33 research outputs found
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
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 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
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
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