Easy and Versatile Synthesis of Bulk Quantities of Highly Enriched 13C-Graphene Materials for Biological and Safety Applications

The preparation of bulk quantities of 13C-labeled graphene materials is relevant for basic investigations and for practical applications. In addition, 13C-labeled graphene materials can be very useful in biological and environmental studies, as they may allow the detection of graphene or its derivatives in cells or organs. In this paper, we describe the synthesis of 13C-labeled graphene materials (few-layer graphene, FLG, and graphene oxide, GO) on a tens of mg scale, starting from 13C-labeled methane to afford carbon fibers, followed by liquid-phase exfoliation (FLG) or oxidation (GO). The materials have been characterized by several analytical and microscopic techniques, including Raman and nuclear magnetic resonance spectroscopies, thermogravimetric analysis, X-ray photoelectron spectroscopy, and X-ray powder diffraction. As a proof of concept, the distribution of the title compounds in cells has been investigated. In fact, the analysis of the 13C/12C ratio with isotope ratio mass spectrometry (IRMS) allows the detection and quantification of very small amounts of material in cells or biological compartments with high selectivity, even when the material has been degraded. During the treatment of 13C-labeled FLG with HepG2 cells, 4.1% of the applied dose was found in the mitochondrial fraction, while 4.9% ended up in the nuclear fraction. The rest of the dose did not enter into the cell and remained in the plasma membrane or in the culture media.La preparación de cantidades a granel de materiales de grafeno marcados con 13C es relevante para investigaciones básicas y para aplicaciones prácticas. Además, los materiales de grafeno marcados con 13C pueden ser muy útiles en estudios biológicos y medioambientales, ya que pueden permitir la detección de grafeno o sus derivados en células u órganos. En este trabajo describimos la síntesis de materiales de grafeno marcados con 13C (grafeno de pocas capas, FLG, y óxido de grafeno, GO) a escala de decenas de mg, partiendo de metano marcado con 13C para obtener fibras de carbono, seguidas de exfoliación en fase líquida (FLG) u oxidación (GO). Los materiales se han caracterizado mediante varias técnicas analíticas y microscópicas, como las espectroscopias Raman y de resonancia magnética nuclear, el análisis termogravimétrico, la espectroscopia de fotoelectrones de rayos X y la difracción de rayos X en polvo. Como prueba de concepto, se ha investigado la distribución de los compuestos del título en las células. De hecho, el análisis de la relación 13C/12C con espectrometría de masas de relación isotópica (IRMS) permite la detección y cuantificación de cantidades muy pequeñas de material en células o compartimentos biológicos con alta selectividad, incluso cuando el material se ha degradado. Durante el tratamiento de FLG marcado con 13C con células HepG2, el 4,1% de la dosis aplicada se encontró en la fracción mitocondrial, mientras que el 4,9% acabó en la fracción nuclear. El resto de la dosis no entró en la célula y permaneció en la membrana plasmática o en el medio de cultivo

Spatial and Temporal Protein Modules Signatures Associated with Alzheimer Disease in 3xTg-AD Mice Are Restored by Early Ubiquinol Supplementation

