1H HR MAS NMR metabolomic and non-destructive 2D NMR relaxometry to assess internal quality in apples.

NMR can be considered a multi-scale multidimensional technology in the sense that it provides both spatial insight at macroscopic (MRI) or microscopic level (relaxometry), together with chemical characterization (HR-MAS). In this study 296 apples (from 4 cultivars) were MRI screened (20 slices per fruit) among which 7 fruits were used for metabolomic study by 1H HR MAS in order to assess various chemical shifts: malic acid, sucrose, glucose, fructose and ethanol. On the first season, tissue samples were taken from the sound and affected apples (near the core, centre and outer part of the mesocarp) belonging to sound and affected locations, while on the second season, tissue samples were focused on the comparison between sound and affected tissue. Beside, MRI and 2D non-destructive relaxometry (on whole fruits, and localized tissue) where performed on 72 and 12 apples respectively in order to compare features at macroscopic (tissue) and microscopic (subcellular) level. HR MAS shows higher content of ?-glucose, ?-glucose, malic acid and aromatic compounds in watercore affected tissues from both seasons, while sound tissue reflects higher sucrose. Microscopic (subcellular) degradation of tissue varies according to disorder development and is in good accordance with macroscopic characterization with MRI

Fluorine Labeling of Nanoparticles and In Vivo 19F Magnetic Resonance Imaging

Fluorinated nanoparticles have increasing applications, but they are still challenging to prepare, especially in the case of water-soluble fluorinated nanoparticles. Herein, a fluorine labeling strategy is presented that is based on the conjugation of custom-made small fluorinated building blocks, obtained by simple synthetic transformations, with carboxylated gold nanoparticles through a convenient phase-transfer process. The synthesis of four fluorinated building blocks with different chemical shifts in 19F nuclear magnetic resonance and varied functionalities is reported, along with their conjugation onto nanoparticles. Fluorinated nanoparticles of small core size obtained by this conjugation methodology and by direct synthesis presented high transverse relaxation times (T2) ranging from 518 to 1030 ms, and a large number of equivalent fluorine atoms per nanoparticle (340-1260 fluorine atoms), which made them potential candidates for 19F magnetic resonance related applications. Finally, nontargeted fluorinated nanoparticles were probed by performing in vivo 19F magnetic resonance spectroscopy (19F MRS) in mice. Nanoparticles were detected at both 1 and 2 h after being injected. 19F MRI images were also acquired after either intravenous or subcutaneous injection. Their fate was studied by analyzing the gold content in tissues by ICP-MS. Thus, the present work provides a general fluorination strategy for nanoparticles and shows the potential use of small fluorinated nanoparticles in magnetic-resonance-related applicationsThis work was supported in part by grants from the Spanish State Research Agency (MCI/PID2019-107449RB-I00/AEI/10.13039/501100011033, RYC-2017-22412, and SAF2017-84494-C2-R), from the Fundación Biofísica Bizkaia, the Basque Excellence Research Centre (BERC) program, and projects IT1196-19 and Grant KK-2019/bmG19 of the Basque Government. We also thank the UE for funding (Nova MRI, H2020-MSCA-ITN-2018-811382). This work was performed under the Maria de Maeztu Units of Excellence Programme Grant MDM-2017-0720 ministry of Science, Innovation and Universities. The authors thank for technical and human support the Materials and Surfaces Service and the Central Analysis Service of Bizkaia provided by SGIker (UPV/EHU) and European funding (ERDF and ESF) for the XPS, TEM and ICP-AES analyses. The MRI service members of CIC biomaGUNE are also acknowledged for their support of the in vivo experiment

Impact of a TAK-1 inhibitor as a single or as an add-on therapy to riociguat on the metabolic reprograming and pulmonary hypertension in the SUGEN5416/hypoxia rat model.

