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Background: High moisture meat analog (HMMA) products processed using extrusion have become increasingly popular in the last few years. Because the formation of disulfide bonds is believed to play a critical role in the texturization mechanism, this study aimed to understand how chemical compounds capable of reducing disulfide bonds, specifically cysteine, sodium metabisulfite, and glutathione, affect the texture and the chemical interactions between the proteins., Method: Wheat protein blended with cysteine, sodium metabisulfite, or glutathione at levels of 0, 0.5, 1.0, 2.5, 5.0, and 7.5 g kg -1 was extruded at three different temperatures (115, 140, and 165 °C) using a co-rotating twin-screw extruder. The feed rate (85 g min -1 ), the moisture content (600 g kg -1 ), and the screw speed (300 rpm) were kept constant. Unextruded and extruded material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polymeric protein fractionation, and sulfhydryl group/disulfide bond analysis. Extruded samples were further analyzed for their hardness and their anisotropic index., Results: The inclusion of reductants significantly affected the structure of the obtained extrudates. Although reducing agents had a relatively small impact on the total amount of disulfide bonds, their action significantly enhanced crosslinking between the proteins. At select conditions, samples with high fibrousness were specifically obtained when cysteine or sodium metabisulfite was included at levels of 5.0 g kg -1 ., Discussion: In the presence of reducing agents, it is believed that disulfide bonds are split earlier during the process without binding to them, giving the protein strands more time to unravel and align, leading to a better flow behavior and more fibrous products. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

We have prepared a novel assembly with copper nanoclusters (CuNCs) and imidazolium-based gemini surfactants (different chain lengths). These novel mimic enzymes formed through the assembly of nanocluster-gemini surfactants have been utilized in creating colorimetric sensors to detect biomolecules. Yet, understanding the method for detecting glutathione (GSH) and its sensing mechanism using this specific assembly-based colorimetric sensor poses a significant challenge. Because of the role of surface ligands, the complexes of cysteine-capped CuNCs (Cys-CuNCs) and gemini surfactants exhibit strong amphiphilicity, enabling them to self-assemble like a molecular amphiphile. We have investigated the kinetics and catalytic capabilities of this Cys-CuNCs@gemini surfactant assembly through peroxidase-like activity. Additionally, a sensitive and simple-to-use colorimetric sensing approach for glutathione (GSH) is also disclosed here, demonstrating a low limit of detection, by using this peroxidase-like activity of Cys-CuNCs@gemini surfactant assemblies. Thus, the remarkable advantages of the Cys-CuNCs@gemini surfactant nanozyme make it suitable for the precise colorimetric detection of GSH, demonstrating excellent sensitivity and reliable selectivity. Additionally, it performs well in detecting GSH in various soft drinks.

Background: Cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging., Results: Here, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 μM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 μg/mL) than classical Coomassie brilliant blue (14.11 μg/mL)., Significance: The outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Biomolecules play vital roles in many biological processes and diseases, making their identification crucial. Herein, we present a colorimetric sensing method for detecting biomolecules like cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). This approach is based on a reaction system whereby colorless 3,3',5,5'-tetramethylbenzidine (TMB) undergoes catalytic oxidation to form blue-colored oxidized TMB (ox-TMB) in the presence of hydrogen peroxide (H 2 O 2 ), utilizing the peroxidase and catalase-mimicking activities of metal-phenolic coordination frameworks (MPNs) of Cu-TA, Co-TA, and Fe-TA nanospheres. The Fe-TA nanospheres demonstrated superior activity, more active sites and enhanced electron transport. Under optimal conditions, the Fe-TA nanospheres were used for the detection of biomolecules. When present, biomolecules inhibit the reaction between TMB and H 2 O 2 , causing various colorimetric responses at low detection limits of 0.382, 0.776 and 0.750 μM for Cys, Hcy and GSH. Furthermore, it was successfully applied to real water samples with good recovery results. The developed sensor not only offers a rapid, portable, and user-friendly technique for multi-target analysis of biomolecules at low concentrations but also expands the potential uses of MPNs for other targets in the environmental field.

Cervical cancer screening is a crucial field of femtech (female technology). In this work, we disclosed a new femtech solution─a simple, straightforward, and on-site applicable urine-based cervical cancer diagnostic method using a fluorescent biothiol probe. Our newly developed nitrobenzene-based fluorescent probe, named NPS-B , effectively differentiates between cysteine and homocysteine within urine samples via controlled Smiles rearrangement. The analysis of emission-based signals offers the potential utility of this method in cervical cancer. NPS-B was designed by considering the substitution effect and structural polarity of the nitrobenzene-based fluorophore. This controlled modification of nitrobenzene-induced substantial intramolecular charge transfer changes in the fluorophore when exposed to biothiols, resulting in significant changes in photophysical properties. NPS-B displayed different emissions of cysteine and homocysteine in clinical human urine (without prior urine treatment). Overall, our findings provide insights not only into fundamental chemical science but also into the broader domain of applied sciences.

Monitoring the level of biothiols in organisms would be beneficial for health inspections. Recently, 3-(2'-nitro vinyl)-4-phenylselenyl coumarin as a fluorescent probe for distinguishing the detection of the small-molecule biothiols cysteine/homocysteine (Cys/Hcy) and glutathione (GSH) was developed. By introducing 4-phenyselenium as the active site, the probe CouSeNO 2 /CouSNO 2 was capable of detecting Cys/Hcy and GSH in dual fluorescence channels. Theoretical insights into the fluorescence sensing mechanism of the probe were provided in this work. The details of the electron excitation process in the probe and sensing products under optical excitation and the fluorescent character were analyzed using the quantum mechanical method. All these theoretical results would provide insight and pave the way for the molecular design of fluorescent probes for the detection of biothiols.

