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Aims: Protein disulfide isomerases (PDIs) are a family of chaperones resident in the endoplasmic reticulum (ER). In addition to holdase function, some members catalyze disulfide bond formation and isomerization, a crucial step for native folding and prevention of aggregation of misfolded proteins. PDIs are characterized by an arrangement of thioredoxin-like domains, with the canonical protein disulfide isomerase A1 (PDIA1) organized as four thioredoxin-like domains forming a horseshoe with two active sites, a and a′, at the extremities. We aimed to clarify important aspects underlying the catalytic cycle of PDIA1 in the context of the full pathways of oxidative protein folding operating in the ER. Results: Using two fluorescent redox sensors, redox green fluorescent protein 2 (roGFP2) and HyPer (circularly permutated yellow fluorescent protein containing the regulatory domain of the H2O2-sensing protein OxyR), either unfolded or native, as client substrates, we identified the N-terminal a active site of PDIA1 as the main oxidant of thiols. From there, electrons can flow to the C-terminal a′ active site, with the redox-dependent conformational flexibility of PDIA1 allowing the formation of an interdomain disulfide bond. The a′ active site then acts as a crossing point to redirect electrons to ER downstream oxidases or back to client proteins to reduce scrambled disulfide bonds. Innovation and Conclusions: The two active sites of PDIA1 work cooperatively as an interdomain redox relay mechanism that explains PDIA1 oxidative activity to form native disulfides and PDIA1 reductase activity to resolve scrambled disulfides. This mechanism suggests a new rationale for shutting down oxidative protein folding under ER redox imbalance. Whether it applies to physiological substrates in cells remains to be shown. [ABSTRACT FROM AUTHOR]

Objectives Synthetic biology has emerged from molecular biology and engineering approaches and aims to develop novel, biologically-inspired systems for industrial and basic research applications ranging from biocomputing to drug production. Surprisingly, redoxin (thioredoxin, glutaredoxin, peroxiredoxin) and other thiol-based redox systems have not been widely utilized in many of these synthetic biology applications. Methods We reviewed thiol-based redox systems and the development of synthetic biology applications that have used thiol-dependent parts. Results The development of circuits to facilitate cytoplasmic disulfide bonding, biocomputing and the treatment of intestinal bowel disease are amongst the applications that have used thiol-based parts. We propose that genetically encoded redox sensors, thiol-based biomaterials and intracellular hydrogen peroxide generators may also be valuable components for synthetic biology applications. Discussion Thiol-based systems play multiple roles in cellular redox metabolism, antioxidant defense and signaling and could therefore offer a vast and diverse portfolio of components, parts and devices for synthetic biology applications. However, factors limiting the adoption of redoxin systems for synthetic biology applications include the orthogonality of thiol-based components, limitations in the methods to characterize thiol-based systems and an incomplete understanding of the design principles of these systems.

Synthetic biology has emerged from molecular biology and engineering approaches and aims to develop novel, biologically-inspired systems for industrial and basic research applications ranging from biocomputing to drug production. Surprisingly, redoxin (thioredoxin, glutaredoxin, peroxiredoxin) and other thiol-based redox systems have not been widely utilized in many of these synthetic biology applications. We reviewed thiol-based redox systems and the development of synthetic biology applications that have used thiol-dependent parts. The development of circuits to facilitate cytoplasmic disulfide bonding, biocomputing and the treatment of intestinal bowel disease are amongst the applications that have used thiol-based parts. We propose that genetically encoded redox sensors, thiol-based biomaterials and intracellular hydrogen peroxide generators may also be valuable components for synthetic biology applications. Thiol-based systems play multiple roles in cellular redox metabolism, antioxidant defense and signaling and could therefore offer a vast and diverse portfolio of components, parts and devices for synthetic biology applications. However, factors limiting the adoption of redoxin systems for synthetic biology applications include the orthogonality of thiol-based components, limitations in the methods to characterize thiol-based systems and an incomplete understanding of the design principles of these systems. [ABSTRACT FROM AUTHOR]

