employed NIRBD-62c for imaging trafficking inside cells and to examine how pharmacological inhibitors can alter the vesicular traffic between intracellular compartments and the plasma membrane., Fundacion Seneca 21124/SF/19 MCIN/AEI PID2020-114256RB-I00, Spanish Government, European Commission PID2019-107991RB-I00 European Research Council (ERC) European Commission 771443, Royal Society of London, European Commission RG160289

The transient outward potassium current (

Three achiral polycyclic aromatic fluorophores─namely, 1-pyrenecarboxylic acid, 9-anthracenecarboxylic acid, and perylene-3,9-dicarboxylic acid─were chosen based on their desired properties before being incorporated into the construction of a K

Here, we report the synthesis of two isomeric BODIPY-based fluorescent molecules with a diethylamino donor group and an acetyl acceptor group at positions 5 and 2 (or 3), respectively, on the boron dipyrromethene core. The structure of the new BODIPY dyes is similar to Prodan, probably the most widely used solvatochromic probe, but with the donor and acceptor groups attached to an electron-poor heteroaromatic core. Interestingly, these BODIPY dyes exhibit unusual spectroscopic and photophysical properties, involving inverse solvatochromic behavior. An in-depth study of the solvent effects and quantum-chemical calculations have been performed to assess the basis for such spectroscopic properties and the effect of the position of the substitution. This work points towards a rational design of new fluorescent probes, based on understanding their spectroscopic and photophysical behavior.

This work was supported by grant CTQ2017–85658-R funded by MCIN/AEI/10.13039/501100011033/ FEDER “Una manera de hacer Europa”, and grants PID2019–104366RB-C22 and PID2020–114256RB-I00 funded by MCIN/AEI/10.13039/501100011033 . Funding for open access charge: Universidad de Granada / CBUA. We acknowledge the Centro de Instrumentación Científica (CIC) of Universidad de Granada for use of the TEM facilities. Á.R.-A. thanks the Spanish Ministerio de Educación y Formación Profesional for a FPU Ph.D. studentship., The presence of neuritic plaques and amyloid fibrils arising from the misfolding of certain proteins is the principal molecular indicator of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Methodologies for studying the early stages of amyloid aggregation are rapidly arising to provide a better understanding of the mechanism of fibrillization and cytotoxicity and to identify potential targets for diagnosis and therapy. The method presented here involves the simultaneous use of two different fluorophores, a quinolimide derivative and Nile Blue A. These are capable of interacting with and reporting on the formation of preamyloid aggregates and fibrils of apoferritin through fluorescence resonance energy transfer (FRET), which occurs between them, thus maximizing the contrast in detection and quantitative information of such amyloid species by using multidimensional fluorescence lifetime imaging microscopy (FLIM)., CBUA, Spanish Ministerio de Educación y Formación, Universidad de Granada, European Regional Development Fund 104366RB-C22, 114256RB-I00, MCIN/AEI/10.13039/501100011033, PID2019, PID2019–104366RB-C22, PID2020, PID2020–114256RB-I00

This work was supported by grants CTQ2017-85658-R, BFU2015-67284-R, and PID2019-104366RB-C22 funded by MCIN/AEI/10.13039/501100011033/FEDER "Una manera de hacer Europa"; grant PID2020-114256RB-I00 funded by MCIN/AEI/10.13039/501100011033; grant A-FQM-386-UGR20 funded by FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, and the CSIC grant 201580E073. Funding for open access charge: Universidad de Granada/CBUA., The small molecule 8-methoxy-2-oxo-1,2,4,5- tetrahydrocyclopenta[de]quinoline-3-carboxylic acid (2b) behaves as a reactive non-fluorescent Michael acceptor, which after reaction with thiols becomes fluorescent, and an efficient Eu3+ antenna, after self-assembling with this cation in water. This behavior makes 2b a highly selective GSH biosensor, which has demonstrated high potential for studies in murine and human cells of the immune system (CD4+ T, CD8+ T, and B cells) using flow cytometry. GSH can be monitored by the fluorescence of the product of addition to 2b (445 nm) or by the luminescence of Eu3+ (592 nm). 2b was able to capture baseline differences in GSH intracellular levels among murine and human CD4+ T, CD8+ T, and B cells. We also successfully used 2b to monitor intracellular changes in GSH associated with the metabolic variations governing the induction of CD4+ naiv̈ e T cells into regulatory T cells (TREG)., MCIN/AEI/FEDER "Una manera de hacer Europa" CTQ2017-85658-R BFU2015-67284-R PID2019-104366RB-C22, MCIN/AEI PID2020-114256RB-I00, FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades A-FQM-386-UGR20, CSIC grant 201580E073, Universidad de Granada/CBUA