Bacillithiol (BSH) replaces glutathione (GSH) as the most prominent low-molecular-weight thiol in many low G + C gram-positive bacteria. BSH plays roles in metal binding, protein/enzyme regulation, detoxification, redox buffering, and bacterial virulence. Given the small amounts of BSH isolated from natural sources and relatively lengthy chemical syntheses, the reactions of BSH with pertinent reactive oxygen, nitrogen, and sulfur species remain largely unexplored. We prepared BSH and exposed it to nitroxyl (HNO), a reactive nitrogen species that influences bacterial sulfur metabolism. The profile of this reaction was distinct from HNO oxidation of GSH, which yielded mixtures of disulfide and sulfinamide. The reaction of BSH and HNO (generated from Angeli's salt) gives only sulfinamide products, including a newly proposed cyclic sulfinamide. Treatment of a glucosamine-cysteine conjugate, which lacks the malic acid group, with HNO forms disulfide, implicating the malic acid group in sulfinamide formation. This finding supports a mechanism involving the formation of an N -hydroxysulfenamide intermediate that dehydrates to a sulfenium ion that can be trapped by water or internally trapped by an amide nitrogen to give the cyclic sulfinamide. The biological relevance of BSH reactivity toward HNO is provided through in vivo experiments demonstrating that Bacillus subtilis exposed to HNO shows a growth phenotype, and a strain unable to produce BSH shows hypersensitivity toward HNO in minimal medium cultures. Thiol analysis of HNO-exposed cultures shows an overall decrease in reduced BSH levels, which is not accompanied by increased levels of BSSB, supporting a model involving the formation of an oxidized sulfinamide derivative, identified in vivo by high-pressure liquid chromatography/mass spectrometry. Collectively, these findings reveal the unique chemistry and biology of HNO with BSH in bacteria that produce this biothiol.

Low molecular-weight thiols as glutathione and cysteine are an important part of the cell's redox regulation system. Previously, we have shown that inactivation of ADP-heptose synthesis in Escherichia coli with a gmhA deletion induces the oxidative stress. It is accompanied by rearrangement of thiol homeostasis and increased sensitivity to antibiotics. In our study, we found that restriction of cysteine metabolism (ΔcysB and ΔcysE) and inhibition of glutathione synthesis (ΔgshAB) lead to a decrease in the sensitivity of the ΔgmhA mutant to antibiotics but not to its expected increase. At the same time, blocking of the export of cysteine (ΔeamA) or increasing import (Ptet-tcyP) into cells of the oxidized form of cysteine-cystine leads to an even greater increase in the sensitivity of gmhA-deleted cells to antibiotics. In addition, there is no correlation between the cytotoxic effect of antibiotics and the level of reactive oxygen species (ROS), the total pool of thiols, or the viability of the initial cell population. However, a correlation between the sensitivity to antibiotics and the level of oxidized glutathione in cells was found in our study. Apparently, a decrease in the content of low-molecular-weight thiols saves NADPH equivalents and limits the processes of protein redox modification. This leads to increasing of resistance of the ΔgmhA strain to antibiotics. An increase in low-molecular-weight thiols levels requires a greater expenditure of cell resources, leads to an increase in oxidized glutathione and induces to greater increase in sensitivity of the ΔgmhA strain to antibiotics.

Clickable glutathione is a glutathione-derived chemical probe designed to identify and analyze protein S-glutathionylation, a major cysteine oxidation in redox signaling. An engineered glutathione synthetase mutant (GS M4) is used to synthesize clickable glutathione in cells or in vitro, which affords utility via click chemistry to detect, identify, and quantify glutathionylation on individual or global proteins in biochemical and mass spectrometric analyses. The clickable glutathione approach is valuable for the unequivocal identification of glutathionylated cysteines, among many reversible cysteine oxoforms, via the direct enrichment and detection of glutathionylated proteins or peptides. Clickable glutathione, in combination with GS M4, has demonstrated utility in the mass-spectrometry-based discovery and profiling of new proteins and cysteines for glutathionylation in cell lines in response to physiologic and oxidative stress. The approach is versatile and applicable to validating the glutathionylation of proteins and cysteines in other biochemical analysis beside mass spectrometry. Here, we describe the applications of clickable glutathione and provide detailed protocols for the identification, profiling, and detection of glutathionylated proteins and cysteines. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of glutathionylated cysteine in individual proteins in vitro Basic Protocol 2: Proteomic identification and quantification of glutathionylation Basic Protocol 3: Biochemical validation of glutathionylation in cells., (© 2023 Wiley Periodicals LLC.)

Considering the biological and ecological importance of Cu-thiol interactions and the discrepancies in previous research, this study focuses on Cu interactions with biologically and ecologically relevant thiols: glutathione (GSH), L-cysteine (L-cys), 3-mercaptopropionic acid (MPA), and thioacetic acid (TAA) in aqueous solution. The addition of Cu(II) to a thiol-containing solution led to a rapid reduction of Cu(II) and the formation of a Cu(I)-thiol complex. The mechanism of Cu(II) reduction and Cu(I) complex formation as well as the kinetics of Cu(I) oxidation strongly depend on the structural properties of the individual thiols investigated. The reducing power of the investigated thiols can be summarized as follows: L-cys ≅ GSH > MPA > TAA. The reaction order, with respect to Cu(I) oxidation, also changes over the time of the reaction course. The deviation of the reaction kinetics from the first order with respect to Cu(I) in the later stages of the reaction course can be attributed to a Fenton-like reaction occurring under low thiol concentration conditions. At high Cu:thiol ratios, in the case of GSH, L-cys, and MPA, the early stage of the reaction course is characterized by high Cu(I) stability, most likely as a result of Cu(I) complexation by the thiols present in excess in the reaction mixture.