Background: Despite increasing evidence suggesting that pulmonary arterial hypertension (PAH) is a complex disease involving vasoconstriction, thrombosis, inflammation, metabolic dysregulation and vascular proliferation, all the drugs approved for PAH mainly act as vasodilating agents. Since excessive TGF-β signaling is believed to be a critical factor in pulmonary vascular remodeling, we hypothesized that blocking TGFβ-activated kinase 1 (TAK-1), alone or in combination with a vasodilator therapy (i.e., riociguat) could achieve a greater therapeutic benefit. Methods: PAH was induced in male Wistar rats by a single injection of the VEGF receptor antagonist SU5416 (20 mg/kg) followed by exposure to hypoxia (10%O2) for 21 days. Two weeks after SU5416 administration, vehicle, riociguat (3 mg/kg/day), the TAK-1 inhibitor 5Z-7-oxozeaenol (OXO, 3 mg/kg/day), or both drugs combined were administered for 7 days. Metabolic profiling of right ventricle (RV), lung tissues and PA smooth muscle cells (PASMCs) extracts were performed by magnetic resonance spectroscopy, and the differences between groups analyzed by multivariate statistical methods. Results: In vitro, riociguat induced potent vasodilator effects in isolated pulmonary arteries (PA) with negligible antiproliferative effects and metabolic changes in PASMCs. In contrast, 5Z-7-oxozeaenol effectively inhibited the proliferation of PASMCs characterized by a broad metabolic reprogramming but had no acute vasodilator effects. In vivo, treatment with riociguat partially reduced the increase in pulmonary arterial pressure (PAP), RV hypertrophy (RVH), and pulmonary vascular remodeling, attenuated the dysregulation of inosine, glucose, creatine and phosphocholine (PC) in RV and fully abolished the increase in lung IL-1β expression. By contrast, 5Z-7-oxozeaenol significantly reduced pulmonary vascular remodeling and attenuated the metabolic shifts of glucose and PC in RV but had no effects on PAP or RVH. Importantly, combined therapy had an additive effect on pulmonary vascular remodeling and induced a significant metabolic effect over taurine, amino acids, glycolysis, and TCA cycle metabolism via glycine-serine-threonine metabolism. However, it did not improve the effects induced by riociguat alone on pulmonary pressure or RV remodeling. None of the treatments attenuated pulmonary endothelial dysfunction and hyperresponsiveness to serotonin in isolated PA. Conclusion: Our results suggest that inhibition of TAK-1 induces antiproliferative effects and its addition to short-term vasodilator therapy enhances the beneficial effects on pulmonary vascular remodeling and RV metabolic reprogramming in experimental PAH.This work was supported by the Instituto de Salud Carlos III-ISCIII (Grant numbers: PI15/01100 and PI19/01616 to LM), the Spanish Ministry of Science and Innovation MCIN (Grant numbers: PID 2019-107363RB-I00 to FP-V, PID 2020-117939RBI00 to AC and PID 2021-123238OB-I00, PDC 2021-121696-I00 to JRC and PID2019-106564RJ-I00 to JI-G), the Comunidad de Madrid-CAM (CM S2017/BMD-3727 to AC and LM and B2017/ BMD3875 to JI-G) and, as appropriate, by “ERDF A way of making Europe”, co-funded by the “European Union”. FP-V received funding from Fundación Contra la Hipertensión Pulmonar (Empathy grant) and JR-C from La Caixa Foundation (Health Research Call 2020: HR20-00075). This work was performed under the Maria de Maeztu Units of Excellence Programme–Grant MDM-2017-0720 funded by MCIN/AEI/10.13039/501100011033.S

Mild and short-term caloric restriction prevents obesity-induced cardiomyopathy in young zucker rats without changing in metabolites and fatty acids cardiac profile