Selenium‐containing monomer (p‐phenylseleno) styrene (p‐PhSeSt) is polymerized by reversible addition‐fragmentation chain transfer polymerization. Polymer, (P(p‐PhSeSt)), with controlled molecular weight and narrow molecular weight is obtained. The selenide moiety in obtained P(p‐PhSeSt) can be selectively oxidized to selenoxide or selenone groups by H2O2 or NaClO, respectively. These oxidized groups can be further reduced to selenide by Na2S2O4. The structure changing of polymers during such redox cycle is characterized by nuclear magnetic resonance, X‐ray photoelectron spectroscopy, and size exclusion chromatography. Properties, such as thermal performance, glass transition temperature, water contact angles, and refractive indices, of the resulting polymers are systematically investigated before and after oxidation. In addition, SiO2 inverse opal photonic crystal (IOPC) is fabricated by sacrificial polymer colloidal template method. Owing to changes of the RIs of P(p‐PhSeSt) after selective oxidation, the predictable change of PC bandgap as a redox‐responsive PC sensor is successfully realized, which provides new perspectives for modulating photonic crystals. [ABSTRACT FROM AUTHOR]

Significance: Redox homeostasis consists of an intricate network of reactions in which reactive molecular species, redox modifications, and redox proteins act in concert to allow both physiological responses and adaptation to stress conditions. Recent Advances: This review highlights established and novel thiol-based regulatory pathways underlying the functional facets and significance of redox biology in photosynthetic organisms. In the last decades, the field of redox regulation has largely expanded and this work is aimed at giving the right credit to the importance of thiol-based regulatory and signaling mechanisms in plants. Critical Issues: This cannot be all-encompassing, but is intended to provide a comprehensive overview on the structural/molecular mechanisms governing the most relevant thiol switching modifications with emphasis on the large genetic and functional diversity of redox controllers (i.e., redoxins). We also summarize the different proteomic-based approaches aimed at investigating the dynamics of redox modifications and the recent evidence that extends the possibility to monitor the cellular redox state in vivo. The physiological relevance of redox transitions is discussed based on reverse genetic studies confirming the importance of redox homeostasis in plant growth, development, and stress responses. Future Directions: In conclusion, we can firmly assume that redox biology has acquired an established significance that virtually infiltrates all aspects of plant physiology. [ABSTRACT FROM AUTHOR]

Significance: Cellular reactive oxygen species (ROS) mediate redox signaling cascades that are critical to numerous physiological and pathological processes. Analytical methods to monitor cellular ROS levels and proteomic platforms to identify oxidative post-translational modifications (PTMs) of proteins are critical to understanding the triggers and consequences of redox signaling. Recent Advances: The prevalence and significance of redox signaling has recently been illuminated through the use of chemical probes that allow for sensitive detection of cellular ROS levels and proteomic dissection of oxidative PTMs directly in living cells. Critical Issues: In this review, we provide a comprehensive overview of chemical probes that are available for monitoring ROS and oxidative PTMs, and we highlight the advantages and limitations of these methods. Future Directions: Despite significant advances in chemical probes, the low levels of cellular ROS and low stoichiometry of oxidative PTMs present challenges for accurately measuring the extent and dynamics of ROS generation and redox signaling. Further improvements in sensitivity and ability to spatially and temporally control readouts are essential to fully illuminate cellular redox signaling. [ABSTRACT FROM AUTHOR]

Cellular redox status affects diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus, individual differences in redox status can give rise to distinct sub-populations even among cells with identical genetic backgrounds. Here, we have created a novel methodology to track redox status at single cell resolution using the redox-sensitive probe Grx1-roGFP2. Our method allows identification and sorting of sub-populations with different oxidation levels in either the cytosol, mitochondria or peroxisomes. Using this approach, we defined a redox-dependent heterogeneity of yeast cells and characterized growth, as well as proteomic and transcriptomic profiles of distinctive redox subpopulations. We report that, starting in late logarithmic growth, cells of the same age have a bi-modal distribution of oxidation status. A comparative proteomic analysis between these populations identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis.

