Synthesis of lipid-linked oligosaccharides by a compartmentalized multi-enzyme cascade for the in vitro N-glycosylation of peptides.

A wide range of glycoproteins can be recombinantly expressed in aglycosylated forms in bacterial and cell-free production systems. To investigate the effect of glycosylation of these proteins on receptor binding, stability, efficacy as drugs, pharmacodynamics and pharmacokinetics, an efficient glycosylation platform is required. Here, we present a cell-free synthetic platform for the in vitro N-glycosylation of peptides mimicking the endoplasmic reticulum (ER) glycosylation machinery of eukaryotes. The one-pot, two compartment multi-enzyme cascade consisting of eight recombinant enzymes including the three Leloir glycosyltransferases, Alg1, Alg2 and Alg11, expressed in E. coli and S. cerevisiae, respectively, has been engineered to produce the core lipid-linked (LL) oligosaccharide mannopentaose-di-(N-acetylglucosamine) (LL-Man5). Pythanol (C20H42O), a readily available alcohol consisting of regular isoprenoid units, was utilized as the lipid anchor. As part of the cascade, GDP-mannose was de novo produced from the inexpensive substrates ADP, polyphosphate and mannose. To prevent enzyme inhibition, the nucleotide sugar cascade and the glycosyltransferase were segregated into two compartments by a cellulose ester membrane with 3.5 kDa cut-off allowing for the effective diffusion of GDP-mannose across compartments. Finally, as a proof-of-principle, pythanyl-linked Man5 and the single-subunit oligosaccharyltransferase Trypanosoma brucei STT3A expressed in Sf9 insect cells were used to in vitro N-glycosylate a synthetic peptide of ten amino acids bearing the eukaryotic consensus motif N-X-S/T.

Medical students' perceptions of stress due to academic studies and its interrelationships with other domains of life: a qualitative study.

BACKGROUND: Medical students have been found to experience considerable stress due to their academic studies. The high demands associated with academic studies may interfere with demands in other domains of life. Conversely, demands in those other domains of life may conflict with academic studies. OBJECTIVE: We aimed to better understand the potential inter-relationships between the demands related to academic studies and in other domains of life. DESIGN: A total of 68 medical students from a medical school in Germany participate in eight focus groups. Sessions were structured by a topic guide and were recorded, transcribed and content-analyzed. RESULTS: Embarking on one's medical studies was perceived to be associated with important personal challenges, such as living alone for the first time and finding a new social role in one's peer group. Permanent stress was perceived to result in emotional exhaustion, which spilled over in other domains of life. Students reported to feel guilty if they did not spend their limited spare time on learning. Consequently, they felt to have little time for leisure time activities and a healthy lifestyle. Feelings of social isolation, especially during exam phases, were reported. Leisure activities were perceived to facilitate recovery from academic stress. Social ties were subjectively able to reduce stress, but also to increase stress due to demands. Side jobs were perceived to increase stress and to be time-consuming and were thus perceived to result in poorer academic performance. Certain personality characteristics seemed to amplify those perceptions. For instance, high levels of conscientiousness were felt to relate to higher expectations regarding one's academic performance and thus increased stress. CONCLUSION: The demands associated with medical studies, the demands in private life, lacking resources for recovery and certain personal traits as well as interactions between those domains can contribute to stress among medical students and reduce well-being.