Healthy lifestyle, metabolomics and incident type 2 diabetes in a population-based cohort from Spain

This work was supported by the Strategic Action for Research in Health sciences [PI10/0082, PI13/01848, PI14/00874, PI16/01402, PI11/00726, PI16/609, PI16/1512, PI18/287, PI19/319 and PI20/00896], the GUTMOM Project (JPI-A Healthy Diet for a Healthy Life INTIMIC-085, State Secretary of R + D + I PCIN-2017-117), the Cátedra de Epidemiología y Control del Riesgo Cardiovascular at UAM (#820024), the State Agency for Research (PID2019-108973RB-C21 and C22), the Valencia Government (GRUPOS 03/101; PROMETEO/2009/029 and ACOMP/2013/039), the Castilla-Leon Government (GRS/279/A/08) and European Network of Excellence Ingenious Hypercare (EPSS- 037093) from the European Commission; CIBER Fisiopatología Obesidad y Nutrición (CIBEROBN) (CIBER-02-08-2009, CB06/03 and CB12/03/30016). MSP holds a Ramón y Cajal contract (RYC-2018-025069-I) from the Ministry of Science, Innovation and Universities. MDV holds a “Predoctoral Training in Health Research” contract (FI20/00162) from the Carlos III Health Institute. MGP and ADR received the support of a fellowship from “la Caixa” Foundation (ID 100010434, fellowship codes LCF/BQ/IN18/11660001, and LCF/BQ/DR19/11740016, respectively). PO received the support of a Sara Borrell contract from the Carlos III Health Institute (reference CD16/00255). The Strategic Action for Research in Health Sciences, CIBEROBN are initiatives from Carlos III Health Institute Madrid and co-funded by the European Social Fund “The ESF - investing in your future”. The State Agency for Research and Carlos III Health Institute belong to the Spanish Ministry of Science and Innovation. The funding bodies had no role in the study design, data collection and analysis, interpretation of results, manuscript preparation or in the decision to submit this manuscript for publication.Background: The contribution of metabolomic factors to the association of healthy lifestyle with type 2 diabetes risk is unknown. We assessed the association of a composite measure of lifestyle with plasma metabolite profiles and incident type 2 diabetes, and whether relevant metabolites can explain the prospective association between healthy lifestyle and incident type 2 diabetes. Methods: A Healthy Lifestyle Score (HLS) (5-point scale including diet, physical activity, smoking status, alcohol consumption and BMI) was estimated in 1016 Hortega Study participants, who had targeted plasma metabolomic determinations at baseline examination in 2001–2003, and were followed-up to 2015 to ascertain incident type 2 diabetes. Results: The HLS was cross-sectionally associated with 32 (out of 49) plasma metabolites (2.5% false discovery rate). In the subset of 830 participants without prevalent type 2 diabetes, the rate ratio (RR) and rate difference (RD) of incident type 2 diabetes (n cases = 51) per one-point increase in HLS was, respectively, 0.69 (95% CI, 0.51, 0.93), and − 8.23 (95% CI, − 16.34, − 0.13)/10,000 person-years. In single-metabolite models, most of the HLS-related metabolites were prospectively associated with incident type 2 diabetes. In probit Bayesian Kernel Machine Regression, these prospective associations were mostly driven by medium HDL particle concentration and phenylpropionate, followed by small LDL particle concentration, which jointly accounted for ~ 50% of the HLS-related decrease in incident type 2 diabetes. Conclusions: The HLS showed a strong inverse association with incident type 2 diabetes, which was largely explained by plasma metabolites measured years before the clinical diagnosis.CIBER Fisiopatología Obesidad y NutriciónCastilla-Leon Government GRS/279/A/08European Network of Excellence Ingenious Hypercare EPSS- 037093State Agency for Research ACOMP/2013/039, GRUPOS 03/101, PROMETEO/2009/029Strategic Action for Research in Health sciences INTIMIC-085, PCIN-2017-117Ministerio de Ciencia, Innovación y Universidades RYC-2018-025069-IEuropean Commission EPSS-037093Instituto de Salud Carlos III CB06/03, CB12/03/30016, CD16/00255, CIBER-02-08-2009, PI10/0082, PI11/00726, PI13/01848, PI14/00874, PI16/01402, PI16/1512, PI16/609, PI18/287, PI19/319, PI20/00896Universidad Autónoma de Madrid 820024Agencia Estatal de Investigación PID2019-108973RB-C21“La Caixa” Foundation 100010434, LCF/BQ/DR19/11740016, LCF/BQ/IN18/1166000

Conformación tridimensional y reconocimiento molecular de biopolímeros : aplicación de RMN multidimensional y desarrollo de metodología de cálculo y estructural

