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The catalytic generation of H2 in living cells provides a method for antioxidant therapy.In this study,an[FeFe]-hydrogenase mimic[Ru+Fe2S2@F127(80)]was synthesized by self-assembling polymeric plur-onic F-127,catalytic[Fe2S2]sites,and photosensitizer Ru(bpy)32+.Under blue light irradiation,hydrated protons were photochemically reduced to H2,which increased the local pH in living cells(HeLa cells).The generated H2 was subsequently used as an antioxidant to decrease reactive oxygen species(ROS)levels in living cells(HEK 293T,HepG2,MCF-7,and HeLa cells).Our findings revealed that the proliferation of HEK 293T cells increased by a factor of about six times,relative to that of other cells(HepG2,MCF-7,and HeLa cells).Intracellular ROS and pH levels were then monitored using fluorescent cell imaging.Our study showed that cell imaging can be used to evaluate the ability of Ru+Fe2S2@F127 to eliminate oxidative stress and prevent ROS-related diseases.
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The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions. However, the molecular machinery underlying these hierarchically organized three-dimensional (3D) chromatin architecture and dynamics remains poorly understood. Here by combining imaging and sequencing, we studied the role of lamin B1 in chromatin architecture and dynamics. We found that lamin B1 depletion leads to detachment of lamina-associated domains (LADs) from the nuclear periphery accompanied with global chromatin redistribution and decompaction. Consequently, the inter-chromosomal as well as inter-compartment interactions are increased, but the structure of topologically associating domains (TADs) is not affected. Using live-cell genomic loci tracking, we further proved that depletion of lamin B1 leads to increased chromatin dynamics, owing to chromatin decompaction and redistribution toward nucleoplasm. Taken together, our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance, chromatin compaction, genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics, supporting their crucial roles in chromatin higher-order structure and chromatin dynamics.
Subject(s)
Humans , Chromatin , Chromosomes , Genome , Lamin Type B/geneticsABSTRACT
Genetic code expansion (GCE) allows the incorporation of unnatural amino acids into proteins via using stop codons. GCE may achieve site-specific labeling of proteins in combination with the click reaction. Compared with other labeling tools such as fluorescent proteins and tagged antibodies, the compound molecules used in protein labeling by GCE technology are smaller, and therefore, may less interfere the conformational structure of proteins. In addition, through click reaction, GCE allows a 1:1 stoichiometric ratio of the target protein molecule and the fluorescent dye, and the protein can be quantified based on the fluorescence intensity. Thus, GCE technology has great advantages in the researches that require the exposition of living cells under high laser power for longer time, for example, in the context of single molecule tracing and super-resolution microscopic imaging. Meanwhile, this technology lays the foundation for improving the accuracy of positioning and molecule counting in the imaging process of living cells. This review summarized the GCE technology and its recent applications in functionally characterizing, labeling and imaging of proteins.
Subject(s)
Amino Acids/chemistry , Fluorescent Dyes/chemistry , Genetic Code , Proteins/chemistryABSTRACT
The biological behavior of carbon dots, especially the mechanism of cellular uptake and intracellular distribution, is the basis of its biomedical applications. In this paper, blue fluorescent carbon quantum dots were synthesized by hydrothermal method with Poria cocos polysaccharide as raw material, and the specific biological behavior of carbon dots entering cells was explored to evaluate its biological activity. It was characterized by transmission electron microscopy, UV-vis absorption spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction and X-ray photoelectron spectroscopy. Two different cell lines, immunocytes-RAW264.7 cells (mouse mononuclear macrophages cells) and cancer cells-4T1 cells (mouse breast cancer cells), were used as the research objects to study the uptake kinetics, uptake pathway, distribution and efflux of polysaccharide carbon dots in cells. The results showed that the carbon dots have a size distribution of 2 to 10 nm, and the average size was 6.85 nm. The carbon dots were mainly composed of C, O and N elements, with abundant surface functional groups such as -OH, C=O, C-N and C=C, and the fluorescence quantum yield was 4.72%. Carbon dots enter cells in a certain concentration and time dependence. Different cell lines have different uptake pathways. RAW264.7 cells enter the cells mainly by macrophage-specific phagocytosis, and a small part of the endocytosis is mediated by caveolin, while 4T1 cells are mainly mediated by grid protein endocytosis and giant cell drinking process. In summary, the synthesized carbon dots have good fluorescence properties, low cytotoxicity and excellent biocompatibility, which can be used for cell imaging applications.
