Highly Sensitive Labeling, Clickable Functionalization, and Glycoengineering of the MUC1 Neighboring System

This study introduces a novel wash-type affinity-primed proximity labeling (WAPL) strategy for labeling and surface engineering of the MUC1 protein neighboring system. The strategy entails the utilization of peroxidase in conjunction with a MUC1-selective aptamer, facilitating targeted binding to MUC1 and inducing covalent labeling of the protein neighboring system. This study reveals a novel finding that the WAPL strategy demonstrates superior labeling efficiency in comparison to nonwash-type affinity-primed proximity labeling, marking the first instance of such observations. The WAPL strategy provides signal amplification by converting a single recognition event into multiple covalent labeling events, thereby improving the detection sensitivity for subtle changes in MUC1. The WAPL platform employs two levels of labeling upgrades, modifying the biotin handles of the conventional labeling substrate, biotin–phenol. The first level involves a range of clickable molecules, facilitating dibenzoazacyclooctynylation, alkynylation, and trans-cyclooctenylation of the protein neighboring system. The second level utilizes lactose as a post-translational modification model, enabling rapid and reliable glycoengineering of the MUC1 neighboring system while remaining compatible with cell-based assays. The implementation of the WAPL strategy in protein neighboring systems has resulted in the establishment of a versatile platform that can effectively facilitate diverse monitoring and regulation techniques. This platform offers valuable insights into the regulation of relevant signaling pathways and promotes the advancement of novel therapeutic approaches, thereby bringing substantial implications for human health

Hapten-Grafted Programmed Probe as a Corecognition Element for a Competitive Immunosensor to Detect Acetamiprid Residue in Agricultural Products

We have developed an effective competitive electrochemical immunosensor assay based on hapten-grafted programmed probe (HGPP) as a corecognition element for highly sensitive and selective detection of acetamiprid. Starting with the synthesis of hapten, HGPP was prepared using carboxyl group in the hapten and amino group in the 5′ end of the programmed probe through covalent conjugation. Acetamiprid present in samples competes with HGPP to bind with capture antibody on the electrodes by specific recognition interaction. Methylene blue probe (MBP) was used as the electrochemical redox probe to capture the hybridized HGPP on the electrodes. The competitive reaction changes in accordance with the quantity of the target acetamiprid in the sample, as the amounts of the hybridized HGPP and the immobilized antibody are constant, i.e., the more acetamiprid samples are added, the less MBP is combined on the electrodes. In the optimal conditions, thus, biosensor output showed a linear relationship from 5 to 10⁵ ng L^–1 for the acetamiprid assay with a detecting limit of 3.2 ng L^–1. The biosensor was successful in quantifying the amount of acetamiprid in spiked strawberry and cabbage extracts. This competitive immunosensor assay represents a rapid and sensitive technology for acetamiprid assay or other small molecule targets in food

Multifunctional Proximity Labeling Strategy for Lipid Raft-Specific Sialic Acid Tracking and Engineering

Lipid raft-specific glycosylation has been implicated in many biological processes, including intracellular trafficking, cell adhesion, signal transduction, and host–pathogen interactions. The major predicament in lipid raft-specific glycosylation research is the unavailability of tools for tracking and manipulating glycans on lipid rafts at the microstructural level. To overcome this challenge, we developed a multifunctional proximity labeling (MPL) platform that relies on cholera toxin B subunit to localize horseradish peroxidase on lipid rafts. In addition to the prevailing electron-rich amino acids, modified sialic acid was included in the horseradish peroxidase-mediated proximity labeling substrate via purposefully designed chemical transformation reactions. In combination with sialic acid editing, the self-renewal of lipid raft-specific sialic acid was visualized. The MPL method enabled tracking of lipid raft dynamics under methyl-β-cyclodextrin and mevinolin treatments; in particular, the alteration of lipid rafts markedly affected cell migration. Furthermore, we embedded functional molecules into the method and implemented raft-specific sialic acid gradient engineering. Our novel strategy presents opportunities for tailoring lipid raft-specific sialic acids, thereby regulating interactions associated with lipid raft regions (such as cell–virus and cell–microenvironment interactions), and can aid in the development of lipid raft-based therapeutic regimens for tumors

Additional file 1 of Association of pre-pregnancy low-carbohydrate diet with maternal oral glucose tolerance test levels in gestational diabetes