El objetivo principal de la Tesis ha sido la exploración, el análisis y el perfeccionamiento de las técnicas más habituales utilizadas en la determinación de la estructura y dinámica de biopolímeros en disolución. En concreto, se han utilizado métodos teóricos y experimentales para obtener las estructuras de dos biopolímeros entre los grupos más numerosos de ellos: proteínas y ácidos nucleicos. La obtención de la estructura de una proteína de interés bioquímico por sus peculiaridades cinéticas mediante dos técnicas diferentes ha sido uno de los objetivos principales en la Tesis. La aplicación de las técnicas de modelización por homología y la realización de un análisis exhaustivo sobre los factores que afectan a la calidad del método permitirá aportar la obtención de mejores estructuras de proteínas mediante el uso de esta técnica. La determinación de la estructura de la Plastocianina mediante un método teórico como es la modelización por homología y otro experimental como es la RMN permitirá evaluar el grado de convergencia entre ambas y sopesar la validez de las técnicas teóricas como medio de ampliación de las estructuras de proteínas caracterizadas. La aplicación del formalismo libre de modelo de Lipari-Szabo al análisis de los procesos de relajación en RMN mediante la confección de un programa escrito en C permitirá aportar mayor objetividad al estudio de la dinámica de proteína mediante técnicas de RMN. El estudio de sistemas cuya dinámica ha sido analizada previamente mediante el uso de otros programas diferentes y la comparación con los resultados obtenidos permitirá conocer la validez tanto de la aproximación empleada como del algoritmo programado. El análisis de la dinámica de una proteína tan bien estudiada como el wt-BPTI (Inhibidor Básico de la Tripsina de Páncreas Bovina en forma nativa) frente a uno de sus mutantes constituye un ejemplo de análisis exhaustivo de la dinámica de una proteína mediante relajación en RMN. El desarrollo de las rutinas aplicadas en el programa HYPER para la obtención de restricciones de ángulos diedros en la cadena principal y en las cadenas laterales mediante la utilización de información de NOEs y constantes de acoplamiento escalar constituye un ejemplo de la dirección a seguir en el refinamiento de estructuras de RMN. La novedosa metodología utilizada en la determinación del ángulo diedro mediante el análisis de intensidades TOCSY (espectroscopía de correlación total) frente al tiempo de mezcla del experimento, incluso para residuos sin dos protones metilénicos como las treoninas y su aplicación a una proteína cuya estructura ya ha sido determinada, permite evaluar la importancia del desarrollo de metodologías similares en el refinamiento de estructuras por RMN. Por último, la determinación de la estructura de un oligonucleótido en disolución mediante RMN utilizando las técnicas de simulación de picos DQF-COSY (Espectroscopía de Correlación Escalar con Filtro de Doble Cuanto) y su comparación con la estructura determinada mediante rayos X, confirma el interés que presentan las estructuras en disolución de bio-polímeros y las sustanciales diferencias que pueden presentar frente a las estructuras cristalinas determinadas mediante difracción de rayos X. Asimismo, la estructura del d(CCGCGG) es un ejemplo de la influencia de la secuencia nucleotídica en la conformación local de cadenas de ADN

Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR

Echistatin is a potent antagonist of the integrins αvβ3, α5β1 and αIIbβ3. Its full inhibitory activity depends on an RGD (Arg-Gly- Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The fulllength C-terminal polypeptide is visible as a β-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30◦. Internal angular motions in the 100–300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin- recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin– αvβ3 docking model suggests that echistatin’s C-terminal amino acids may contact αv-subunit residues and provides new insights to delineate structure–function [email protected]; [email protected]; [email protected]; [email protected]

Peripheral blood mitochondrial DNA content in relation to circulating metabolites and inflammatory markers: a population study

Mitochondrial DNA (mtDNA) content might undergo significant changes caused by metabolic derangements, oxidative stress and inflammation that lead to development and progression of cardiovascular diseases. We, therefore, investigated in a general population the association of peripheral blood mtDNA content with circulating metabolites and inflammatory markers. We examined 310 subjects (50.6% women; mean age, 53.3 years) randomly selected from a Flemish population. Relative mtDNA content was measured by quantitative real-time PCR in peripheral blood cells. Peak circulating metabolites were quantified using nuclear magnetic resonance spectroscopy. The level of inflammation was assessed via established inflammatory markers. Using Partial Least Squares analysis, we constructed 3 latent factors from the 44 measured metabolites that explained 62.5% and 8.5% of the variance in the contributing metabolites and the mtDNA content, respectively. With adjustments applied, mtDNA content was positively associated with the first latent factor (P = 0.002). We identified 6 metabolites with a major impact on the construction of this latent factor including HDL3 apolipoproteins, tyrosine, fatty acid with αCH2, creatinine, β-glucose and valine. We summarized them into a single composite metabolite score. We observed a negative association between the composite metabolic score and mtDNA content (P = 0.001). We also found that mtDNA content was inversely associated with inflammatory markers including hs-CRP, hs-IL6, white blood cell and neutrophil counts as well as neutrophil-to-lymphocyte ratio (P≤0.0024). We demonstrated that in a general population relative peripheral blood mtDNA content was associated with circulating metabolites indicative of perturbed lipid metabolism and with inflammatory biomarkers

