Pyruvate cellular uptake and enzymatic conversion probed by dissolution DNP-NMR: the impact of overexpressed membrane transporters.

Pyruvate membrane crossing and its lactate dehydrogenase-mediated conversion to lactate in cells featuring different levels of expression of membrane monocarboxylate transporters (MCT4) were probed by dissolution dynamic nuclear polarization-enhanced NMR. Hyperpolarized 13 C-1-labeled pyruvate was transferred to suspensions of rodent tumor cell carcinoma, cell line 39. The pyruvate-to-lactate conversion rate monitored by dissolution dynamic nuclear polarization-NMR in carcinoma cells featuring native MCT4 expression level was lower than the rate observed for cells in which the human MCT4 gene was overexpressed. The enzymatic activity of lactate dehydrogenase was also assessed in buffer solutions, following the real-time pyruvate-to-lactate conversion speeds at different enzyme concentrations. Copyright © 2016 John Wiley & Sons, Ltd.

Long-lived states to monitor protein unfolding by proton NMR.

The relaxation of long-lived states (LLS) corresponds to the slow return to statistical thermal equilibrium between symmetric and antisymmetric proton spin states. This process is remarkably sensitive to the presence of external spins and can be used to obtain information about partial unfolding of proteins. We detected the appearance of a destabilized conformer of ubiquitin when urea is added to the protein in its native state. This conformer shows increased mobility in the C-terminus, which significantly extends the lifetimes of proton LLS magnetisation in Ser-65. These changes could not be detected by conventional measurements of T(1) and T(2) relaxation times of protons, and would hardly be sensed by carbon-13 or nitrogen-15 relaxation measurements. Conformers with similar dynamic and structural features, as revealed by LLS relaxation times, could be observed, in the absence of urea, in two ubiquitin mutants, L67S and L69S.

Early Use of Mother's Own Raw Milk, Maternal Satisfaction, and Breastfeeding Continuation in Hospitalised Neonates: A Prospective Cohort Study.

BACKGROUND: Despite the critical importance of breast milk for preterm and sick neonates, there is no consensus regarding the use of raw mother's own milk (MOM) in neonatal units. OBJECTIVES: This study aimed to describe the use of raw MOM in hospitalised neonates before day 7 (early use), and to investigate: (i) related factors, (ii) maternal satisfaction, and (iii) the association with breastfeeding continuation. METHODS: This prospective cohort included 516 neonates intended to be breastfed in 2 French neonatal units. Neonates receiving raw MOM before day 7 were compared to those who did not. The association between early use of MOM and breastfeeding continuation at hospital discharge, and up to 6 months later, was measured by logistic regression. RESULTS: More than one-third (36.2%) of breastfed neonates did not receive any MOM during their first week, mainly due to organisational constraints and staff reluctance. Maternal satisfaction related to early raw MOM use was high (96%), and was coupled with a more frequent maternal feeling of being supported in breastfeeding (p = 0.003). There was a significant association between early use of MOM and breastfeeding continuation at discharge (OR 2.92, 95% CI 1.94-4.40, p < 0.0001), which persisted 6 months later (OR 2.70, 95% CI 1.21-6.03, p = 0.023). This association appeared independent in multivariable analyses (at discharge: aOR 2.03, 95% CI 1.27-3.25, p = 0.003; 6 months later: aOR 2.46, 95% CI 1.02-5.92, p = 0.045). CONCLUSION: While the early use of raw MOM in hospitalised neonates can be limited by multiple factors, it appears supportive for mothers, and might represent a simple opportunity to improve breastfeeding in neonatal units.

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR.

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.