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

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

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

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

Simple and non-charged long-lived fluorescent intracellular organelle trackers

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 AndalucíaMinisterio de Economía y Competitivida

Programming Skeletal Muscle Metabolic Flexibility in Offspring of Male Rats in Response to Maternal Consumption of Slow Digesting Carbohydrates during Pregnancy

Skeletal muscle plays a relevant role in metabolic flexibility and fuel usage and the associated muscle metabolic inflexibility due to high-fat diets contributing to obesity and type 2 diabetes. Previous research from our group indicates that a high-fat and rapid-digesting carbohydrate diet during pregnancy promotes an excessive adipogenesis and also increases the risk of non-alcoholic fatty liver disease in the offspring. This effect can be counteracted by diets containing carbohydrates with similar glycemic load but lower digestion rates. To address the role of the skeletal muscle in these experimental settings, pregnant rats were fed high-fat diets containing carbohydrates with similar glycemic load but different digestion rates, a high fat containing rapid-digesting carbohydrates diet (HF/RD diet) or a high fat containing slow-digesting carbohydrates diet (HF/SD diet). After weaning, male offspring were fed a standard diet for 3 weeks (weaning) or 10 weeks (adolescence) and the impact of the maternal HF/RD and HF/SD diets on the metabolism, signaling pathways and muscle transcriptome was analyzed. The HF/SD offspring displayed better muscle features compared with the HF/RD group, showing a higher muscle mass, myosin content and differentiation markers that translated into a greater grip strength. In the HF/SD group, metabolic changes such as a higher expression of fatty acids (FAT/CD36) and glucose (GLUT4) transporters, an enhanced glycogen content, as well as changes in regulatory enzymes such as muscle pyruvate kinase and pyruvate dehydrogenase kinase 4 were found, supporting an increased muscle metabolic flexibility and improved muscle performance. The analysis of signaling pathways was consistent with a better insulin sensitivity in the muscle of the HF/SD group.This research was funded by European Union’s Seventh Framework Programme (FP7/2007–2013): project Early Nutrition, under grant agreement no. 289346

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

Intake of slow-digesting carbohydrates is related to changes in the microbiome and its functional pathways in growing rats with obesity induced by diet

Introduction: The main cause of insulin resistance in childhood is obesity, which contributes to future comorbidities as in adults. Although high-calorie diets and lack of exercise contribute to metabolic disease development, food quality rather than the quantity of macronutrients is more important than food density. The purpose of the present study was to examine the effects of changing the quality of carbohydrates from rapidly to slowly digestible carbohydrates on the composition of the gut microbiota and the profiles of the functional pathways in growing rats with obesity due to a high-fat diet (HFD). Methods: During the course of 4 weeks, rats growing on an HFD-containing carbohydrates with different digestive rates were fed either HFD-containing carbohydrates with a rapid digestion rate (OBE group) or HFD-containing carbohydrates with a slow digestion rate (OBE-ISR group). A non-obese group (NOB) was included as a reference, and rats were fed on a rodent standard diet (AIN93G). An analysis of gut microbiota was conducted using 16S rRNA-based metagenomics; a linear mixed-effects model (LMM) was used to determine changes in abundance between baseline and 4 weeks of treatment, and functional pathways were identified. Gut microbiota composition at bacterial diversity and relative abundance, at phylum and genus levels, and functional profiles were analyzed by integrating the Integrated Microbial Genomes (IMG) database. Results: The groups showed comparable gut microbiota at baseline. At the end of the treatment, animals from the ISR group exhibited differences at the phylum levels by decreasing the diversity of Fisher’s index and Firmicutes (newly named as Bacillota), and increasing the Pielou’s evenness and Bacteroidetes (newly named as Bacteroidota); at the genus level by increasing Alistipes, Bifidobacterium, Bacteroides, Butyricimonas, Lachnoclostridium, Flavonifractor, Ruminiclostridium 5, and Faecalibaculum and decreasing Muribaculum, Blautia, and Ruminiclostridium 9. Remarkably, relative abundances of genera Tyzzerella and Angelakisella were higher in the OBE group compared to NOB and OBE-ISR groups. In addition, some microbiota carbohydrate metabolism pathways such as glycolysis, glucuronic acid degradation, pentose phosphate pathway, methanogenesis, and fatty acid biosynthesis exhibited increased activity in the OBE-ISR group after the treatment. Higher levels of acetate and propionate were found in the feces of the ISR group compared with the NOB and OBE groups. Conclusion: The results of this study demonstrate that replacing rapidly digestible carbohydrates with slowly digestible carbohydrates within an HFD improve the composition of the gut microbiota. Consequently, metabolic disturbances associated with obesity may be prevented.Abbott Laboratories S.A"Fundacion Ramon Areces", Madrid, Spai

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