Caloric restriction (CR) ameliorates cardiac dysfunction associated with obesity. However, most of the studies have been performed under severe CR (30-65% caloric intake decrease) for several months or even years in aged animals. Here, we investigated whether mild (20% food intake reduction) and short-term (2-weeks) CR prevented the obese cardiomyopathy phenotype and improved the metabolic profile of young (14 weeks of age) genetically obese Zucker fa/fa rats. Heart weight (HW) and HW/tibia length ratio was significantly lower in fa/fa rats after 2 weeks of CR than in counterparts fed ad libitum. Invasive pressure measurements showed that systolic blood pressure, maximal rate of positive left ventricle (LV) pressure, LV systolic pressure and LV end-diastolic pressure were all significantly higher in obese fa/fa rats than in lean counterparts, which were prevented by CR. Magnetic resonance imaging revealed that the increase in LV end-systolic volume, stroke volume and LV wall thickness observed in fa/fa rats was significantly lower in animals on CR diet. Histological analysis also revealed that CR blocked the significant increase in cardiomyocyte diameter in obese fa/fa rats. High resolution magic angle spinning magnetic resonance spectroscopy analysis of the LV revealed a global decrease in metabolites such as taurine, creatine and phosphocreatine, glutamate, glutamine and glutathione, in obese fa/fa rats, whereas lactate concentration was increased. By contrast, fatty acid concentrations in LV tissue were significantly elevated in obese fa/fa rats. CR failed to restore the LV metabolomic profile of obese fa/fa rats. In conclusion, mild and short-term CR prevented an obesity-induced cardiomyopathy phenotype in young obese fa/fa rats independently of the cardiac metabolic profile.This study was supported by grants from Spanish Ministry of Science and Innovation (BFU2011-25303), Spanish Institute of Health Carlos III (CP15/00129), UCM groups (GR-921641), SESCAMET, Fundación Mutua Madrileña, Fundación Eugenio Rodríguez Pascual and Fondos FEDER.Peer Reviewe

Impact of a TAK-1 inhibitor as a single or as an add-on therapy to riociguat on the metabolic reprograming and pulmonary hypertension in the SUGEN5416/hypoxia rat model

Background: Despite increasing evidence suggesting that pulmonary arterial hypertension (PAH) is a complex disease involving vasoconstriction, thrombosis, inflammation, metabolic dysregulation and vascular proliferation, all the drugs approved for PAH mainly act as vasodilating agents. Since excessive TGF-β signaling is believed to be a critical factor in pulmonary vascular remodeling, we hypothesized that blocking TGFβ-activated kinase 1 (TAK-1), alone or in combination with a vasodilator therapy (i.e., riociguat) could achieve a greater therapeutic benefit.Methods: PAH was induced in male Wistar rats by a single injection of the VEGF receptor antagonist SU5416 (20 mg/kg) followed by exposure to hypoxia (10%O2) for 21 days. Two weeks after SU5416 administration, vehicle, riociguat (3 mg/kg/day), the TAK-1 inhibitor 5Z-7-oxozeaenol (OXO, 3 mg/kg/day), or both drugs combined were administered for 7 days. Metabolic profiling of right ventricle (RV), lung tissues and PA smooth muscle cells (PASMCs) extracts were performed by magnetic resonance spectroscopy, and the differences between groups analyzed by multivariate statistical methods.Results:In vitro, riociguat induced potent vasodilator effects in isolated pulmonary arteries (PA) with negligible antiproliferative effects and metabolic changes in PASMCs. In contrast, 5Z-7-oxozeaenol effectively inhibited the proliferation of PASMCs characterized by a broad metabolic reprogramming but had no acute vasodilator effects. In vivo, treatment with riociguat partially reduced the increase in pulmonary arterial pressure (PAP), RV hypertrophy (RVH), and pulmonary vascular remodeling, attenuated the dysregulation of inosine, glucose, creatine and phosphocholine (PC) in RV and fully abolished the increase in lung IL-1β expression. By contrast, 5Z-7-oxozeaenol significantly reduced pulmonary vascular remodeling and attenuated the metabolic shifts of glucose and PC in RV but had no effects on PAP or RVH. Importantly, combined therapy had an additive effect on pulmonary vascular remodeling and induced a significant metabolic effect over taurine, amino acids, glycolysis, and TCA cycle metabolism via glycine-serine-threonine metabolism. However, it did not improve the effects induced by riociguat alone on pulmonary pressure or RV remodeling. None of the treatments attenuated pulmonary endothelial dysfunction and hyperresponsiveness to serotonin in isolated PA.Conclusion: Our results suggest that inhibition of TAK-1 induces antiproliferative effects and its addition to short-term vasodilator therapy enhances the beneficial effects on pulmonary vascular remodeling and RV metabolic reprogramming in experimental PAH