This study was supported by project PID2020-118498GB-I00 funded by MCIN/AEI/10.13039/501100011033, Spain and by project CTQ2017-85658-R funded by MCIN/AEI/10.13039/501100011033/FEDER "Una manera de hacer Europa", Spain, and by FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades (Spain) project P18-FR-3533. CGV and MCMT acknowledge respectively grants FPU17/00491 and PRE2018083773 funded by MCIN/AEI/10.13039/501100011033 and FSE "El FSE invierte en tu futuro", Spain. Thanks go to the CIC personnel of the University of Granada for technical assistance. We thank the Centro de Servicios de Informatica y Redes de Comunicaciones (CSIRC), Universidad de Granada, for providing the computing time. The authors acknowledge Dr Rosario Herranz and Dr Francisco Fueyo-Gonzalez for the fluorophore 9-azetidinyl-5-butyl-quinolimide (AQui) used in this study, which was synthesized at Instituto de Quimica Medica-CSIC (IQM-CSIC) with the support of the Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion grant FU201567284-R., We have investigated the co-self-assembly, in water and at room temperature, of different aromatic short peptides containing Fmoc- (fluorenylmethyloxycarbonyl-) and Nap- (2-(naphthalen-2-yloxy)acetyl) groups having also different chirality. Using a combination of spectroscopy and microscopy techniques we have shown that mixtures of peptides have a stronger preference to form co-assemblies giving rise to different types of fibrils of well-defined morphology. Kinetic analysis of fluorescence resonance energy transfer (FRET) between Fmoc- and Nap- side groups reported more information about the process of self-assembly between different dipeptides. We have shown that when peptides are mixed in an equimolar ratio, the kinetics of co-aggregation is faster than that occurring when the proportion is unbalanced. Moreover, following the emission band of Nap-excimers we have shown that these peptides form co-assemblies in an alternate fashion at an equimolar ratio. The mechanism of self-assembly has been studied by molecular dynamics and monitored by differential scanning calorimetry. The mechanical properties of the resulting composite hydrogels have been evaluated by rheology. These results show that the formation of co-assemblies is promoted by π–π interactions between the different aromatic groups resulting in accelerating polymerization due to destabilization of the intermediates., MCIN/AEI, Spain PID2020-118498GB-I00 FPU17/00491, MCIN/AEI/FEDER "Una manera de hacer Europa", Spain CTQ2017-85658-R, FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades (Spain) P18-FR-3533, FSE "El FSE invierte en tu futuro", Spain PRE2018083773, Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion grant

This study was supported by projects CTQ2017-86568-R and FIS2017-85954-R (Ministerio de Economia, Industria y Competitividad, MINECO, and Agencia Estatal de Investigacion, AEI, Spain, cofunded by Fondo Europeo de Desarrollo Regional, FEDER, European Union), and by FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades (Spain) project P18-FR-3533. We want to thank "Unidad de Excelencia Quimica aplicada a Biomedicina y Medioambiente" of the University of Granada. Thanks go to the CIC personnel of the University of Granada for technical assistance. We thank the Centro de Servicios de Informatica y Redes de Comunicaciones (CSIRC), Universidad de Granada, for providing the computing time. Authors acknowledge to Dr Rosario Herranz and Dr Francisco Fueyo-Gonzalez for the fluorophore 9-azetidinyl-5-butyl-quinolimide (AQui) used in this study, which was synthesized at Instituto de Quimica Medica-CSIC (IQM-CSIC) with the support of the Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion grant FU2015-67284-R., Making use of the combination of multiparametric Fluorescence Lifetime Imaging Microscopy (FLIM) and single-molecule Fluorescence Lifetime Correlation Spectroscopy (FLCS), we have been able to study for the early stages of the fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF) self-assembly process with single-molecule resolution, the kinetics of fiber formation, the packaging of the peptides within the fibers and the capacity of the peptides to reassemble after disruption (self-healing) in the presence of different metallic cations. Other techniques such as FTIR, TEM, DSC and DFT calculations support our findings. The impact that the mechanism of self-assembly has on the physical (rigidity and self-healing) properties of the resulting gels have also been evaluated by rheology. Calcium ions are able to promote the self-assembly of Fmoc-FF faster and more efficiently, forming more rigid hydrogels than do cesium ions. The reasons behind this effect may be explained by the different capacities that these two cations have to coordinate with the peptide, modulate its hydrophobicity and stabilize the water-solute interphase. These findings shed light on the impact that small changes have on the process of self-assembly and can help to understand the influence of the environmental conditions on the in vivo uncontrolled self-assembly of certain proteins., Ministerio de Economia, Industria y Competitividad, MINECO CTQ2017-86568-R FIS2017-85954-R, Agencia Estatal de Investigacion, AEI, Spain CTQ2017-86568-R FIS2017-85954-R, Fondo Europeo de Desarrollo Regional, FEDER, European Union CTQ2017-86568-R FIS2017-85954-R, FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades (Spain) P18-FR-3533, Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion FU2015-67284-R

