Cellular O-Glycome Reporter/Amplification to explore O-glycans of living cells

Protein O-glycosylation has key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe an approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method used to amplify and profile mucin-type O-glycans synthesized by living cells. Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS). Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases

Automated left ventricular diastolic function evaluation from phase-contrast cardiovascular magnetic resonance and comparison with Doppler echocardiography

International audienceBACKGROUND: Early detection of diastolic dysfunction is crucial for patients with incipient heart failure. Although this evaluation could be performed from phase-contrast (PC) cardiovascular magnetic resonance (CMR) data, its usefulness in clinical routine is not yet established, mainly because the interpretation of such data remains mostly based on manual post-processing. Accordingly, our goal was to develop a robust process to automatically estimate velocity and flow rate-related diastolic parameters from PC-CMR data and to test the consistency of these parameters against echocardiography as well as their ability to characterize left ventricular (LV) diastolic dysfunction. RESULTS: We studied 35 controls and 18 patients with severe aortic valve stenosis and preserved LV ejection fraction who had PC-CMR and Doppler echocardiography exams on the same day. PC-CMR mitral flow and myocardial velocity data were analyzed using custom software for semi-automated extraction of diastolic parameters. Inter-operator reproducibility of flow pattern segmentation and functional parameters was assessed on a sub-group of 30 subjects. The mean percentage of overlap between the transmitral flow segmentations performed by two independent operators was 99.7 ± 1.6%, resulting in a small variability ( 0.71) and receiver operating characteristic (ROC) analysis revealed their ability to separate patients from controls, with sensitivity > 0.80, specificity > 0.80 and accuracy > 0.85. Slight superiority in terms of correlation with echocardiography (r = 0.81) and accuracy to detect LV abnormalities (sensitivity > 0.83, specificity > 0.91 and accuracy > 0.89) was found for the PC-CMR flow-rate related parameters. CONCLUSIONS: A fast and reproducible technique for flow and myocardial PC-CMR data analysis was successfully used on controls and patients to extract consistent velocity-related diastolic parameters, as well as flow rate-related parameters. This technique provides a valuable addition to established CMR tools in the evaluation and the management of patients with diastolic dysfunction

Remote patient management for home dialysis patients

Remote Patient Management (RPM) offers renal health care providers and patients with end stage kidney disease (ESKD) opportunities to embrace home dialysis therapies with greater confidence and the potential to obtain better clinical outcomes. Barriers and evidence required to increase adoption of RPM by the nephrology community need to be clearly defined. Ten healthcare providers from specialties including nephrology, cardiology, pediatrics, epidemiology, nursing, and health informatics with experience in home dialysis and the use of RPM systems gathered in Vienna, Austria to discuss opportunities, barriers, and system requirements of RPM as it applies to the home dialysis patient. While improved outcomes and cost effectiveness of RPM have been demonstrated in patients with diabetes mellitus and heart disease, only observational data on RPM has been gathered in patients on dialysis. The current review focused on RPM systems currently in use, on how RPM should be integrated into future care, and the evidence needed for optimized implementation in order to improve clinical and economic outcomes. Randomized controlled trials and/or large observational studies could inform the most effective and economical use of RPM in home dialysis. These studies are needed to establish the value of existing and/or future RPM models among patients, policy makers, and healthcare providers

Inhibition of N-glycanase1 induces autophagic clearance of protein aggregates

Quality control of protein folding is crucial to the maintenance of cellular homeostasis. Impairment of these systems and the related degradative pathways involved in the clearance of misfolded proteins can result in severe and varied pathologies. Peptide N-glycanase (EC 3.5.1.52) is an endoglycosidase which cleaves N-linked glycans from incorrectly folded glycoproteins exported from the endoplasmic reticulum and occurs prior to degradation by the 26S proteasome and is important for the degradation of misfolded glycoproteins during ER-associated degradation. Mutations in this enzyme are responsible for the rare disorder, N-GLY1, a congenital multi-system disorder which results in a build-up of protein aggregates in the cell cytosol. Using a pharmacological inhibitor of peptide N-glycanase, carbobenzoxy-valyl-ananyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk), we have found that inhibition of Nglycanase using 50 µM Z-VAD-fmk resulted in an increase in Thioflavin T fluorescence intensity after 48 hours which decreases to near basal levels after 72 hours. Thioflavin T is a fluorescent dye that exhibits enhanced fluorescence upon binding to β-sheets, characteristic of aggregated structures and fibril formation. Changes in characteristic ER stress markers were also observed; variation in the expression of BiP (GRP78), which is increased during the unfolded protein response, was observed to correlate with the changes in Thioflavin T fluorescence. Using a GFP-LC3 reporter in HEK cells we found an increase in autophagy after 72 hours of peptide N-glycanase inhibition. The increase in autophagy corresponded to the decrease in Thioflavin T fluorescence and variation in BiP levels, suggesting that protein aggregates were removed by the induction of autophagy when deglycosylation is impaired. In an autophagy deficient cell line, ATG13-/- MEFs, we found inhibition of peptide N-glycanase by 50 µM Z-VAD-fmk lead to a 45 % decrease in cell viability within 24 hours with no loss of viability seen in the corresponding wild type MEFs or HEK cells. These results show that autophagy is essential for removal of protein aggregates resulting from the inhibition of peptide N-glycanase. Further work will focus on the investigation of the autophagic machinery linked to clearance of protein aggregates caused by peptide N-glycanase inhibition