Cys play an important physiological role in the human body. Abnormal Cys concentration can cause many diseases. Therefore, it is of great significance to detect Cys with high selectivity and sensitivity in vivo. Because homocysteine (Hcy) and glutathione (GSH) have similar reactivity and structure to cysteine, few fluorescent probes have been reported to be specific and efficient for cysteine. In this study, we designed and synthesized an organic small molecule fluorescent probe ZHJ-X based on cyanobiphenyl, which can be used to specifically recognize cysteine. The probe ZHJ-X exhibits specific selectivity for cysteine, high sensitivity, short reaction response time, good anti-interference ability, and has a low detection limit of 3.8 × 10 -6 M. The probe ZHJ-X was successfully applied to the visualization of Cys in living cells and had great application prospects in cell imaging and detection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cysteine (Cys), one of the biological thiols, which plays critical roles in biological system regulating the balance of redox homeostasis. In order to monitor the level of Cys in the living cells and organisms, a chromogenic fluorescence probe Rhocl-Cys based on Rhodamine chloride exhibiting the preferable performance of fluorescence turn-on response reacting with Cys was presented. Rhocl-Cys responded rapidly to Cys within 20 min, and had stable fluorescence intensity within pH 6.0-10.0, high selectivity towards Cys and the anti-inference capability with a low detection limit of 0.80 μM. In particular, Rhocl-Cys could qualitatively and quantitatively monitor the level of endogenous and exogenous Cys in living cells and successfully apply to zebrafish detecting Cys. Therefore, these results might further provide the basis exploring the role of Cys in biological system and facilitate as clinical diagnostic molecular tools., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Oxidative stress is a hallmark of a range of diseases including cancer, HIV-AIDS and cardiovascular disorders. Blood plasma cysteine (Cys) is an established biomarker for oxidative stress, rendering the development of sensitive and technically straightforward approaches for its estimation extremely desirable. Herein, we achieve this goal by developing a series of Michael addition-elimination-cyclization based turn-on fluorescence (MADELCY TOF) probes that selectively detect Cys over other amino acids and biothiols such as homocysteine (Hcy) and glutathione (GSH). Cysteine detection by these compounds involves cascaded fluorophore release via the Michael addition of the cysteinyl thiol followed by the elimination of a leaving group and lactamization via the cysteinyl amine. Conveniently, the Cys-reactivity of these probes can be tuned via both inductive and mesomeric effects imparted by their β' position-substituents. One of our probes that contains the p -nitrophenyl group at its β' position demonstrated rapid kinetics ( t 1/2 = 2.9 min) with excellent sensitivity (detection limit = 8.2 nM) towards Cys. This probe also enabled the sensitive (detection limit = 9.5 pM) estimation of the blood plasma levels of the cancer and liver cirrhosis biomarker, the aminoacylase-1 (ACY-1) enzyme. Furthermore, another probe of our library enabled live cell imaging of cellular Cys allowing us to develop an imaging-based assay to monitor hydrogen peroxide-induced oxidative stress in mammalian cells.

Theaflavin, a polyphenol responsible for the bright orange color and various bioactivities of black tea exudates, is susceptible to autoxidation at neutral and mild alkaline pH, changing its color to brown. In the presence of cysteine (Cys), glutathione (GSH), or N-acetyl cysteine (NAC), the browning of theaflavin solution was inhibited concomitantly with time-dependent decreases in the concentrations of both theaflavin and thiol group. The rank order of the decrease was Cys ≅ GSH > NAC, suggesting the relevance of the nucleophilic property of the thiol group to its reaction with theaflavin. LC-MS analysis of the reaction products indicated formation of novel derivatives that were mono- or di-molecular adducts of thiol compounds. We determined the chemical structures of theaflavin-Cys and theaflavin-GSH adducts by NMR and proposed the reaction mechanisms. It was found that the theaflavin-Cys adduct was not a simple adduct, to which a new cyclic structure was added., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cysteine (Cys), an essential biological amino acid, participates several crucial functions in various physiological and pathological processes. The sensitive and specific detection of Cys is of great significance for understanding its biological function to disease diagnosis. Herein, we designed and synthesized a simple fluorescence sensor 2-(benzothiophen-2-yl)-4-oxo-4H-chromen-3-yl acrylate (BTCA) composed of a flavonol skeleton as the fluorophore and acrylic ester group as the recognition receptor. Probe BTCA displayed high selectivity and extremely fast response toward Cys in phosphate buffer solution in the presence of other competitive species even Homocysteine (Hcy) and Glutathione (GSH) owing to a specific conjugate addition-cyclization reaction between the acrylate moiety and Cys. The photoluminescence mechanism of probe BTCA toward Cys was modulated by excited state intramolecular proton transfer (ESIPT) process. The sensing property for Cys was studied by UV-Visible, fluorescence spectrophotometric analyses and time-dependent density functional theory (TD-DFT) calculations, those results indicated that probe BTCA possessed excellent sensitivity, higher specificity, dramatically "naked-eye" fluorescence enhancement (30-fold), high anti-interference ability, especially immediate response speed (within 40 s). Additionally, the practicability of sensor BTCA in exogenous and endogenous Cys imaging in living cells and zebrafish was elucidated as well, suggesting that it has remarkedly diagnostic significance in physiological and pathological process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

