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At least 163 records · Page 9

Chlamydomonas as a model for reactive oxygen species signaling and thiol redox regulation in the green lineage

While the maintenance of redox balance is important for all organisms, the energy balance in the chloroplast is particularly tightly linked to the overall cellular redox status in photosynthetic organisms. Excess light can cause overreduction of the photosynthetic electron transport chain, which, when unchecked, results in the generation of reactive oxygen species (ROS) that can oxidize cellular components including nucleic acids, proteins, and lipids (Li et al., 2009). A photosynthetic cell possesses multiple layers of defense against oxidative stress, such as antioxidant enzymes (Foyer and Noctor, 2005) and alternative electron transfer pathways (Alric and Johnson, 2017). At the same time, ROS can function as important signals that mediate responses to biotic and abiotic stresses (Apel and Hirt, 2004). Reversibly oxidizable small molecules and proteins can serve as ROS sinks and signals and are re-reduced by multiple mechanisms. For example, the ascorbate–glutathione (GSH) cycle is central in controlling ROS levels and regenerating/reducing oxidized cellular redox agents (Figure 1A; reviewed by Foyer and Noctor, 2011). The functions of many proteins can be regulated by the redox state of cysteine residues, such as the formation or reduction of disulfides by thioredoxins (TRX) (Buchanan, 1991) and glutaredoxins (Michelet et al., 2013b; Figure 1, B and C).

59 BASIC BIOLOGICAL SCIENCES↗

Triiodide Attacks the Organic Cation in Hybrid Lead Halide Perovskites: Mechanism and Suppression

Molecular I 2 can be produced from iodide-based lead perovskites under thermal stress; triiodide, I 3 − , is formed from this I 2 and I − . Triiodide attacks protic cation MA + - or FA + -based lead halide perovskites (MA + , methylammonium; FA + , formamidinium) as explicated through solution-based nuclear magnetic resonance (NMR) studies: triiodide has strong hydrogen-bonding affinity for MA + or FA + , which leads to their deprotonation and perovskite decomposition. Triiodide is a catalyst for this decomposition that can be obviated through perovskite surface treatment with thiol reducing agents. In contrast to methods using thiol incorporation into perovskite precursor solutions, no penetration of the thiol into the bulk perovskite is observed, yet its surface application stabilizes the perovskite against triiodide-mediated thermal stress. Thiol applied to the interface between FAPbI 3 and Spiro-OMeTAD (“Spiro”) prevents oxidized iodine species penetration into Spiro and thus preserves its hole-transport efficacy. Surface-applied thiol affects the perovskite work function; it ameliorates hole injection into the Spiro overlayer, thus improving device performance. It helps to increase interfacial adhesion (“wetting”): fewer voids are observed at the Spiro/perovskite interface if thiols are applied. Perovskite solar cells (PSCs) incorporating interfacial thiol treatment maintain over 80% of their initial power conversion efficiency (PCE) after 300 h of 85 °C thermal stress.

36 MATERIALS SCIENCE↗

Application of Atmospheric Pressure Photoionization H/D-exchange Mass Spectrometry for Speciation of Sulfur-containing Compounds

Herein we report the observation of atmospheric pressure in-source hydrogen–deuterium exchange (HDX) of thiol group for the first time. The HDX for thiol group was optimized for positive atmospheric pressure photoionization (APPI) mass spectrometry (MS). The optimized HDX-MS was applied for 31 model compounds (thiols, thiophenes, and sulfides) to demonstrate that exchanged peaks were observed only for thiols. The optimized method has been successfully applied to the isolated fractions of sulfur-rich oil samples. The exchange of one and two thiol hydrogens with deuterium was observed in the thiol fraction; no HDX was observed in the other fractions. Thus, the results presented in this study demonstrate that the HDX-MS method using APPI ionization source can be effective for speciation of sulfur compounds. This method has the potential to be used to access corrosion problems caused by thiol-containing compounds. .