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

Protein O-fucosyltransferase 1 (PoFUT1) is a GT-B fold enzyme that fucosylates proteins containing EGF-like repeats. GT-B glycosyltransferases have shown a remarkable grade of plasticity adopting closed and open conformations as a way of tuning their catalytic cycle, a feature that has not been observed for PoFUT1. Here, we analyzed Caenorhabditis elegans PoFUT1 (CePoFUT1) conformational behavior in solution by atomic force microscopy (AFM) and single-molecule fluorescence resonance energy transfer (SMF-FRET). Our results show that this enzyme is very flexible and adopts mainly compact conformations and to a lesser extend a highly dynamic population that oscillates between compact and highly extended conformations. Overall, our experiments illustrate the inherent complexity of CePoFUT1 dynamics, which might play a role during its catalytic cycle., ARAID: MEC (CTQ2013-44367-C2-2-P, BFU2016-75633-P and PID2019-105451GBI00 to RH-G, CTQ2017-85658-R and CTQ2014-56370-R to AO), Gobierno de Aragón (E35_R20 and LMP58_18), FEDER (2014-2020) funds for ‘Building Europe from Aragón’, Juan de la Cierva fellowship IJCI-2017-32874

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.

M.C.G.-G. thanks MICIU/AEI for a predoctoral fellowship. We thank Juan M. Cuerva and Delia Miguel (University of Granada, Spain) for the synthesis of the acridone dyes, Rafael Salto and Maria D. Giron (University of Granada, Spain) for providing the cultured cells, and Amparo Navarro and Tomas Peña-Ruiz (University of Jaen, Spain) for computational studies of the acridone dyes. M.C.G.-G. thanks MICIU/AEI for a predoctoral fellowship. We thank Juan M. Cuerva and Delia Miguel (University of Granada, Spain) for the synthesis of the acridone dyes, Rafael Salto and Maria D. Giron (University of Granada, Spain) for providing the cultured cells, and Amparo Navarro and Tomas Peña-Ruiz (University of Jaen, Spain) for computational studies of the acridone dyes., The precise knowledge of intracellular polarity, a physiological parameter that involves complex and intertwined intracellular mechanisms, may be relevant in the study of important diseases like cancer or Alzheimer's. In this technical note, we illustrate our recently developed, accurate method for obtaining intracellular polarity maps employing potent fluorescence microscopy techniques. Our method is based on the selection of appropriate luminescent probes, in which several emission properties vary with microenvironment polarity, specifically spectral shifts and luminescence lifetime. A multilinear calibration is performed, correlating polarity vs. spectral shift vs. luminescence lifetime, to generate a powerful and error-free 3D space for reliable interpolation of microscopy data. Multidimensional luminescence microscopy is then used to obtain simultaneously spectral shift and luminescence lifetime images, which are then interpolated in the 3D calibration space, resulting in accurate, quantitative polarity maps., Spanish Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and the European Regional Development Fund CTQ2017-85658-R.

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.

The aberrant aggregation of certain peptides and proteins, forming extracellular plaques of fibrillar material, is one of the hallmarks of amyloid diseases, such as Alzheimer's and Parkinson's. Herein, we have designed a new family of solvatochromic dyes based on the 9-amino-quinolimide moiety capable of reporting during the early stages of amyloid fibrillization. We have rationally improved the photophysical properties of quinolimides by placing diverse amino groups at the 9-position of the quinolimide core, leading to higher solvatochromic and fluorogenic character and higher lifetime dependence on the hydrophobicity of the environment, which represent excellent properties for the sensitive detection of prefibrillar aggregates. Among the different probes prepared, the 9-azetidinyl-quinolimide derivative showed striking performance in the following β-amyloid peptide (Aβ) aggregation in solution in real time and identifying the formation of different types of early oligomers of Aβ, the most important species linked to cytotoxicity, using novel, multidimensional fluorescence microscopy, with one- or two-photon excitation. Interestingly, the new dye allowed the visualization of proteinaceous inclusion bodies in a zebrafish model with neuronal damage induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Our results support the potential of the novel fluorophores as powerful tools to follow amyloid aggregation using fluorescence microscopy

