The analysis of the skeletal muscle metabolism is crucial for designing optimal exercise paradigms in type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease that commonly results from a high-calorie diet and sedentary lifestyle, leading to insulin resistance and glucose homeostasis perturbation. Physical activity is recommended as one first-line treatment in T2DM, but it leads to contrasted results. We hypothesized that, instead of applying standard exercise protocols, the prescription of personalized exercise programs specifically designed to reverse the potential metabolic alterations in skeletal muscle could result in better results. METHODS: To test this hypothesis, we drew the metabolic signature of the fast-twitch quadriceps muscle, based on a combined unbiased NMR spectroscopy and RT-qPCR study, in several T2DM mouse models of different genetic background (129S1/SvImJ, C57Bl/6J), sex and aetiology (high-fat diet (HFD) or HFD/Streptozotocin (STZ) induction or transgenic MKR (FVB-Tg Ckm-IGF1R*K1003R)1Dlr/J) mice. Three selected mouse models with unique muscular metabolic signatures were submitted to three different swimming-based programs, designed to address each metabolic specificity. RESULTS: We found that depending on the genetic background, the sex, and the mode of T2DM induction, specific muscular adaptations occurred, including depressed glycolysis associated with elevated PDK4 expression, shift to ß-oxidation, or deregulation of amino-acid homeostasis. Interestingly, dedicated swimming-based exercises designed to restore specific metabolic alterations in muscle were found optimal in improving systemic T2DM hallmarks, including a significant reduction in insulin resistance, the improvement of glucose homeostasis, and a delay in sensorimotor function alterations. CONCLUSION: The muscle metabolism constitutes an important clue for the design of precision exercises with potential clinical implications for T2DM patients.

Perspectives and perception of haemophilia gene therapy by French patients.

INTRODUCTION AND AIM: A national survey was initiated by representatives of French patients with haemophilia (AFH) and the French reference centre for haemophilia, in order to appreciate the awareness and knowledge of these patients regarding haemophilia gene therapy (HGT) and understand better their position about this innovative treatment that will soon become available. RESULTS: Of 143 answers received, 137 could be analysed, representing about 3.5% of patients with severe or moderate haemophilia over 16year-old. They were 80.3% with haemophilia A and 19.7 % with haemophilia B, with a severe form of the disease for 80.3 % of them. Curiosity for HGT was formulated by 64.2% of the participants, 33.6 % being interested by this approach as soon as it will be available and 38.7 % preferring to wait until more patients have been treated. Only 3.6 % of the participants would never consider receiving HGT. The level of awareness and knowledge was estimated to be limited by 39.5 % of the patients. More than 60 % of them declared having never or almost never discussed HGT with the team of their haemophilia centre. Before deciding to get HGT, 54.4 % of the participants considered that it will be very important to compare it with their current treatment and 53.7 % would like to be better informed by their care providers. CONCLUSIONS: These results highlight the need for training and education for patients, but also for professionals at haemophilia centres, about HGT and the shared decision-making process. Objective, unbiased and transparent information must be available for patients about this very promising therapy which nonetheless carries more uncertainty and unknowns compared to other haemophilia treatments.

Revisiting the nature and pharmacodynamics of tacrolimus metabolites.

The toxicity of tacrolimus metabolites and their potential pharmacodynamic (PD) interactions with tacrolimus might respectively explain the surprising combination of higher toxicity and lower efficacy of tacrolimus despite normal blood concentrations, described in extensive metabolizers. To evaluate such interactions, we produced tacrolimus metabolites in vitro and characterized them by high resolution mass spectrometry (HRMS, for all) and nuclear magnetic resonance (NMR, for the most abundant, M-I). We quantified tacrolimus metabolites and checked their structure in patient whole blood and peripheral blood mononuclear cells (PBMC). We explored the interactions of M-I with tacrolimus in silico, in vitro and ex vivo. In vitro metabolization produced isoforms of tacrolimus and of its metabolites M-I and M-III, whose HRMS fragmentation suggested an open-ring structure. M-I and M-III open-ring isomers were also observed in patient blood. By contrast, NMR could not detect these open-ring forms. Transplant patients expressing CYP3A5 exhibited higher M-I/TAC ratios in blood and PBMC than non-expressers. Molecular Dynamics simulations showed that: all possible tacrolimus metabolites and isomers bind FKPB12; and the hypothetical open-ring structures induce looser binding between FKBP12 and calcineurins, leading to lower CN inhibition. In vitro, tacrolimus bound FKPB12 with more affinity than purified M-I, and the pool of tacrolimus metabolites and purified M-I had only weak inhibitory activity on IL2 secretion and not at all on NFAT nuclear translocation. M-I showed no competitive effect with tacrolimus on either test. Finally, M-I or the metabolite pool did not significantly interact with tacrolimus MLR suppression, thus eliminating a pharmacodynamic interaction.