Despite its robust proteopathic nature, the spatiotemporal signature of disrupted protein modules in sporadic Alzheimer's disease (AD) brains remains poorly understood. This considered oxidative stress contributes to AD progression and early intervention with coenzyme Q10 or its reduced form, ubiquinol, delays the progression of the disease. Using MALDI-MSI and functional bioinformatic analysis, we have developed a protocol to express how deregulated protein modules arise from hippocampus and cortex in the AD mice model 3xTG-AD in an age-dependent manner. This strategy allowed us to identify which modules can be efficiently restored to a non-pathological condition by early intervention with ubiquinol. Indeed, an early deregulation of proteostasis-related protein modules, oxidative stress and metabolism has been observed in the hippocampus of 6-month mice (early AD) and the mirrored in cortical regions of 12-month mice (middle/late AD). This observation has been validated by IHC using mouse and human brain sections, suggesting that these protein modules are also affected in humans. The emergence of disrupted protein modules with AD signature can be prevented by early dietary intervention with ubiquinol in the 3xTG-AD mice model.A pesar de su robusta naturaleza proteopática, la firma espaciotemporal de los módulos de proteínas interrumpidos en los cerebros de la enfermedad de Alzheimer (EA) esporádica sigue siendo poco conocida. Este considerado estrés oxidativo contribuye a la progresión de la EA y la intervención precoz con coenzima Q10 o su forma reducida, el ubiquinol, retrasa la progresión de la enfermedad. Usando MALDI-MSI y análisis bioinformático funcional, hemos desarrollado un protocolo para expresar cómo surgen módulos de proteína desregulados del hipocampo y la corteza en el modelo de ratones AD 3xTG-AD de una manera dependiente de la edad. Esta estrategia nos permitió identificar qué módulos se pueden restaurar de manera eficiente a una condición no patológica mediante una intervención temprana con ubiquinol. De hecho, una desregulación temprana de los módulos proteicos relacionados con la proteostasis, Se ha observado estrés oxidativo y metabolismo en el hipocampo de ratones de 6 meses (EA temprana) y se refleja en regiones corticales de ratones de 12 meses (EA media/tardía). Esta observación ha sido validada por IHC utilizando secciones de cerebro humano y de ratón, lo que sugiere que estos módulos de proteína también se ven afectados en humanos. La aparición de módulos de proteínas interrumpidos con la firma AD puede prevenirse mediante una intervención dietética temprana con ubiquinol en el modelo de ratones 3xTG-AD

CoQ10 reduces glioblastoma growth and infiltration through proteome remodeling and inhibition of angiogenesis and inflammation

Purpose: Most monotherapies available against glioblastoma multiforme (GBM) target individual hallmarks of this aggressive brain tumor with minimal success. In this article, we propose a therapeutic strategy using coenzyme Q10 (CoQ10) as a pleiotropic factor that crosses the blood-brain barrier and accumulates in cell membranes acting as an antioxidant, and in mitochondrial membranes as a regulator of cell bioenergetics and gene expression. Methods: Xenografts of U251 cells in nu/nu mice were used to assay tumor growth, hypoxia, angiogenesis, and inflammation. An orthotopic model was used to explore microglial infiltration, tumor growth, and invasion into the brain parenchyma. Cell proliferation, migration, invasion, proteome remodeling, and secretome were assayed in vitro. Conditioned media were used to assay angiogenesis, monocyte chemoattraction, and differentiation into macrophages in vitro. Results: CoQ10 treatment decreased tumor volume in xenografts and orthotopic models, although its effect on tumor cell proliferation was not direct. Tumors from mice treated with CoQ10 were less hypoxic and vascularized, having less infiltration from inflammatory cells. Treatment-induced downregulation of HIF-1α and NF-kB led to a complete remodeling of the tumor cells proteome and secretome, impacting angiogenesis, monocyte infiltration, and their differentiation into macrophages. Besides, tumor cell migration and invasion were drastically restricted by mechanisms involving modulation of the actin cytoskeleton and downregulation of matrix metalloproteases (MMPs). Conclusions: CoQ10 has a pleiotropic effect on GBM growth, targeting several hallmarks simultaneously. Thus, its integration into current treatments of this fatal disease should be considered. Keywords: Angiogenesis; Coenzyme Q10; Glioblastoma; Inflammation; Invasion.Propósito: La mayoría de las monoterapias disponibles contra el glioblastoma multiforme (GBM) se dirigen a las características individuales de este tumor cerebral agresivo con un éxito mínimo. En este artículo proponemos una estrategia terapéutica utilizando la coenzima Q 10 (CoQ 10 ) como factor pleiotrópico que atraviesa la barrera hematoencefálica y se acumula en las membranas celulares actuando como antioxidante, y en las membranas mitocondriales como regulador de la bioenergética celular y gen expresión. Métodos: Se utilizaron xenoinjertos de células U251 en ratones nu/nu para analizar el crecimiento tumoral, la hipoxia, la angiogénesis y la inflamación. Se utilizó un modelo ortotópico para explorar la infiltración microglial, el crecimiento tumoral y la invasión del parénquima cerebral. Se ensayaron in vitro la proliferación celular, la migración, la invasión, la remodelación del proteoma y el secretoma. Se usaron medios acondicionados para analizar la angiogénesis, la quimioatracción de monocitos y la diferenciación en macrófagos in vitro. Resultados: el tratamiento con CoQ 10 disminuyó el volumen tumoral en xenoinjertos y modelos ortotópicos, aunque su efecto sobre la proliferación de células tumorales no fue directo. Los tumores de ratones tratados con CoQ 10 eran menos hipóxicos y vascularizados, con menos infiltración de células inflamatorias. La regulación a la baja inducida por el tratamiento de HIF-1α y NF-kB condujo a una remodelación completa del proteoma y el secretoma de las células tumorales, lo que impactó en la angiogénesis, la infiltración de monocitos y su diferenciación en macrófagos. Además, la migración e invasión de células tumorales se vieron drásticamente restringidas por mecanismos que involucran la modulación del citoesqueleto de actina y la regulación a la baja de las metaloproteasas de matriz (MMP). Conclusiones: CoQ 10 tiene un efecto pleiotrópico en el crecimiento de GBM, apuntando a varios sellos simultáneamente. Por lo tanto, se debe considerar su integración en los tratamientos actuales de esta enfermedad mortal

