Genetically Encoded Bioluminescent Indicator for ERK2 Dimer in Living Cells

In this study, a genetically encoded bioluminescent indicator for ERK2 dimer was developed with the split Renilla luciferase complementation method, in which the formation of ERK2 dimer induces a spontaneous emission of bioluminescence in living cells. In response to extracellular stimuli, such as epidermal growth factor (EGF) or 17β‐estradiol (E2), extracellular signal‐regulated kinase 2 (ERK2) is phosphorylated by its upstream kinase MEK, and also phosphorylates its substrates in various regions of the cell, including the nucleus. Phosphorylated ERK2 is led to form its dimer, thereby transporting itself into the nucleus. We demonstrated with the indicator that stimulation with EGF or E2 induces the formation of ERK2 dimer in living MCF‐7 cells. The dynamics of this dimer formation was examined and discussed.     

Localized In Vivo Activation of a Photoactivatable Doxorubicin Prodrug in Deep Tumor Tissue

Sparing sensitive healthy tissue from chemotherapy exposure is a critical challenge in the treatment of cancer. The work described here demonstrates the localized in vivo photoactivation of a new chemotherapy prodrug of doxorubicin (DOX). The DOX prodrug (DOX‐PCB) was 200 times less toxic than DOX and was designed to release pure DOX when exposed to 365 nm light. This wavelength was chosen because it had good tissue penetration through a 1 cm diameter tumor, but had very low skin penetration, due to melanin absorption, preventing uncontrolled activation from outside sources. The light was delivered specifically to the tumor tissue using a specialized fiber‐optic LED system. Pharmacokinetic studies showed that DOX‐PCB had an α circulation half‐life of 10 min which was comparable to that of DOX at 20 min. DOX‐PCB demonstrated resistance to metabolic cleavage ensuring that exposure to 365 nm light was the main mode of in vivo activation. Tissue extractions from tumors exposed to 365 nm light in vivo showed the presence of DOX‐PCB as well as activated DOX. The exposed tumors had six times more DOX concentration than nearby unexposed control tumors. This in vivo proof of concept demonstrates the first preferential activation of a photocleavable prodrug in deep tumor tissue.     

细胞电化学的研究进展

本文从细胞化学组成的电化学研究、细胞生物生理行为的电化学研究、细胞分析传感器、细胞的电场和电磁场生化效应及其应用等4 个方面介绍细胞电化学的原理和方法。综述了近年来细胞电化学的研究内容和最新进展, 探讨了细胞电化学的发展方向。     

Illuminating Molecular Processes in Living Cells

Lately, scientists have explored approaches to developing fluorescent and/or bioluminescent indicators to pinpoint cellular processes in single living cells. These analytical methods have become a key technology for visualizing and detecting what was otherwise unseen in live cells. The target signaling included second messengers, protein phosphorylations, protein–protein interactions, and protein localizations.     

Design and Optimization of a Biosensor Surface Functionalization to Effectively Capture Urinary Extracellular Vesicles

For this study, we tested and optimized silicon surface functionalization procedures for capturing urinary extracellular vesicles (uEVs). The influence of the silane type (APTES or GOPS) and protein concentration on the efficiency of uEVs binding was investigated. Human lactadherin protein (LACT) was used to capture uEVs. We applied surface characterization techniques, including ellipsometry, atomic force microscopy, and time-of-flight secondary ion mass spectrometry, to observe changes in the biosensor surface after each functionalization step. uEVs were purified by a low-vacuum filtration method and concentrated by ultracentrifugation. The physical parameters of uEVs after the isolation procedure, such as morphology and size distribution, were determined using transmission electron microscopy and tunable resistive pulse sensing methods. We observed a gradual growth of the molecular layer after subsequent stages of modification of the silicon surface. The ToF-SIMS results showed no changes in the mean intensities for the characteristic peaks of amino acids and lipids in positive and negative polarization, in terms of the surface-modifying silane (APTES or GOPS) used. The most optimal concentration of LACT for the tested system was 25 µg/mL.     