One touch is all it takes: the supramolecular interaction between ubiquitin and lanthanide complexes revisited by paramagnetic NMR and molecular dynamics.

The supramolecular interaction between lanthanide complexes and proteins is at the heart of numerous chemical and biological studies. Some of these complexes have demonstrated remarkable interaction properties with proteins or peptides in solution and in the crystalline state. Here we have used the paramagnetism of lanthanide ions to characterize the affinity of two lanthanide complexes for ubiquitin. As the interaction process is dynamic, the acquired NMR data only reflect the time average of the different steps. We have used molecular dynamics (MD) simulations to get a deeper insight into the detailed interaction scenario at the microsecond scale. This NMR/MD approach enabled us to establish that the tris-dipicolinate complex interacts specifically with arginines and lysines, while the crystallophore explores the protein surface through weak interactions with carboxylates. These observations shed new light on the dynamic interaction properties of these complexes, which will ultimately enable us to propose a crystallization mechanism.

NMR metabolomics study of chronic low-dose exposure to a cocktail of persistent organic pollutants.

Nowadays, exposure to endocrine-disrupting chemicals (EDCs), including persistent organic pollutants (POPs), is one of the most critical threats to public health. EDCs are chemicals that mimic, block, or interfere with hormones in the body's endocrine system and have been associated with a wide range of health issues. This innovative, untargeted metabolomics study investigates chronic low-dose internal exposure to a cocktail of POPs on multiple tissues that are known to accumulate these lipophilic compounds. Interestingly, the metabolic response differs among selected tissues/organs in mice. In the liver, we observed a dynamic effect according to the exposure time and the doses of POPs. In the brain tissue, the situation is the opposite, leading to the conclusion that the presence of POPs immediately gives a saturated effect that is independent of the dose and the duration of exposure studied. By contrast, for the adipose tissues, nearly no effect is observed. This metabolic profiling leads to a holistic and dynamic overview of the main metabolic pathways impacted in lipophilic tissues by a cocktail of POPs.

A Toolbox for Glutamine Use in Dissolution Dynamic Nuclear Polarization: from Enzymatic Reaction Monitoring to the Study of Cellular Metabolic Pathways and Imaging.

Glutamine is under scrutiny regarding its metabolic deregulation linked to energetic reprogramming in cancer cells. Many analytical techniques have been used to better understand the impact of the metabolism of amino acids on biological processes, however only a few are suited to work with complex samples. Here, we report the use of a general dissolution dynamic nuclear polarization (D-DNP) formulation using an unexpensive radical as a multipurpose tool to study glutamine, with insights from enzymatic modelling to complex metabolic networks and fast imaging. First, hyperpolarized [5-13 C] glutamine is used as molecular probe to study the kinetic action of two enzymes: L-asparaginase that has been used as an anti-metabolic treatment for cancer, and glutaminase. These results are also compared with those acquired with another hyperpolarized amino acid, [1,4-13 C] asparagine. Second, we explored the use of hyperpolarized (HP) substrates to probe metabolic pathways by monitoring metabolic profiles arising from hyperpolarized glutamine in E. coli extracts. Finally, a highly concentrated sample formulation is proposed for the purpose of fast imaging applications. We think that this approach can be extended to formulate other amino acids as well as other metabolites and provide complementary insights into the analysis of metabolic networks.

Evaluation of the care pathway in the context of the dispensing of emicizumab (Hemlibra) in community and hospital pharmacies in France: A patient satisfaction survey.