An enigmatic and never described hyper-reactivity of most of the cysteines resident in the reduced, molten globule-like intermediate of a few proteins has been recently discovered. In particular, all ten cysteines of chymotrypsinogen showed hundred times increased reactivity against hydrophobic reagents. A single cysteine (Cys1) was also found thousand times more reactive toward GSSG, making speculate that a single glutathionylation could represent the primordial event of its oxidative folding. In the present study, we compare these kinetic properties with those present in trypsinogen taken in its reduced, molten globule-like intermediate and identify the origin of these unusual properties. Despite the divergent evolution of these two proteins, the different amount of disulfides and the very different 3D localization of three disulfides, their hyper-reactivity toward hydrophobic thiol reagents and disulfides is very similar. Mass spectrometry identifies two cysteines in trypsinogen, Cys148 and Cys197, 800 times more reactive toward GSSG than an unperturbed protein cysteine. These results point toward a stringent and accurate preservation of these peculiar kinetic properties during a divergent evolution suggesting some important role, which at the present can only be hypothesized. Similar extraordinary hyper-reactivity has been found also in albumin, ribonuclease, and lysozyme confirming that it cannot be considered a kinetic singularity of a single protein. Interestingly, the very flexible and fluctuating structures like those typical of the molten globule status prove capable of enabling sophisticated actions typical of enzymes such as binding to GSSG with relevant specificity and high affinity (K D  = 0.4 mm) and accelerating the reaction of its cysteines by thousands of times., (© 2021 Federation of European Biochemical Societies.)

The presence of reduced aminothiols, including homocysteine (Hcy), cysteine (Cys), cysteinyl-glycine (CG), and glutathione (GSH), is significantly increased in the pathological state. However, there have been no reports on the relationship between reduced aminothiols (Hcy, Cys, CG, and GSH) and different genders, ages, and drug combinations in human blood. The accurate quantification of these reduced thiols in biological fluids is important for monitoring some special pathological conditions of humans. However, the published methods typically not only require cumbersome and technically challenging processing procedures to ensure reliable measurements, but are also laborious and time-consuming, which may disturb the initial physiological balance and lead to inaccurate results. We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation coupled with a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and used it to determine four reduced aminothiols (Hcy, Cys, CG, and GSH) in human blood for the first time. A total of 96 clinical patients were enrolled in our study. The influence of different genders, ages, and drug combinations on the levels of four reduced thiols in human blood was also discussed by SPSS 24.0. The sample preparation was simplified to a single 5 min centrifugation step in a sealed system that did not disturb the physiological environment. The validation parameters for the methodological results were excellent. The procedure was successfully applied to monitoring the concentrations of four reduced aminothiols (Hcy, Cys, CG, and GSH) in 96 clinical blood samples. There were no significant differences in Hcy, Cys, CG, or GSH for the different genders, ages, or combinations with methotrexate or vancomycin (P > 0.05). However, there was a significant increase in Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy (p=0.0007). It is advisable to measure reduced Hcy level in patients taking valproic acid. The developed HFCF-UF method was simple and accurate. It can be easily applied in clinical research to evaluate oxidative stress in further study., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

A novel fluorescent probe (RA), based on the rosamine skeleton bearing acrylate group, has been reasonably designed and prepared, which employed an addition-cyclization-elimination sequence reaction mechanism to detect cysteine. RA displayed rapid response to cysteine within 1.5 min, and exhibited satisfactory selectivity for cysteine over H 2 S, glutathione (Glu), and homocysteine (Hcy), due to the formation of seven-membered lactam favored kinetically. Fluorescence ratio was utilized to detect cysteine from 6.0 to 20.0 μM with a detection limit of 0.29 μM. More, RA was used to monitor cysteine in BSA, water, milk, milk powder, cabbage, radish, apple, and pear., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Surface chemistry control is a key means to improve substrate selectivity and enhance catalytic activity of nanozymes, a kind of novel artificial enzymes. Herein, we demonstrated that apart from chemical properties of functional groups, their spatial distance to the catalytic sites is also very important to improve the catalytic performance of nanozymes. Using cetyltrimethylammonium bromide (CTAB) coated gold nanorods (AuNR) as the example, we showed that cysteine (Cys) surface modification can greatly enhance the peroxidase activity of AuNR for the oxidation of substrate 3,3',5,5'-tetramethylbenzidine (TMB). By using cysteine derivatives, the key role of the carboxylic group in cysteine is revealed in improving substrate binding and activity enhancement. The electrostatic interactions of carboxylic groups from adsorbed cysteine molecules with protonated amino groups of TMB bring TMB molecules to the surface Au active sites and thus markedly increase catalytic activity. In contrast, despite having two carboxylic groups, glutathione (GSH) surface modification only leads to quite limited improvement of catalytic activity. We speculated that due to large molecular size of GSH, the spatial distance between TMB-GSH and Au is larger than that between TMB-Cys and Au. Furthermore, Raman characterization indicated that at high Cys coverage, they form patches on rod surface via zwitterionic interactions, which may give additional benefits by decreasing the steric hindrance of TMB diffusion to surface Au atom sites. In all, our study highlights the importance of fine surface tuning in the design of nanozymes., (© 2021 IOP Publishing Ltd.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (M pro ), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. M pro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show M pro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated M pro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated M pro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated M pro exists as a monomer. Tryptic and chymotryptic digestions of M pro as well as experiments using a C300S M pro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon M pro glutathionylation. These findings indicate that M pro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for M pro activity, and provides a novel target for the development of agents to block M pro dimerization and activity. This feature of M pro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (M pro ), a dimeric enzyme necessary for viral replication. Most work thus far developing M pro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for M pro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits M pro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.

