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109 Evaluation of the Efficacy and Safety of Fractional Picosecond 1064-nm Laser Treatment for Skin Rejuvenation
摘要： Gold (Au) colloids are becoming ubiquitous across biomedical engineering, solar energy conversion, and nano-optics. Such universality has originated from the exotic plasmonic effect of Au colloids (i.e., localized surface plasmon resonance (LSPRs)) in conjunction with the versatile access to their synthetic routes. Herein, we introduce a previously undiscovered usage of Au colloids for advancing cryoprotectants with significant ice recrystallization inhibition (IRI). Oligopeptides inspired by the antifreeze protein (AFP) and antifreeze glycoprotein (AFGP) are attached onto the surface of well-defined Au colloids with the same sizes but different shapes. These AF(G)P-inspired Au colloids can directly adsorb onto a growing ice crystal via the synergistic interplay between hydrogen bonding and hydrophobic groups, in stark contrast to their bare Au counterparts. Dark-field optical microscopy analyses, benefitting from LSPR, allow us to individually trace the in situ movement of the antifreezing Au colloids during ice growth/recrystallization and clearly evidence their direct adsorption onto the growing ice crystal, which is consistent with theoretical predictions. With the assistance of molecular dynamics (MD) simulations, we evidently attribute the IRI of AF(G)P-inspired Au colloids to the Kelvin effect. We also exploit the IRI dependence on the Au colloidal shapes; indeed, the facet contacts between ice and Au colloids can be better than the point-like counterparts in terms of IRI. The design principles and predictive theory outlined in this work will be of broad interest not only for the fundamental exploration of the inhibition of ice growth but also for enriching the application of Au colloids.
关键词： gold colloids,antifreezing proteins,dark-field microspectroscopy,oligopeptides,Ice recrystallization inhibition
更新于2025-09-11 14:15:04
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Genetically encoded FRET-based optical sensor for Hg2+ detection and intracellular imaging in living cells
摘要： Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg2+ are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg2+ at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg2+ with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (Kd) of 5.09 × 10?7 M, thus providing a detection range for Hg2+ quantification between 0.210?μM and 1.196?μM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intra- cellular Hg2+ concentration with a highly responsive saturation curve, proving its potential application in cellular systems.
关键词： Genetically encoded,FRET,Fluorescent proteins,Mercury,Nanosensors
更新于2025-09-11 14:15:04
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Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism
摘要： Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signaling processes. These proteins often gain structure upon binding to their target proteins in multistep reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain and the disordered interaction domain from activator of thyroid hormone and retinoid receptors. We demonstrate that helix formation in a rapid binding reaction can be followed by stopped-flow synchrotron-radiation circular dichroism (CD) spectroscopy and describe the design of such a beamline. Fluorescence-monitored binding experiments of activator of thyroid hormone and retinoid receptors and nuclear coactivator binding domain display several kinetic phases, including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time-resolved CD experiments show that almost all helicity is formed upon initial association of the proteins or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron-radiation CD for mechanistic studies of protein-protein interactions.
关键词： coupled folding and binding,synchrotron-radiation circular dichroism,protein-protein interactions,Intrinsically disordered proteins,helix formation
更新于2025-09-11 14:15:04
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X-ray irradiation effects on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells investigated by Raman micro-spectroscopy
摘要： Raman micro-spectroscopy was performed in vitro on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). The acquired spectra were analyzed by principal component analysis (PCA) and interval-PCA (i-PCA) methods. Biochemical changes occurring in the different regions of single cells as a consequence of the radiation exposure were observed in cells fixed immediately after the irradiation. The most relevant effects arose from the analysis of the spectra from the cell nucleus region. The observed changes were discussed in terms of the modifications in the cell cycle, resulting in an increase in the DNA-related signal, a protein rearrangement and changes in lipid and carbohydrates profiles within the nucleus. Potential markers of an apoptotic process in cell population irradiated with 6 and 8-Gy X-ray doses could have been singled out. No significant effects were found in spectra from cells fixed 24 h after the irradiation, thus suggesting the occurrence of repairing processes of the X-ray induced damage.
