ez-ADiCon: A novel glyco-remodeling based strategy that enables preparation of homogenous antibody-drug conjugates via one-step enzymatic transglycosylation with payload-preloaded bi-antennary glycan complexes.

The conserved N-linked glycan at the Fc domain of recombinant monoclonal antibodies is an attractive target for site-specific payload conjugation for preparation of homogenous antibody-drug conjugates (ADCs). Here, we report a novel ADC constructing strategy, named "ez-ADiCon", that is achieved by one-step enzymatic transglycosylation of a payload-preloaded bi-antennary glycan oxazoline onto a deglycosylated antibody. In this method, a mixture of different glycoforms of the Fc-glycan is replaced with a pre-defined payload-linked glycan. Since two payloads are linked on each donor glycan substrate, efficient conjugation results in a highly homogenous ADC with mostly-four drug molecules per antibody, facilitating hydrophobic interaction chromatography analysis and purification. We validated this conjugation strategy using Monomethyl auristatin E (MMAE) and an anti-Human epidermal growth factor receptor 2 (anti-Her2) antibody as the model ADC components and demonstrated its target-specific in vitro cytotoxicity. Our novel conjugation strategy, ez-ADiCon, provides a new approach for the preparation of next generation ADCs.

HNF4α Is a Covalent Bond-Forming Receptor.

HNF4α is a nuclear receptor whose ligands are fatty acids. HNF4α is a target molecule for drug discovery research and thus we tested its covalent binding ability to investigate the possible development of covalent modifiers of HNF4α. Oxidized polyunsaturated fatty acids (oxo-PUFAs) have moderate flexibility and possess a Michael acceptor that participates in conjugate addition reactions with nucleophilic amino acid residues. Thus, oxo-PUFAs were used as probes and their covalent binding abilities to HNF4α were verified. Several oxo-PUFAs, such as 4-oxoDHA, were shown to be covalent modifiers of HNF4α and therefore we concluded that HNF4α can form covalent bonds to ligands.

Construction of a series of intermediates in the ß-oxidation pathway from THA to EPA via DHA in free acid form.

ß-Oxidation of most fatty acids occurs in the mitochondria. However, ß-oxidation for ω-3 polyunsaturated fatty acids (PUFAs) is distinct from abundant fatty acids and occurs in the peroxisomes. Since little is known about peroxisomal ß-oxidation, here we report the synthesis of proposed intermediates of ω-3 PUFA ß-oxidation steps in free fatty acid form having a conjugated double bond, a ß-hydroxyl group, a ß-olefin and a ß-carbonyl group. These fatty acids can serve as authentic samples for biological experiments.

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

Fabrication of bright and thin Zn2SiO4 luminescent film for electron beam excitation-assisted optical microscope.

We fabricated a bright and thin Zn2SiO4 luminescent film to serve as a nanometric light source for high-spatial-resolution optical microscopy based on electron beam excitation. The Zn2SiO4 luminescent thin film was fabricated by annealing a ZnO film on a Si3N4 substrate at 1000 °C in N2. The annealed film emitted bright cathodoluminescence compared with the as-deposited film. The film is promising for nano-imaging with electron beam excitation-assisted optical microscopy. We evaluated the spatial resolution of a microscope developed using this Zn2SiO4 luminescent thin film. This is the first report of the investigation and application of ZnO/Si3N4 annealed at a high temperature (1000 °C). The fabricated Zn2SiO4 film is expected to enable high-frame-rate dynamic observation with ultra-high resolution using our electron beam excitation-assisted optical microscopy.