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Spontaneous Charge Generation in Flowing Albumin Solutions at 35 °C and 38 °C
摘要： The time dependence of a charge accumulation in a 10?15 M albumin solution, flowing through a measuring cell of an analytical flow system injector, had a nonlinear character under certain conditions, within a human physiological temperature range. Sharp charge increases depended on albumin concentration. This effect must be taken into consideration when generating models that describe electrokinetic phenomena in flowing protein solutions and when developing analytical flow systems for the registration of biomolecules in low concentration ranges.
关键词： flowing protein solution,charge accumulation,analytical flow-through systems,physiological temperature,serum albumin
更新于2025-09-23 15:22:29
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Live-cell super-resolution microscopy reveals a primary role for diffusion in polyglutamine-driven aggresome assembly
摘要： The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed in vitro. Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that increasing surface area of the expanding aggresome increases the rate of accretion due to diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies.
关键词： molecular modelling,protein aggregation,molecular imaging,passive transport,amyloid protein,aggresome formation,transport,live cell SIM,protein misfolding,molecular dynamics
更新于2025-09-23 15:21:21
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Luminescent silica mesoparticles for protein transduction
摘要： Unlike silica nanoparticles, the potential of silica mesoparticles (SMPs) (i.e. particles of submicron size) for biological applications in particular the in vitro (let alone in vivo) cellular delivery of biological cargo has so far not been sufficiently studied. Here we examine the potential of luminescent (namely, octahedral molybdenum cluster doped) SMPs synthesised by a simple one-pot reaction for the labelling of cells and for protein transduction into larynx carcinoma (Hep-2) cells using GFP as a model protein. Our data demonstrates that the SMPs internalise into the cells within half an hour. This results in cells that detectably luminesce via conventional methods. In addition, the particles are non-toxic both in darkness and upon photo-irradiation. The SMPs were modified to allow their functionalisation by a protein, which then delivered the protein (GFP) efficiently into the cells. Thus, the luminescent SMPs offer a cheap and trackable alternative to existing materials for cellular internalisation of proteins, such as the HIV TAT protein and commercial protein delivery agents (e.g. Pierce?).
关键词： protein transduction,cytotoxicity,silica,Octahedral molybdenum cluster,cellular uptake
更新于2025-09-23 15:21:21
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Quantification of Cellular Proteostasis in Live Cells by Fluorogenic Assay Using the AgHalo Sensor
摘要： Proper cellular proteostasis is essential to cellular ?tness and viability. Exogenous stress conditions compromise proteostasis and cause aggregation of cellular proteins. We have developed a ?uorogenic sensor (AgHalo) to quantify stress-induced proteostasis de?ciency. The AgHalo sensor uses a destabilized HaloTag variant to represent aggregation-prone cellular proteins and is equipped with a series of ?uorogenic probes that exhibit a ?uorescence increase when the sensor forms either soluble oligomers or insoluble aggregates. Herein, we present protocols that describe how the AgHalo sensor can be employed to visualize and quantify proteome stress in live cells using a direct ?uorescence read-out and visualization with a ?uorescence microplate reader and a microscope. Additionally, protocols for using the AgHalo sensor in combination with ?uorogenic probes and commercially available HaloTag probes to enable two-color imaging experiments are described. These protocols will enable use of the AgHalo sensor to visualize and quantify proteostasis in live cells, a task that is dif?cult to accomplish using previous, always-?uorescent methods.
关键词： protein aggregation,?uorescence intensity,proteostasis,?uorogenic sensor
更新于2025-09-23 15:21:21
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Biomolecular interaction and kinematics differences between P25 and E171 TiO2 nanoparticles
摘要： Titanium dioxide (TiO2) nanoparticles (NPs) are used abundantly as food additives (E171). For the purpose of risk assessment, it is imperative to understand the behavior of these nanoparticles in a food relevant environment, and their consequent toxicology impacts. However, most of such studies use model TiO2 NPs (P25) as substitutes for E171. To understand the suitability of this approach, we investigated the functional behavior of E171 and P25 in solutions of bovine serum albumin (BSA) and sucrose as model food ingredients. Our data showed that E171 were better dispersed in BSA than P25. In sucrose, E171 displayed a reduction in agglomerated size while P25 agglomerated extensively. Adsorption studies showed that P25 attracted more pronounced corona formation per unit mass of material compared to E171. In vitro sedimentation, diffusion and dosimetry (ISDD) results demonstrated that the time-weighted dosage of E171 was more than two-folds higher than P25, implying that any test performed using P25 to model E171 would underestimate actual dosage and potential toxicity. Taken collectively, this study demonstrated the specificity of TiO2 nanoparticle interaction with food ingredients, and the importance of using food-grade E171 TiO2 for food-relevant toxicological assessments.