关键词： X-ray effects on DNA, lipids, proteins and carbohydrates,Single SH-SY5Y human cancer cells,Raman micro-spectroscopy,Cellular nucleus and membrane,Multivariate analysis
更新于2025-09-11 14:15:04
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Photoexcitation Dynamics of Thiocyanate-Bound Heme Proteins Using Femtosecond Infrared Spectroscopy
摘要： Myoglobin (Mb) and hemoglobin (Hb) have been used as model systems to understand the relationships between protein function, structure, and dynamics. These studies have primarily been carried out by observing the (re)binding between ferrous heme proteins and neutral ligands. Ferric heme proteins also bind to various anionic ligands, such as CN?, N3?, OCN?, and SCN?. Only a few anion-bound ferric heme proteins have been investigated for ligand-binding dynamics.1–4 Although CN?-bound Mb (MbCN) was reported to be photostable, i.e., not photodeligated by a visible photon, recent study using Raman spectroscopy claimed a photodeligation quantum yield for MbCN of 0.75.5 Thus, the photostability of ?-bound MbCN is still in question. At room temperature, N3?-bound Mb (MbN3) exists in both high spin (HS) and low spin (LS) complexes. When MbN3 absorbs a visible photon, the ligand is not dissociated; instead the excited MbN3 undergoes a thermal relaxation process involving a spin transition.3 Recently, we have shown that photoexcited OCN?-bound Mb and OCN?-bound Hb are also photostable, losing their excess energy via thermal relaxation after rapid electronic relaxation.4 Here, to determine the general excitation properties of anion-bound heme proteins after absorption of a visible photon, we have extended our study of photoexcitation dynamics to SCN?-bound ferric Mb (MbNCS) and Hb (HbNCS).
关键词： Femtosecond vibrational spectroscopy,Thiocyanate-bound myoglobin,Electronic relaxation,Ferric heme proteins,Thermal relaxation
更新于2025-09-10 09:29:36
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[Methods in Molecular Biology] FOXO Transcription Factors Volume 1890 (Methods and Protocols) || Immunofluorescence Analysis by Confocal Microscopy for Detecting Endogenous FOXO
摘要： Cancer cells are known to inactivate tumor suppressor proteins by triggering their anomalous subcellular location. It has been well established that the aberrant location of FOXO proteins is linked to tumor formation, progression of the same, or resistance to anti-neoplastic treatment. Furthermore, the abnormal location of FOXO has also been considered a potential biomarker for diabetic complications or longevity in different organisms. Here, we describe the immunodetection of endogenous FOXO by confocal microscopy, which can be used as a chemical tool to quantify FOXO expression levels, its cellular location, and even its active/inactive forms with relevant antibodies.
关键词： Immunodetection,Nuclear translocation,FOXO proteins,Confocal microscopy,Biomarker
更新于2025-09-10 09:29:36
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[Methods in Enzymology] Intrinsically Disordered Proteins Volume 611 || Accurate Transfer Efficiencies, Distance Distributions, and Ensembles of Unfolded and Intrinsically Disordered Proteins From Single-Molecule FRET
摘要： Intrinsically disordered proteins (IDPs) sample structurally diverse ensembles. Characterizing the underlying distributions of conformations is a key step toward understanding the structural and functional properties of IDPs. One increasingly popular method for obtaining quantitative information on intramolecular distances and distributions is single-molecule F?rster resonance energy transfer (FRET). Here we describe two essential elements of the quantitative analysis of single-molecule FRET data of IDPs: the sample-specific calibration of the single-molecule instrument that is required for determining accurate transfer efficiencies, and the use of state-of-the-art methods for inferring accurate distance distributions from these transfer efficiencies. First, we illustrate how to quantify the correction factors for instrument calibration with alternating donor and acceptor excitation measurements of labeled samples spanning a wide range of transfer efficiencies. Second, we show how to infer distance distributions based on suitably parameterized simple polymer models, and how to obtain conformational ensembles from Bayesian reweighting of molecular simulations or from parameter optimization in simplified coarse-grained models.
