Genetically encoded red fluorescent pH ratiometric sensor: Application to measuring pH gradient abnormalities in cystic fibrosis cells

Genetically encoded probes to measure in vivo pH are challenging. They must be chloride insensitive and require normalization of the transfection efficiency. Furthermore, probes that emit in the red or far red are advisable to promote in vivo use. The mBeRFP D162T fluorescent protein presents two emission bands with different pH sensitivities. When the probe was cytosolic-expressed in HeLa as control cells and CFBE41o, an epithelial cell line that carries an F508del mutation in the CFTR transporter, the ratiometric measurement between both emission bands allows us to determine the pH, demonstrating that mBeRFP D162T can be used to accurately measure the cytosolic pH differences of these cell lines. Furthermore, we have located the sensor inside or outside the lysosomal membrane to investigate the lysosomal pH gradient. In HeLa cells, our probe detected pH gradient changes under conditions known to alter lysosomal pH. In the CFBE41o cells, which mimic cystic fibrosis disease, we observed a complete loss of lysosomal acidification. Using lumacaftor, a drug that restores functioning CFTR protein, partially brings back the pH gradient. In conclusion, mBeRFP D162T is a valuable tool for measuring in vivo intracellular pH values and lysosomal pH gradient dynamics in physiological or pathological conditions

Poly(ethylene-imine)-Functionalized Magnetite Nanoparticles Derivatized with Folic Acid: Heating and Targeting Properties

Magnetite nanoparticles (MNPs) coated by branched poly (ethylene-imine) (PEI) were synthesized in a one-pot. Three molecular weights of PEI were tested, namely, 1.8 kDa (sample MNP-1), 10 kDa (sample MNP-2), and 25 kDa (sample MNP-3). The MNP-1 particles were further functionalized with folic acid (FA) (sample MNP-4). The four types of particles were found to behave magnetically as superparamagnetic, with MNP-1 showing the highest magnetization saturation. The particles were evaluated as possible hyperthermia agents by subjecting them to magnetic fields of 12 kA/m strength and frequencies ranging between 115 and 175 kHz. MNP-1 released the maximum heating power, reaching 330 W/g at the highest frequency, in the high side of reported values for spherical MNPs. In vitro cell viability assays of MNP-1 and MNP-4 against three cell lines expressing different levels of FA receptors (FR), namely, HEK (low expression), and HeLa (high expression), and HepG2 (high expression), demonstrated that they are not cytotoxic. When the cells were incubated in the presence of a 175 kHz magnetic field, a significant reduction in cell viability and clone formation was obtained for the high expressing FR cells incubated with MNP-4, suggesting that MNP-4 particles are good candidates for magnetic field hyperthermia and active targeting.Spanish Institutions: Ministerio de Ciencia, Innovación y Universidades (PGC2018-098770-B-I00 and CTQ2017-86125-P)Junta de Andalucía (ProgramaOperativo FEDER 2014-2020, grants B-FQM-141-UGR18, A1-FQM-341-UGR-18, C-FQM-497-UGR18

Transmissible spongiform encephalopathies: molecular biology, diagnosis and therapeutic approaches

Las encefalopatías espongiformes transmisibles constituyen un grupo de enfermedades neurodegenerativas que están asociadas a la presencia en el tejido nervioso de agregados insolubles constituidos por una isoforma anómala de una proteína denominada prión. Esta isoforma se produce por un cambio conformacional en una molécula que puede transmitirse a otras proteínas priónicas normales. Las proteínas modificadas pierden su actividad biológica, desencadenándose la muerte de las neuronas por apoptosis. Los cambios conformacionales de los priones que derivan en enfermedad pueden deberse a la existencia de mutaciones que disminuyan la estabilidad de las formas celulares. Existe susceptibilidad genética, por tanto, a padecer tipos hereditarios de la enfermedad o adquiridos por infección con isoformas priónicas anormales. En la actualidad se están perfeccionando métodos sensibles de diagnóstico basados en la detección de las isoformas anormales de la proteína priónica. Todavía no existen tratamientos curativos para estas enfermedades aunque se están diseñando métodos terapéuticos que bloqueen los cambios conformacionales que conducen a la precipitación de la proteína priónica.Transmissible spongiform encephalopaties constitute a group of neurodegenerative diseases associated to the presence in nervous tissue of insoluble aggregates originated by an anomalous isoform of a protein called prion. This isoform is produced by a conformational change in a molecule which is transmitted to normal prion proteins. Modified proteins loose their biological activity, provoking apoptotic death of neurons. Conformational changes of prions occasioning the illness can be due to the existence of mutations diminishing the stability of cellular forms. There is genetic susceptibility for suffering any type of the illness, either hereditary or acquired by infection with abnormal prion isoforms. New sensitive diagnostic methods are presently being developed, based on detection of abnormal isoforms of prion protein. There are no curative treatments for these illnesses, although new therapeutic strategies based on the blockage of conformational changes driving to protein precipitation are being designed

