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Redox state mediates embryonic stem cell (ESC) differentiation and thus offers an important complementary approach to understanding the pluripotency of stem cells. NADH redox ratio (NADH/ (Fp + NADH)), where NADH is the reduced fo...
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Redox state mediates embryonic stem cell (ESC) differentiation and thus offers an important complementary approach to understanding the pluripotency of stem cells. NADH redox ratio (NADH/ (Fp + NADH)), where NADH is the reduced form of nicotinamide adenine dinucleotide and Fp is the oxidized flavoproteins, has been established as a sensitive indicator of mitochondrial redox state. In this paper, we report our redox imaging data on the mitochondrial redox state of mouse ESC (mESC) colonies and the implications thereof. The low-temperature NADH/Fp redox scanner was employed to image mESC colonies grown on a feeder layer of gamma-irradiated mouse , embryonic fibroblasts (MEFs) on glass cover slips. The result showed significant heterogeneity in the mitochondrial redox state within individual mESC colonies (size: ～200-440μm), exhibiting a core with a more reduced state than the periphery. This more reduced state positively correlates with the expression pattern of Oct4, a well-established marker of pluripotency. Our observation is the first to show the heterogeneity in the mitochondrial redox state within a mESC colony, suggesting that mitochondrial redox state should be further investigated as a potential new bio-marker for the sternness of embryonic stem cells.
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Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism as well as redox signaling events. Under oxidative stress conditions, the tripeptide glutath...
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Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism as well as redox signaling events. Under oxidative stress conditions, the tripeptide glutathione can switch from its reduced form (GSH) to oxidized glutathione disulfide (GSSG), and thus, forms an important cellular redox buffer. GSSG is normally reduced to GSH by 2 glutathione reductase (GR) isoforms encoded in the Arabidopsis genome, cytosolic GR1 and GR2 dual-targeted to chloroplasts and mitochondria. Measurements of total GR activity in leaf extracts of wild-type and 2 gr1 deletion mutants revealed that ≈65% of the total GR activity is attributed to GR1, whereas ≈35% is contributed by GR2. Despite the lack of a large share in total GR activity, gr1 mutants do not show any informative phe-notype, even under stress conditions, and thus, the physiological impact of GR1 remains obscure. To elucidate its role in plants, glutathione-specific redox-sensitive GFP was used to dynamically measure the glutathione redox potential (E_(GSH)) in the cytosol. Using this tool, it is shown that £gsh in gr1 mutants is significantly shifted toward more oxidizing conditions. Surprisingly, dynamic reduction of GSSG formed during induced oxidative stress in gr1 mutants is still possible, although significantly delayed compared with wild-type plants. We infer that there is functional redundancy in this critical pathway. Integrated biochemical and genetic assays identify the NADPH-dependent thioredoxin system as a backup system for GR1. Deletion of both, NADPH-dependent thioredoxin reductase A and GR1, prevents survival due to a pollen lethal phenotype.
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Although conventional radiotherapy can directly damage DNA and other organic molecules within cells, most of the damage and the cytotoxicity of such ionising radiation, comes from the production of ions and free radicals produced ...
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Although conventional radiotherapy can directly damage DNA and other organic molecules within cells, most of the damage and the cytotoxicity of such ionising radiation, comes from the production of ions and free radicals produced via interactions with water. This 'indirect effect', a form of oxidative stress, can be modulated by a variety of systems within cells that are in place to, in normal situations, maintain homeostasis and redox balance. If cancer cells express high levels of antioxidant redox proteins, they may be more resistant to radiation and so targeting such systems may be a profitable strategy to increase therapeutic efficacy of conventional radiotherapy. An overview, with exemplars, of the main systems regulating redox homeostasis is supplied and discussed in relation to their use as prognostic and predictive biomarkers, and how targeting such proteins and systems may increase radiosensitivity and, potentially, improve the radiotherapeutic response.
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Magnetic Resonance Imaging (MRI) is a powerful imaging tool that combined with the use of contrast agents (CA) provides meaningful and crucial information both in the research and clinical settings. In recent years, strides have b...