关键词： Intrinsically disordered proteins,Coarse-grained models,Conformational ensembles,Polymer models,Single-molecule FRET,Distance distributions,Bayesian inference
更新于2025-09-10 09:29:36
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ROLE OF CYCLIC AND PSEUDO-CYCLIC ELECTRON TRANSPORT IN RESPONSE TO DYNAMIC LIGHT CHANGES IN PHYSCOMITRELLA PATENS
摘要： Photosynthetic organisms support cell metabolism by harvesting sunlight and driving the electron transport chain at the level of thylakoid membranes. Excitation energy and electron flow in the photosynthetic apparatus is continuously modulated in response to dynamic environmental conditions. Alternative electron flow around photosystem I plays a seminal role in this regulation contributing to photo-protection by mitigating over-reduction of the electron carriers. Different pathways of alternative electron flow coexist in the moss Physcomitrella patens, including cyclic electron flow mediated by the PGRL1/PGR5 complex and pseudo-cyclic electron flow mediated by the flavodiiron proteins FLV. In this work we generated P. patens plants carrying both pgrl1 and flva knock-out (KO) mutations. A comparative analysis of the WT, pgrl1, flva and pgrl1 flva lines suggests that cyclic and pseudo-cyclic processes have a synergic role in the regulation of photosynthetic electron transport. However, while both contribute to photosystem I protection from over-reduction by modulating electron flow following changes in environmental conditions, FLV activity is particularly relevant in the first seconds after a light change while PGRL1 has a major role upon sustained strong illumination.
关键词： Photosynthetic Reaction Center Complex Proteins,Photosynthesis,photoprotection,Energy metabolism,Evolution, Molecular,Bryophyta
更新于2025-09-10 09:29:36
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Transition metal complexes based aptamers as optical diagnostic tools for disease proteins and biomolecules
摘要： Aptamers are powerful recognition elements that can bind a large number of target molecules, including metal ions, small molecules, proteins, enzymes, even complex targets like cancer cells, etc., with high affinity and specificity. Hence, aptamer-based biosensors (hereafter named "aptasensors") have been extensively utilized in the field of clinical diagnostics and biomedical applications. In contrast to organic luminophores and quantum dots, luminescent transition metal complexes offer many desirable and wide-ranging properties, including tunable emission throughout the visible to NIR regions, long lifetime with a large Stockes shift, high quantum yield, good thermal, chemical and photochemical stability and metabolic inertness for biosensing applications. The incorporation of biomolecules or lipophilic entities into the metal complexes could overcome problems associated with membrane permeability and uptake by cells. Especially, Ru(II) and Ir(III) complexes are promising candidates for these potential applications. This review describes an overview of recent progress in the emerging area of aptasensors utilizing Ru(II) and Ir(III) transition metal complexes. To date, though aptasensors have been used in a wide variety of detection techniques, we have focused mainly on the luminescence approach in this article. Numerous aptasensors have illustrated promising detection results, even in complicated biological environments. If more rigorous research is continued on this area, it is hoped that in the future transition metal complexes based aptamers may show tremendous applications in biomedical research, especially diagnostics, imaging and drug delivery.
关键词： Proteins,Biosensor,Metal complexes,Luminescence,Aptamer
更新于2025-09-10 09:29:36
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Photoactivatable Reaction for Covalent Nanoscale Patterning of Multiple Proteins
摘要： This article describes a photochemical approach for independently patterning multiple proteins to an inert substrate, particularly for studies of cell adhesion. A photoactivatable chloropyrimidine ligand was employed for covalent immobilization of SnapTag fusion proteins on self-assembled monolayers of alkanethiolates on gold. A two-step procedure was used: first, patterned UV illumination of the surface activated protein capture ligands, and second, incubation with a SnapTag fusion protein bound to the surface in illuminated regions. Two different fluorescent proteins were patterned in registry with features of 400 nm in size over a 1 mm2 area. An example is given wherein an anti-carcinoembryonic antigen (anti-CEA) scFv antibody was patterned to direct the selective attachment of a human cancer cell line that express the CEA antigen. This method enables the preparation of surfaces with control over the density and activity of independently patterned proteins.
关键词： immobilization,photochemistry,monolayers,surface chemistry,proteins
更新于2025-09-10 09:29:36