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Obtaining comprehensive, untargeted metabolic profiles for complex solid samples, e.g., animal tissues, requires sample preparation and access to information-rich analytical methodologies such as mass spectrometry (MS). Here we describe a practical two-step process for tissue samples that is based on extraction into 'aqueous' and 'organic' phases for polar and nonpolar metabolites. Separation methods such as ultraperformance liquid chromatography (UPLC) in combination with MS are needed to obtain sufficient resolution to create diagnostic metabolic profiles and identify candidate biomarkers. We provide detailed protocols for sample preparation, chromatographic procedures, multivariate analysis and metabolite identification via tandem MS (MS/MS) techniques and high-resolution MS. By using these optimized approaches, analysis of a set of samples using a 96-well plate format would take ∼48 h: 1 h for system setup, 8–10 h for sample preparation, 34 h for UPLC-MS analysis and 2–3 h for preliminary/exploratory data processing, representing a robust method for untargeted metabolic screening of tissue samples.

Metabolomic approaches are increasingly used to identify new disease biomarkers, yet normal values of many plasma metabolites remain poorly defined. The aim of this study was to define the \"normal\" metabolome in healthy volunteers. We included 800 French volunteers aged between 18 and 86, equally distributed according to sex, free of any medication and considered healthy on the basis of their medical history, clinical examination and standard laboratory tests. We quantified 185 plasma metabolites, including amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, sphingomyelins and hexose, using tandem mass spectrometry with the Biocrates AbsoluteIDQ p180 kit. Principal components analysis was applied to identify the main factors responsible for metabolome variability and orthogonal projection to latent structures analysis was employed to confirm the observed patterns and identify pattern-related metabolites. We established a plasma metabolite reference dataset for 144/185 metabolites. Total blood cholesterol, gender and age were identified as the principal factors explaining metabolome variability. High total blood cholesterol levels were associated with higher plasma sphingomyelins and phosphatidylcholines concentrations. Compared to women, men had higher concentrations of creatinine, branched-chain amino acids and lysophosphatidylcholines, and lower concentrations of sphingomyelins and phosphatidylcholines. Elderly healthy subjects had higher sphingomyelins and phosphatidylcholines plasma levels than young subjects. We established reference human metabolome values in a large and well-defined population of French healthy volunteers. This study provides an essential baseline for defining the \"normal\" metabolome and its main sources of variation.

Mutationsin epidermal growth factor receptor (EGFR) are found in approximately 48% of Asian and 19% of Western patients with lung adenocarcinoma (LUAD), leading to aggressive tumor growth. While tyrosine kinase inhibitors (TKIs) like gefitinib and osimertinib target this mutation, treatments often face challenges such as metastasis and resistance. To address this, we developed physiologically based pharmacokinetic (PBPK) models for both drugs, simulating their distribution within the primary tumor and metastases following oral administration. These models, combined with a mechanistic knowledge-based disease model of EGFR-mutated LUAD, allow us to predict the tumor’s behavior under treatment considering the diversity within the tumor cells due to different mutations. The combined model reproduces the drugs’ distribution within the body, as well as the effects of both gefitinib and osimertinib on EGFR-activation-induced signaling pathways. In addition, the disease model encapsulates the heterogeneity within the tumor through the representation of various subclones. Each subclone is characterized by unique mutation profiles, allowing the model to accurately reproduce clinical outcomes, including patients’ progression, aligning with RECIST criteria guidelines (version 1.1). Datasets used for calibration came from NEJ002 and FLAURA clinical trials. The quality of the fit was ensured with rigorous visual predictive checks and statistical tests (comparison metrics computed from bootstrapped, weighted log-rank tests: 98.4% (NEJ002) and 99.9% (FLAURA) similarity). In addition, the model was able to predict outcomes from an independent retrospective study comparing gefitinib and osimertinib which had not been used within the model development phase. This output validation underscores mechanistic models’ potential in guiding future clinical trials by comparing treatment efficacies and identifying patients who would benefit most from specific TKIs. Our work is a step towards the design of a powerful tool enhancing personalized treatment in LUAD. It could support treatment strategy evaluations and potentially reduce trial sizes, promising more efficient and targeted therapeutic approaches. Following its consecutive prospective validations with the FLAURA2 and MARIPOSA trials (validation metrics computed from bootstrapped, weighted log-rank tests: 94.0% and 98.1%, respectively), the model could be used to generate a synthetic control arm.

