- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors
摘要： DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20–25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.
关键词： DNA-PAINT,microfluidics,super-resolution microscopy,fluorescent proteins,molecular localization,multi-color imaging,multiplexing,single domain antibodies (sdAb),linkage error,nanobodies
更新于2025-09-19 17:15:36
-
Occurrence and mechanism of visual phosphenes in external photon beam radiation therapy and how to influence them
摘要： Background and purpose: Two plausible mechanisms to explain the appearance of visual phosphenes are: direct activation of the photochemicals in the retina and the generation of Cherenkov radiation in the vitreous humour. In this clinical trial we investigated the occurrence of visual phosphenes in external photon beam radiation therapy. Material and methods: Logistic regression analysis is used to examine whether seeing light flashes and seeing steady light depended on the ambient light intensity and the dose. Results: In total, 465 treatments of 25 patients were analysed. The odds of seeing light flashes multiply by 0,926 as the ambient light intensity increases by 10 lux. Similarly, the odds multiply by 1,604 as the dose to the retina increases by 10 cGy. The odds of seeing steady light multiply by 1,540 as the dose to the vitreous humour increases by 10 cGy. Conclusions: We postulate that one should reduce the dose rate, instruct patients to keep the eyes open and increase the illuminance in the treatment room to reduce the probability of experiencing visual phosphenes. We hypothesize that melanopsin is involved in the visual phosphenes and that fatigue of patients might be correlated with the observation of visual phosphenes.
关键词： Light flashes,Visual phosphenes,Photoreceptor proteins,Cherenkov radiation,Photon beam radiation therapy
更新于2025-09-19 17:15:36
-
Fluorescence resonance energy transfer links membrane ferroportin, hephaestin but not ferroportin, amyloid precursor protein complex with iron efflux
摘要： Iron efflux from mammalian cells is supported by the synergistic actions of the ferrous iron efflux transporter, ferroportin (Fpn) and a multicopper ferroxidase, that is, hephaestin (Heph), ceruloplasmin (Cp) or both. The two proteins stabilize Fpn in the plasma membrane and catalyze extracellular Fe3+ release. The membrane stabilization of Fpn is also stimulated by its interaction with a 22-amino acid synthetic peptide based on a short sequence in the extracellular E2 domain of the amyloid precursor protein (APP). However, whether APP family members interact with Fpn in vivo is unclear. Here, using cyan fluorescent protein (CFP)-tagged Fpn in conjunction with yellow fluorescent protein (YFP)-fusions of Heph and the APP family members APP, APLP1, and APLP2 in HEK293T cells we used fluorescence and surface biotinylation to quantify Fpn membrane occupancy and also measured 59Fe efflux. We demonstrate that Fpn and Heph co-localize, and FRET analysis indicated that the two proteins form an iron-efflux complex. In contrast, none of the full-length, cellular APP proteins exhibited Fpn co-localization or FRET. Moreover, iron supplementation increased surface expression of the iron-efflux complex, and copper depletion knocked down Heph activity and decreased Fpn membrane localization. Whereas cellular APP species had no effects on Fpn and Heph localization, addition of soluble E2 elements derived from APP and APLP2, but not APLP1, increased Fpn membrane occupancy. We conclude that a ferroportin-targeting sequence, K/REWEE, present in APP and APLP2, but not APLP1, helps modulate Fpn-dependent iron efflux in the presence of an active multicopper ferroxidase.
关键词： cell surface protein,hephaestin,iron metabolism,membrane transport,metal transport,Ferroportin,multicopper ferroxidase,APP-like proteins,iron efflux,amyloid precursor protein
更新于2025-09-19 17:15:36
-
Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in <i>Caenorhabditis elegans</i>
摘要： CRISPR-based genome editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry, or wrmScarlet in Caenorhabditis elegans. This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, ssDNA donors (≤200 bp) are used to insert the 5’ and 3’ fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a dsDNA donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal crRNAs and PCR products. We have also used Nested CRISPR in a non-essential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic sgRNAs, but did not observe a significant increase in efficiency. To streamline the approach, we combined all Step 1 and Step 2 reagents in a single injection and were successful in 3 of 5 loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.