The Protective Effect of Ubiquinone against the Amyloid Peptide in Endothelial Cells Is Isoprenoid Chain Length-Dependent

Vascular brain pathology constitutes a common feature in neurodegenerative diseases that could underlie their development. Indeed, vascular dysfunction acts synergistically with neurodegenerative changes to exacerbate the cognitive impairment found in Alzheimer’s disease. Different injuries such as hypertension, high glucose, atherosclerosis associated with oxidized low-density lipoprotein or inflammation induce NADPH oxidase activation, overproduction of reactive oxygen species, and apoptosis in endothelial cells. Since it has been shown that pretreatment of cultured endothelial cells with the lipophilic antioxidant coenzyme Q10 (CoQ10) displays a protective effect against the deleterious injuries caused by different agents, this study explores the cytoprotective role of different CoQs homologues against Aβ25–35-induced damage and demonstrates that only pretreatment with CoQ10 protects endothelial brain cells from Aβ25–35-induced damage. Herein, we show that CoQ10 constitutes the most effective ubiquinone in preventing NADPH oxidase activity and reducing both reactive oxygen species generation and the increase in free cytosolic Ca2+ induced by Aβ25–35, ultimately preventing apoptosis and necrosis. The specific cytoprotective effect of CoQ with a side chain of 10 isoprenoid units could be explained by the fact that CoQ10 is the only ubiquinone that significantly reduces the entry of Aβ25–35 into the mitochondria

Biological Significance of the Protein Changes Occurring in the Cerebrospinal Fluid of Alzheimer’s Disease Patients: Getting Clues from Proteomic Studies

The fact that cerebrospinal fluid (CSF) deeply irrigates the brain together with the relative simplicity of sample extraction from patients make this biological fluid the best target for biomarker discovery in neurodegenerative diseases. During the last decade, biomarker discovery has been especially fruitful for the identification new proteins that appear in the CSF of Alzheimer’s disease (AD) patients together with amyloid-β (Aβ42), total tau (T-tau), and phosphorylated tau (P-tau). Thus, several proteins have been already stablished as important biomarkers, due to an increase (i.e., CHI3L1) or a decrease (i.e., VGF) in AD patients’ CSF. Notwithstanding this, only a deep analysis of a database generated with all the changes observed in CSF across multiple proteomic studies, and especially those using state-of-the-art methodologies, may expose those components or metabolic pathways disrupted at different levels in AD. Deep comparative analysis of all the up- and down-regulated proteins across these studies revealed that 66% of the most consistent protein changes in CSF correspond to intracellular proteins. Interestingly, processes such as those associated to glucose metabolism or RXR signaling appeared inversely represented in CSF from AD patients in a significant manner. Herein, we discuss whether certain cellular processes constitute accurate indicators of AD progression by examining CSF. Furthermore, we uncover new CSF AD markers, such as ITAM, PTPRZ or CXL16, identified by this study

A theranostic water-based platform made of carbon nanotubes and nanocellulose for selective colon cancer killing