Diseño de un sistema de bobinas de RF para aplicaciones de imagen de resonancia magnética en flúor

De acuerdo con las estimaciones de la Organización Mundial de la Salud (OMS), las enfermedades pulmonares, cardiovasculares y neurovasculares son la principal causa de mortalidad en el mundo desarrollado. Las técnicas de diagnóstico por imagen desempeñan un papel fundamental en la detección precoz de estas patologías. La imagen de resonancia magnética (IRM) es una modalidad de imagen médica relativamente reciente, la primera aplicación data de 1973. Ha demostrado ser una poderosa técnica de imagen que goza de una gran notoriedad debido a su flexibilidad y sensibilidad a una amplia gama de propiedades de los tejidos y a la naturaleza no invasiva de los campos magnéticos. Dado que no requiere la exposición del sujeto a la radiación ionizante, generalmente se considera más segura que otras técnicas de imagen y una de las más apropiadas para la detección de tejido blando. Las aplicaciones de la IRM siguen expandiéndose gracias a los desarrollos en secuencias rápidas y a los avances en instrumentación, con equipos de IRM de mayor campo magnético y nuevas configuraciones en bobinas de radiofrecuencia (RF). Todo ello culmina en una mejora de la relación señal-ruido (SNR) de las imágenes de IRM. Normalmente, la imagen de RM utiliza los protones de los núcleos atómicos de hidrógeno debido a su abundancia natural. Ahora bien, todos estos adelantos en la RM, junto con los desarrollos en nuevos materiales inteligentes como, por ejemplo, derivados del perfluorocarbono (19F) incorporado en nanoemulsiones, hacen que la experimentación en X-núcleo y más concretamente en 19F, se haya incrementado considerablemente. La imagen de RM de 19F juega un papel fundamental en aquellas zonas donde la imagen de 1H está limitada, como es el caso de la detección de la estructura y función pulmonar a causa de las características propias del pulmón. Ante la escasez de bobinas de RF comerciales para 19F y teniendo en cuenta las necesidades del grupo de investigación donde se circunscribe este trabajo, el objetivo principal de esta tesis fue la implementación de bobinas de RF en 19F que maximizasen la relación señal-ruido para experimentación animal. A tal efecto, y previo a la implementación de cualquier bobina de RF, se examinó la viabilidad de cada tipo de bobinas de RF, centrando el estudio en obtener máxima SNR. El trabajo desarrollado en esta tesis doctoral expone el estudio, diseño y fabricación de tres configuraciones de bobinas de RF diferentes sobre las que se valora y compara la SNR. Primeramente, se implementó una bobina de RF de volumen birdcage paso alto de doble sintonía 1H/19F optimizada para animales pequeños, tipo rata, y para equipos de imagen de RM de 7 T. Dada su versatilidad se puede emplear tanto como bobina de RF transmisora y receptora de doble sintonía, como bobina de RF solo transmisora de doble sintonía. Fue la que presentó mayor homogeneidad en B1, pero menor SNR. A pesar de este menor rendimiento, presenta una sensibilidad capaz de detectar concentraciones tan bajas como de 3 mM en tan solo 6 minutos. A continuación, se diseñó una bobina de RF receptora consistente en un conjunto de ellas o array sintonizado en 19F, formado por cuatro elementos receptores. Se implementó para el mismo equipo de RM de 7 T y se optimizó para animales de peso no superior a 300 g. La singularidad de este array es la disposición de sus cuatro elementos alrededor de un cilindro, otorgándole forma volumétrica, en lugar de lineal que es lo habitual. El análisis de las imágenes demuestra un incremento en la SNR cuatro veces superior a la bobina de RF de volumen birdcage, resultado similar al que presentan las bobinas de RF comerciales. La ventaja de esta disposición volumétrica radica en la alta sensibilidad en el centro de la imagen, muy por encima de cualquier otra configuración. Asimismo, la sensibilidad es muy superior a la bobina de RF de volumen birdcage. Por consiguiente, esta disposición volumétrica permitiría la realización de imagen torácica con alta SNR para la visualización de estructuras internas que con cualquier otra configuración sería prácticamente imposible. Por último, se realizó otra configuración adicional para comparar los diferentes diseños. En este caso, se seleccionó una bobina de RF superficial circularmente polarizada de doble sintonía 1H/19F para el mismo equipo de RM de 7 T y optimizada para animales más pequeños, tipo ratón. Por un lado, se seleccionó la configuración de una bobina de RF superficial porque presentan mayor SNR que las bobinas de RF de volumen. Por otro lado, se eligió que fuera circularmente polarizada porque esta configuración aporta un incremento de √2 en la SNR. Los resultados obtenidos indicaron que es la configuración que proporciona mayor SNR en la proximidad de la bobina de RF. En cambio, en el centro de la imagen decae en comparación con el array, si bien es superior a la bobina de RF de volumen birdcage. Ahora bien, el nivel de sensibilidad es semejante al array. Las tres configuraciones se han optimizado para el 19F, por ser el núcleo de menor sensibilidad, y en ningún caso se ha optado por incluir elementos discretos, dado que estos degradan la SNR debido a la resistencia que presentan. No obstante, las imágenes adquiridas en 1H son de la suficiente