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

High methylation potential of mercury complexed with mixed thiolate ligands by Geobacter sulfurreducens PCA

We report some thiols, such as cysteine (CYS) at moderate concentrations (10–500 µM), can enhance methylmercury (MeHg) formation by Geobacter sulfurreducens PCA, whereas others such as dithiol 2,3-dimercaptopropanesulfonate (DMPS) and 2,3-dimercaptosuccinic acid (DMSA) abolish mercury [Hg(II)] methylation. Little is known, however, about whether Hg(II) methylation could be enhanced or inhibited by the presence of mixed thiol ligands at low concentrations observed in the environment. Surprisingly we found that mixing CYS (1 µM) with DMPS (0.025–0.5 µM) or DMSA (0.025–1 µM) substantially increased MeHg production by 1.5–3.5-fold, compared to the no-thiol control, whereas complexation with a single DMPS, or DMSA, or CYS (1 µM) strongly inhibited Hg(II) methylation. Pre-equilibration between Hg(II) and thiols before the addition of cells was necessary to observe enhanced methylation. Spectroscopic analyses indicated the formation of mixed or heteroleptic coordinated Hg(II)-S 3 /S 4 complexes, which likely facilitated exchange of Hg(II) with cells and its uptake and internal transfer to the HgcAB proteins required for methylation. These results suggest that the effects of thiols on Hg(II) methylation were more complex than previously thought (using a single thiol) and thus underscore the importance of understanding how mixed thiols and their interactions with Hg(II) may ultimately influence MeHg production in the natural environment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Striped gold nanoparticles: New insights from molecular dynamics simulations

Recent simulations have improved our knowledge of the molecular-level structure and hydration properties of mixed self-assembled monolayers (SAMs) with equal and unequal alkyl thiols at three different arrangements, namely, random, patchy, and Janus. In our previous work [V. Vasumathi et al., J. Phys. Chem. C 119, 3199–3209 (2015)], we showed that the bending of longer thiols over shorter ones clearly depends on the thiols’ arrangements and chemical nature of their terminal groups. In addition, such a thiol bending revealed to have a strong impact on the structural and hydration properties of SAMs coated on gold nanoparticles (AuNPs). In this paper, we extend our previous atomistic simulation study to investigate the bending of longer thiols by increasing the stripe thickness of mixed SAMs of equal and unequal lengths coated on AuNPs. We study also the effect of stripe thickness on the structural morphology and hydration of the coated SAMs. Our results show that the structural and hydration properties of SAMs are affected by the stripe thickness for mixtures of alkyl thiols with unequal chain length but not for equal length. Hence, the stability of the stripe configuration depends on the alkyl’s chain length, the length difference between the thiol mixtures, and solvent properties.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A deep redox proteome profiling workflow and its application to skeletal muscle of a Duchenne Muscular Dystrophy model

Perturbation to the redox state accompanies many diseases and its effects are viewed through oxidation of biomolecules, including proteins, lipids, and nucleic acids. The thiol groups of protein cysteine residues undergo an array of redox post-translational modifications (PTMs) that are important for regulation of protein and pathway function. To better understand what proteins are redox regulated following a perturbation, it is important to be able to comprehensively profile protein thiol oxidation at the proteome level. Herein, we report a deep redox proteome profiling workflow and demonstrate its application in measuring the changes in thiol oxidation along with global protein expression in skeletal muscle from mdx mice, a model of Duchenne Muscular Dystrophy (DMD). In depth coverage of the thiol proteome was achieved with >18,000 Cys sites from 5608 proteins in muscle being quantified. Compared to the control group, mdx mice exhibit markedly increased thiol oxidation, where ~2% shift in the median oxidation occupancy was observed. Further, pathway analysis for the redox data revealed that coagulation system and immune-related pathways were among the most susceptible to increased thiol oxidation in mdx mice, whereas protein abundance changes were more enriched in pathways associated with bioenergetics. This study illustrates the importance of deep redox profiling in gaining greater insight into oxidative stress regulation and pathways/processes that are perturbed in an oxidizing environment.

