Light-activated g-C3N4 photoelectrodes with a selective molecular sieve for in vivo quantification of oxygen levels in the living mouse brain.

A novel micro-photoelectrode with a selective molecular sieve was created for in vivo monitoring of O2 levels in the mouse brain. An ITO optical fiber modified by graphitized carbon nitride (g-C3N4) in situ was employed as the light activated substrate to provide rich photo-induced electrons for the catalytic reduction of O2. Meanwhile, the porous hybrid layer composed of zeolitic imidazolate framework-8 and polysulfone was constructed over the g-C3N4 surface as the molecular sieve to synergically enhance the selectivity of O2 detections. By advantage of this useful tool, the real time variation of the O2 level was successfully determined in the mouse brain upon ischemia.

Fluorescent proteins and genetically encoded biosensors.

The genetically encoded fluorescent sensors convert chemical and physical signals into light. They are powerful tools for the visualisation of physiological processes in living cells and freely moving animals. The fluorescent protein is the reporter module of a genetically encoded biosensor. In this study, we first review the history of the fluorescent protein in full emission spectra on a structural basis. Then, we discuss the design of the genetically encoded biosensor. Finally, we briefly review several major types of genetically encoded biosensors that are currently widely used based on their design and molecular targets, which may be useful for the future design of fluorescent biosensors.

Fluid lubricated polishing based on shear thickening.

With the development of short wavelength optics, high requirements are put forward for the full frequency errors of optical elements, while the processing efficiency and surface quality of traditional polishing methods are difficult to meet their requirements. In this paper, a fluid lubricated polishing method is proposed by combining non-Newtonian fluid with traditional polishing methods. According to Preston equation and shear thickening principle, the tool influence function of fluid lubricated polishing is established and verified by experiments. The results show that the fluid lubricated polishing has a very good convergence ability to the full frequency error of the workpiece. In addition, the convergence rate of fluid lubricated polishing on roughness is about twice that of chemical mechanical polishing. Finally, fluid lubricated polishing extends Preston from Newtonian fluid polishing to non-Newtonian fluid polishing.

Real-Time Monitoring of Neurotransmitters in the Brain of Living Animals.

Neurotransmitters, as important chemical small molecules, perform the function of neural signal transmission from cell to cell. Excess concentrations of neurotransmitters are often closely associated with brain diseases, such as Alzheimer's disease, depression, schizophrenia, and Parkinson's disease. On the other hand, the release of neurotransmitters under the induced stimulation indicates the occurrence of reward-related behaviors, including food and drug addiction. Therefore, to understand the physiological and pathological functions of neurotransmitters, especially in complex environments of the living brain, it is urgent to develop effective tools to monitor their dynamics with high sensitivity and specificity. Over the past 30 years, significant advances in electrochemical sensors and optical probes have brought new possibilities for studying neurons and neural circuits by monitoring the changes in neurotransmitters. This Review focuses on the progress in the construction of sensors for in vivo analysis of neurotransmitters in the brain and summarizes current attempts to address key issues in the development of sensors with high selectivity, sensitivity, and stability. Combined with the latest advances in technologies and methods, several strategies for sensor construction are provided for recording chemical signal changes in the complex environment of the brain.

A CDR-based approach to generate covalent inhibitory antibody for human rhinovirus protease.

Covalent target modulation with small molecules has been emerging as a promising strategy for drug discovery. However, covalent inhibitory antibody remains unexplored due to the lack of efficient strategies to engineer antibody with desired bioactivity. Herein, we developed an intracellular selection method to generate covalent inhibitory antibody against human rhinovirus 14 (HRV14) 3C protease through unnatural amino acid mutagenesis along the heavy chain complementarity-determining region 3 (CDR-H3). A library of antibody mutants was thus constructed and screened in vivo through co-expression with the target protease. Using this screening strategy, six covalent antibodies with proximity-enabled bioactivity were identified, which were shown to covalently target HRV14-3C protease with high inhibitory potency and exquisite selectivity. Compared to structure-based rational design, this library-based screening method provides a simple and efficient way for the discovery and engineering of covalent antibody for enzyme inhibition.

Suzuki Cross-Coupling Reaction with Genetically Encoded Fluorosulfates for Fluorogenic Protein Labeling.