Amphiphilic-like carbon dots as antitumoral drug vehicles and phototherapeutical agents

The work was financially supported by the Spanish institutions Ministerio de Ciencia, Innovacion y Universidades (PGC2018-098770-B-I00 and CTQ2017-86125-P) and Junta de Andalucia (ProgramaOperativo FEDER 2014-2020, grants B-FQM-141-UGR18, A1-FQM-341-UGR-18, C-FQM-497-UGR18).Water-insoluble carbon dots are recognized as promising materials, although their applications in nanomedicine are rarely explored, despite their lipophilic character and foreseen compatibility with biological membranes. In this article, we exploit the anhydride functionalization of carbon dots obtained by thermolysis of citric acid to synthesize amphiphilic-like carbon dots (LCDs) by reaction with alkyl amines. A differential feature of this approach is that the hydrophobicity of LCDs is a balance between the ionization of the carboxylic groups resulting from the reaction and the hydrophobicity from the grafted amines. The alkyl chains allow LCDs to entrap hydrophobic molecules and the ionization of the carboxylic groups increases the hydrosolubility, permitting the transfer between organic and aqueous phases. The biomedical interest of these features is illustrated by analyzing the application of LCDs as carriers of the drug campothecin and their evaluation on a battery of cancer cell lines, as well as the transformation of LCDs into a phototherapeutic agent by the formation of a complex with IR780 dye. Results demonstrate that LCDs behave as nanocarriers in a manner that resembles other supramolecular hosts with two differential features: (i) the length of the alkyl chains determines the size of the hosted guest, and (ii) the hydrosolubility of the complex can be modulated by pH.Ministerio de Ciencia, Innovacion y Universidades PGC2018-098770-B-I00 CTQ2017-86125-PJunta de Andalucia B-FQM-141-UGR18 A1-FQM-341-UGR-18 C-FQM-497-UGR1

Dynamic Excimer (DYNEX) Imaging of Lipid Droplets

This work was funded by grants CTQ2017-85658-R (MICIU/AEI/ERDF), FQM-337 (Junta de Andalucía), and PIUJA 2019-20 (Universidad de Jaén). We acknowledge the Universidad de Granada (Spain) microscopy central facilities (CIC-UGR) and computing time from CSIRC-UGR. M.C.G.-G. thanks MICIU/AEI for a predoctoral fellowship. Funding for open access charge: Universidad de Granada/CBUA. We are indebted to Prof. Reinhard Jahn for his generous support.Unraveling cellular physiological processes via luminescent probes that target specific cellular microenvironments is quite challenging due to the uneven distribution of probes. Herein, we designed a new dynamic excimer (DYNEX) imaging method that involves the sensitive detection of nanosecond-scale dynamic molecular contacts of a fluorescent acridone derivative and reveals the cell microenvironment polarity. Using our method, we specifically tracked cell lipid droplets in fibroblast colon carcinoma cells. These organelles play a central role in metabolic pathways, acting as energy reservoirs in regulatory processes. DYNEX imaging provides the inner polarity of cell lipid droplets, which can be related to lipid contents and metabolic dysfunctions. This new methodology will inspire development of novel multidimensional fluorescent sensors that are able to provide target-specific and orthogonal information at the nanosecond scale.CBUAMICIUUniversidad de GranadaUniversidad de JaénAgencia Estatal de InvestigaciónEuropean Regional Development Fund FQM-337Junta de Andalucía PIUJA 2019-2

Beneficial Effects of Bovine Milk Exosomes in Metabolic Interorgan Cross-Talk

Extracellular vesicles are membrane-enclosed secreted vesicles involved in cell-to-cell communication processes, identified in virtually all body fluids. Among extracellular vesicles, exosomes have gained increasing attention in recent years as they have unique biological origins and deliver different cargos, such as nucleic acids, proteins, and lipids, which might mediate various health processes. In particular, milk-derived exosomes are proposed as bioactive compounds of breast milk, which have been reported to resist gastric digestion and reach systemic circulation, thus being bioavailable after oral intake. In the present manuscript, we critically discuss the available evidence on the health benefits attributed to milk exosomes, and we provide an outlook for the potential future uses of these compounds. The use of milk exosomes as bioactive ingredients represents a novel avenue to explore in the context of human nutrition, and they might exert important beneficial effects at multiple levels, including but not limited to intestinal health, bone and muscle metabolism, immunity, modulation of the microbiota, growth, and development