60 APPLIED LIFE SCIENCES↗

Intracellular Hg(0) Oxidation in Desulfovibrio desulfuricans ND132

The disposal of elemental mercury (Hg(0)) wastes in mining and manufacturing areas has caused serious soil and groundwater contamination issues. Under anoxic conditions, certain anaerobic bacteria can oxidize dissolved elemental mercury and convert the oxidized Hg to neurotoxic methylmercury. In this study, we conducted experiments with the Hg-methylating bacterium Desulfovibrio desulfuricans ND132 to elucidate the role of cellular thiols in anaerobic Hg(0) oxidation. The concentrations of cell-surface and intracellular thiols were measured, and specific fractions of D. desulfuricans ND132 were examined for Hg(0) oxidation activity and analyzed with extended X-ray absorption fine structure (EXAFS) spectroscopy. The experimental data indicate that intracellular thiol concentrations are approximately six times higher than those of the cell wall. Cells reacted with a thiol-blocking reagent were severely impaired in Hg(0) oxidation activity. Spheroplasts lacking cell walls rapidly oxidized Hg(0) to Hg(II), while cell wall fragments exhibited low reactivity toward Hg(0). EXAFS analysis of spheroplast samples revealed that multiple different forms of Hg-thiols are produced by the Hg(0) oxidation reaction and that the local coordination environment of the oxidized Hg changes with reaction time. Here, the results of this study indicate that Hg(0) oxidation in D. desulfuricans ND132 is an intracellular process that occurs by reaction with thiol-containing molecules.

54 ENVIRONMENTAL SCIENCES↗

Characterization of cellular oxidative stress response by stoichiometric redox proteomics

The thiol redox proteome refers to all proteins whose cysteine thiols are subjected to various redox-dependent posttranslational modifications (PTMs) including S-glutathionylation (SSG), S-nitrosylation (SNO), S-sulfenylation (SOH), and S-sulfhydration (SSH). These modifications can impact various aspects of protein function, including activity, binding, conformation, localization, and interactions with other molecules. It is highly desirable to have robust redox proteomics methods that can provide global, site-specific, and stoichiometric quantification of redox PTMs in order to elucidate the complex redox landscape and identify novel redox regulators in signaling and regulation. Mass spectrometry-based redox proteomics has emerged as the primary platform for enabling the broad characterization of thiol PTMs in cells and tissues. Herein we review recent advances in MS-based redox proteomics approaches for quantitative profiling of redox PTMs in cells and tissues at physiological or oxidative stress conditions and highlight some recent applications using these enabling techniques. Considering the relative maturity of available methods, a primary emphasis will be on two types of modifications: 1) total oxidation (i.e., all reversible thiol modifications), the level of which represents the overall redox state, and 2) S-glutathionylation, a major form of reversible thiol oxidation. We also discuss the significance of stoichiometric measurements of thiol PTMs as well as future perspectives towards a better understanding of cellular redox regulatory networks in cells and tissues.

59 BASIC BIOLOGICAL SCIENCES↗

Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis

Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of relevant pathways/networks. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol-reactive probes with a flexible click chemistry functional group to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. We observe qualitative differences in protein thiol profiles by SDS-PAGE analysis when detection by iodoacetamide vs maleimide probe chemistries are compared, and pretreatment of cells with hydrogen peroxide eliminates detection of the majority of SDS-PAGE bands. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent donors, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and this deficiency was confirmed by Western blot. Redox probes revealed additional protein thiol differences between GDFs and NHDFs, including radiation responsive annexin family members. Our results indicate a multifactorial basis for the unusual sensitivity of Gorlin syndrome to radiation carcinogenesis, and the pathways identified have plausible implications for radiation health effects.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Mercury Speciation via Direct Mercury Analyzer