calidad para permitir los ajustes previos y la localización de la zona anatómica de interés. Se concluye que el objetivo propuesto al principio de la tesis de diseñar bobinas de RF que maximicen la SNR para experimentación animal fue cumplido, en especial con la nueva disposición de los cuatro elementos del array proporcionándoles forma volumétrica que posibilitaría la realización de imagen torácica con alta SNR. ABSTRACT According to World Health Organization, pulmonary, cardiovascular, and neurovascular diseases are the leading cause of death in the world. Diagnostic imaging techniques play a crucial role in the early diagnosis of these pathologies. Magnetic resonance imaging (MRI) is a relatively recent medical imaging modality, the first application dating back to 1973. It has proven to be a powerful imaging technique that enjoys incredible notoriety due to its flexibility and sensitivity to a wide range of tissue properties and the noninvasive nature of magnetic fields. Since it does not require exposure of the subject to ionizing radiation, it is generally considered safer than other imaging techniques and one of the most appropriate for soft tissue detection. MR imaging applications continue to expand, thanks to developments in fast sequencing and advanced instrumentation, with higher magnetic field MR imaging equipment and new radiofrequency (RF) coil configurations. All of this results in improved signal-to-noise ratio (SNR) of MR images. Typically, MR imaging uses the protons of hydrogen atomic nuclei due to their natural abundance. However, all these advances in MRI, together with developments in new smart materials such as, for example, perfluorocarbon (19F) derivatives incorporated in nanoparticles or nanoemulsions, mean that experimentation in X-nuclei, and more specifically in 19F, has increased considerably. 19F MR imaging plays a fundamental role in those areas where 1H imaging is limited, as is the case in detecting lung structure and function due to the characteristics of the lung. Given the few commercial RF coils for 19F and considering the needs of the research group where this work is circumscribed, the main objective of this thesis was the implementation of RF coils in 19F that maximize the signal-to-noise ratio for animal experimentation. To this end, and before the construction of any RF coil, the feasibility of each type of RF coil was examined, focusing the study on obtaining maximum SNR. The work developed in this doctoral thesis presents the analysis, design, and fabrication of three different RF coil configurations on which the SNR is evaluated and compared. First, a 1H/19F dual-tuned high-pass birdcage coil optimized for small animals, such as rats, and for a 7 T MR scanner was implemented. It has the characteristic of being used both as a dual-tuned transmitter and receiver coil and as a dual-tuned transmitter-only coil. It was the one that presented higher homogeneity in B1 but lower SNR. Despite this lower performance, it has a sensitivity capable of detecting concentrations as low as 3 mM in as little as 6 minutes. Next, a receiver coil consisting of a 19F-tuned array coil consisting of four receiver elements was designed. It was implemented for the same 7 T scanner and optimized for animals weighing no more than 300 g. The uniqueness of this array is the arrangement of its four elements around a cylinder, giving it a volumetric shape instead of the usual linear one. The analysis of the images shows an increase in SNR four times higher than the birdcage coil, a result similar to commercial coils. The advantage of this volumetric arrangement lies in the high sensitivity in the center of the image, far above any other configuration. Also, the sensitivity is far superior to the birdcage coil. Consequently, this volumetric arrangement allows thoracic imaging with high SNR visualize internal structures that would be practically impossible with any other configuration. Finally, an additional setup is performed to compare the different designs. In this case, a 1H/19F dual-tuned circularly polarized surface coil is selected for the same 7 T scanner and optimized for smaller, mouse-type animals. On the one hand, the surface coil configuration was chosen because it presents higher SNR than volume coils. On the other hand, it was selected to be circularly polarized because this configuration provides a √2 increase in SNR. The results indicate that this format offers the highest SNR in the vicinity of the coil. On the other hand, in the center of the image, it declines compared to the array, although it is superior to the birdcage coil. However, the sensitivity level is similar to the array coil. The three configurations have been optimized for the 19F, the lowest sensitivity core. In no case have discrete elements been included since they degrade the SNR due to the resistance they present. Nevertheless, the images acquired in 1H are of sufficient quality to allow for presetting and localization of the anatomical area of interest. Therefore, it is concluded that the objective proposed at the beginning of the thesis to design coils that maximize SNR for animal experimentation has been fulfilled. This is especially true for the volumetrically distributed new four-element array that makes it possible to perform thoracic imaging with high SNR