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BACKGROUND: Various cell therapy products have been approved by clinical trials worldwide, and cell therapies such as stem cell therapy and adoptive immunotherapy have attracted much attention. Real-time observation and imaging in vivo can visualize the distribution of cells, track cell movement, monitor cell viability, and observe the cell migration and growth. Many imaging technologies can visualize cells in vivo, such as ultrasound, optics, MRI and nuclear imaging, and these methods need to correspond to different labeling and detection strategies. Each strategy has its own advantages and disadvantages. OBJECTIVE: To review the principle and development of different tracking methods, and their application in animals and humans. METHODS: PubMed, Google Scholar, Web of Science and CNKI databases were searched with the keywords of “cell tracking, in vivo cell tracking, PET imaging, MRI, optical imaging.” The articles published in the past 5-10 years were preferred. The contents of the articles mainly describe the principle of different tracking methods, and their application in animal models and patients. RESULTS AND CONCLUSION: In the past 20 years, cell tracking has developed into a multifarious discipline, not only establishing a variety of robust methods in animal models, but also proving the feasibility of clinical transformation in some human studies. The development of the non-invasive detection methods, such as PET and MRI, and new contrast agents provides strong support for the application of cell therapy in clinical and scientific researches.
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Secondary ion mass spectrometry ( SIMS) as a powerful surface analysis technique has been widely applied in semiconductor industry and geology research. Recently, with the development of instrumental technology, SIMS is attracting more and more attention in life sciences. SIMS can provide surface MS spectra, 2D/3D chemical images and depth profiling of substances simultaneously. The minimal lateral resolution of 2D SIMS imaging is 80 to 100 nm, and the longitudinal resolution of 3D SIMS imaging is about 1-5 nm. However, due to lack of specific ions to render the structures of organelles, SIMS imaging for single cells still has great challenges. Optical microscopy, in particular laser scanning confocal microscopy ( LSCM) , has been emerged to be an indispensable technique for single cell imaging and can obtain high spatial 2D/3D imaging to visualize the structures of organelles. Thus, the combinational use of SIMS and LSCM, which takes advantages of SIMS for molecular imaging and LSCM for morphological imaging, has greatly extended the application of SIMS imaging and ensured its accuracy at single cells level, providing novel insights into better understanding of the biological events inside cells. In this review, we focus on the development and application of SIMS imaging and the correlated SIMS and LSCM imaging in the research of cell biology and drug discovery. We anticipate that the combinational use of SIMS and LSCM imaging has promising future in biomedicine and life sciences.
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Aim To compare high-content cell imaging system and other methods in detecting cell proliferation, including the traditional thymidine ( 3H-TdR) incorporation method, methyl thiazolyl tetrazolium ( MTT ) method and cell counting kit-8 (CCK-8) method. Methods The fibroblast-like synovial cells (FLS) were used as the study object to observe the sensitivity and stability of FLS proliferation in different methods by using the usual proliferative stimulant tumor necrosis factor-α ( TNF-α) and the known proliferation inhibitor methotrexate at different concentrations. Results The 3H-TdR method and high-content cell imaging could detect a significant inhibitory effect of 1 nmol ·L-1 MTX on FLS cell proliferation, MTT assay and CCK-8 method could detect the significant inhibitory effect of 10 nmol· L-1 MTX on FLS cell proliferation. 3H-TdR method was found to have a large degree of discretization in the data set, with a standard deviation of 32.61% ~61.36% , and the MTT method was 11.9% ~ 17.8% , the CCK-8 method was 17.15% ~32.88% , and the high-content cell imaging system method was 12.66% ~26.54%. Conclusion The method of high-content cell imaging system is more accurate and stable for detecting cell proliferation.
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A water-soluble fluorescent probe (7-diethyl amino-3-formaldehyde coumarin) for Fe3+ detection was designed and synthesiZed, and its structure was confirmed by 1 HNMR, 13 CNMR and MS spectra.This probe showed high emission intensity at 471 nm.With the continuous addition of Fe3+, the emission intensities at 471 nm decreased gradually, and showed an excellent linearity with Fe3+in the range of 0.02-60 μmol/L, and the regression equation was I=322.56-4.86CFe3+.This probe was able to detect Fe3+ qualitatively and quantitatively with the detection limit as low as 22 nmol/L.Besides, this probe showed high sensitivity to Fe3+over other metal ions.The detection process was reversible, which could be recycled for the Fe3+detection.In terms of good optical properties and the strong fluorescence in physiological pH, the probe was successfully applied in imaging Fe3+ of living Ramos cells.