Plants often face combinatorial stresses in their natural environment. Here, arsenic (As) toxicity was combined with hypoxia (Hpx) in the roots of Arabidopsis thaliana as it often occurs in nature. Arsenic inhibited growth of both roots and leaves, whereas root growth almost entirely ceased in Hpx. Growth efficiently resumed, and Hpx marker transcripts decreased upon reaeration. Compromised recovery from HpxAs treatment following reaeration indicated some persistent effects of combined stresses despite lower As accumulation. Root glutathione redox potential turned more oxidized in Hpx and most strongly in HpxAs. The more oxidizing root cell redox potential and the lowered glutathione amounts may be conducive to the growth arrest of plants exposed to HpxAs. The stresses elicited changes in elemental and transcriptomic composition. Thus, calcium, magnesium, and phosphorous amounts decreased in rosettes, but the strongest decline was seen for potassium. The reorganized potassium‐related transcriptome supports the conclusion that disturbed potassium homeostasis contributes to the growth phenotype. In a converse manner, photosynthesis‐related parameters were hardly affected, whereas accumulated carbohydrates under all stresses and anthocyanins under Hpx exclude carbohydrate limitation. The study demonstrates the existence of both synergistic since mutually aggravating effects and antagonistic effects of single and combined stresses. The present study evaluates the physiological, biochemical, and transcriptomic changes induced by co‐occurrence of arsenic toxicity and hypoxia. The severe decline in nutrient contents especially K and S along with the perturbations in redox homeostasis leads to growth arrest as well as compromised recovery in combined stressed plants. The functional characterization of transcripts, which showed stress combination‐specific alterations, provides information on differences in signalling patterns between individual stresses and stress combination. The existence of both synergistic since aggravating effects and antagonistic changes of single and combined stresses is demonstrated. [ABSTRACT FROM AUTHOR]

Cellular heterogeneity is a widespread phenomenon, existing across organisms and serving a crucial role in evolution and cell survival. Genetically identical cells may as a result present in a variety of forms with different gene and protein expressions, as well as oxidation level. As a result, a wide range of methodologies and techniques for dissecting different types of genetic, proteomic, and phenotypic heterogeneous traits have emerged in recent years in an effort to better understand how diversity exists within a single population and its effects therein. A key area of interest seeks to establish the ways in which cellular heterogeneity and aging processes interact with each other. Here, we discuss recent developments in defining cellular heterogeneity, specifically focusing on redox-dependent heterogeneity, its characterization, quantification, and behavior. We further expand on potential applications of a cell sorting-based methodology for distinguishing between cells harboring different redox statuses. As an example, we use organelle-specific fluorescence protein-based probes to examine the crosstalk between cytosol and mitochondria in a yeast strain lacking glutathione reductase. Together, these may have wide-reaching implications for future research into redox-associated factors, as well as mechanisms of redox-dependent heterogeneity and its influence on organelles and the cell at large. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H2O2) is an important target material for detecting biomolecules including acetylcholine (ACh), glutamate (Glu), and glucose. In this study, we report on H2O2 detection under biological environments based on the redox reaction. The redox potential change caused by the reaction between the electron mediators of ferrocenes and H2O2 catalyzed by horseradish peroxidase (HRP) was measured using a gold electrode connected to a source follower circuit. The mediators were either dissolved in sample solutions using ferrocenyl methanol (FcMeOH) or immobilized on the sensor surface in the form of 11-ferrocenyl-1- undecanethiol (11-FUT). H2O2 detection under biological environments was demonstrated in both samples. The overall outputs in the 11-FUT-immmobilzed electrodes were lower than those in the samples with dissolved FcMeOH. The detection range of H2O2 was from 10-5 to 10-3 M for the samples with dissolved FcMeOH, while it was from 10-4 to 10-2 M for the 11-FUT-immobilized electrodes. It was suggested that the oxidation of the mediators by H2O2 insufficiently took place in the 11-FUT-immobilized electrodes, leading to the lower outputs. [ABSTRACT FROM AUTHOR]