Selective Activation of a Prodrug by Thioredoxin Reductase Providing a Strategy to Target Cancer Cells

Elevated reactive oxygen species and antioxidant defense systems have been recognized as one of the hallmarks of cancer cells. As a major regulator of the cellular redox homeostasis, the selenoprotein thioredoxin reductase (TrxR) is increasingly considered as a promising target for anticancer drug development. The current approach to inhibit TrxR predominantly relies on the modification of the selenocysteine residue in the C‐terminal active site of the enzyme, in which it is hard to avoid the off‐target effects. By conjugating the anticancer drug gemcitabine with a 1,2‐dithiolane scaffold, an unprecedented prodrug strategy is disclosed that achieves a specific release of gemcitabine by TrxR in cells. As overexpression of TrxR is frequently found in different types of tumors, the TrxR‐dependent prodrugs are promising for further development as cancer chemotherapeutic agents.     

Selective Fluorescence Labeling of Lipids in Living Cells

Click chemistry in vivo : Three phosphatidic acid derivatives with alkyne groups in their fatty acid chains were synthesized and incorporated into mammalian cell membranes. Copper(I)‐catalyzed and strain‐promoted azide–alkyne cycloaddition reactions were used for their visualization (see schematic representation and fluorescence microscopic image).

     

活细胞内单个分子荧光检测

简要介绍了单个分子荧光检测在活细胞体系研究中的优势,介绍并讨论了单个分子荧光检测的主要技术和标记、检测方法,并回顾了目前单个分子荧光检测技术在生命科学研究中所取得的进展。     

Visualization of Redox-Controlled Protein Fold in Living Cells

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Design and Synthesis of Glycosylated Cholera Toxin B Subunit as a Tracer of Glycoprotein Trafficking in Organelles of Living Cells

Glycosylation of proteins is known to be essential for changing biological activity and stability of glycoproteins on the cell surfaces and in body fluids. Delivering of homogeneous glycoproteins into the endoplasmic reticulum (ER) and the Golgi apparatus would enable us to investigate the function of asparagine-linked (N-) glycans in the organelles. In this work, we designed and synthesized an intentionally glycosylated cholera toxin B-subunit (CTB) to be transported to the organelles of mammalian cells. The heptasaccharide, the intermediate structure of various complex-type N-glycans, was introduced to the CTB. The synthesized monomeric glycosyl-CTB successfully entered mammalian cells and was transported to the Golgi and the ER, suggesting the potential use of synthetic CTB to deliver and investigate the functions of homogeneous N-glycans in specific organelles of living cells.     

基于纳米粒子的光学细胞传感器在癌细胞研究中的应用

作为一种极为灵敏、快速的新型生物检测技术,光学细胞传感器在生物医学领域的研究应用备受关注,成为当今生物分析化学领域的研究前沿和热点.它是以细胞作为传感元件来研究信号识别、传导和指示的过程,在毒性物质、病原体等外界条件作用下,研究细胞中活性分子及其生理条件的变化,通过光学信号的变化定量分析细胞膜表面分子、胞内酶分子及微环...     

A Microfluidic Approach to Encapsulate Living Cells in Uniform Alginate Hydrogel Microparticles

A microfluidic technique is described to encapsulate living cells in alginate hydrogel microparticles generated from monodisperse double‐emulsion templates. A microcapillary device is used to fabricate double emulsion templates composed of an alginate drop surrounded by a mineral oil shell. Hydrogel formation begins when the alginate drop separates from the mineral oil shell and comes into contact with Ca²⁺ ions in the continuous phase. Alginate hydrogel microparticles with diameters ranging from 60 to 230 µm are obtained. 65% of the cells encapsulated in the alginate microparticles were viable after one week. The technique provides a useful means to encapsulate the living cells in monodisperse hydrogel microparticles.