We thank the company Nanogetic S.L. (Granada, Spain) for support with the synthesis of the SS peptides and the Centro de Instrumentacion Cientifica (CIC) of the Universidad de Granada for use of the TEM facilities., The main role of mitochondria, as pivotal organelles for cellular metabolism, is the production of energy (ATP) through an oxidative phosphorylation system. During this process, the electron transport chain creates a proton gradient that drives the synthesis of ATP. One of the main features of tumoral cells is their altered metabolism, providing alternative routes to enhance proliferation and survival. Hence, it is of utmost importance to understand the relationship between mitochondrial pH, tumoral metabolism, and cancer. In this manuscript, we develop a highly specific nanosensor to accurately measure the intramitochondrial pH using fluorescence lifetime imaging microscopy (FLIM). Importantly, we have applied this nanosensor to establish differences that may be hallmarks of different metabolic pathways in breast cancer cell models, leading to the characterization of different metabophenotypes., Spanish Ministerio de Ciencia, Innovacion y Universidades CTQ2014-56370-R CTQ2017-86568-R, European Union (EU), Spanish Agencia Estatal de Investigacion, Fundacion Ramon Areces

Two new families of lanthanide antennas are described. 8-Methoxy- 4,5-dihydrocyclopenta[de]quinolin-2(1H)-one phosphonates or carboxylates behave as selective antennas exhibiting Eu3+ luminescence in organic solvents, while quinolin-2(1H)-one analogues selectively sensitize the Tb3+ emission. These emissions are quenched by H2O addition. Based on this behaviour, the new lanthanide antennas can be used as highly sensitive water sensors., Spanish Ministerio de Economia y Competividad SAF2012-32209 FU2015-67284-R, Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion/European Regional Development Fund CTQ2017-85658-R CTQ2015-63997-C2, Consejo Superior de Investigaciones Cientificas (CSIC) 201580E073

The aberrant aggregation of certain peptides and proteins, forming extracellular plaques of fibrillar material, is one of the hallmarks of amyloid diseases, such as Alzheimer’s and Parkinson’s. Herein, we have designed a new family of solvatochromic dyes based on the 9-aminoquinolimide moiety capable of reporting during the early stages of amyloid fibrillization. We have rationally improved the photophysical properties of quinolimides by placing diverse amino groups at the 9-position of the quinolimide core, leading to higher solvatochromic and fluorogenic character and higher lifetime dependence on the hydrophobicity of the environment, which represent excellent properties for the sensitive detection of prefibrillar aggregates. Among the different probes prepared, the 9-azetidinylquinolimide derivative showed striking performance in the following β-amyloid peptide (Aβ) aggregation in solution in real time and identifying the formation of different types of early oligomers of Aβ, the most important species linked to cytotoxicity, using novel, multidimensional fluorescence microscopy, with one- or two-photon excitation. Interestingly, the new dye allowed the visualization of proteinaceous inclusion bodies in a zebrafish model with neuronal damage induced by the neurotoxin 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine. Our results support the potential of the novel fluorophores as powerful tools to follow amyloid aggregation using fluorescence microscopy in vivo, revealing heterogeneous populations of different types of aggregates and, more broadly, to study protein interactions., The work was supported by the Spanish Ministerio de Economía y Competividad grants SAF2012-32209, FU2015-67284-R, CTQ2017-85658-R, Ministerio de Ciencia, Innovación y Univer-sidades grant PGC2018-094644-B-C22, CSIC grant 201580E073, and Comunidad Autónoma de Madrid grant P2018/EMT-4329 AIRTEC-CM.