关键词： ISDD,Nanoparticles,Titanium dioxide,Sucrose,Corona,Protein
更新于2025-09-23 15:21:21
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Identification of spherical and non-spherical protein by solid-state nanopore
摘要： Three-dimensional structure of a protein plays an important role in protein dynamics in biological system of human. By now, it remains challenge to characterize and quantify the shape of a protein at single-molecule level. Nanopore, as a novel single-molecule sensor, has been widely applied in many fields such as DNA sequencing and human diseases diagnosis. In this paper, we investigated the translocation of sphere-like con.A and the prolate Bovine Serum Albumin (BSA) under electric field by solid-state nanopore. By analyzing the ionic current, the con.A and the BSA could be characterized and differentiated due to their intrinsic shape difference. Because the prolate BSA will have the preferred orientations for higher electric field when it is residing inside the nanopore, thus multiple ionic current blockade levels will be observed. While for the spherical con.A, there is only one ionic current blockade level. This method presented here will be potentially applied to fingerprint a single protein as a new method having features of low-cost and high-throughput in the near future.
关键词： orientation,solid-state nanopore,shape,electric field,ionic current,protein
更新于2025-09-23 15:21:21
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Sensors Based on Metal Nanoclusters Stabilized on Designed Proteins
摘要： Among all new nanomaterials, metal nanoclusters (NCs) have attracted special attention due to their interesting optical properties, among others. Metal NCs have been recently studied and used as sensors for different analytes. However, there is a need to explore the potential of these new sensors in a systematic manner and to develop new systems to broaden the possibilities that sensing offers to the industry. In this work, we show the potential use of repeat protein scaffolds as versatile templates for the synthesis and stabilization of various metal NCs, speci?cally Au, Ag, and CuNCs. The resulting protein-metal NCs hybrids are evaluated as sensors for different stimuli such as temperature, ions, or reactive oxygen species (ROS). Among the three protein-metal NCs, all performed nicely as temperature sensors, AuNCs responded to metal ions, and AgNCs were able to detect ROS.
关键词： nanosensor,ROS sensors,metal nanocluster,protein design,temperature sensor,metal sensors,?uorescent probe
更新于2025-09-23 15:21:21
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Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy
摘要： We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.
关键词： imaging,single-protein,real-time,scattering,dynamics,cellular secretion,label-free,iSCAT
更新于2025-09-23 15:21:21
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Rapid Detection of Proteins on One-Dimensional Polyacrylamide Gel Electrophoresis Using an Inexpensive Fluorescent Optical Brightener
摘要： A commercial fabric fluorescent optical brightener Ranipal? (F-OB), has been successfully employed to stain proteins on native and SDS-1D-PAGE. The F-OB was purified by using a biphasic solvent system of dichloromethane and water. The Rf value (0.63) of purified and crude F-OB were comparable on TLC. The mass spectrometry of purified F-OB indicated base peak at 414 (m/z). Absorption and emission maxima of F-OB were found to be 350 nm and 430 nm, respectively. The F-OB could stain the proteins both pre- and post-electrophoretic run, on native gel. Post-electrophoretic staining was rapid and required 20 min to visualize the stained proteins. On the other hand, in SDS gels, an additional 20 min was required for the extraction of SDS before staining the proteins with F-OB. SDS was found to interfere with binding of F-OB to proteins. Varying concentrations of molecular weight markers were loaded and their fluorescent intensity was plotted against the concentration of the proteins. The r2 values ranged from 0.965 to 0.997 indicating excellent linearity. The detection for carbonic anhydrase was in the range of 8.0-800 ng. Unlike most of the dyes used for protein staining, staining with F-OB could be carried out in tank buffer (2 mg/100 mL) and was also reversible. The F-OB, perhaps would be the most cost-effective fluorescent dye to stain the proteins (US $ 0.04/25.0 g). The F-OB was found to be simple, safe, sensitive, less time consuming and economical fluorescent dye as an alternative, for staining proteins on polyacrylamide gels.
关键词： Fluorescent optical brightener,1D-Electrophoresis,CCD-based digital image based analysis system,Protein staining
更新于2025-09-23 15:21:21
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Identification of Protein Targets of Bioactive Small Molecules Using Randomly Photomodified Probes
摘要： Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe re-synthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.
关键词： bioactive small molecules,target isolation,protein targets,target identification,stochastic modification,target visualization,photoactivatable phenyldiazirine linker,biotin,fluorophore,hydrophilic copolymer
更新于2025-09-23 15:21:21