A palladium-catalyzed cross-coupling reaction with aryl halide functionalities has recently emerged as a valuable tool for protein modification. Herein, a new fluorogenic modification methodology for proteins, with genetically encoded fluorosulfate-l-tyrosine, which exhibits high efficiency and biocompatibility in bacterial cells as well as in aqueous medium, is described. Furthermore, the cross-coupling of 4-cyanophenylboronic acid on green fluorescent protein was shown to possess a unique fluorogenic property, which could open up the possibility of a responsive "off/on" switch with great potential to enable spectroscopic imaging of proteins with minimal background noise. Taken together, a convenient and efficient catalytic system has been developed that may provide broad utilities in protein visualization and live-cell imaging.

Protein labeling approach to improve lysosomal targeting and efficacy of antibody-drug conjugates.

An anti-EGFR nanobody was labeled at the C-terminus with a lysosome-sorting NPGY (Asn-Pro-Gly-Tyr) motif via sortase-mediated ligation to enhance the engagement of the clathrin-mediated endocytosis. The synergistic effects of NPGY motif and nona-arginine peptide were found to induce robust internalization and lysosomal trafficking, which in turn improved anti-tumor activity of an antibody-drug conjugate.

Site-specific labeling of an anti-MUC1 antibody: probing the effects of conjugation and linker chemistry on the internalization process.

Antibody-drug conjugates (ADCs) have recently received enormous attention as an attractive approach for cancer therapy. Although ADC design has been believed to be important for the relative efficacy of ADCs, it remains unexplored how the structural characteristics of ADCs would impact the internalization process and intracellular trafficking of the molecules. Herein, we report our efforts in investigating the cellular endocytosis implications of the conjugation and linker chemistry in designing antibody-based agents. A series of anti-MUC1 single-chain variable fragment (scFv-SM3) conjugates were designed with unique structural characteristics ranging from conjugation methods, sites of attachment and linker chemistry. In vitro confocal imaging showed that both random lysine-conjugation and site-specific conjugation, including C-terminus modification or internal site conjugation, could afford antibody conjugates with similar binding affinity and cellular uptake to target-expressing cells. Time-course internalization studies demonstrated that SM3-conjugates with short polyethylene glycol linkers outcompeted those that lack any hydrophilic linkers for higher cellular uptake and faster internalization rate. The SM3-conjugates with the highest affinity and internalization rate were also tested in mouse xenograft models using MUC1-overexpressing tumor cells. Our results indicate that the linker and conjugation chemistry play an important role in the internalization process of antibody conjugates, and this in turn could impact the therapeutic effects of ADCs.

Notch ligand Delta-like1 enhances degranulation and cytokine production through a novel Notch/Dok-1/MAPKs pathway in vitro.

Food allergy includes sensitization phase and effect phase, and effect cells degranulate and secrete cytokines in the effect phase, causing allergic clinical symptoms. We have demonstrated that Notch signaling plays an important role in the sensitization phase, but its role in effect phases still remains unclear. In this study, we investigated the role of Notch signaling in degranulation and cytokine production of the effect phase response. A RBL-2H3 cell model was used and Notch signaling was induced by priming with Notch ligands. Our results showed after priming with Notch ligand, Delta-like1(Dll1)-Fc, ß-hexosaminidase release, and cytokines production, including TGF-ß, IL-1ß, IL-4, IL-6, and IL-13, were increased significantly, and the enhancement was abolished after DAPT treatment, a γ-secretase inhibitor, indicating that Dll1 Notch signaling enhanced RBL-2H3 cell degranulation and cytokine production. Western blot analysis showed that Dll1 Notch signaling augmented high-affinity IgE receptors-mediated phosphorylation of MAPKs through suppressing the expression of downstream tyrosine kinases 1 (Dok-1). Besides, a passive systemic anaphylaxis mouse model was used to confirm the role of Notch signaling. And our data showed that allergic clinical features of mice were alleviated, and the level of degranulation was decreased significantly after inhibiting Notch signaling in vivo. Therefore, we demonstrated Notch ligand Dll1 enhanced RBL-2H3 cell degranulation and cytokine production through a novel Notch/Dok-1/MAPKs pathway, suggesting Notch signaling played a key role in the effect phase of food allergy.