关键词： fluorescent proteins,CRISPR,genome editing,C. elegans,Cas9
更新于2025-09-19 17:15:36
-
Blue Light Switchable Cell-Cell Interactions Provide Reversible and Spatiotemporal Control Towards Bottom-Up Tissue Engineering
摘要： Controlling cell–cell interactions is central for understanding key cellular processes and bottom-up tissue assembly from single cells. The challenge is to control cell–cell interactions dynamically and reversibly with high spatiotemporal precision noninvasively and sustainably. In this study, cell–cell interactions are controlled with visible light using an optogenetic approach by expressing the blue light switchable proteins CRY2 or CIBN on the surfaces of cells. CRY2 and CIBN expressing cells form specific heterophilic interactions under blue light providing precise control in space and time. Further, these interactions are reversible in the dark and can be repeatedly and dynamically switched on and off. Unlike previous approaches, these genetically encoded proteins allow for long-term expression of the interaction domains and respond to nontoxic low intensity blue light. In addition, these interactions are suitable to assemble cells into 3D multicellular architectures. Overall, this approach captures the dynamic and reversible nature of cell–cell interactions and controls them noninvasively and sustainably both in space and time. This provides a new way of studying cell–cell interactions and assembling cellular building blocks into tissues with unmatched flexibility.
关键词： bottom-up tissue engineering,spatiotemporal control,photoswitchable proteins,cell adhesion,cell–cell interactions
更新于2025-09-19 17:15:36
-
Raman spectroscopic analysis of high molecular weight proteins in solution – considerations for sample analysis and data pre-processing
摘要： This study explores the potential of Raman spectroscopy, coupled with multivariate regression techniques and a protein separation technique (ion exchange chromatography), to quantitatively monitor diagnostically relevant changes in high molecular weight proteins in liquid plasma. Measurement protocols to detect the imbalances in plasma proteins as an indicator of various diseases using Raman spectroscopy are optimised, such that strategic clinical applications for early stage disease diagnostics can be evaluated. In a simulated plasma protein mixture, concentrations of two proteins of identified diagnostic potential (albumin and fibrinogen) were systematically varied within physiologically relevant ranges. Scattering from the poorly soluble fibrinogen fraction is identified as a significant impediment to the accuracy of measurement of mixed proteins in solution, although careful consideration of pre-processing methods allows construction of an accurate multivariate regression prediction model for detecting subtle changes in the protein concentration. Furthermore, ion exchange chromatography is utilised to separate fibrinogen from the rest of the proteins and mild sonication is used to improve the dispersion and therefore quality of the prediction. The proposed approach can be expeditiously employed for early detection of pathological disorders associated with high or low plasma/serum proteins.
关键词： sonication,pre-processing,disease diagnostics,Raman spectroscopy,plasma proteins,ion exchange chromatography,multivariate regression
更新于2025-09-19 17:15:36
-
Preparation of Highly Stable and Photoluminescent Cadmiuma??Free InP/GaP/ZnS Core/Shell Quantum Dots and Application to Quantitative Immunoassay
摘要： Indium phosphide (InP) quantum dots (QDs) are ideal substitutes for widely used cadmium-based QDs and have great application prospects in biological fields due to their environmentally benign properties and human safety. However, the synthesis of InP core/shell QDs with biocompatibility, high quantum yield (QY), uniform particle size, and high stability is still a challenging subject. Herein, high quality (QY up to 72%) thick shell InP/GaP/ZnS core/shell QDs (12.8 ± 1.4 nm) are synthesized using multiple injections of shell precursor and extension of shell growth time, with GaP serving as the intermediate layer and 1-octanethiol acting as the new S source. The thick shell InP/GaP/ZnS core/shell QDs still keep high QY and photostability after transfer into water. InP/GaP/ZnS core/shell QDs as fluorescence labels to establish QD-based fluorescence-linked immunosorbent assay (QD-FLISA) for quantitative detection of C-reactive protein (CRP), and a calibration curve is established between fluorescence intensity and CRP concentrations (range: 1–800 ng mL?1, correlation coefficient: R2 = 0.9992). The limit of detection is 2.9 ng mL?1, which increases twofold compared to previously reported cadmium-free QD-based immunoassays. Thus, InP/GaP/ZnS core/shell QDs as a great promise fluorescence labeling material, provide a new route for cadmium-free sensitive and specific immunoassays in biomedical fields.