Two-Week Aflibercept or Erlotinib Administration Does Not Induce Changes in Intestinal Morphology in Male Sprague-Dawley Rats But Aflibercept Affects Serum and Urine Metabolic Profiles

Gastrointestinal toxicity is a frequently observed adverse event during cancer treatment with traditional chemotherapeutics. Currently, traditional chemotherapeutics are often combined with targeted biologic agents. These biologics, however, possess a distinct toxicity profile, and they may also exacerbate the adverse effects of traditional chemotherapeutics. In this study, we aimed to characterize the gastrointestinal and metabolic changes after a 2-week treatment period with aflibercept, an antiangiogenic VEGFR decoy, and with erlotinib, a tyrosine-kinase inhibitor. Male rats were treated either with aflibercept or erlotinib for 2 weeks. During the 2-week treatment period, the animals in the aflibercept group received twosubcutaneous doses of 25 mg/kg aflibercept. The erlotinib group got 10 mg/kg of erlotinib by oral gavage every other day. The control groups were treated similarly but received either saline injections or oral gavage of water. Intestinal toxicity was assessed by measuring intestinal permeability and by histological analyses of intestinal tissues. Metabolic changes were measured with H-1 nuclear magnetic resonance in serum and urine. Neither aflibercept nor erlotinib induced changes in intestinal permeability or intestinal tissue morphology. However, aflibercept treatment resulted in stunted body weight gain and altered choline, amino acid, and lipid metabolism. Two-week treatment with aflibercept or erlotinib alone does not induce observable changes in gastrointestinal morphology and function. However, observed aflibercept-treatment related metabolic changes suggest alterations in intestinal microbiota, nutrient intake, and adipose tissue function. The metabolic changes are also interesting in respect to the systemic effects of aflibercept and their possible associations with adverse events caused by aflibercept administration.Peer reviewe

Convex non-negative matrix factorization for brain tumor delimitation from MRSI data

Background: Pattern Recognition techniques can provide invaluable insights in the field of neuro-oncology. This is because the clinical analysis of brain tumors requires the use of non-invasive methods that generate complex data in electronic format. Magnetic Resonance (MR), in the modalities of spectroscopy (MRS) and spectroscopic imaging (MRSI), has been widely applied to this purpose. The heterogeneity of the tissue in the brain volumes analyzed by MR remains a challenge in terms of pathological area delimitation. Methodology/Principal Findings: A pre-clinical study was carried out using seven brain tumor-bearing mice. Imaging and spectroscopy information was acquired from the brain tissue. A methodology is proposed to extract tissue type-specific sources from these signals by applying Convex Non-negative Matrix Factorization (Convex-NMF). Its suitability for the delimitation of pathological brain area from MRSI is experimentally confirmed by comparing the images obtained with its application to selected target regions, and to the gold standard of registered histopathology data. The former showed good accuracy for the solid tumor region (proliferation index (PI)>30%). The latter yielded (i) high sensitivity and specificity in most cases, (ii) acquisition conditions for safe thresholds in tumor and non-tumor regions (PI>30% for solid tumoral region; ≤5% for non-tumor), and (iii) fairly good results when borderline pixels were considered. Conclusions/Significance: The unsupervised nature of Convex-NMF, which does not use prior information regarding the tumor area for its delimitation, places this approach one step ahead of classical label-requiring supervised methods for discrimination between tissue types, minimizing the negative effect of using mislabeled voxels. Convex-NMF also relaxes the non-negativity constraints on the observed data, which allows for a natural representation of the MRSI signal. This should help radiologists to accurately tackle one of the main sources of uncertainty in the clinical management of brain tumors, which is the difficulty of appropriately delimiting the pathological area

Magnetic Resonance Microscopy at 14 Tesla and Correlative Histopathology of Human Brain Tumor Tissue