Work presented at the Metals and Water 2021 Virtual Zaragoza. IV International Congress in Water Soluble Metal Complexes Applications, 24th-25th June 2021.For their large surface area and versatile chemical reactivity, single-walled carbon nanotubes (SWCNTs) are regarded as the basis of new pharmacological complexes. Here, SWCNTs are chemically functionalized with fluorescein, folic acid, and capecitabine drug, being the latter commonly used against colorectal cancer. Functionalized SWCNTs are dispersed in water through their synergistic interaction with type-II nanocrystalline cellulose (II-NCC), and the resulting colloidal system is tested in vitro on both normal and cancer human colon cells (Caco-2). The bare SWCNT/II-NCC hybrid presented a selective effect against cancer Caco-2 cells, so the study was extended also to the functionalized analogues. The functionalized SWCNT/II-NCC hybrid shows a higher activity than the reference capecitabine drug against the cancer Caco-2 cell line. However, the effect appears to be intrinsically associated to the SWCNT/II-NCC complex, particularly boosted by fluorescein, while the presence of the capecitabine chemical group is not required. In addition, fluorescence imaging is shown on cell cultures by confocal microscopy, highlighting the enormous potential of this nanohybrid platform towards colon cancer theranostics

Las ciencias como herramienta de motivación e inclusión académica: INCLUCIENCIA

Resumen del póster presentado en el XVI Simposio de Investigadores Jóvenes de la Real Sociedad Española de Química RSEQ-Sigma Aldrich (Merck), Valencia, 4-7 noviembre 2019.-- P19.INCLUCIENCIA es un proyecto de divulgación científica e innovación educativa que tiene como meta convertirse en una herramienta eficaz para ayudar a reducir el fracaso y abandono escolar, así como combatir la falta de motivación de alumnos adolescentes por materias científicas. El proyecto ha sido diseñado por un grupo multidisciplinar de profesionales de edades y formaciones muy variadas. El principal reto de INCLUCIENCIA se ha basado en si un colectivo adolescente (centrado en la edad crítica de los 14-16 años) podría verse positivamente motivado, en los planos académico y personal, tras participar activamente en actividades programadas de contenido científico divulgativo y experimental. El fracaso escolar puede verse motivado por circunstancias tan ajenas al modelo educativo como el desarraigo, las dificultades lingüísticas, los problemas familiares o el bullying; por ello se está actuando sobre grupos heterogéneos de estudiantes, de distintos colectivos sociales. Como elemento diferencial de otras iniciativas similares, INCLUCIENCIA ha apostado por la implicación activa del alumnado y la relación directa entre las posibles salidas y metas profesionales con las actividades didácticas realizadas, con el fin de despertar vocaciones y pasiones, y que los jóvenes utilicen ese estímulo para sobreponerse a las adversidades que les alejan del éxito académico, decidiendo seguir con sus estudios. La metodología empleada en INCLUCIENCIA consta de toda una serie de actividades interactivas, donde se estimula la curiosidad del alumnado por distintas ramas científicas, utilizando en la mayor parte de los casos el recurso de la gamificación, haciendo así divertido a la vez que riguroso, tanto el concepto científico que se desee transmitir, como todas aquellas salidas profesionales que deriven de los mismos. INCLUCIENCIA cuenta con su propio método de medición del impacto. El análisis global de la información obtenida hasta la fecha, tras aplicar metodología científica y estadística, ha revelado contundentes resultados. El 87% del alumnado consideró que era necesario estudiar; hasta un 57% del conjunto mostraron interés por la ciencia, y más de la mitad quedaron positivamente sorprendidos con las actividades presentadas. Es importante destacar que el 61% del alumnado acaba considerando que las actividades realizadas les servirían de ayuda para elegir las asignaturas de su futuro itinerario académico; y el 57% estimó que se les habían planteado salidas profesionales que anteriormente desconocían. INCLUCIENCIA puede servir también como vía para combatir los sesgos de género en ciencia ya que, entre el alumnado adolescente, el impacto de las actividades es en promedio mayor en chicas que en chicos.Peer reviewe

Human amyloid-β enriched extracts: evaluation of in vitro and in vivo internalization and molecular characterization