INTRODUCTION: Since June 2021 in France, patients with haemophilia A with anti-factor VIII inhibitors and patients with severe haemophilia A without anti-factor VIII inhibitors, and treated with emicizumab (Hemlibra), have to choose the dispensing circuit community or hospital pharmacy. AIM: To evaluate satisfaction of patients whether they choose dispensation from a community pharmacy or retained dispensation from the hospital pharmacy, to understand the main motivation for choosing the community or the hospital pharmacy. METHODS: All patients living in France, regardless of age, were eligible to participate. Between September 13, 2022, and January 9, 2023, 175 respondents answered the satisfaction survey, including 123 in community pharmacy and 52 in hospital pharmacy. RESULTS: Eighteen months after availability in community pharmacies, treatment accessibility is improved for the benefit of the patient. The door-to-door travel times are significantly reduced to the community pharmacy with an average gain of 16.5 min saved from the place of residence. Patients are mostly satisfied with the new dispensing circuit especially concerning the overall satisfaction (p < .0001), the travel time (p < .0001) and the strong relationship with the pharmacist (p = .0022) compared to hospital pharmacy. CONCLUSION: Innovation in care pathways is showing its full potential in improving access to medication, made possible by the implementation of a rigorous organization accompanied by training to enable healthcare professionals involved in primary care to provide appropriate management.

Determinants of adherence and consequences of the transition from adolescence to adulthood among young people with severe haemophilia (TRANSHEMO): A multicentric French national observational cross-sectional study based on the FranceCoag registry.

INTRODUCTION: It is necessary to gain insights into adherence to healthcare in people with severe haemophilia (PwSH), especially during the transition from paediatric to adult care, which is an important phase in lives of young people with childhood chronic disease. This adherence can be considered as a marker of successful transition. OBJECTIVES: The main objective of the quantitative phase of the TRANSHEMO project was to compare the adherence to healthcare between adolescents and young adults (YAs) with severe haemophilia. The secondary objective was to identify the determinants (facilitators and barriers) of this adherence and associations between these determinants. METHODS: A multicentre, observational, cross-sectional study was conducted in 2017-2019 on PwSH aged between 14 and 17 years (adolescents) or between 20 and 29 years (YAs), included in the FranceCoag registry and having completed the questionnaires. The adherence to healthcare (treatment regimens and clinical follow-up) was compared between adolescents and YAs using the chi-squared test. The determinants of this adherence were analysed by structural equation modelling. RESULTS: There were 277 participants, 107 adolescents, and 170 YAs. The rate of adolescents adhering to healthcare was 82.2%, while the rate of YAs was 61.2% (p < .001). The barriers to the adherence to healthcare were being YA, having repeated at least one school grade and presenting mental health concerns. CONCLUSION: Adolescents had better adherence to healthcare than YAs. According to the determinants enlightened in this project, targeted supportive strategies and adapted therapeutic education programs can be developed for young PwSH to facilitate their adherence to healthcare.

The Crying Need for a Better Response Assessment in Rectal Cancer.

OPINION STATEMENT: Since total neoadjuvant treatment achieves almost 30% pathologic complete response, organ preservation has been increasingly debated for good responders after neoadjuvant treatment for patients diagnosed with rectal cancer. Two organ preservation strategies are available: a watch and wait strategy and a local excision strategy including patients with a near clinical complete response. A major issue is the selection of patients according to the initial tumor staging or the response assessment. Despite modern imaging improvement, identifying complete response remains challenging. A better selection could be possible by radiomics analyses, exploiting numerous image features to feed data characterization algorithms. The subsequent step is to include baseline and/or pre-therapeutic MRI, PET-CT, and CT radiomics added to the patients' clinicopathological data, inside machine learning (ML) prediction models, with predictive or prognostic purposes. These models could be further improved by the addition of new biomarkers such as circulating tumor biomarkers, molecular profiling, or pathological immune biomarkers.

Present and future of pure shift NMR in metabolomics.