关键词： cadmium-free,quantum dot-based fluorescence-linked immunosorbent assays,thick shell InP core/shell quantum dots,C-reactive proteins,quantitative detection
更新于2025-09-19 17:13:59
-
N-glycosylation state of TRPM8 protein revealed by terahertz spectroscopy and molecular modelling
摘要： TRPM8 member of the TRP superfamily of membrane proteins participates to various cellular processes ranging from Ca2+ uptake and cold sensation to cellular proliferation and migration. TRPM8 is a large tetrameric protein with more than 70% of its residues located in the cytoplasm. TRPM8 is N-glycosylated, with a single site per subunit. This work focuses on the N-glycosylation of TRPM8 channel that was previously studied by our group in relation to proliferation and migration of tumoral cells. Here, experimental data performed with deglycosylating agents assess that the sole glycosylation site contains complex glycans with a molecular weight of 2.5 kDa. The glycosylation state of TRPM8 in cells untreated and treated with a deglycosylating agent was addressed with Terahertz (THz) spectroscopy. Results show a clear difference between cells comprising glycosylated and deglycosylated TRPM8, the first presenting an increased THz absorption. Human TRPM8 was modelled using as templates the available TRPM8 and other TRPM channels structures. Glycosylations were modelled by considering two glycan structures with molecular weight close to the experiment: shorter and branched at the first sugar unit (glc1) and longer and unbranched (glc2). Simulation of THz spectra based on the molecular dynamics of unglycosylated and the two glycosylated TRPM8 models in lipid membrane and solvation box showed that glycan structure strongly influences the THz spectrum of the channel and of other components from the simulation system. Only spectra of TRPM8 with glc1 glycans were in agreement with the experiment, leading to the validation of glc1 glycan structure.
关键词： N-glycosylation,Terahertz spectroscopy,TRPM8,Molecular modelling,Membrane proteins
更新于2025-09-19 17:13:59
-
[IEEE 2019 IEEE International Conference on Consumer Electronics - Asia (ICCE-Asia) - Bangkok, Thailand (2019.6.12-2019.6.14)] 2019 IEEE International Conference on Consumer Electronics - Asia (ICCE-Asia) - Development of Automated LED Light Compensation System for Lycopersicon Esculentum
摘要： The cells in an organism emit different amounts of proteins according to their clinical state (healthy/pathological, for instance). The resulting proteomic pro?le can be used for early detection, diagnosis, and therapy planning. In this paper, we study the classi?cation of a proteomic sample from the point of view of an inverse problem with a joint Bayesian solution, called inversion-classi?cation. We propose a hierarchical physical forward model and present encouraging results from both simulation and clinical data.
关键词： proteins,classi?cation algorithms,proteomics,Statistical signal processing,probability,liquid chromatography,mass spectrometry,selective reaction monitoring,inverse problems,mathematical modelling
更新于2025-09-19 17:13:59
-
[IEEE 2019 IEEE International Symposium on Olfaction and Electronic Nose (ISOEN) - Fukuoka, Japan (2019.5.26-2019.5.29)] 2019 IEEE International Symposium on Olfaction and Electronic Nose (ISOEN) - Odorant-binding protein-based optoelectronic tongue and nose for sensing volatile organic compounds
摘要： We developed an array of odorant-binding protein mutants with various binding properties. The same design is suitable for the detection and identification of volatile organic compounds (VOCs) both in the liquid phase and in the gas phase by surface plasmon resonance imaging. The obtained optoelectronic tongue is highly selective at low concentrations of VOCs with a low detection limit, but a narrow linear range. In comparison, the optoelectronic nose gives a much higher signal to noise ratio, but the discrimination of VOCs from different chemical classes requires kinetic data to get rid of non-specific signals. This work shows that these optoelectronic tongue and nose are promising for numerous applications, each system having its own advantages.
关键词： electronic tongue,surface plasmon resonance imaging,electronic nose,Odorant-binding proteins,volatile organic compound
更新于2025-09-19 17:13:59