Magnetic Resonance Microscopy (MRM) can provide high microstructural detail in excised human lesions. Previous MRM images on some experimental models and a few human samples suggest the large potential of the technique. The aim of this study was the characterization of specific morphological features of human brain tumor samples by MRM and correlative histopathology. We performed MRM imaging and correlative histopathology in 19 meningioma and 11 glioma human brain tumor samples obtained at surgery. To our knowledge, this is the first MRM direct structural characterization of human brain tumor samples. MRM of brain tumor tissue provided images with 35 to 40 µm spatial resolution. The use of MRM to study human brain tumor samples provides new microstructural information on brain tumors for better classification and characterization. The correlation between MRM and histopathology images allowed the determination of image parameters for critical microstructures of the tumor, like collagen patterns, necrotic foci, calcifications and/or psammoma bodies, vascular distribution and hemorrhage among others. Therefore, MRM may help in interpreting the Clinical Magnetic Resonance images in terms of cell biology processes and tissue patterns. Finally, and most importantly for clinical diagnosis purposes, it provides three-dimensional information in intact samples which may help in selecting a preferential orientation for the histopathology slicing which contains most of the informative elements of the biopsy. Overall, the findings reported here provide a new and unique microstructural view of intact human brain tumor tissue. At this point, our approach and results allow the identification of specific tissue types and pathological features in unprocessed tumor samples

Metabolic Profile of chronic liver disease by NMR spectroscopy of human biopsies

Abstract Among the different processes occurring during the evolution of liver disease, fibrosis has a predominant role. Liver fibrosis mechanisms are fairly constant irrespective of the underlying etiology. Cirrhosis is the end-stage of this reaction. Metabolic profiles, which are affected by many physiological and pathological processes, may provide further insight into the metabolic consequences of this severe liver disease. The aim of this study was to demonstrate the applicability of 1H high resolution magic angle spinning (HR-MAS) NMR spectroscopy in the biochemical profile determination of human liver needle biopsy samples for the characterization of metabolic alterations related to the severity of liver disease. We recorded and analyzed HR-MAS spectra of 68 liver tissue samples obtained by needle biopsy from patients with chronic liver disease. Multivariate analysis was applied to these data to obtain discrimination patterns and to reveal relevant metabolites. The metabolic characterization of liver tissue from needle biopsies by HR-MAS NMR spectroscopy provided differential patterns for cirrhotic and non-cirrhotic chronic liver disease tissue. Metabolites closely related to the liver metabolism such as some fatty acids, glucose and amino acids show differences between the two groups. Phospholipid precursors, which have been previously correlated with hepatic lesions also show differences. Furthermore, the correlation between histologically assessed liver disease stages and the levels of the most discriminative metabolites show that liver dysfunction is present at the initial stages of chronic hepatic lesions. Overall, this work suggests that the additional information obtained by NMR metabolomics applied to needle biopsies of human liver may be useful for assessing metabolic alterations and liver dysfunction in chronic liver disease

Gut-derived metabolites mediating cognitive development in 5-year-old children: Early-life transplant in mice has lasting effects throughout adulthood

The gut microbiota has been causally linked to cognitive development. We aimed to identify metabolites mediating its effect on cognitive development, and foods or nutrients related to most promising metabolites. Faeces from 5-year-old children (DORIAN-PISAC cohort, including 90 general population families with infants, 42/48 females/males, born in 2011-2014) were transplanted (FMT) into C57BL/6 germ-free mice. Children and recipient mice were stratified by cognitive phenotype, or based on protective metabolites. Food frequency questionnaires were obtained in children. Cognitive measurements in mice included five Y-maze tests until 23 weeks post-FMT, and (at 23 weeks) PET-CT for brain metabolism and radiodensity, and ultrasound-based carotid vascular indices. Children (faeces, urine) and mice (faeces, plasma) metabolome was measured by 1H NMR spectroscopy, and the faecal microbiota was profiled in mice by 16S rRNA amplicon sequencing. Cognitive scores of children and recipient mice were correlated. FMT-dependent modifications of brain metabolism were observed. Mice receiving FMT from high-cognitive or protective metabolite-enriched children developed superior cognitive-behavioural performance. A panel of metabolites, namely xanthine, hypoxanthine, formate, mannose, tyrosine, phenylalanine, glutamine, was found to mediate the gut-cognitive axis in donor children and recipient mice. Vascular indices partially explained the metabolite-to-phenotype relationships. Children's consumption of legumes, whole-milk yogurt and eggs, and intake of iron, zinc and vitamin D appeared to support protective gut metabolites. Overall, metabolites involved in inflammation, purine metabolism and neurotransmitter synthesis mediate the gut-cognitive axis, and holds promise for screening. The related dietary and nutritional findings offer leads to microbiota-targeted interventions for cognitive protection, with long-lasting effects