A genome-edited N. benthamiana line for industrial-scale production of recombinant glycoproteins with targeted N-glycosylation.

Control over glycosylation is an important quality parameter in recombinant protein production. Here, we demonstrate the generation of a marker-free genome edited Nicotiana benthamiana N-glycosylation mutant (NbXF-KO) carrying inactivated ß1,2-xylosyltransferase and α1,3-fucosyltransferase genes. The knockout of seven genes and their stable inheritance was confirmed by DNA sequencing. Mass spectrometric analyses showed the synthesis of N-glycans devoid of plant-specific ß1,2-xylose and core α 1,3-fucose on endogenous proteins and a series of recombinantly expressed glycoproteins with different complexities. Further transient glycan engineering towards more diverse human-type N-glycans resulted in the production of recombinant proteins decorated with ß1,4-galactosylated and α2,6-sialylated structures, respectively. Notably, a monoclonal antibody expressed in the NbXF-KO displayed glycosylation-dependent activities. Collectively, the engineered plants grow normally and are well suited for upscaling, thereby meeting industrial and regulatory requirements for the production of high-quality therapeutic proteins.

Age-related modification of antioxidant enzyme activities in relation to cardiovascular risk factors.

BACKGROUND: Since oxidative stress might cause and promote cardiovascular risk factors such as oxidized low-density lipoproteins (oxLDL), apolipoprotein(a) [apo(a)], asymmetric dimethylarginine (ADMA) and fetuin A, we investigated antioxidant enzyme activities in relation to the vascular redox balance and these risk factors in elderly people. MATERIALS AND METHODS: For this observational study, a total of 102 subjects were recruited and divided into three groups: A (70-74 years/n = 48), B (75-79 years/n = 35) and C (≥ 80 years/n = 19). Activities of the erythrocyte antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were determined photometrically oxLDL, apo(a), ADMA and fetuin A by ELISA. Plasma concentrations of the lipid peroxidation products malondialdehyde (MDA) and conjugated dienes (CD) were analysed with HPLC. RESULTS: There were no significant age-associated alterations in apo(a) levels, but there was a significant age-related decrease in activities of SOD (A>C, B>C: P < 0·01), CAT (A>C: P < 0·05) and GSH-Px (A>C: P < 0·05), accompanied by a significant increase in oxLDL (AC: P < 0·01; B>C: P < 0·05). Consequently, all groups showed significant negative age-associated correlations between CAT and MDA (A, B, C: P < 0·05), GSH-Px and CD (A, C: P < 0·01; B: P < 0·05), SOD and oxLDL (A, B: P < 0·05; C: P < 0·01), and fetuin A and MDA (A: P < 0·01; B, C: P < 0·05), and a significant positive correlation between oxLDL and ADMA (A, B: P < 0·05; C: P < 0·01). CONCLUSIONS: This study indicates a significant age-related decrease in antioxidant enzyme activities accompanied by significantly increased systemic oxidative stress, which promotes the cardiovascular risk factors oxLDL, ADMA and fetuin A in elderly people.

Intake of medication and vitamin status in the elderly.

BACKGROUND/AIMS: An inadequate vitamin status is associated with higher morbidity and frailty in the elderly and might be due to medication. This study aimed to evaluate the status of several vitamins in relation to regular intake of medication in this population. METHODS: A total of 102 non-institutionalized subjects aged 70-90 years were recruited. Plasma levels of vitamins A, D, E, K and C were determined by HPLC. The functional parameters of vitamins B(1), B(2) and B(6), i.e. the activities of the erythrocyte enzymes transketolase, glutathione reductase and glutamic oxaloacetic transaminase were analyzed photometrically; plasma folate and vitamin B(12) were determined by RIA. RESULTS: The status of vitamins A, E and C was generally satisfactory. Eighty-eight percent and 42% of participants were deficient in vitamins D and K, respectively, as were 29% in B(6); up to 10% were deficient in vitamins B(1), B(2), B(12) and folate. A considerable percentage of participants was, however, at risk for vitamin deficiencies (vitamins B(1), B(6), B(12) and folate: 20-30%; vitamin B(2): 60%). Regular intake of maximally 2 drugs per day was not adversely related to the status of several vitamins; intake of ≥ 3 drugs per day was significantly negatively associated with the status of vitamins D, K, B(6) and folate. CONCLUSION: Daily intake of ≥ 3 drugs was found to be adversely associated with the status of some vitamins in the elderly. Hence, the medication schedule and nutritional status of these subjects should be monitored closely to ensure that the daily micronutrient requirement is fulfilled.