Significance: Genetically encoded hydrogen peroxide (H2O2) sensors, based on fusions between thiol peroxidases and redox-sensitive green fluorescent protein 2 (roGFP2), have dramatically broadened the available “toolbox” for monitoring cellular H2O2 changes. Recent Advances: Recently developed peroxiredoxin-based probes such as roGFP2-Tsa2ΔCR offer considerably improved H2O2 sensitivity compared with previously available genetically encoded sensors and now permit dynamic, real-time, monitoring of changes in endogenous H2O2 levels. Critical Issues: The correct understanding and interpretation of probe read-outs is crucial for their meaningful use. We discuss probe mechanisms, potential pitfalls, and best practices for application and interpretation of probe responses and highlight where gaps in our knowledge remain. Future Directions: The full potential of the newly available sensors remains far from being fully realized and exploited. We discuss how the ability to monitor basal H2O2 levels in real time now allows us to re-visit long-held ideas in redox biology such as the response to ischemia-reperfusion and hypoxia-induced reactive oxygen species production. Further, recently proposed circadian cycles of peroxiredoxin hyperoxidation might now be rigorously tested. Beyond their application as H2O2 probes, roGFP2-based H2O2 sensors hold exciting potential for studying thiol peroxidase mechanisms, inactivation properties, and the impact of post-translational modifications, in vivo. Antioxid. Redox Signal. 29, 552–568. [ABSTRACT FROM AUTHOR]

Summary Glutathione is the major low-molecular weight thiol of eukaryotic cells. It is central to one of the two major NADPH-dependent reducing systems and is likely to play a role in combating oxidative stress, a process suggested to play a key role in Alzheimer’s disease (AD). However, the nature and relevance of redox changes in the onset and progression of AD are still uncertain. Here, we combine genetically encoded redox sensors with our Drosophila models of amyloid-beta (Aβ) aggregation. We find that changes in glutathione redox potential (E GSH ) closely correlate with disease onset and progression. We observe this redox imbalance specifically in neurons, but not in glia cells. E GSH changes and Aβ 42 deposition are also accompanied by increased JNK stress signaling. Furthermore, pharmacologic and genetic manipulation of glutathione synthesis modulates Aβ 42 -mediated neurotoxicity, suggesting a causal relationship between disturbed glutathione redox homeostasis and early AD pathology. [ABSTRACT FROM AUTHOR]

Preclinical Research Cancer metastasis is the major cause of cancer ‐ related mortality. Accumulated evidence has shown that high ‐ metastasis potential cancer cells have more reactive oxygen species (ROS) accumulation compared with low ‐ metastasis potential cancer cells. ROS can function as second messengers to regulate multiple cancer metastasis ‐ related signaling pathways via reversible oxidative posttranslational modifications of cysteine in key redox ‐ sensitive proteins, which leads to the structural and functional change of these proteins. Because ROS can promote cancer metastasis, therapeutic strategies aiming at inducing/reducing cellular ROS level or targeting redox sensors involved in metastasis hold great potential in developing new efficient approaches for anticancer therapy. In this review, we summarize recent findings on regulation of tumor metastasis by key redox sensors and describe the potential of targeting redox signaling pathways for cancer therapy. [ABSTRACT FROM AUTHOR]

Abstract: In this work a theoretical and experimental analysis of the spectrum of methylene blue is made in order to clarify the nature of the shoulder appearing at 620nm. This shoulder has been several times attributed to the existence of an hypothetical methylene blue dimer. The results here obtained do not agree with the existence of such a dimeric specie, and point out differences in the ionic strength of the solution as the phenomenon responsible for the variations observed in these spectra. [Copyright &y& Elsevier]