NMR is one of the most powerful techniques for the analysis of biological samples in the field of metabolomics. However, the high complexity of fluids, tissues, or other biological materials taken from living organisms is still a challenge for state-of-the-art pulse sequences, thereby limiting the detection, the identification, and the quantification of metabolites. In this context, the resolution enhancement provided by broadband homonuclear decoupling methods, which allows for simplifying 1 H multiplet patterns into singlets, has placed this so-called pure shift technique as a promising approach to perform metabolic profiling with unparalleled level of detail. In recent years, the many advances achieved in the design of pure shift experiments has paved the way to the analysis of a wide range of biological samples with ultra-high resolution. This review leads the reader from the early days of the main pure shift methods that have been successfully developed over the last decades to address complex samples, to the most recent and promising applications of pure shift NMR to the field of NMR-based metabolomics.

Ultrahigh-Resolution NMR with Water Signal Suppression for a Deeper Understanding of the Action of Antimetabolic Drugs on Diffuse Large B-Cell Lymphoma.

Ultrahigh-resolution NMR has recently attracted considerable attention in the field of complex samples analysis. Indeed, the implementation of broadband homonuclear decoupling techniques has allowed us to greatly simplify crowded 1H spectra, yielding singlets for almost every proton site from the analyzed molecules. Pure shift methods have notably shown to be particularly suitable for deciphering mixtures of metabolites in biological samples. Here, we have successfully implemented a new pure shift pulse sequence based on the PSYCHE method, which incorporates a block for solvent suppression that is suitable for metabolomics analysis. The resulting experiment allows us to record ultrahigh-resolution 1D NOESY 1H spectra of biofluids with suppression of the water signal, which is a crucial step for highlighting metabolite mixtures in an aqueous phase. We have successfully recorded pure shift spectra on extracellular media of diffuse large B-cell lymphoma (DLBCL) cells. Despite a lower sensitivity, the resolution of pure shift data was found to be better than that of the standard approach, which provides a more detailed vision of the exo-metabolome. The statistical analyses carried out on the resulting metabolic profiles allow us to successfully highlight several metabolic pathways affected by these drugs. Notably, we show that Kidrolase plays a major role in the metabolic pathways of this DLBCL cell line.

Occupational integration of adults with severe haemophilia (INTHEMO): A study based on the FranceCoag registry.

INTRODUCTION: Health of people with severe haemophilia (PwSH) improves thanks to the advancements in haemophilia care, giving them more opportunities in occupational integration. However, there is little literature on the occupational integration of PwSH. OBJECTIVES: The main objective of our study was to assess the occupational integration of PwSH and to compare it with that of the general population. The secondary objective was to study the association between individual characteristics (sociodemographic, clinical and psycho-behavioural) and occupational integration of PwSH. METHODS: A multicentre, non-interventional, cross-sectional study was conducted in 2018-2020 on PwSH, aged over 18 and under 65 years and included in the FranceCoag registry. Measurements included indicators of occupational integration, sociodemographic, clinical and psycho-behavioural characteristics. The indicators of occupational integration were compared with those of the general population, using indirect standardization. The data of the general population were available from the National Institute of Statistics and Economic Studies (INSEE). Determinants of occupational integration were explored using structural equation modelling. RESULTS: Of 1262 eligible people, 588 were included. PwSH had a lower employment rate than the general population (standardized ratio, .85; 95% CI, .77-.94). There were more PwSH at tertiary education level than expected (standardized ratio, 1.38; 95% CI, 1.17-1.61). HIV infection, poor physical health and mental health concerns were associated with a higher risk of unemployment in PwSH. CONCLUSION: Employment rate of PwSH is lower than that of the general population despite their higher education level. Target interventions focusing on determinants of difficult occupational integration could be helpful for PwSH.

Synthesis and characterization of novel conformers of (E)-2-(3-nitro-H-imidazo[1,2-a]pyridin-2-ylthio)-N'-benzylideneacetohydrazide derivatives.

