FUNCTIONAL MODULATION OF HUMAN PLURIPOTENT STEM CELLS BY O-LINKED N-ACETYLGUCOSAMINE

Ph.DDOCTOR OF PHILOSOPH

Excess of O-GlcNAc modifies human pluripotent stem cells differentiation

International audienc

Excess of O-GlcNAc modifies human pluripotent stem cells differentiation

International audienc

Coupled fluid flow, heat transfer, phase transformation, stress and deformation numerical model for gas quenching

International audienc

RING1B O-GlcNAcylation regulates gene targeting of polycomb repressive complex 1 in human embryonic stem cells

International audienceO-linked-N-acetylglucosamine (O-GlcNAc) post-translationally modifies and regulates thousands of proteins involved in various cellular mechanisms. Recently, O-GlcNAc has been linked to human embryonic stem cells (hESC) differentiation, however the identity and function of O-GlcNAc proteins regulating hESC remain unknown. Here, we firstly identified O-GlcNAc modified human stem cell regulators such as hnRNP K, HP1γ, and especially RING1B/RNF2. Thereafter, we focused our work on RING1B which is the catalytic subunit of the polycomb repressive complex 1 (PRC1) a major epigenetic repressor essential for pluripotency maintenance and differentiation. By point-mutation, we show that T 250 /S 251 and S 278 RING1B residues are bearing O-GlcNAc, and that T 250 /S 251 O-GlcNAcylation decreases during differentiation. O-GlcNAc seems to regulate RING1B-DNA binding as suggested by our ChIP-sequencing results. Non-O-GlcNAcylated RING1B is found to be enriched near cell cycle genes whereas O-GlcNAcylated RING1B seems preferentially enriched near neuronal genes. Our data suggest that during hESC differentiation, the decrease of RING1B O-GlcNAcylation might enable PRC1 to switch its target to induce neuro

Multiple Reaction Monitoring Mass Spectrometry for the Discovery and Quantification of O‑GlcNAc-Modified Proteins

O-linked <i>N</i>-acetylglucosamine (O-GlcNAc) is a post-translational modification regulating proteins involved in a variety of cellular processes and diseases. Unfortunately, O-GlcNAc remains challenging to detect and quantify by shotgun mass spectrometry (MS) where it is time-consuming and tedious. Here, we investigate the potential of Multiple Reaction Monitoring Mass Spectrometry (MRM-MS), a targeted MS method, to detect and quantify native O-GlcNAc modified peptides without extensive labeling and enrichment. We report the ability of MRM-MS to detect a standard O-GlcNAcylated peptide and show that the method is robust to quantify the amount of O-GlcNAcylated peptide with a method detection limit of 3 fmol. In addition, when diluted by 100-fold in a trypsin-digested whole cell lysate, the O-GlcNAcylated peptide remains detectable. Next, we apply this strategy to study glycogen synthase kinase-3 beta (GSK-3β), a kinase able to compete with O-GlcNAc transferase and modify identical site on proteins. We demonstrate that GSK-3β is itself modified by O-GlcNAc in human embryonic stem cells (hESC). Indeed, by only using gel electrophoresis to grossly enrich GSK-3β from whole cell lysate, we discover by MRM-MS a novel O-GlcNAcylated GSK-3β peptide, bearing 3 potential O-GlcNAcylation sites. We confirm our finding by quantifying the increase of O-GlcNAcylation, following hESC treatment with an O-GlcNAc hydrolase inhibitor. This novel O-GlcNAcylation could potentially be involved in an autoinhibition mechanism. To the best of our knowledge, this is the first report utilizing MRM-MS to detect native O-GlcNAc modified peptides. This could potentially facilitate rapid discovery and quantification of new O-GlcNAcylated peptides/proteins

Bedside prediction of intradialytic hemodynamic instability in critically ill patients: the SOCRATE study

International audienceBACKGROUND:Despite improvements in intermittent hemodialysis management, intradialytic hemodynamic instability (IHI) remains a common issue that could account for increased mortality and delayed renal recovery. However, predictive factors of IHI remain poorly explored. The objective of this study was to evaluate the relationship between baseline macrohemodynamic, tissue hypoperfusion parameters and IHI occurrence.METHODS:Prospective observational study conducted in a 18-bed medical ICU of a tertiary teaching hospital. Cardiovascular SOFA score, index capillary refill time (CRT) and lactate level were measured just before (T0) consecutive intermittent hemodialysis sessions performed for AKI. The occurrence of IHI requiring a therapeutic intervention was recorded.RESULTS:Two hundred eleven sessions, corresponding to 72 (34%) first sessions and 139 (66%) later sessions, were included. As IHI mostly occurred during first sessions (43% vs 12%, P  2 mmol/L (68% vs 29%, P = 0.0018). Moreover, the occurrence of IHI increased with the number of macrohemodynamic and tissue perfusion impaired parameters, named SOCRATE score (cardiovascular SOFA, index CRT and lactATE): 10% (95% CI [3%, 30%]), 33% (95% CI [15%, 58%]), 55% (95% CI [35%, 73%]) and 80% (95% CI [55%, 93%]) for 0, 1, 2 and 3 parameters, respectively (AUC = 0.79 [0.69-0.89], P < 0.0001). These results were confirmed by analyzing the 139 later sessions included in the study.CONCLUSIONS:The SOCRATE score based on 3 easy-to-use bedside parameters correlates with the risk of IHI. By improving risk stratification of IHI, this score could help clinicians to manage intermittent hemodialysis initiation in critically ill AKI patients