Redox indicators were employed to monitor redox status in a bioaugmented, sediment-packed column during the dechlorination of tetrachloroethene (PCE) to ethene (ETH). The speciation of the indicators thionine and cresyl violet, immobilized on transparent films, was spectrometrically monitored with a flow sensor based on circulating the column solution through a specially constructed flow cell placed in a conventional spectrometer. A fiber optic redox probe based on immobilized azure C was constructed. A 75-cm column with 4 sets of ports along the column axis at regular intervals was constructed and packed with aquifer material. These ports enabled sampling to determine concentrations of chlorinated ethene species and allowed for in situ or non-invasive monitoring of redox conditions with negligible O2 contamination. The flow sensor and fiber optic sensor showed similar responses to redox conditions in the column. After 60 days, complete conversion of PCE to ETH occurred by the end of the column and the redox level indicated by the indicators was consistent throughout the column. Significant formation of vinyl chloride or ETH was observed only after significant reduction of cresyl violet. [ABSTRACT FROM AUTHOR]

Two-component regulatory systems that respond to changes in redox potential have recently been discovered in bacteria. 'Redox sensors' are defined as electron carriers which initiate control of gene expression upon oxidation or reduction. 'Redox response regulators' are defined as DNA-binding proteins which modify gene expression as a result of the action of redox sensors. Redox sensors and redox response regulators may comprise a mechanism for feedback control of redox potential in photosynthetic electron transport chains, thereby protecting plants, algae and photosynthetic bacteria from damage caused by electrochemistry operating on inappropriate electron donors and acceptors. Chloroplast redox sensors and redox response regulators, themselves encoded in the nucleus, may place chloroplast gene expression under redox regulatory control. This may account for the persistence, in evolution, of chloroplast genomes, and for the constancy of the sub-set of chloroplast proteins encoded and synthesised in situ. These and other predictions are discussed. [ABSTRACT FROM AUTHOR]

Genetically encoded redox-sensitive fluorescent proteins are very useful tools for investigating the occurrence and consequences of reactive species (RS) in living cells. However, light-induced side effects are serious confounding factors [Edwards, A.M. & Silva, E.,2001; Eichler, M. et al.,2005]. Therefore, we have previously introduced a genetically engineered redox-sensitive sodium channel (rNaV1.4 mutant M1305C; roNaV1) as a non-photonic tool for measuring membrane-delimited cellular RS [Ojha, N.K. et al.,2014]. Here we introduce a second-generation NaV-channel based RS sensor, containing selenocysteine in its inactivation motif (rNaV1.4 mutant M1305U; roNaV2), with strongly increased RS sensitivity. Kinetics of loss of channel inactivation was assayed for application of 1 µM chloramine T: While the ratiometric signal of roNaV1 was basically insensitive to that low concentration, roNaV2 responded with a time constant of 300 s. Likewise, roNaV2 responded rapidly to 500 µM H 2 O 2 (ca. 50 s). Most importantly, roNaV2 responds to blue light (470 nm) induced chemical modification with a time constant of about 300 ms, while roNaV1 was 1,000- and wild-type NaV1.4 even 10,000-times slower. Thus, roNaV2 allows for the assessment of chemical modification induced in fluorescence microscopy settings with high sensitivity and time resolution. [ABSTRACT FROM AUTHOR]

Glutathione is the major low-molecular weight thiol of eukaryotic cells. It is central to one of the two major NADPH-dependent reducing systems and is likely to play a role in combating oxidative stress, a process suggested to play a key role in Alzheimer's disease (AD). However, the nature and relevance of redox changes in the onset and progression of AD are still uncertain. Here, we combine genetically encoded redox sensors with our Drosophila models of amyloid-beta (Aβ) aggregation. We find that changes in glutathione redox potential (E GSH ) closely correlate with disease onset and progression. We observe this redox imbalance specifically in neurons, but not in glia cells. E GSH changes and Aβ 42 deposition are also accompanied by increased JNK stress signaling. Furthermore, pharmacologic and genetic manipulation of glutathione synthesis modulates Aβ 42 -mediated neurotoxicity, suggesting a causal relationship between disturbed glutathione redox homeostasis and early AD pathology., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)