A Slow-Digesting Carbohydrate Diet during Rat Pregnancy Protects O spring from Non-Alcoholic Fatty Liver Disease Risk through the Modulation of the Carbohydrate-Response Element and Sterol Regulatory Element Binding Proteins

High-fat (HF) and rapid digestive (RD) carbohydrate diets during pregnancy promote excessive adipogenesis in o spring. This e ect can be corrected by diets with similar glycemic loads, but low rates of carbohydrate digestion. However, the e ects of these diets on metabolic programming in the livers of o spring, and the liver metabolism contributions to adipogenesis, remain to be addressed. In this study, pregnant insulin-resistant rats were fed high-fat diets with similar glycemic loads but di erent rates of carbohydrate digestion, High Fat-Rapid Digestive (HF–RD) diet or High Fat-Slow Digestive (HF–SD) diet. O spring were fed a standard diet for 10 weeks, and the impact of these diets on the metabolic and signaling pathways involved in liver fat synthesis and storage of o spring were analyzed, including liver lipidomics, glycogen and carbohydrate and lipid metabolism key enzymes and signaling pathways. Livers from animals whose mothers were fed an HF–RD diet showed higher saturated triacylglycerol deposits with lower carbon numbers and double bond contents compared with the HF–SD group. Moreover, the HF–RD group exhibited enhanced glucose transporter 2, pyruvate kinase (PK), acetyl coenzyme A carboxylase (ACC) and fatty acid (FA) synthase expression, and a decrease in pyruvate carboxylase (PyC) expression leading to an altered liver lipid profile. These parameters were normalized in the HF–SD group. The changes in lipogenic enzyme expression were parallel to changes in AktPKB phosphorylation status and nuclear expression in carbohydrate-response element and sterol regulatory element binding proteins. In conclusion, an HF–RD diet during pregnancy translates to changes in liver signaling and metabolic pathways in o spring, enhancing liver lipid storage and synthesis, and therefore non-alcoholic fatty liver disease (NAFLD) risk. These changes can be corrected by feeding an HF–SD diet during pregnancy.This research was funded by the European Union’s Seventh Framework Programme (FP7/2007–2013): project EarlyNutrition, under grant agreement no. 289346

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.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

Single chain variable fragment fused to maltose binding protein: a modular nanocarrier platform for the targeted delivery of antitumorals

This work was supported by grants CTQ2014-55474-C2-1-R, CTQ2014-55474-C2-2-R and CTQ2017-86125-P from the Ministerio Economia, Industria y Competitividad (co-financed by FEDER funds). SP is supported by a FPU fellowship (FPU17/ 04749). We acknowledge the University of Granada (Spain) cell culture, animal and microscopy central facilities (CIC-UGR).The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due to their smaller size, low immunogenicity, and low-cost production. Although covalent strategies for the preparation of such ScFv-based therapeutic conjugates are prevalent, this approach is not straightforward, as it requires prior chemical activation and/or modification of both the ScFv and the therapeutics for the application of robust chemistries. A non-covalent alternative based on ScFv fused to maltose-binding protein (MBP) acting as a binding adapter is proposed for active targeted delivery. MBP-ScFv proves to be a valuable modular platform to synergistically bind maltose-derivatized therapeutic cargos through the MBP, while preserving the targeting competences provided by the ScFv. The methodology has been tested by using a mutated maltose-binding protein (MBP I334W) with an enhanced affinity toward maltose and an ScFv coding sequence toward the human epidermal growth factor receptor 2 (HER2). Non-covalent binding complexes of the resulting MBP-ScFv fusion protein with diverse maltosylated therapeutic cargos (a near-infrared dye, a maltosylated supramolecular beta-cyclodextrin container for doxorubicin, and non-viral polyplex gene vector) were easily prepared and characterized. In vitro and in vivo assays using cell lines that express or not the HER2 epitope, and mice xenografts of HER2 expressing cells demonstrated the capability and versatility of MBP-ScFv for diagnosis, imaging, and drug and plasmid active targeted tumor delivery. Remarkably, the modularity of the MBP-ScFv platform allows the flexible interchange of both the cargos and the coding sequence for the ScFv, allowing ad hoc solutions in targeting delivery without any further optimization since the MBP acts as a pivotal element.Ministerio Economia, Industria y Competitividad - FEDER funds CTQ2014-55474-C2-1-R CTQ2014-55474-C2-2-R CTQ2017-86125-PSpanish Government FPU17/0474

Seeding and Growth of β-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 β-amyloid peptide (Aβ) 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 Aβ 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 Aβ 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 Aβ 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.Spanish Ministerio de Ciencia, Innovacion y Universidades CTQ2014-56370-R CTQ2017-86568-R CTQ2017-86125-PSpanish Agencia Estatal de InvestigacionEuropean Union (EU