Cytochrome P450 enzymes (CYPs) are of great interest to pharmaceutical industry due to their major role in drug metabolism and drug-drug interactions in man. As CYPs also act on endogenous substrates and are regulated by nuclear receptors involved in biochemical pathways, their profiles are likely to affect endogenous metabolic profiles. The work in this thesis aimed to establish baseline rat liver CYP levels and assess relationships between CYP profiles and corresponding endogenous metabolic profiles, before introducing CYP inducers in subsequent projects to identify early biomarkers of CYP induction. An efficient and reproducible protocol was first developed and optimised for liver untargeted metabolic profiling by ultra performance liquid chromatography-mass spectrometry (UPLC-MS). Comparison of technical and biological variation for this liver protocol then demonstrated that sample preparation and UPLC-MS variability was mostly small compared to inter-animal variability. Subsequently, an in vivo study was designed including 20 male and 20 female Wistar rats. Liver mRNA of 81 CYPs was quantified, of which 23 exhibited significant gender differences with a gender ratio > 2. Clear gender differences were also observed in serum, urine and liver UPLC-MS metabolic profiles, e.g for metabolites belonging to steroid, triglyceride and phospholipid families. Multivariate models constructed to investigate relationships between CYP and metabolic profiles mainly highlighted the latent variable “gender”. Separate investigations for each gender yielded good prediction of CYP mRNA profiles from male liver aqueous metabolic profiles and female urine metabolic profiles. For prediction of CYP activities, the best models were obtained from serum metabolic profiles. In conclusion, this work improved our knowledge of rat basal CYP and metabolic profiles, and provides a strong basis for subsequent studies on CYP inducers. Importantly, strategies developed for UPLC-MS sample preparation, study design and data pre-processing/analysis are now routinely employed in our laboratory and have applications in many metabolic profiling studies.

Objectives: Investigate factors contributing to the effective management of age-related hearing loss (ARHL) rehabilitation. Methods: A systematic review was conducted following PRISMA guidelines. The protocol was registered in PROSPERO (CRD42022374811). Articles were identified through systematic searches in the Scopus, PubMed, Web of Science, and Cochrane databases in May 2024. Only articles published between January 2005 and May 2024 were included. Studies were assessed for eligibility by two independent researchers and evaluated using the Crowe Critical Appraisal Tool v1.4 (CCAT). Results: Of the 278 articles identified, 54 were included. Three factors explain effective HA use. First, hearing aid signal processing, with directional microphones and noise reduction, improves user comfort and understanding regarding noise. Second, there is hearing aid fitting, with the NAL prescription rules as the gold standard, and bilateral, high-level HA performance for spatial localization and noise comprehension. Third, there is a patient-centered approach, using patient-related outcome measures (PROMs), questionnaires, counseling, and regular follow-up to involve patients in their therapeutic rehabilitation. Conclusions: Reaching a consensus on acoustic parameters is challenging due to variability in audiological results. Involving patients in their rehabilitation, addressing their needs and expectations, and offering individualized care are crucial.

Aims It has recently been shown that an allele in the glucokinase regulatory protein (GCKR) gene was associated with increased liver fat content in obese children. In this study, we set out to determine whether GCKR rs1260326 polymorphism was associated with liver fat content in patients with type 2 diabetes. Methods Three hundred and eight patients with type 2 diabetes were included in this study. Liver fat content was evaluated using 1H-MR spectroscopy. Results In our population, carriers of the rs1260326 minor T allele had a higher liver fat content than did carriers of the C allele homozygote (12.4 ± 9.6 vs. 10.3 ± 9.1 %, p  = 0.03). The number of patients with steatosis was significantly higher in minor T allele carriers than in C allele homozygote carriers (70.7 vs. 55.4 %; p  = 0.008). In multivariate analysis, the predictive variables for steatosis were BMI [odds ratio (OR) 1.08; 95 % confidence interval (CI) 1.03–1.13; p  = 0.002], statin therapy (yes) [OR 0.54; 95 % CI 0.31–0.94; p  = 0.03], metformin therapy (yes) [OR 2.67; 95 % CI 1.50–4.75; p  < 0.001], and rs1260326 GCKR polymorphism (TT+CT) [OR 1.99; 95 % CI 1.14–3.47; p  = 0.01]. Conclusions This study shows that in patients with type 2 diabetes who were not selected for liver abnormalities, liver fat content was related to GCKR rs1260326 polymorphism independent of BMI, triglyceride levels, and age.

Abstract Event-related potentials (ERPs) associated with the involuntary orientation of (bottom-up) attention towards an unexpected sound are of larger amplitude in high dream recallers (HR) than in low dream recallers (LR) during passive listening, suggesting different attentional functioning. We measured bottom-up and top-down attentional performance and their cerebral correlates in 18 HR (11 women, age = 22.7 ± 4.1 years, dream recall frequency = 5.3 ± 1.3 days with a dream recall per week) and 19 LR (10 women, age = 22.3, DRF = 0.2 ±0.2) using EEG and the Competitive Attention Task. Between-group differences were found in ERPs but not in behavior. The results confirm that HR present larger ERPs to distracting sounds than LR during active listening, suggesting enhanced bottom-up processing of irrelevant sounds. HR also presented a larger contingent negative variation during target expectancy and a larger P3b response to target sounds than LR, speaking for an enhanced recruitment of top-down attention. Enhancement of both top-down and bottom-up processes in HR leads to an apparently preserved attentional balance since similar performance were observed in the two groups. Therefore, different neurophysiological profiles can result in similar cognitive performance, with some profiles possibly costlier in term of resource/energy consumption. Competing Interest Statement The authors have declared no competing interest.