Using the MitoB method to assess levels of reactive oxygen species in ecological studies of oxidative stress

In recent years evolutionary ecologists have become increasingly interested in the effects of reactive oxygen species (ROS) on the life-histories of animals. ROS levels have mostly been inferred indirectly due to the limitations of estimating ROS from in vitro methods. However, measuring ROS (hydrogen peroxide, H2O2) content in vivo is now possible using the MitoB probe. Here, we extend and refine the MitoB method to make it suitable for ecological studies of oxidative stress using the brown trout Salmo trutta as model. The MitoB method allows an evaluation of H2O2 levels in living organisms over a timescale from hours to days. The method is flexible with regard to the duration of exposure and initial concentration of the MitoB probe, and there is no transfer of the MitoB probe between fish. H2O2 levels were consistent across subsamples of the same liver but differed between muscle subsamples and between tissues of the same animal. The MitoB method provides a convenient method for measuring ROS levels in living animals over a significant period of time. Given its wide range of possible applications, it opens the opportunity to study the role of ROS in mediating life history trade-offs in ecological settings

Mitochondrial chaotic dynamics: Redox-energetic behavior at the edge of stability

Mitochondria serve multiple key cellular functions, including energy generation, redox balance, and regulation of apoptotic cell death, thus making a major impact on healthy and diseased states. Increasingly recognized is that biological network stability/instability can play critical roles in determining health and disease. We report for the first-time mitochondrial chaotic dynamics, characterizing the conditions leading from stability to chaos in this organelle. Using an experimentally validated computational model of mitochondrial function, we show that complex oscillatory dynamics in key metabolic variables, arising at the “edge” between fully functional and pathological behavior, sets the stage for chaos. Under these conditions, a mild, regular sinusoidal redox forcing perturbation triggers chaotic dynamics with main signature traits such as sensitivity to initial conditions, positive Lyapunov exponents, and strange attractors. At the “edge” mitochondrial chaos is exquisitely sensitive to the antioxidant capacity of matrix Mn superoxide dismutase as well as to the amplitude and frequency of the redox perturbation. These results have potential implications both for mitochondrial signaling determining health maintenance, and pathological transformation, including abnormal cardiac rhythms.publishedVersionKembro, Jackelyn Melissa. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; Argentina.Kembro, Jackelyn Melissa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina.Cortassa, Sonia. National Institutes of Health. NIH · NIA Intramural Research Program; Estados Unidos.Lloyd, David. Cardiff University. School of Biosciences 1; Inglaterra.Sollot, Steven. Johns Hopkins University. Laboratory of Cardiovascular Science; Estados Unidos.Sollot, Steven. Johns Hopkins University. Laboratory of Cardiovascular Science; Estados Unidos

Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes

In obesity, there is an increase in reactive oxygen species (ROS) within adipose tissue caused by increases in inflammation and overnutrition. Hormone sensitive lipase (HSL) is part of the canonical lipolytic pathway and critical for complete lipolysis. This study hypothesizes that ROS is a signal that integrates regulation of lipolysis by targeting HSL. Experiments were performed with human differentiated adipocytes from the subcutaneous depot. Antioxidants were employed as a tool to decrease ROS, and it was found that scavenging ROS with diphenyliodonium, N-acetyl cysteine, or resveratrol decreased lipolysis in adipocytes. HSL phosphorylation of a key serine residue, Ser552, as well as translocation of this enzyme from the cytosol to the lipid droplet upon lipolytic stimulation were both abrogated by scavenging ROS. The phosphorylation status of other serine residues on HSL were not affected. These findings are significant because they document that ROS contributes to the physiological regulation of lipolysis via an effect on translocation. Such regulation could be useful in developing new obesity therapies

The role of reactive oxygen species in adipogenic differentiation

Interest in reactive oxygen species and adipocyte differentiation/adipose tissue function is steadily increasing. This is due in part to a search for alternative avenues for combating obesity, which results from the excess accumulation of adipose tissue. Obesity is a major risk factor for complex disorders such as cancer, type 2 diabetes, and cardiovascular diseases. The ability of mesenchymal stromal/stem cells (MSCs) to differentiate into adipocytes is often used as a model for studying adipogenesis in vitro. A key focus is the effect of both intra- and extracellular reactive oxygen species (ROS) on adipogenesis. The consensus from the majority of studies is that ROS, irrespective of the source, promote adipogenesis. The effect of ROS on adipogenesis is suppressed by antioxidants or ROS scavengers. Reactive oxygen species are generated during the process of adipocyte differentiation as well as by other cell metabolic processes. Despite many studies in this field, it is still not possible to state with certainty whether ROS measured during adipocyte differentiation are a cause or consequence of this process. In addition, it is still unclear what the exact sources are of the ROS that initiate and/or drive adipogenic differentiation in MSCs in vivo. This review provides an overview of our understanding of the role of ROS in adipocyte differentiation as well as how certain ROS scavengers and antioxidants might affect this process.The South African Medical Research Council in terms of the SAMRC's Flagship Award Project SAMRC-RFA-UFSP-01-2013/STEM CELLS, the SAMRC Extramural Unit for Stem Cell Research and Therapy and the Institute for Cellular and Molecular Medicine of the University of Pretoria.http://www.springer.comseries/5584hj2019GeneticsImmunologyOral Pathology and Oral Biolog