Mercury has no known biological role, but is present in every living thing and widespread in the environment. As mercury enters an aqueous system, it is subject to methylation or demethylation. Methylmercury selectively partitions into biota and biomagnifies through the food chain. Nearly all methylmercury exposures in the U.S. occur through eating fish and shellfish that contain higher levels of methylmercury. Methyl mercury and other organomercury compounds are more toxic than inorganic mercury. Thus, monitoring and understanding mercury speciation are key to effective environmental management. It is particularly important to be able to separate and measure bioaccumulative-toxic species of mercury such as methyl mercury. We are developing a protocol for utilizing the Direct Mercury Analyzer (DMA-80) for mercury speciation of water samples and to monitor methylmercury (meHg). Objectives: - Advance the understanding of the performance of the direct mercury analysis (DMA) under a range of DOE relevant water chemistry conditions to quantify total mercury: - Demonstrate the applicability of DMA to analysis of typical DOE high level liquid 'tank' waste samples using nonradioactive simulants; Test innovative sorbents to extend the range of the direct mercury analyzer to environmental concentration levels. - Perform scoping tests to demonstrate proof of principle for the chemical reduction amalgamation modular microcolumns for mercury speciation: Examine various microcolumn configurations and document speciation performance for typical DOE high level liquid 'tank' waste samples using nonradioactive simulants; Perform tests to examine extending the speciation paradigm to environmental concentration levels. General Method: all samples analyzed using a direct mercury analyzer (DMA 80) which automates the following steps: 1) pyrolysis to release the mercury, 2) catalysis to convert all mercury to elemental, 3) amalgamation to trap the mercury, 4) desorption to release the mercury as a pulse and 5) spectroscopy to detect the mercury.. Instrument analyzes all mercury species in solids and liquids. The DMA 'eliminates' sample preparation, reduces labor, decreases waste, and results in more practical and cost effective methods. To test the performance of the DMA for DOE relevant conditions, we analyzed samples representative of at high (mg/L) tank waste concentrations in water and in diluted tank waste simulant and analyzed samples representative of environmental (ng/L levels) in water. High concentration samples were analyzed directly in the instrument. Low concentration samples were analyzed by concentrating the mercury onto sorbents such as silica thiol or a mixture of silica thiol and copper. The concentration was performed using microcolumns and placing the columns into the DMA (see below) or by mixing the sorbent into the bulk sample and then filtering ad analyzing the filter in the DMA. Speciation Methods: The speciation tests were based on the selective reduction of inorganic mercury ions - Hg{sup (+} {sup or} {sup 2+)} - to elemental mercury - Hg(0). The elemental mercury is then trapped by amalgamation into the metal. These reactions do not impact methyl mercury allowing for separation. All reactions were performed using powdered elemental copper. To demonstrate proof-of-principle for the selective collection of inorganic mercury, samples were exposed to different amounts of powdered copper (and a control with no copper). This was done using microcolumns and a vacuum box. The columns were placed directly on the DMA for analysis. All materials and conditions were tested using solutions of both inorganic mercury and methyl mercury and all trials were in triplicate. Results and Observations: Validated DMA for high concentration Hg samples such as DOE tank waste: - Liquid inHg - samples were stable on autosampler carousel and results were accurate; - Liquid meHg - samples required a stabilizer. Si-Thiol (0.02 g) in the sample boat generated reliable meHg results. Stabilizer can be added for routine operations. Successful 'proof of principal' in extending DMA range to near environmental levels - using both microcolumns and filtration; - Filtration - Adding Si-Thiol (0.5 g) to sample extracted both inHg and meHg and preserved sample. Handling filters was labor intensive. - Microcolumns - best performance using a combination of copper and Si-Thiol - columns clogged and flowed slowly in some cases; - Practical limit may be in the range of 1 to 5 ng/L due to blanks; - Path Forward - Test additional packing materials and methods to improve flow and consistency, and approaches to minimize blanks. Successful 'proof of principal' for Hg speciation using chemical reduction and amalgamation; - Copper microcolumns (0.5 g Cu) selectively trapped inHg for high concentration mercury samples; - Copper microcolumns (0.5 g Cu selectively trapped inHg for large volume samples at environmental concentrations; - Some sorption of me Hg so that separations not quantitative; - Path Forward - test additional packing materials and alternate eluents to better trap inHg and minimize meHg retention in column.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