A Contribution to the Harmonization of Non-targeted NMR Methods for Data-Driven Food Authenticity Assessment

Spectroscopic non-targeted methods are gaining ever-growing importance in quality control and authenticity assessment of food products because of their strong potential for identification of specific features of the products by data-driven classifiers. One of the factors hampering the diffusion of spectroscopic non-targeted methods and data-driven classifiers is the lack of harmonized guidelines for their development and validation. In particular, to date, neither conditions to directly compare spectra recorded by different spectrometers nor studies demonstrating the statistical equivalence of the spectra are available. Among the spectroscopic analytical techniques suitable for the development of non-targeted methods, nuclear magnetic resonance (NMR) offers the unique opportunity to generate statistically equivalent signals. In this paper, the feasibility of NMR spectroscopy to generate statistically equivalent NMR signals from a number of different spectrometers was demonstrated for complex mixtures (aqueous extracts of wheat and flour) by organizing an inter-laboratory comparison involving 36 NMR spectrometers. Univariate statistics along with multivariate analysis were exploited to establish unbiased criteria for assessing the statistical equivalence of the NMR signals. The aspects affecting the signal equivalence were investigated, and possible solutions to reduce the extent of the human error were proposed and applied with satisfactory results. This study furnishes the scientific community with an appropriate and easy procedure to validate non-targeted NMR methods and provides error values to be used as a reference for future studies