Identification of molecular signatures specific for distinct cranial sensory ganglia in the developing chick

Background The cranial sensory ganglia represent populations of neurons with distinct functions, or sensory modalities. The production of individual ganglia from distinct neurogenic placodes with different developmental pathways provides a powerful model to investigate the acquisition of specific sensory modalities. To date there is a limited range of gene markers available to examine the molecular pathways underlying this process. Results Transcriptional profiles were generated for populations of differentiated neurons purified from distinct cranial sensory ganglia using microdissection in embryonic chicken followed by FAC-sorting and RNAseq. Whole transcriptome analysis confirmed the division into somato- versus viscerosensory neurons, with additional evidence for subdivision of the somatic class into general and special somatosensory neurons. Cross-comparison of distinct ganglia transcriptomes identified a total of 134 markers, 113 of which are novel, which can be used to distinguish trigeminal, vestibulo-acoustic and epibranchial neuronal populations. In situ hybridisation analysis provided validation for 20/26 tested markers, and showed related expression in the target region of the hindbrain in many cases. Results One hundred thirty-four high-confidence markers have been identified for placode-derived cranial sensory ganglia which can now be used to address the acquisition of specific cranial sensory modalities.</p

Crosstalk between reactive oxygen species and pro-inflammatory markers in developing various chronic diseases: a review

The inflammation process in the human body plays a central role in the pathogenesis of many chronic diseases. In addition, reactive oxygen species (ROS) exert potentially a decisive role in human body, particularly in physiological and pathological process. The chronic inflammation state could generate several types of diseases such as cancer, atherosclerosis, diabetes mellitus and arthritis, especially if it is concomitant with high levels of pro-inflammatory markers and ROS. The respiratory burst of inflammatory cells during inflammation increases the production and accumulation of ROS. However, ROS regulate various types of kinases and transcription factors such nuclear factor-kappa B which is related to the activation of pro-inflammatory genes. The exact crosstalk between pro-inflammatory markers and ROS in terms of pathogenesis and development of serious diseases is still ambitious. Many studies have been attempting to determine the mechanistic mutual relationship between ROS and pro-inflammatory markers. Therefore hereby, we review the hypothetical relationship between ROS and pro-inflammatory markers in which they have been proposed to initiate cancer, atherosclerosis, diabetes mellitus and arthritis

In vivo parameters influencing 2-Cys Prx oligomerization: The role of enzyme sulfinylation

Abstract2-Cys Prxs are H2O2-specific antioxidants that become inactivated by enzyme hyperoxidation at elevated H2O2 levels. Although hyperoxidation restricts the antioxidant physiological role of these enzymes, it also allows the enzyme to become an efficient chaperone holdase. The critical molecular event allowing the peroxidase to chaperone switch is thought to be the enzyme assembly into high molecular weight (HMW) structures brought about by enzyme hyperoxidation. How hyperoxidation promotes HMW assembly is not well understood and Prx mutants allowing disentangling its peroxidase and chaperone functions are lacking. To begin addressing the link between enzyme hyperoxidation and HMW structures formation, we have evaluated the in vivo 2-Cys Prxs quaternary structure changes induced by H2O2 by size exclusion chromatography (SEC) on crude lysates, using wild type (Wt) untagged and Myc-tagged S. cerevisiae 2-Cys Prx Tsa1 and derivative Tsa1 mutants or genetic conditions known to inactivate peroxidase or chaperone activity or altering the enzyme sensitivity to hyperoxidation. Our data confirm the strict causative link between H2O2-induced hyperoxidation and HMW formation/stabilization, also raising the question of whether CP hyperoxidation triggers the assembly of HMW structures by the stacking of decamers, which is the prevalent view of the literature, or rather, the stabilization of preassembled stacked decamers

HyPer-3: A Genetically Encoded H(2)O(2) Probe with Improved Performance for Ratiometric and Fluorescence Lifetime Imaging.

High-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. Here, we present HyPer-3, a genetically encoded fluorescent indicator for intracellular H(2)O(2) exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrate this performance by in vivo imaging of tissue-scale H(2)O(2) gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for single-wavelength fluorescent lifetime imaging of H(2)O(2) levels both in vitro and in vivo