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There has been a concern that sodium–glucose cotransporter 2 (SGLT2) inhibitors could reduce skeletal muscle mass and function. Here, we examine the effect of canagliflozin (CANA), an SGLT2 inhibitor, on slow and fast muscles from nondiabetic C57BL/6J mice. In this study, mice were fed with or without CANA under ad libitum feeding, and then evaluated for metabolic valuables as well as slow and fast muscle mass and function. We also examined the effect of CANA on gene expressions and metabolites in slow and fast muscles. During SGLT2 inhibition, fast muscle function is increased, as accompanied by increased food intake, whereas slow muscle function is unaffected, although slow and fast muscle mass is maintained. When the amount of food in CANA-treated mice is adjusted to that in vehicle-treated mice, fast muscle mass and function are reduced, but slow muscle was unaffected during SGLT2 inhibition. In metabolome analysis, glycolytic metabolites and ATP are increased in fast muscle, whereas glycolytic metabolites are reduced but ATP is maintained in slow muscle during SGLT2 inhibition. Amino acids and free fatty acids are increased in slow muscle, but unchanged in fast muscle during SGLT2 inhibition. The metabolic effects on slow and fast muscles are exaggerated when food intake is restricted. This study demonstrates the differential effects of an SGLT2 inhibitor on slow and fast muscles independent of impaired glucose metabolism, thereby providing new insights into how they should be used in patients with diabetes, who are at a high risk of sarcopenia.

As reported in the previous commentary (Ishii-Watabe et al., J Pharm Sci 2017), the Japanese biopharmaceutical research group is promoting collaborative multilaboratory studies to evaluate and standardize new methodologies for biopharmaceutical characterization and quality control. We have conducted the studies and held 2 annual meetings in 2018 and 2019. At the 2018 meeting, Dr. Rukman DeSilva of the U.S. Food and Drug Administration and Dr. Srivalli Telikepalli of the National Institute of Standards and Technology participated as guest speakers. At the 2019 meeting, we invited Prof. John Carpenter of the University of Colorado, Prof. Gerhard Winter and Prof. Wolfgang Friess of Ludwig Maximilian University of Munich, and Dr. Tim Menzen of Coriolis Pharma Research, as guest commentators. In both meetings, the main research topic was strategies for the characterization and control of protein aggregates/subvisible particles in drug products. Specifically, the use of the light obscuration method for insoluble particulate matter testing with reduced injection volumes, and a comparison of analytical performance between flow imaging and light obscuration were discussed. Other topics addressed included host cell protein analysis, bioassay, and quality control strategies. In this commentary, the recent achievements of the research group, meeting discussions, and future perspectives are summarized.

Parkinson’s disease (PD) is a common progressive neurodegenerative disease. It can manifest with a wide range of motor and non-motor signs. Among the most disabling PD symptoms are gait impairments and freezing of gait (FOG). External cueing is currently accepted as an effective strategy to improve gait parameters and overcome FOG. However, some patients show a weak response to external cues or even the worsening of gait under cueing condition. In this paper we propose a new walking assist robotic system UPS-PD, which employs pneumatically-actuated artificial muscle and simple control system. The working principle of UPS-PD is based on inducing spontaneous synchronization in PD patients. That allows them to maintain a stable gait pattern while walking at their own pace. UPS-PD was tested in two PD patients and in one of them a significant improvement of gait features was demonstrated. Our results suggest that UPS-PD might improve walking performance in PD patients with highly variable and asymmetric gait patterns.

The antibody drug market is rapidly expanding, and various antibody engineering technologies are being developed to create antibodies that can provide better benefit to patients. Although bispecific antibody drugs have been researched for more than 30 years, currently only a limited number of bispecific antibodies have achieved regulatory approval. Of the few successful examples of industrially manufacturing a bispecific antibody, the “common light chain format” is an elegant technology that simplifies the purification of a whole IgG–type bispecific antibody. Using this IgG format, the bispecific function can be introduced while maintaining the natural molecular shape of the antibody. In this article, we will first introduce the outline, prospects, and limitations of the common light chain format. Then, we will describe the identification and optimization process for ERY974, an anti-glypican-3 × anti-CD3e T cell–redirecting bispecific antibody with a common light chain. This format includes one of Chugai’s proprietary technologies, termed ART-Ig technology, which consists of a method to identify a common light chain, isoelectric point (pI) engineering to purify the desired bispecific IgG antibody from byproducts, and Fc heterodimerization by an electrostatic steering effect. Furthermore, we describe some tips for de-risking the antibody when engineering a T cell redirecting antibody.

