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Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future.

ABSTRACT: Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O₂^̇̄), hydrogen peroxide, lipid hydroperoxides, peroxynitrite, and hypochlorous and hypobromous acids play a key role in many pathophysiological processes. Recent studies have focused on mitochondrial ROS as redox signaling species responsible for promoting cell division, modulating and regulating kinases and phosphatases, and activating transcription factors. Many ROS also stimulate cell death and senescence. The extent to which these processes occur is attributed to ROS levels (low or high) in cells. However, the exact nature of ROS remains unknown. Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria. However, because most of these redox-active probes (untargeted and mitochondria-targeted) are oxidized by several ROS species, attributing redox probe oxidation to specific ROS species is difficult. It is conceivable that redox-active probes are oxidized in common one-electron oxidation pathways, resulting in a radical intermediate that either reacts with another oxidant (including oxygen to produce O₂^̇̄) and forms a stable fluorescent product or reacts with O₂^̇̄ to form a fluorescent marker product. Here, we propose the use of multiple probes and complementary techniques (HPLC, LC-MS, redox blotting, and EPR) and the measurement of intracellular probe uptake and specific marker products to identify specific ROS generated in cells. The low-temperature EPR technique developed to investigate cellular/mitochondrial oxidants can easily be extended to animal and human tissues.

SUBMITTER: Cheng G

PROVIDER: S-EPMC6028982 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Movement trajectories are usually recorded as a sequence of discrete movement events described by two parameters: step length (distance) and turning angle (bearing). One of the most widespread methods to record the geocoordinates of each step is by a GPS device. Such devices have limited suitability for recording fine movements of species with low dispersal ability including flightless carabid beetles at small spatio-temporal scales. As an alternative, the distance-bearing approach can avoid the measurement error of GPS units since it uses directly measured distances and compass azimuths. As no quantification of measurement error between distance-bearing and GPS approaches exists so far, we generated artificial fine-scale trajectories and in addition radio-tracked living carabids in a temperate forest and recorded each movement step by both methods. Trajectories obtained from distance-bearing were compared to those obtained by a GPS device in terms of movement parameters. Consequently, both types of trajectories were segmented by state-switching modeling into two distinct movement stages typical for carabids: random walk and directed movement. We found that the measurement error of GPS compared to distance-bearing was 1.878 m (SEM = 0.181 m) for distances and 31.330° (SEM = 2.066°) for bearings. Moreover, these errors increased under dense forest canopy and rainy weather. Distance error did not change with increasing distance recorded by distance-bearing but bearings were significantly more sensitive to error at short distances. State-switching models showed only slight, not significant, differences in movement states between the two methods in favor of the random walk in the distance-bearing approach. However, the shape of the GPS-measured trajectories considerably differed from those recorded by distance-bearing caused especially by bearing error at short distances. Our study showed that distance-bearing could be more appropriate for recording movement steps not only of ground-dwelling beetles but also other small animals at fine spatio-temporal scales.

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Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future.

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