RESUMO
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.
Assuntos
Antineoplásicos , Imunoconjugados , Imunoconjugados/química , Antineoplásicos/química , Anticorpos Monoclonais/química , Interações Hidrofóbicas e Hidrofílicas , Polissacarídeos/químicaRESUMO
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.
Assuntos
Ácidos Docosa-Hexaenoicos/química , Ácido Eicosapentaenoico/química , Ácidos Graxos Insaturados/química , Ácidos Docosa-Hexaenoicos/síntese química , Ligantes , Oxirredução , EstereoisomerismoRESUMO
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.
RESUMO
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.
Assuntos
Ácidos Graxos Insaturados , Fator 4 Nuclear de Hepatócito , Ácidos Graxos , Fator 4 Nuclear de Hepatócito/genética , Receptores Citoplasmáticos e NuclearesRESUMO
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.