     

Cephalosporins Determination with a Novel Microbial Biosensor Based on Permeabilized Pseudomonas aeruginosa Whole Cells

A new potentiometric microbial biosensor based on Pseudomonas aeruginosa was developed in this study for detecting the cephalosporin group of antibiotics. Preliminary results with the biosensor indicated that P. aeruginosa cells, when treated with lysozyme, showed more efficiency in detecting cephalosporin C in a wide concentration range of 0.1-11 mM with high sensitivity compared to the normal cells. Optimization of the three important biosensor design parameters permeabilized cell contents, quantities of gelatin, and glutaraldehyde resulted in high performance of the biosensor. The optimized values of the above parameters were cell contents 2.5 mg/cm(2), gelatin 8.5 mg/cm(2), and 0.25% glutaraldehyde. The assay conditions, namely phosphate buffer pH, ionic strength, and temperature, were optimized for best performance of the biosensor. The specificity test of the biosensor towards detecting different beta-lactam antibiotics showed good response only for the cephalosporins. The operational and storage stability in detecting cephalosporin C indicated very good potential of the biosensor in detecting cephalosporins with high accuracy.     

Thermophoresis in Nanoliter Droplets to Quantify Aptamer Binding

Biomolecule interactions are central to pharmacology and diagnostics. These interactions can be quantified by thermophoresis, the directed molecule movement along a temperature gradient. It is sensitive to binding induced changes in size, charge, or conformation. Established capillary measurements require at least 0.5 μL per sample. We cut down sample consumption by a factor of 50, using 10 nL droplets produced with acoustic droplet robotics (Labcyte). Droplets were stabilized in an oil–surfactant mix and locally heated with an IR laser. Temperature increase, Marangoni flow, and concentration distribution were analyzed by fluorescence microscopy and numerical simulation. In 10 nL droplets, we quantified AMP‐aptamer affinity, cooperativity, and buffer dependence. Miniaturization and the 1536‐well plate format make the method high‐throughput and automation friendly. This promotes innovative applications for diagnostic assays in human serum or label‐free drug discovery screening.     

Optochemical Nanosensors and Subcellular Applications in Living Cells

What may be the smallest anthropogenic devices to date, spherical sensors (wireless and fiberless) with radii as small as 10?nm have been produced. This class of optochemical PEBBLE (Probe Encapsulated By Biologically Localized Embedding) sensors covers a wide range of analytes (pH, calcium, oxygen and potassium included here) with excellent spatial, temporal and chemical resolution. Examples of such sensors for the monitoring of intracellular analytes are given. Methods, such as pico-injection, liposomal delivery and gene gun bombardment, are used to inject PEBBLE sensors into single cells. These PEBBLEs have caused minimal perturbation when delivered and operated inside single mammalian cells, such as human neuroblastoma, mouse oocytes or rat alveolar macrophage.     

Excluded‐Volume Effects in Living Cells

Biomolecules evolve and function in densely crowded and highly heterogeneous cellular environments. Such conditions are often mimicked in the test tube by the addition of artificial macromolecular crowding agents. Still, it is unclear if such cosolutes indeed reflect the physicochemical properties of the cellular environment as the in‐cell crowding effect has not yet been quantified. We have developed a macromolecular crowding sensor based on a FRET‐labeled polymer to probe the macromolecular crowding effect inside single living cells. Surprisingly, we find that excluded‐volume effects, although observed in the presence of artificial crowding agents, do not lead to a compression of the sensor in the cell. The average conformation of the sensor is similar to that in aqueous buffer solution and cell lysate. However, the in‐cell crowding effect is distributed heterogeneously and changes significantly upon cell stress. We present a tool to systematically study the in‐cell crowding effect as a modulator of biomolecular reactions.     

Incorporation of Functionalized Calcium Phosphate Nanoparticles in Living Cells

Journal of Cluster Science - Intracellular calcium (Ca2+) is a key signaling element that is involved in a great variety of fundamental biological processes. Thus, Ca2+ deregulation would be...     

A Rapidly Reversible Chemical Dimerizer System to Study Lipid Signaling in Living Cells

Chemical dimerizers are powerful tools for non‐invasive manipulation of enzyme activities in intact cells. Here we introduce the first rapidly reversible small‐molecule‐based dimerization system and demonstrate a sufficiently fast switch‐off to determine kinetics of lipid metabolizing enzymes in living cells. We applied this new method to induce and stop phosphatidylinositol 3‐kinase (PI3K) activity, allowing us to quantitatively measure the turnover of phosphatidylinositol 3,4,5‐trisphosphate (PIP₃) and its downstream effectors by confocal fluorescence microscopy as well as standard biochemical methods.