Production and N-glycan engineering of Varlilumab in Nicotiana benthamiana.

N-glycan engineering has dramatically evolved for the development and quality control of recombinant antibodies. Fc region of IgG contains two N-glycans whose galactose terminals on Fc-glycan have been shown to increase the stability of CH2 domain and improve effector functions. Nicotiana benthamiana has become one of the most attractive production systems for therapeutic antibodies. In this study, Varlilumab, a CD27-targeting monoclonal antibody, was transiently produced in fresh leaves of soil-grown and hydroponic-grown N. benthamiana, resulted in the yield of 174 and 618 µg/gram, respectively. However, the IgG produced in wild-type N. benthamiana lacked the terminal galactose residues in its N-glycan. Therefore, N-glycan engineering was applied to fine-tune recombinant antibodies produced in plant platforms. We further co-expressed IgG together with murine ß1,4-galactosyltransferase (ß1,4-GALT) to modify plant N-glycan with ß1,4-linked Gal residue(s) and Arabidopsis thaliana ß1,3-galactosylatransferase (ß1,3-GALT) to improve galactosylation. The co-expression of IgG with each of GALTs successfully resulted in modification of N-glycan structures on the plant-produced IgG. Notably, IgG co-expressed with murine ß1,4-GALT in soil-grown N. benthamiana had 42.5% of N-glycans variants having galactose (Gal) residues at the non-reducing terminus and 55.3% of that in hydroponic-grown N. benthamiana plants. Concomitantly, N-glycan profile analysis of IgG co-expressed with ß1,3-GALT demonstrated that there was an increased efficiency of galactosylation and an enhancement in the formation of Lewis a structure in plant-derived antibodies. Taken together, our findings show that the first plant-derived Varlilumab was successfully produced with biantennary ß1,4-galactosylated N-glycan structures.

A water-soluble supramolecular complex that mimics the heme/copper hetero-binuclear site of cytochrome c oxidase.

In mitochondria, cytochrome c oxidase (CcO) catalyses the reduction of oxygen (O2) to water by using a heme/copper hetero-binuclear active site. Here we report a highly efficient supramolecular approach for the construction of a water-soluble biomimetic model for the active site of CcO. A tridentate copper(ii) complex was fixed onto 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(iii) (FeIIITPPS) through supramolecular complexation between FeIIITPPS and a per-O-methylated ß-cyclodextrin dimer linked by a (2,2':6',2''-terpyridyl)copper(ii) complex (CuIITerpyCD2). The reduced FeIITPPS/CuITerpyCD2 complex reacted with O2 in an aqueous solution at pH 7 and 25 °C to form a superoxo-type FeIII-O2-/CuI complex in a manner similar to CcO. The pH-dependent autoxidation of the O2 complex suggests that water molecules gathered at the distal Cu site are possibly involved in the FeIII-O2-/CuI superoxo complex in an aqueous solution. Electrochemical analysis using a rotating disk electrode demonstrated the role of the FeTPPS/CuTerpyCD2 hetero-binuclear structure in the catalytic O2 reduction reaction.

Reference values for hand grip strength in Japanese community-dwelling elderly: a meta-analysis.

OBJECTIVES: Reference values for hand grip strength in Japanese community-dwelling elderly independent in activities of daily living (ADLs) were calculated by meta-analysis. METHODS: Papers reporting data on hand grip strength in elderly Japanese adults ≥60 years of age and independent in ADLs were retrieved from electronic databases. Data were extracted from the selected papers and the weighted mean for hand grip strength by sex was calculated by random effect model. The association of hand grip strength with age and body weight was also analyzed using meta-regression analysis. RESULTS: Data for 15,784 individuals (5216 men and 10,568 women; mean age 67.0-79.8 years) were extracted from 97 sets of data from 33 papers. The weighted mean for hand grip strength was calculated as 33.11 kg [95 % CI 32.27-33.96] in men and 20.92 kg [95 % CI 20.45-21.39] in women. A significant negative correlation was also seen between hand grip strength and age. CONCLUSIONS: The mean hand grip strength of elderly people calculated in this study can be used as a reference value for the hand grip strength of Japanese community-dwelling elderly who are independent in ADLs. However, age needs to be considered in reference values for hand grip strength.

Aperture correction with an asymmetrically trimmed Gaussian weight in SPECT with a fan-beam collimator.

OBJECTIVES: The aim of the study is to improve the spatial resolution of SPECT images acquired with a fan-beam collimator. METHODS: The aperture angle of a hole in the fan-beam collimator depends on the position of the collimator. To correct the aperture effect in an iterative image reconstruction, an asymmetrically trimmed Gaussian weight was used for a model. To confirm the validity of our method, point source phantoms and brain phantom were used in the simulation, and we applied the method to the clinical data. RESULTS: The results of the simulation showed that the spatial resolution of point sources improved from about 6 to 2 pixels full width at half maximum, and the corrected point sources were isotropic. The results of the simulation with the brain phantom showed that our proposed method could improve the spatial resolution of the phantom, and our method was effective for different fan-beam collimators with different focal lengths. The results of clinical data showed that the quality of the reconstructed image was improved with our proposed method. CONCLUSIONS: Our proposed aperture correction method with the asymmetrically trimmed Gaussian weighting function was effective in improving the spatial resolution of SPECT images acquired with the fan-beam collimator.

Derangement of ghrelin secretion after long-term high-fat diet feeding in rats.

AIM: Appetite control is an important goal for the management of non-alcoholic fatty liver disease, diabetes mellitus and obesity; however, little is known about how hormones concerning appetite regulation are affected by long-term consumption of a high-fat diet. We investigated the effect of high-fat diet on secretory regulation of ghrelin and leptin in rats. METHODS: Rats were fed a control or a high-fat diet for 18 weeks and then killed. Before being killed, a glucose tolerance test was performed. Weight, total calorie intake and blood glucose levels were measured, and the plasma levels of total and active ghrelin, and leptin were analyzed by enzyme-linked immunosorbent assay. RESULTS: Body and fat weight and total calorie intake were significantly higher in the high-fat diet group than in the control, although blood glucose levels did not differ. Plasma leptin was significantly higher in the high-fat diet group, and a significant positive correlation was observed between bodyweight and leptin levels in both groups. The levels of active and total ghrelin were not significantly changed by high-fat diet, and active ghrelin levels in the control group significantly correlated negatively with bodyweight, while its correlation was lost in the high-fat diet group. The glucose tolerance test showed that ghrelin levels were significantly higher than those of controls even 60 min after glucose loading. CONCLUSION: These results indicate that secretion of ghrelin, but not leptin, are deranged by consumption of a high-fat diet, and active ghrelin levels lose their correlation with bodyweight and food intake.