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Magnetic Resonance Imaging (MRI) is a powerful imaging tool that combined with the use of contrast agents (CA) provides meaningful and crucial information both in the research and clinical settings. In recent years, strides have been taken towards the development of new CAs with enhanced capabilities (specificity, responsiveness, multimodality ...) to better diagnose and monitor physiological and pathological conditions in the body. Despite recent development, there are still challenges in the use of MRI to quantify specific functional or metabolic processes. The application of a ratiometric approach, in which images are acquired from the same sample using two different MR acquisitions, has potential to greatly improve the utility of MRI. In order to make this ratiometric approach work, a new generation of ratiometric probes are being developed which will be discussed in this review, classifying them based on the biological parameter under study as well as on the ratiometric analysis approach. Finally, the advantages and feasibility of using this methodology in a routine way will also be discussed. (C) 2021 Elsevier B.V. All rights reserved.
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Anthraquinone has found diverse use within the sphere of coordination chemistry. The sheer breadth of application in this context has been driven by the remarkable physical and biological properties that are known for a wide varie...
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Anthraquinone has found diverse use within the sphere of coordination chemistry. The sheer breadth of application in this context has been driven by the remarkable physical and biological properties that are known for a wide variety of anthraquinone derivatives. This review discusses three main areas of research and development: (ⅰ) the coordination chemistry of substituted anthraquinones, including self-assembled systems, coordination polymers and metal organic frameworks; (ⅱ) the incorporation of anthraquinone into ligand structures and their resultant chemistry with both d- and/-block metal ions; and (ⅲ) the application of metal-anthraquinone species to biological and chemosensing disciplines. Key aspects regarding synthetic approaches and physical characteristics are discussed throughout, with particular attention and focus on the electronic and redox properties of the species in question.
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Living organisms produce hydrogen peroxide (H_2O_2) to kill invading pathogens and for cellular signaling, but aberrant generation of this reactive oxygen species is a hallmark of oxidative stress and inflammation in aging, injury...
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Living organisms produce hydrogen peroxide (H_2O_2) to kill invading pathogens and for cellular signaling, but aberrant generation of this reactive oxygen species is a hallmark of oxidative stress and inflammation in aging, injury, and disease. The effects of H_2O_2 on the overall health of living animals remain elusive, in part owing to a dearth of methods for studying this transient small molecule in vivo. Here we report the design, synthesis, and in vivo applications of Peroxy Caged Luciferin-1 (PCL-1), a chemoselective bioluminescent probe for the real-time detection of H_2O_2 within living animals. PCL-1 is a boronic acid-caged firefly luciferin molecule that selectively reacts with H_2O_2 to release firefly luciferin, which triggers a bioluminescent response in the presence of firefly luciferase. The high sensitivity and selectivity of PCL-1 for H_2O_2, combined with the favorable properties of bioluminescence for in vivo imaging, afford a unique technology for real-time detection of basal levels of H_2O_2 generated in healthy, living mice. Moreover, we demonstrate the efficacy of PCL-1 for monitoring physiological fluctuations in H_2O_2 levels by directly imaging elevations in H_2O_2 within testosterone-stimulated tumor xenografts in vivo. The ability to chemoselectively monitor H_2O_2 fluxes in real time in living animals offers opportunities to dissect H_2O_2's disparate contributions to health, aging, and disease.
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Turbid tissues pose serious problems of strong absorption and scattering that make steady state fluorescence imaging methods less successful in imaging tissue layers deeper than a few tens of micrometers. Complications arise as on...
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Turbid tissues pose serious problems of strong absorption and scattering that make steady state fluorescence imaging methods less successful in imaging tissue layers deeper than a few tens of micrometers. Complications arise as one progresses from imaging cells to tissues to whole animal—which include enormous autofluores-cence background in tissues and poor signal from regions of interest. Since the steady state, intensity-based methods cannot discriminate the photons arising from the fluorophores and the autofluorescence background, it is almost impractical to isolate these two signals. We describe multiphoton fluorescence lifetime imaging methods in the time domain to demonstrate fluorescence lifetime contrast in discriminating autofluorescence background from the fluorescent signals. Since the photophysical schemes of the fluorophore and autofluorescence contributions are distinct, it is feasible to isolate these two contributions in every pixel based only on their decay constants without compromising the SNR. We present preliminary lifetime measurements to characterize autofluorescence in various cell lines and ex vivo tissues obtained from mouse models. Together, these results suggest a novel direction in obtaining quantitative information from endogenous tissue fluorescence without any exogenous staining. The prospects for this approach in metabolic redox imaging and disease diagnosis are discussed.