Additional file 1: STable 1. Recommended range of macronutrient intakes from Chinese Dietary Reference Intakes. STable 2. Carbohydrate intake habit of LC/GDM grou

DataSheet_1_Ubiquitination-mediated molecular pathway alterations in human lung squamous cell carcinomas identified by quantitative ubiquitinomics.zip

Abnormal ubiquitination is extensively associated with cancers. To investigate human lung cancer ubiquitination and its potential functions, quantitative ubiquitinomics was carried out between human lung squamous cell carcinoma (LSCC) and control tissues, which characterized a total of 627 ubiquitin-modified proteins (UPs) and 1209 ubiquitinated lysine sites. Those UPs were mainly involved in cell adhesion, signal transduction, and regulations of ribosome complex and proteasome complex. Thirty three UPs whose genes were also found in TCGA database were significantly related to overall survival of LSCC. Six significant networks and 234 hub molecules were obtained from the protein-protein interaction (PPI) analysis of those 627 UPs. KEGG pathway analysis of those UPs revealed 47 statistically significant pathways, and most of which were tumor-associated pathways such as mTOR, HIF-1, PI3K-Akt, and Ras signaling pathways, and intracellular protein turnover-related pathways such as ribosome complex, ubiquitin-mediated proteolysis, ER protein processing, and proteasome complex pathways. Further, the relationship analysis of ubiquitination and differentially expressed proteins shows that ubiquitination regulates two aspects of protein turnover - synthesis and degradation. This study provided the first profile of UPs and molecular networks in LSCC tissue, which is the important resource to insight into new mechanisms, and to identify new biomarkers and therapeutic targets/drugs to treat LSCC.</p

Cell-Selective Multifunctional Surface Covalent Reconfiguration Using Aptamer-Enabled Proximity Catalytic Labeling

Cell surface engineering provides access to custom-made cell interfaces with desirable properties and functions. However, cell-selective covalent labeling methods that can simultaneously install multiple molecules with different functions are scarce. Herein, we report an aptamer-enabled proximity catalytic covalent labeling platform for multifunctional surface reconfiguration of target cells in mixed cell populations. By conjugating peroxidase with cell-selective aptamers, the probes formed can selectively bind target cells and catalyze target-cell-localized covalent labeling in situ. The universal applicability of the platform to different phenol-modified functional molecules allows us to perform a variety of manipulations on target cells, including labeling, tracking, assembly regulation, and surface remodeling. In particular, the platform has the ability of multiplexed covalent labeling, which can be used to install two mutually orthogonal click reactive molecules simultaneously on the surface of target cells. We thus achieve “multitasking” in complex multicellular systems: programming and tracking specific cell–cell interactions. We further extend the functional molecules to carbohydrates and perform ultrafast neoglycosylation on target living cells. These newly introduced sugars on the cell membrane can be recognized and remodeled by a glycan-modifying enzyme, thus providing a method package for cell-selective engineering of the glycocalyx

Interception Proximity Labeling for Interrogating Cell Efflux Microenvironment

The difficulty in elucidating the microenvironment of extracellular H2O2 efflux has led to the lack of a critical extracellular link in studies of the mechanisms of redox signaling pathways. Herein, we mounted horseradish peroxidase (HRP) to glycans expressed globally on the living cell surface and constructed an interception proximity labeling (IPL) platform for H2O2 efflux. The release of endogenous H2O2 is used as a “physiological switch” for HRP to enable proximity labeling. Using this platform, we visualize the oxidative stress state of tumor cells under the condition of nutrient withdrawal, as well as that of macrophages exposed to nonparticulate stimuli. Furthermore, in combination with a proteomics technique, we identify candidate proteins at the invasion interface between fungal mimics (zymosan) and macrophages by interception labeling of locally accumulated H2O2 and confirm that Toll-like receptor 2 binds zymosan in a glycan-dependent manner. The IPL platform has great potential to elucidate the mechanisms underlying biological processes involving redox pathways

Supplementary Table 2 from R-Ketorolac Targets Cdc42 and Rac1 and Alters Ovarian Cancer Cell Behaviors Critical for Invasion and Metastasis

Biochemical properties of Cdc42 and Rac1 nucleotide binding</p

Supplementary Figure S2 from R-Ketorolac Targets Cdc42 and Rac1 and Alters Ovarian Cancer Cell Behaviors Critical for Invasion and Metastasis

Cell characterization of primary ovarian cancer cells by immunofluorescence.</p