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Hydrogen sulfide ( H2S ) has been confirmed as a significant endogenous gaseous signaling molecule involved in various physiological processes.To monitor H2S in living cells, a F?rster resonance energy transfer ( FRET) ratiometric probe based on quantum dot-cresyl violet was developed.In this work, quantum dot nanospheres ( QDS) were firstly synthesiZed via a facile ultrasonication emulsion strategy, and the mixture chloroform solution containing hydrophobic quantum dots and COOH-functionaliZed amphiphilic polymer were successfully transferred into the oil-in-water micelle.The negatively charged quantum dot nanospheres with quantum dots embedded in the polymer matrixes were successfully fabricated after the evaporation of chloroform.And then, these quantum dot nanospheres were condensed with positively charged cresyl violet-aZide ( CV-N3 ) via electrostatic interaction to obtain the QDS-N3 complexes.The as-prepared QDS-N3 complexes were monodispersed nanospheres with an average diameter of about 120 nm.These complexes were taken up by the cell through endocytosis, and they were still stable even in wide pH range.In addition, the QDS-N3 complexes exhibited no cellular toxicity which was verified by MTT assay.In this ratiometric probe, CV-N3 as a FRET acceptor was conjugated to quantum dot nanospheres.The quantum dots emitted at 591 nm and served as the FRET donor;once the aryl aZide on the CV-N3 was reduced to aniline by H2S, the probe emitted at 620 nm.The ratiometric probe allowed the elimination of interference of excitation intensity, intracellular environment and other factors.Furthermore, this method also offered a general protocol for preparing nanosensors for monitoring various small molecular in living cells.
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A ball milling method which is green with simple-manipulation and low-cost was used to prepare graphene as precursor for graphene quantum dots (GQDs) synthesis.Subsequently, GQDs with good dispersibility, uniform size distribution, average diameter of (4.80 ± 0.20) nm and 1-3 layers were prepared by one-step hydrothermal method.The morphology, structure and optical properties of the GQDs were characterized by high-resolution transmission electron microscopy (HRTEM), atomic force microscope (AFM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), UV-Vis absorption and fluorescence spectroscopy.Furthermore, the GQDs were used in label-free and specific detection of ferric ion (Fe.3+) with broad linear ranges of 2.0×10.-6-7.0×10.-4 mol/L and low detection limit of 1.8 × 10.6 mol/L (S/N=3).The possible mechanism was also discussed and the application of GQDs for Fe.3+ detection in tap water was demonstrated.Finally, based on their low cytotoxicity and excellent biocompatibility, the as-prepared GQDs were successfully applied to efficient cell imaging.This work provides a new way for preparation of carbon-based nanomaterials and build a foundation for deepening applications of GQDs in bio-/chem-analysis, bioimaging, etc.
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Polyacrylic acid was firstly grafted by N-amino-4-N-methylpiperazine-1,8-naphthlimide (AMN) to prepare a amphiphilic polymer, which was self-assembled in water producing nanoparticles called as PAAMN.Then the morphology, structure and fluorescence properties of PAAMN were investigated by various methods including transmission electron microscopy, dynamic light scattering, ultraviolet-visible spectroscopy (UV-Vis), nuclear magnetic resonance spectroscopy (HNMR) and fluorescence spectroscopy.MTT assay was carried out to assess the cell compatibility of PAAMN.Finally, the fluorescence from PAAMN self and HeLa cells incubated with PAAMN was observed by fluorescence microscope.The results revealed that PAAMN had spherical structure, in which naphthlimide fluorphores were immobilized in the polyacrylic acid matrix with the degree of substitution of 4.1%.Under the physiological pH condition, PAAMN excited at 390 nm could emit strong and stable fluorescence at 534 nm.In the range of pH 4.0-10.0, its excitation and emission wavelengths had no obvious change.The fluorescence intensity of PAAMN increased with the decrease of pH values, but the pH sensitivity of PAAMN was much lower than that of AMN.PAAMN had good cell compatibility.From the pictures of fluorescence imaging, it was found that both PAAMN self and cells-engulfed PAAMN could emit green fluorescence upon excited at 390 nm, indicating the potential of the developed nanoparticle for cell imaging.
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Superoxide anion radical ( O·-2 ) is the first generated reactive oxygen species ( ROS) and plays essential function in life processes. Normal level of O·-2 as important signaling molecular can regulate redox equilibrium, cellular proliferation and differentiation. However, abnormal level of O·-2 is closely associated with diseases, such as cancer, neurodegenerative diseases and diabetes. Hence it is significant to uncover diseases mechanism by exploring dynamic regulation of O·-2 . Considering the advantages of fluorescence imaging method, the key factor is to develop O·-2 probes with highly selective and sensitive properties for revealing the molecular mechanism of diseases. Recently, with the development of fluorescence microscopy, many fluorescent probes have been constructed and applied for imaging analysis of O·-2 . In this review, we mainly summarized the progress of O·-2 fluorescent probes according to the different probe structure and prospected the development directions of O·-2 probes.