Eighteen new N-acylhydrazones (9a-r) containing the imidazo[1,2-a]pyridine scaffold were synthesized through a seven steps reaction sequence, ending with a condensation of 2-(3-nitro-H-imidazo[1,2-a]pyridin-2-ylthio)acetohydrazide with various benzaldehyde derivatives (8a-r). All synthesized compounds were characterized by 1D NMR (1 H and 13 C NMR) and 2D NMR (NOESY) spectroscopic analyses and high-resolution mass spectrometry (HRMS). The analysis of 1 H NMR data performed at room temperature in deuterated dimethylsulfoxide (DMSO-d6 ) revealed the presence of (E)-2-(3-nitro-H-imidazo[1,2-a]pyridin-2-ylthio)-N'-benzylideneacetohydrazide (9a-r) as a mixture of two conformers, namely, syn-periplanar E (sp E) and anti-periplanar E (ap E). For all N-acylhydrazones that were synthesized, the sp E conformer was found to be the major form except in the case of hydrazone derived from o-hydroxybenzaldehyde.

Structural analysis of unstable norbixin isomers guided by pure shift nuclear magnetic resonance.

We report the analysis of complex samples obtained during the microwave irradiation/heating of norbixin, which has been identified as a potential therapeutic target for age-related macular degeneration (AMD). In this context, identifying the different isomers that are obtained during its degradation is of primary importance. However, this characterization is challenging because, on the one hand, some of these isomers are unstable, and on the other hand, the 1 H spectra of these isomeric mixtures are poorly resolved. We could successfully apply 1D pure shift experiments to obtain ultrahigh-resolution 1 H nuclear magnetic resonance (NMR) spectra of the norbixin isomer samples and exploit their information content to analyze complementary 2D NMR data and describe accurately their isomeric composition.

Capturing the dynamic association between a tris-dipicolinate lanthanide complex and a decapeptide: a combined paramagnetic NMR and molecular dynamics exploration.

In the realm of biomolecules, peptides can present a large diversity of structures. Our study sheds new light on the structural interplay between a tris-dipicolinate lanthanide probe and a decapeptide SASYKTLPRG. Although a rather trivial, electrostatically driven interaction was expected, the combination of paramagnetic NMR and molecular dynamics simulations reveals a highly dynamic association process and allows for providing extensive insights into the interaction sites and their occupancy. This study highlights the importance of a large conformational sampling to reconcile characteristic time in NMR with molecular dynamics simulations, where sampling in the microsecond range is needed. This study opens the door for a detailed mechanistic elucidation of the early steps of lanthanide complex-peptide or lanthanide complex-protein interaction or self-assembly processes.

Highly Accurate Quantitative Analysis Of Enantiomeric Mixtures from Spatially Frequency Encoded 1H NMR Spectra.

We propose an original concept to measure accurately enantiomeric excesses on proton NMR spectra, which combines high-resolution techniques based on a spatial encoding of the sample, with the use of optically active weakly orienting solvents. We show that it is possible to simulate accurately dipolar edited spectra of enantiomers dissolved in a chiral liquid crystalline phase, and to use these simulations to calibrate integrations that can be measured on experimental data, in order to perform a quantitative chiral analysis. This approach is demonstrated on a chemical intermediate for which optical purity is an essential criterion. We find that there is a very good correlation between the experimental and calculated integration ratios extracted from G-SERF spectra, which paves the way to a general method of determination of enantiomeric excesses based on the observation of 1H nuclei.

Paramagnetic DOSY: An Accurate Tool for the Analysis of the Supramolecular Interactions between Lanthanide Complexes and Proteins.

Diffusion ordered NMR is implemented to determine accurately the mobility of paramagnetic tris-dipicolinate lanthanide complexes that are versatile probes of protein structure. It is shown that diffusion coefficient ratios can be measured with an accuracy of 1 % using a standard BPPLED pulse sequence, which allows for observing significant, though weak, variations when different species are interacting with the paramagnetic compound. We demonstrate that this approach is complementary to classical chemical shift titration experiments, and that it can be applied successfully to probe the supramolecular dynamic interactions between lanthanide complexes and small molecules on the one hand, or to determine rapidly their affinity for a targeted protein.

Correction: Achieving high resolution and optimizing sensitivity in spatial frequency encoding NMR spectroscopy: from theory to practice.

Correction for 'Achieving high resolution and optimizing sensitivity in spatial frequency encoding NMR spectroscopy: from theory to practice' by Bertrand Plainchont et al., Phys. Chem. Chem. Phys., 2016, 18, 22827-22839.