The photophysical properties of naphthalimide-based fluorophores can be easily tuned by chemical manipulation of the substituents on that privileged scaffold. Replacement of a OMe group at position 6 in 2-(hydroxyl)ethyl-naphthalimide derivatives by diverse amines, including 2-(hydroxyl)ethylamine, trans-(4-acetamido)cyclohexylamine and azetidine increases the solvatochromic (ICT) character, while this replacement in 2-(dimethylamino)ethyl-naphthalimide analogues (PET fluorophores) decrease their solvent polarity sensitivity or even reversed them to solvatochromic fluorophores. These fluorophores resulted macrophage nucleus imaging probes, which bind DNA as intercalants and showed low cytotoxicity in human cancer cells, The work was supported by grants SAF2012-32209, FU2015-67284-R, CTQ2017-85658-R form the Spanish Ministerio de Economía y Competividad, Agencia Estatal de Investigación and the European Regional Development Fund; and the CSIC grant 201580E073

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

Three new boron dipyrromethene (BODIPY) dyes substituted with a benzylamino group at the 3-position have been synthesized from halogenated BODIPYs by nucleophilic substitution. The spectroscopic and photophysical properties have been explored in different solvents and have been compared with an analogous 8- benzylaminoBODIPY derivative. The position of the benzylamino group has a significant influence on the spectral band positions, fluorescence quantum yields, and fluorescence lifetimes. The 8- benzylaminoBODIPY emits in the blue range, whereas the 3- substituted shows green fluorescence. Additionally, the extension of the conjugation at the 5-position of 3-benzylaminoBODIPY produces a bathochromic shift of the absorption and emission spectra. The solvent effect on their spectroscopic features has been investigated using the generalized Catalan solvent scales. Quantum-chemical calculations have been performed to assess the effect of the position of the substitution on the spectroscopic properties. Finally, the BODIPY dyes have been employed as probes in fluorescence lifetime imaging of living cells, demonstrating their high potential for bioimaging.

In this manuscript, we report the first synthesis of an organic monomolecular emitter, which behaves as a circularly polarized luminescence (CPL)-based ratiometric probe. The enantiopure helical ortho-oligo(phenylene)ethynylene ( o-OPE) core has been prepared by a new and efficient macrocyclization reaction. The combination of such o-OPE helical skeleton and a pyrene couple leads to two different CPL emission features in a single structure whose ratio linearly responds to silver(I) concentration.

This work was supported by the Spanish Ministerio de Economia y Competitividad (grant SAF2015-66690-R), the Spanish Ministerio de Ciencia, Innovaci on y Universidades (MCIU/AEI/FEDER, UE; grants RTI2018-093940-B-I00 to CD, and CTQ2017-85658-R to AO) and the Japan Society for the promotion of Science (JSPS grant- 17F17420 to GUE). MAU is funded through a studentship from the Petroleum Technology Development Fund (PTDF), Abuja, Nigeria. IAA was funded through a Ph.D. studentship from the Ministry of Health of Saudi Arabia.We thank Dr. Jos e Cumella for the synthesis of the 1,14-dibromotetradecane precursor and to Prof. Ibon Alkorta for his help with DFT calculations. We also thank Professor Fred Opperdoes and Dr Alena Zíkov a for their insightful discussions on T. brucei energy metabolism., We have recently reported on the development and trypanocidal activity of a class of inhibitors of Trypanosome Alternative Oxidase (TAO) that are targeted to the mitochondrial matrix by coupling to lipophilic cations via C14 linkers to enable optimal interaction with the enzyme’s active site. This strategy resulted in a much-enhanced anti-parasite effect, which we ascribed to the greater accumulation of the compound at the location of the target protein, i.e. the mitochondrion, but to date this localization has not been formally established. We therefore synthesized a series of fluorescent analogues to visualize accumulation and distribution within the cell. The fluorophore chosen, julolidine, has the remarkable extra feature of being able to function as a viscosity sensor and might thus additionally act as a probe of the cellular glycerol that is expected to be produced when TAO is inhibited. Two series of fluorescent inhibitor conjugates incorporating a cationic julolidine-based viscosity sensor were synthesized and their photophysical and biological properties were studied. These probes display a red emission, with a high signal-to-noise ratio (SNR), using both single- and two-photon excitation. Upon incubation with T. brucei and mammalian cells, the fluorescent inhibitors 1a and 2a were taken up selectively in the mitochondria as shown by live-cell imaging. Efficient partition of 1a in functional isolated (rat liver) mitochondria was estimated to 66 ± 20% of the total. The compounds inhibited recombinant TAO enzyme in the submicromolar (1a, 2c, 2d) to low nanomolar range (2a) and were effective against WT and multidrug-resistant trypanosome strains (B48, AQP1-3 KO) in the submicromolar range. Good selectivity (SI > 29) over mammalian HEK cells was observed. However, no viscosity-related shift could be detected, presumably because the glycerol was produced cytosolically, and released through aquaglyceroporins, whereas the probe was located, virtually exclusively, in the trypanosome’s mitochondrion., Spanish Ministerio de Economia y Competitividad (grant SAF2015-66690-R), Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU/AEI/FEDER, UE; grants RTI2018-093940-B-I00 to CD, and CTQ2017-85658-R to AO), Japan Society for the promotion of Science (JSPS grant- 17F17420 to GUE), Petroleum Technology Development Fund (PTDF), Abuja, Nigeria, Ph.D. studentship from the Ministry of Health of Saudi Arabia