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Overhauser-enhanced MRI (OMRI) enables visualization of free radicals in animals based on dynamic nuclear polarization. Real-time data of tissue redox status gathered from kinetic images of redox-sensitive nitroxyl radical probes ...
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Overhauser-enhanced MRI (OMRI) enables visualization of free radicals in animals based on dynamic nuclear polarization. Real-time data of tissue redox status gathered from kinetic images of redox-sensitive nitroxyl radical probes using OMRI provided both anatomic and physiological information. Phantom experiments demonstrated the linear correlation between the enhancement factor and the concentration of a membrane-impermeable probe, carboxy-PROXYL (3-carboxy-2,2,5,5-tetramethyl- pyrrolidine-1-oxyl). Whole-body OMRI images illustrated the in vivo kinetics of carboxy-PROXYL for 25 min. Initial distribution was observed in lung, heart, liver, and kidney, but not brain, corresponding to its minimal lipophilicity. Based on these images (pixel size, 1.33×1.33 mm; slice thickness, 50 mm), a time-concentration curve with low coefficient of variance (<0.21) was created to assess pharmacokinetic behaviors. A biexponential curve showed a distribution phase from 1 to 10 min and an elimination phase from 15 to 25 min. The α rate constant was greater than the β rate constant in ROIs, confirming that its pharmacokinetics obeyed a two-compartment model. As a noninvasive technique, combining OMRI imaging with redox probes to monitor tissue redox status may be useful in acquiring valuable information regarding organ function for preclinical and clinical studies of oxidative diseases.
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Herein we show that faradaic electrochemistry can be confined to a discrete region of a monolithic semiconducting electrode by means of locally addressing the kinetics of electron transfer with a focused light-pointer. A Si(100) e...
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Herein we show that faradaic electrochemistry can be confined to a discrete region of a monolithic semiconducting electrode by means of locally addressing the kinetics of electron transfer with a focused light-pointer. A Si(100) electrode is modified with a self-assembled monolayer to which ferrocene is attached. The extent to which a model heterogeneous electrochemical reaction can be addressed in two-dimensions was explored at Si(100) photoanodes using scanning electrochemical microscopy operating in surface-generation/tip-collection mode. The link between the experimental radial profile of the catalytic current around the site of illumination and the substrate thickness indicates an inverse relationship between thickness and spatial resolution. We show to which extent the diffusion of charge carriers in the substrate governs the light-addressability. A spatial resolutions for our electrocatalytic system can be as low as ca. 385 mu m (ca. 5-times the size of the light pointer) using a non-structured Si (100) photoanode configured as a single-wire device. (C) 2017 Elsevier Ltd. All rights reserved.
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Cytosolic and organelle redox are highly interrelated, and their alterations play critical roles in both physio-logical and pathological cell states. This highly regulated process is crucial in life-death decisions of cells. Among...
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Cytosolic and organelle redox are highly interrelated, and their alterations play critical roles in both physio-logical and pathological cell states. This highly regulated process is crucial in life-death decisions of cells. Among organelles, the mitochondrion is the major source of intracellular-ROS and contributes to oxidation damage -induced cell death. Increase in cytosolic-redox and mitochondrial-redox is evident in cells undergoing diverse forms of cell death, such as apoptosis, necrosis, and necroptosis. The hierarchical profiling of redox signaling at the cytosol and mitochondria in a single cell is important to understand the relative contribution of each species in the initiation and shaping of cell death. Here, we demonstrate the potential application of ratiometric redox GFP (roGFP) and intensity-based redox-sensitive RFP (rxRFP) targeted to mitochondria in revealing both rapid and slow progressing changes in redox during cell division and in cells undergoing multiple modes of cell death. To generate imaging quality signal, single-cell clones stably expressing both roGFP at the cytosol and rxRFP probes targeted to mitochondria were generated. The cells provided sufficient temporal resolution with imaging -ready signal for the real-time visualization of rapidly progressing redox alterations at the cytosol and mito-chondria. The long-time imaging of the cells revealed that a moderate increase in cytosolic ROS marks the di-vision stage. Similarly, distinct forms of cell death trigger a unique and temporally regulated redox change at the cytosol and mitochondria, suggesting the potential utility of the sensor cells to dissect the nature of cell death pathways induced by specific forms of stress.
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