Cysteine (Cys) is the most reactive amino acid participating in a wide range of biological functions. In-silico predictions complement the experiments to meet the need of functional characterization. Multiple Cys function prediction algorithm is scarce, in contrast to specific function prediction algorithms. Here we present a deep neural network-based multiple Cys function prediction, available on web-server (DeepCys) (https://deepcys.herokuapp.com/). DeepCys model was trained and tested on two independent datasets curated from protein crystal structures. This prediction method requires three inputs, namely, PDB identifier (ID), chain ID and residue ID for a given Cys and outputs the probabilities of four cysteine functions, namely, disulphide, metal-binding, thioether and sulphenylation and predicts the most probable Cys function. The algorithm exploits the local and global protein properties, like, sequence and secondary structure motifs, buried fractions, microenvironments and protein/enzyme class. DeepCys outperformed most of the multiple and specific Cys function algorithms. This method can predict maximum number of cysteine functions. Moreover, for the first time, explicitly predicts thioether function. This tool was used to elucidate the cysteine functions on domains of unknown functions belonging to cytochrome C oxidase subunit-II like transmembrane domains. Apart from the web-server, a standalone program is also available on GitHub (https://github.com/vam-sin/deepcys)., (© 2021 Wiley Periodicals LLC.)

For the first time the enzyme mimic activity of iron oxychloride (FeOCl) nanosheets has been studied. The intrinsic peroxidase-mimetic activity of the nanosheets in the presence of H 2 O 2 was approved by the efficient oxidation of tetramethylbenzidine (TMB). The Michaelis-Menten constant of the nanosheets toward TMB was about six times lower than that of natural horseradish peroxidase. The superiority of the nanosheets' catalytic property ascribes to their H 2 O 2 activation ability. Based on the inhibition of the nanozymes' catalytic reaction, an assay was developed for the quantitative measurement of glutathione (GSH) and cysteine (Cys). The linear range for both biomolecules was over the range of 3-33 μM. The LOD values (3σ/slope) for GSH and Cys were 2.23 μM and 2.76 μM, respectively. Importantly, we succeeded in colorimetric discrimination of GSH and Cys kinetically. We achieved high selectivity toward GSH and Cys. This work extends the feasibility of using FeOCl as nanozymes to construct biosensors, colorimetric probes for medical diagnosis, and nanozyme-based cancer therapy.

Glutathionyl hemoglobin is a minor form of hemoglobin with intriguing properties. The measurement of the redox potential of its reactive β- 93 -Cysteine is useful to improve understanding of the response of erythrocytes to transient and chronic conditions of oxidative stress, where the level of glutathionyl hemoglobin is increased. An independent literature experiment describes the recovery of human erythrocytes exposed to an oxidant burst by measuring glutathione, glutathione disulfide and glutathionyl hemoglobin in a two-hour period. This article calculates a value for the redox potential E 0 of the β- 93 -Cysteine, considering the erythrocyte as a closed system at equilibrium described by the Nernst equation and using the measurements of the literature experiment. The obtained value of E 0 of -121 mV at pH 7.4 places hemoglobin as the most oxidizing thiol of the erythrocyte. By using as synthetic indicators of the concentrations the electrochemical potentials of the two main redox pairs in the erythrocytes, those of glutathione-glutathione disulfide and of glutathionyl-hemoglobin, the mechanism of the recovery phase can be hypothesized. Hemoglobin acts as the redox buffer that scavenges oxidized glutathione in the oxidative phase and releases it in the recovery phase, by acting as the substrate of the NAD(P)H-cofactored enzymes.

As a kind of bioactive sulfur species, biothiols (Cys, Hcy, and GSH) play an irreplaceable role in regulating the redox balance of life processes. Because of their similar chemical structures and properties, a sulfydryl group, and an amino group, it is an important challenge to distinguish two or more of them at the same time. Herein, a fluorescent sensor ( NTPC ) based on the coumarin structure was developed to discriminate Cys/Hcy and GSH simultaneously. The sensor has no fluorescence due to the d-PET effect but displays strong fluorescence after its reaction with biothiols. There are two potential reaction sites (nitrophenyl sulfide group and aldehyde group) in the structure of NTPC , resulting in different fluorescent signal changes after reacting with biothiols (green for Cys and Hcy and red for GSH). Under double-wavelength excitation, the sensor shows low background fluorescence, high selectivity, and low detection limits toward biothiols. Moreover, the sensor can be used to discriminate different biothiols (Cys/Hcy and GSH) in cells and zebra fish by different fluorescence signals with low toxicity and might provide a promising tool for studying the roles of different biothiols in various physiological and pathological processes.