A simple method for direct detection of microRNAs (miRs) in human serum without the use of polymerase amplification is presented, achieving low miR-122 concentrations and importantly, discerning effectively single-base sequence mutations. The method is based on the capture of target miRs with synthetic peptide nucleic acid oligomers, dynamic chemical labelling, separation with quaternary amine microplatforms and detection using time-gated fluorescence imaging.

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.

Luminescence emission is a multidimensional phenomenon comprising a time-domain layer defined by its excited-state kinetics and corresponding lifetime, which is specific to each luminophore and depends on environmental conditions. This feature allows for the discrimination of luminescence signals from species with a similar spectral profile but different lifetimes by time-gating (TG) the acquisition of luminescence. This approach represents an efficient tool for removing unwanted, usually short-lived, signals from scattered light and fluorescence interferents using luminophores with a long lifetime. Due to the emergence of time-resolved techniques using rapid excitation and acquisition methods (i.e. pulsed lasers and single-photon timing acquisition) and new long-lifetime luminophores (i.e. acridones, lanthanide complexes, nanoparticles, etc.), TG analyses can be easily applied to relevant chemical and biochemical issues. The successful application of TG to important biomedical topics has attracted the attention of the R&D industry due to its potential in the development and patenting of new probes, methods and techniques for drug discovery, immunoassays, biomarker discovery and biomolecular interactions, etc. Here, we review the technological efforts of innovative companies in the application of TG-based techniques.

Ferritin, a soluble and highly robust protein with subunits packed into well-defined helices, is a key component of the iron regulatory system in the brain and thus is widely recognized as a crucial protein for iron metabolism, but may also bear possible implications in some neurodegenerative disorders. Here, we present evidence of how human recombinant apoferritin can convert into an unusual structure from its folded native state; that is, amyloid fibrils analogue to those found in pathological disorders such as Alzheimer's and Parkinson's diseases. An extensive combination of advanced microscopy, spectroscopy and scattering techniques concur to reveal that apoferritin fibrils possess a common double stranded twisted ribbon structure which can result in a mesoscopic right-handed chirality. We highlight a direct connection between the chirality and morphology of the resulting amyloid fibrils, and the initial protein subunits composition, advancing our understanding on the possible role of misfolding in some ferritin-related pathologies and posing new bases for the design of chiral 1D functional nanostructures.

Recently, research in the field of protein amyloid fibers has gained great attention due to the use of these materials as nanoscale templates for the construction of functional hybrid materials. The formation of apoferritin amyloid-like protein fibers is demonstrated herein for the first time. The morphology, size and stiffness of these one-dimensional structures are comparable to the fibers formed by β-lactoglobulin, a protein frequently used as a model in the study of amyloid-like fibrillar proteins. Nanometer-sized globular apoferritin is capable of self-assembling to form 1D micrometer-sized structures after being subjected to a heating process. Depending on the experimental conditions, fibers with different morphologies and sizes are obtained. The wire-like protein structure is rich in functional groups and allows chemical functionalization with diverse quantum dots (QD), as well as with different Alexa Fluor (AF) dyes, leading to hybrid fluorescent fibers with variable emission wavelengths, from green to near infrared, depending on the QD and AFs coupled. For fibers containing the pair AF488 and AF647, efficient fluorescence energy transfer from the covalently coupled donor (AF488) to acceptor tags (AF647) takes place. Apoferritin fibers are proposed here as a new promising template for obtaining hybrid functional materials., This work was funded by the Junta de Andalucía (Project P11-FQM-8136 and Project P10-FQM-6154), MINECO (Projects CTQ2012-32236, RyC2011-07637, MAT2012-34487, PTA2011- 6702-I, MAT2015-66605-P and CTQ2015-64538) and the Euro- pean Commission (FP7-PEOPLE-2011-CIG no. 303620).