[Background]: Intracerebral inoculation of extracts from post-mortem human Alzheimer’s disease brains into mice produces a prion-like spreading effect of amyloid-β. The differences observed between these extracts and the synthetic peptide, in terms of amyloid-β internalization and seed and cell-to-cell transmission of cytosolic protein aggregates, suggest that brain extracts contain key contributors that enhance the prion-like effect of amyloid-β. Nevertheless, these potential partners are still unknown due to the complexity of whole brain extracts.[Methods]: Herein, we established a method based on sequential detergent solubilization of post-mortem samples of human brains affected by Alzheimer’s disease that strongly enrich amyloid-β aggregates by eliminating 92% of the remaining proteins. Internalization of Aβ1–42 from the enriched AD extracts was evaluated in vitro, and internalization of fluorescent-labeled AD extracts was also investigated in vivo. Furthermore, we carried out a molecular characterization of the Aβ-enriched fraction using label-free proteomics, studying the distribution of representative components in the amygdala and the olfactory cortex of additional human AD brain samples by immunohistochemistry.[Results]: Aβ1–42 from the enriched AD extracts are internalized into endothelial cells in vitro after 48 h. Furthermore, accumulation of fluorescent-labeled Aβ-enriched extracts into mouse microglia was observed in vivo after 4 months of intracerebral inoculation. Label-free proteomics (FDR < 0.01) characterization of the amyloid-β-enriched fraction from different post-mortem samples allowed for the identification of more than 130 proteins, several of which were significantly overrepresented (i.e., ANXA5 and HIST1H2BK; p < 0.05) and underrepresented (i.e., COL6A or FN1; p < 0.05) in the samples with Alzheimer’s disease. We were also able to identify proteins exclusively observed in Alzheimer’s disease (i.e., RNF213) or only detected in samples not affected by the disease (i.e., CNTN1) after the enrichment process. Immunohistochemistry against these proteins in additional tissues revealed their particular distribution in the amygdala and the olfactory cortex in relation to the amyloid-β plaque.[Conclusions]: Identification and characterization of the unique features of these extracts, in terms of amyloid-β enrichment, identification of the components, in vitro and in vivo cell internalization, and tissue distribution, constitute the best initial tool to further investigate the seeding and transmissibility proposed in the prion-like hypothesis of Alzheimer’s disease.Sponsored by the Spanish Ministry of Economy and Competitiveness-FEDER (grant # SAF2016-75768-R) to AMM, MINECO-RETOS (AEI-FEDER) to MDP, and the Autonomous Government of Castilla-La Mancha/FEDER (grant no. SBPLY/ 17/180501/000430) to AMM and DSS.Peer reviewe

Slow diffusion co-assembly as an efficient tool to tune colour emission in alkynyl benzoazoles

11 FigurasWe report here the preparation of co-assembled microcrystals by employing an easy, reproducible and cost-effective technique, namely slow diffusion. 2H-Benzo[d][1,2,3]triazole and benzo[c][1,2,5]thiadiazole were chosen as host and guest skeletons, respectively. Structural similarities allowed the correct co-assembly of the two structures. The co-assemblies were studied by different techniques that included Raman spectroscopy and X-ray diffraction, amongst others. The waveguiding properties and the emission colour of the doped organic microcrystals were also investigated. It was found that changes in the molar ratio of the different doping agents could tune the light emission. Fluorescence microscopy images of the co-assembled microcrystals revealed light colour changes from green to whitish, up to CIE coordinates of (0.370, 0.385). These tunable colour-active materials could be useful in the fields of optoelectronics or lab-on-a-chip for integrated optical circuits at micro-/nanoscale.This work has been financially supported by JCCM-FEDER (project SBPLY/17/180501/000189) and MINECO of Spain (projects CTQ2017-88158-R, CTQ2017-84825-R and RED2018-102331-T). R. Martín and B. Donoso are indebted to MEC for a FPU studentship.Peer reviewe

Sweet graphene: exfoliation of graphite and preparation of glucose-graphene cocrystals through mechanochemical treatments

Mechanochemical treatment with carbohydrates leads to the successful exfoliation of graphite and this can be considered as a green approach to the preparation of graphene. Glucose, fructose and saccharose were used and the former showed the best exfoliation behaviour to generate graphene materials with a relatively low number of defects, as evidenced by Raman spectroscopy. The addition of small amounts of water to the ball milling treatment led to the formation of glucose-graphene co-crystals, which exhibited superior properties in terms of colloidal stability and minimization of cell toxicity