Hsp70 proteins are a family of ancient and conserved chaperones. They play important roles in vital cellular processes, such as protein quality control and the stress response. Hsp70 proteins are a potential drug target for treatment of disease, particularly cancer. PES (2-phenylethynesulfonamide or pifithrin-μ) has been reported to be an inhibitor of Hsp70. However, the mechanism of PES inhibition is still unclear. In this study we found that PES can undergo a Michael addition reaction with Cys-574 and Cys-603 in the SBDα of human HspA1A (hHsp70), resulting in covalent attachment of a PES molecule to each Cys residue. We previously showed that glutathionylation of Cys-574 and Cys-603 affects the structure and function of hHsp70. In this study, PES modification showed similar structural and functional effects on hHsp70 to glutathionylation. Further, we found that susceptibility to PES modification is influenced by changes in the conformational dynamics of the SBDα, such as are induced by interaction with adjacent domains, allosteric changes, and mutations. This study provides new avenues for development of covalent inhibitors of hHsp70., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Targeting to obtain better water solubility and stability and less aggregation-caused quenching effects of quantum dots, two kinds of thiol molecules, glutathione and L-cysteine, were firstly united to offer stabilizing ligands for aqueous synthesized CdS quantum dots, which exhibited sensitive aggregation-induced emission properties. Fluorescent intensity of the CdS quantum dots was enhanced about 5 folds by simple solvent exchange from water to 90 vol% PEG200. Restriction of intramolecular motions in an aggregate state was probably the main cause of the phenomenon. At the same time, fluorescent intensity of CdS quantum dots in the presence of zinc ions was able to be enhanced about 2.2 folds. Based on the researches, a handy metal enhanced fluorescent probe for detecting zinc ions was established. And the detection limit was 0.58 μmol/L. Zinc ions as a bridge among CdS quantum dots to form aggregates limited motions of CdS quantum dots to a certain extent and simultaneously enhanced their fluorescence emission intensities. Meanwhile, activation of surface states of CdS quantum dots also led to emission enhancement. Both of the two factors together contributed to the fluorescence enhancement and ultimately to the sensitivity to zinc ion sample detection.

Vimentin intermediate filaments are a significant component of the cytoskeleton in cells of mesenchymal origin. In vivo, filaments assemble and disassemble and thus participate in the dynamic processes of the cell. Post-translational modifications (PTMs) such as protein phosphorylation regulate the multiphasic association of vimentin from soluble complexes to insoluble filaments and the reverse processes. The thiol side chain of the single vimentin cysteine at position 328 (Cys328) is a direct target of oxidative modifications inside cells. Here, we used atomic force microscopy, electron microscopy and a novel hydrogen-deuterium exchange mass spectrometry (HDex-MS) procedure to investigate the structural consequences of S-nitrosylation and S-glutathionylation of Cys328 for in vitro oligomerisation of human vimentin. Neither modification affects the lateral association of tetramers to unit-length filaments (ULF). However, S-glutathionylation of Cys328 blocks the longitudinal assembly of ULF into extended filaments. S-nitrosylation of Cys328 does not hinder but slows down the elongation. Likewise, S-glutathionylation of preformed vimentin filaments causes their extensive fragmentation to smaller oligomeric species. Chemical reduction of the S-glutathionylated Cys328 thiols induces reassembly of the small fragments into extended filaments. In conclusion, our in vitro results suggest S-glutathionylation as a candidate PTM for an efficient molecular switch in the dynamic rearrangements of vimentin intermediate filaments, observed in vivo, in response to changes in cellular redox status. Finally, we demonstrate that HDex-MS is a powerful method for probing the kinetics of vimentin filament formation and filament disassembly induced by PTMs., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding.

Herein, gold@platinum (Au@Pt) bimetallic nanoparticles with high catalytic ability were in situ decorated onto a molybdenum disulfide (MoS 2 ) surface to obtain nanocomposites (MoS 2 -Au@Pt) with high peroxidase-mimicking activity, which were used to construct a colorimetric sensor for cysteine (Cys) detection. Interestingly, this sensor can efficiently distinguish Cys from homocysteine (Hcy), glutathione (GSH) and 19 other amino acids with high sensitivity. As expected, the colorimetric sensor can determine the Cys content in Cys supplement tablets due to its high stability and repeatability. Finally, the detection mechanism was studied.

Ferroptosis is an iron-dependent form of regulated cell death. In this study, a ratiometric fluorescent probe, gold carbon dots (GCDs) consisting of carbon skeleton and gold nanoclusters, was used for in situ imaging to monitor redox status in biothiols (glutathione and cysteine) and ferric metabolism of cancer cells in ferroptosis. The as-prepared GCDs can selectively respond to biothiols, interestingly, the fluorescence may be switched to sense ferric ions without interference by biothiols under proper conditions. The robust GCDs-probe exhibits excellent photobleaching resistance and can reversibly respond to intracellular biothiols/ferric ion with high temporal resolution. The 8 h real-time imaging of living cells was employed to track the fluctuation of biothiols, showing the change of redox status in ferroptosis. In addition, release of ferric ions in cells was monitored. The real-time imaging of depletion of biothiols and release of ferric ion in cells indicates the GCDs-probe can monitor how the ferroptosis regulates redox status in biothiols and ferric metabolism.

Biothiols and SO 2 derivatives, as essential reactive sulfur species (RSS), play vital roles in various physiological processes and have a close network of generation and metabolic pathways among them. To clarify their complex correlations, fluorescent probes to simultaneously detect GSH, Cys, and SO 2 derivatives are highly desirable. Herein, we develop the first fluorescent probe ( BO-HEM ) to simultaneously discriminate GSH, Cys, and SO 2 derivatives. The fluorescent probe is designed by integration of hemicyanine and BODIPY fluorophores through an ether bond. The ether bond of the probe is rapidly replaced by thiolates through nucleophilic aromatic substitution (S N Ar) to generate hemicyanine with NIR fluorescence and sulfur-BODIPY . The amino groups of Cys but not GSH then further replace the thiolate to form amino-BODIPY . As for SO 3 2- , nucleophilic addition to the double bond of BO-HEM generates adduct O-BODIPY with green fluorescence. To further improve the sensing performance, the nanoprobe with increased reactivity and biocompatibility is constructed by encapsulation of BO-HEM into the polymeric micelle. More importantly, by taking advantage of the hydrophilicity of the reaction products, the spectral discrimination was further enhanced to avoid signal interference. The nanoprobe is applied to discriminate biothiols and SO 2 derivatives in living cells through three-color fluorescence imaging.