Development of a novel host cell DNA assay using PicoGreen is described, which is capable of detecting short double stranded DNAs (ds-DNAs) in cell culture supernatants and process intermediates. Examination of this PicoGreen DNA assay was carried out by determination of the DNA length detection limit, observation of short ds-DNAs in cell culture supernatants and process intermediates, evaluation of dose dependency and a supersensitizing protocol, and comparison of the novel assay with conventional assays for measuring host cell DNA concentration in real samples. The PicoGreen DNA assay was capable of detecting ds-DNAs as short as 20 bp, and the sensitivity of the PicoGreen DNA assay was comparable to that of the Threshold system with application of additional SDS/Proteinase K digesting and DNA concentrating steps. Also, the amount of DNA identified in both cell culture supernatant and process intermediates was clearly underestimated by the Threshold system results when compared with the PicoGreen DNA assay results. The PicoGreen DNA assay clearly provides better accuracy and is a simpler procedure for measuring host cell DNA levels in cell culture supernatant and process intermediates than the conventional method with the Threshold system. This newly developed DNA assay will be prominent among host cell DNA assays for measuring host cell DNA levels in bio-pharmaceuticals.

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

Currently, the standard length measurement method with nanometre resolution is laser interferometry. However, it is difficult to determine an arbitrary length with an accuracy of sub-nanometre or less order using interferometers because they have a nonlinearity problem in fringe interpolation. A phase modulation homodyne interferometer (PMHI) that can be used to determine the optical path difference of an integer multiple of the wavelength (n × λ) with picometre resolution was proposed by IMGC (former Italian Standard Institute). The lattice spacing of approximately 0.246 nm for graphite regular crystalline lattices is uniform and stable over a long range, when the crystals are stress free. These crystals can be used as a reference scale with sub-nanometre resolution. The scanning tunnelling microscope (STM) is emerging as a powerful tool in surface engineering, and enables us to image atoms on a crystalline surface. Therefore, such a crystalline surface can be used as 'the crystalline scale' using the STM.In this study, an instrument for calibrating optical encoders is developed by combining a graphite crystalline lattice as a fine scale and the optical fringe of the PMHI as a coarse scale. The instrument consists of a precise linear X-axis sample stage, on which the reference graphite crystal and the optical encoder scale are set, a head of the STM with a YZ tip scanner and a PMHI. The relative displacement of the X-axis sample stage between optical interference dark fringes (= null points) of the PMHI, which is λ/16 times the integer value in the design, can be measured with picometre resolution using the phase modulation technique. A lattice spacing of 0.246 nm on the graphite crystalline surface is derived as the fine scale from the STM image and the optical fringes of the PMHI. In the experiment, the periodical error of the optical encoder, whose minimum resolution is less than a nanometre, is measured using both the lattice spacing of graphite and the optical fringes of the PMHI. The results show that the proposed instrument has the feasibility to calibrate optical encoders with an uncertainty of 10 pm order.

Well-crystalline LiFePO4 particles were prepared by a hydrothermal reaction. The particles thus prepared by adding polyethylene glycol (PEG) to the starting reaction solution were fine crystalline in the range of 0.5–1.5 μm with a narrow particle size distribution. The initial discharge capacity of the sample as a lithium secondary battery was achieved to 143 mAhg−1 at 0.5 mA cm−2 (corresponding to 35 mA g−1), larger than that of the sample prepared by the hydrothermal method without PEG. The increase in the initial discharge capacity should be correlated with the change in particle size and degree of crystallization.

Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase requires redox co-enzymes, i.e., flavin mononucleotide (FMN) and NAD(P)H, for activity, although it catalyzes a non-redox reaction. Spectrometric studies and enzyme assays under anaerobic conditions indicate that FMN is reduced through the reaction and is sufficient for activity. The sole function of NAD(P)H appears to be the reduction of FMN since it could be replaced by an alternate reducing agent. When the enzyme was reconstructed with a flavin analogue, no activity was detected, suggesting that the isomerase reaction proceeds via a radical transfer mechanism.

Although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. A novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg2+ for activity, similar to its bacterial homologues. Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH.

The mine detection hexapod robot COMET-III is driven by hydraulic power. Because of the strong nonlinear characteristic of the oil pressure system, conventional classic methods produce the flattery delay to the target orbit. In the unknown environment of minefield, the robot leg must follow correctly the walking orbit. The sliding mode control has strong robustness to parameter change or disturbance, and the preview control prevents the flattery delay by using a future target value. In this paper, we design a preview sliding mode controller, which incorporate the advantages of both control theory, and so improves control performance.