It has been suggested that thiol-containing amino acids could be used as biomarkers for diseases associated with oxidative stress. We investigated the thiol-containing amino acids, homocysteine (Hcy), cysteine (Cys), glutathione (GSH) and γ-glutamylcysteine (γ-GluCys), in commercial human serum by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after precolumn derivatization with 4-fluoro-7-sulfobenzofurazan. This method was applied to determine the composition of thiol-containing amino acids in exosomes prepared from the serum. Hcy, Cys, GSH and γ-GluCys could be detected in the exosomal fraction, and the ratio of each thiol-containing amino acid was similar to those in the corresponding native serum. Cys (94.76%) was most enriched in the exosomal fraction, followed by GSH (2.97%), γ-GluCys (1.59%) and Hcy (0.68%). These findings suggest that thiol-containing amino acids, Hcy, Cys, GSH and γ-GluCys, are included in exosomes in human serum., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Sirtuins ( e.g. human Sirt1-7) catalyze the removal of acyl groups from lysine residues in proteins in an NAD + -dependent manner, and loss of sirtuin deacylase activity correlates with the development of aging-related diseases. Although multiple reports suggest that sirtuin activity is regulated by oxidative post-translational modifications of cysteines during inflammation and aging, no systematic comparative study of potential direct sirtuin cysteine oxidative modifications has been performed. Here, using IC 50 and k inact / K I analyses, we quantified the ability of nitrosothiols ( S -nitrosoglutathione and S -acetyl-d,l-penicillamine), nitric oxide, oxidized GSH, and hydrogen peroxide to post-translationally modify and inhibit the deacylase activity of Sirt1, Sirt2, Sirt3, Sirt5, and Sirt6. The inhibition was correlated with cysteine modification and assessed with chemical-probe and blot-based assays for cysteine N -nitrosation, sulfenylation, and glutathionylation. We show that the primarily nuclear sirtuins Sirt1 and Sirt6, as well as the primarily cytosolic sirtuin Sirt2, are modified and inhibited by cysteine S -nitrosation, sulfenylation, and glutathionylation. We show that the primarily nuclear sirtuins Sirt1 and Sirt6, as well as the primarily cytosolic sirtuin Sirt2, are modified and inhibited by cysteine S -nitrosation in response to exposure to both free nitric oxide and nitrosothiols ( k inact / K I ≥ 5 m -1 -nitrosation. Surprisingly, the mitochondrial sirtuins Sirt3 and Sirt5 were resistant to inhibition by cysteine oxidants. Collectively, these results suggest that nitric oxide-derived oxidants may causatively link nuclear and cytosolic sirtuin inhibition to aging-related inflammatory disease development.-1 ), which is the first report of Sirt2 and Sirt6 inhibition by S -nitrosation. Surprisingly, the mitochondrial sirtuins Sirt3 and Sirt5 were resistant to inhibition by cysteine oxidants. Collectively, these results suggest that nitric oxide-derived oxidants may causatively link nuclear and cytosolic sirtuin inhibition to aging-related inflammatory disease development., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Kalous et al.)

Perfluorooctane sulfonyl fluoride (PFOSF) is the precursor of many fluorochemicals that are ubiquitous in the environment. However, its distribution and toxicology are rarely studied. In this work, the oxidability of PFOSF was found. PFOSF can accelerate oxidation of glutathione (GSH) to its oxidized form GSSG, and itself is reduced to a sulfinic acid. The yielded sulfinic acid was prepared and identified with high resolution mass spectrometry and NMR. Similar redox reactions were observed for PFOSF's short chain alternatives. The reduction potentials of perfluoroalkane sulfonyl fluorides (PFASFs) were determined to be -2.13 V vs. SCE with cyclic voltammetry, further demonstrating their oxidability. The peptide mixtures of GSH plus another cysteine-containing peptide were also oxidized by PFASFs to GSSG and an asymmetric disulfide GS-S-PEP. A single short-sequence PEP-SH could be oxidized to the symmetric disulfide PEP-S-S-PEP as the final product. In vitro experiments were carried out by adding PFASFs into rat liver S9 fractions. The turnover ratio of PFASFs were calculated to be about 4-10% by quantification of sulfinic acid with LC-MS/MS. Our work illustrates one of the potential metabolic pathways of PFASFs and demonstrates the oxidation of PEP-SHs by PFASFs, thus providing a preliminary exploration in the toxicology of these fluorochemicals., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Three 5H-benzo[a]phenoxazin-5-one-based (benzoresorufin and nile-red) Cysteine (Cys) detection probes have been comparatively designed and synthesized in this paper. The optical experiments exhibit probe 1b with a crotonoyl group has no response toward Cys; while probes 1a and 1c have the same reaction site (acryloyl group), their optical responses to Cys are quite different. The benzoresorufin-based-probe 1a shows a turn-on fluorescence response (118-fold) to Cys at 631 nm and affords a very low detection limit (DL = 19.8 nM). Compared with probe 1a, the nile-red-based probe 1c displays gradually diminishing fluorescence intensity with increased Cys concentration at 665 nm. And the notable different fluorescence response mechanisms of probes 1a and 1c toward Cys can be interpreted by HRMS and time-dependent density functional theorety (TDDFT) calculations. Furthermore, both of the two probes indicate high sensitivity and selectivity toward Cys over other similar structured amino acids including homocysteine (Hcy) and glutathione (GSH). Further cellular applications of the two probes have been successfully performed in HeLa cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Glutathione (GSH) levels are closely related to the homeostasis of redox state which directly affects human disease occurrence by regulating cell apoptosis. Hence, real-time monitoring of dynamic changes in intracellular GSH levels is urgently needed for disease early diagnosis and evaluation of therapy efficiency. In this study, an endogenous cysteine (Cys)-assisted detection system based on GSH@AgNCs and reduced graphene oxide (rGO) with high sensitivity and specificity was developed for GSH detection. Compared with GSH, GSH@AgNCs with weaker affinity and bonding force was quite easier to extrude from the rGO surface when competing against GSH, leading to the obvious change in fluorescence signal. This phenomenon was termed as "a crowding out effect". Furthermore, the presence of Cys can improve GSH assay sensitivity by enhancing the quenching efficiency of rGO on the GSH@AgNCs. In vitro assay indicated that the efficiency of fluorescence recovery was positively related with GSH concentration in the range from 0 to 10 mM. In addition, the method was employed for real-time monitoring of the dynamic changes in GSH levels regulated by natural drugs. The imaging results showed that the natural compound 3 (C3) can downregulate GSH levels in HepG2 cells, which was accompanied by reactive oxygen species (ROS) release and apoptosis induction. Finally, the method was used to monitor the change of GSH levels in serum samples with chronic hepatitis B (CHB) infection. The results demonstrated that the occurrence and development of CHB may be positively correlated with GSH levels to some extent. Overall, the above results demonstrate the potential application of this new nanosystem in anticancer natural drug screening and clinical assay regarding GSH levels.

Two highly selective OFF-ON isomer fluorescent probes (1 and 2) for homo-/cysteine were designed and synthesized. The pyrene modified tetraphenylethylene derivative with AIE was used as luminescent group while maleimide was used as recognition group. These two isomer probes were found to be nearly nonfluorescent when treated with GSH. However, upon interaction with Cys or Hcy, the fluorescence was enhanced by 2000 folds in a wide pH range from 3 to 10. Experimental results and DFT calculation have demonstrated that the fluorescence OFF-ON switch of such thiol probes is resulted from the termination of the PET (photo-induced electron transfer) effect through the Michael addition reaction of maleimide unit and thiols. In addition, probe 1 and 2 exhibit excellent selectivity and sensitivity towards Cys, Hcy over GSH and other amino acids, which was confirmed by mass MS. We suggested that Michael addition reaction of these probes with GSH was prevented because of the stereo-hindrance effect. Furthermore, these two isomer probes were successfully used for imaging biothiols in living H1299 lung cancer cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The aim of this study was to extract benzyl isothiocyanate (BITC) from green papaya by distillation apparatus without using organic solvents, and to improve the stability of BITC in aqueous solution. The distillation of mashed green papaya successfully yielded BITC as a water solution with more than 80% purity with good reproducibility. The amount of BITC in the distilled water gradually decreased during its storage at 4 °C, whereas it was not significantly changed at -20 °C for a few months. Moreover, the addition of l-cysteine ameliorated the BITC decomposition by the 4 °C-storage, but not affected by N-acetyl-cysteine and glutathione. These results suggested that the combination of BITC extraction by distillation and cysteine supplementation as well as frozen storage might be a useful method for the preparation and storage of the safer grade of BITC-containing extract., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cellular biothiols function crucially and differently in physiological and pathological processes. However, it is still challenging to detect and discriminate thiols within a single one molecule, especially for cysteine (Cys) and homocysteine (Hcy). In this study, a simple two-emission turn-on fluorescent biothiol probe (ICN-NBD) was rationally designed and synthesized through a facile ether bond linking 7-nitro-1,2,3-benzoxadiazole (NBD) and phenanthroimidazole containing a cyano tail. The probe in the presence of Cys elicited two fluorescence responses at 470 nm and 550 nm under excitation at 365 nm and 480 nm, respectively, because of the concomitant generation of both the fluorophore and NBD-N-Cys. In contrast, addition of Hcy and glutathione (GSH) could result in only a blue fluorescence enhancement at 470 nm. which was reasonably attributed to rearrangement from NBD-S-Hcy/GSH to NBD-N-Hcy/GSH as a result of geometrical constraints or solvent effects. Therefore, the fluorescent probe with the NBD scaffold could detect biothiols and simultaneously discriminate Cys from Hcy/GSH in both blue and green channels. The probe has been successfully applied for visualizing biothiols in living cells and zebrafish., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cyclopropenes are an important new addition to the portfolio of functional groups that can be used for bioorthogonal couplings. The inert nature of these highly strained compounds in complex biological systems is almost counterintuitive given their established electrophilic properties in organic synthesis. Here we provide the first demonstration of a cyclopropene that is capable of direct conjugation to protein targets in cells and show that this compound preferentially alkylates the active site cysteine of glutathione S-transferase omega-1 (GSTO1)., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Herein, we report the design of a novel fluorescent probe consisting of a naphthalimide fluorophore and a silicone small molecule for the reversible detection of hypochlorous acid and biothiol amino acids. The response mechanism of BSi-1 is based on the concept of the S-based oxidation/reduction. The probe was found to be suitable for imaging HOCl in HeLa, RAW 264.7 cells and zebrafish, demonstrating its utility in biological applications.

Vildagliptin (VG), a dipeptidyl peptidase-4 inhibitor, is used for treating type 2 diabetes. On rare occasions, VG causes liver injury as an adverse reaction. One case report suggested the involvement of immune responses in the hepatotoxicity, but the underlying mechanisms are unknown. We recently reported that VG binds covalently in vitro to l-cysteine to produce a thiazoline acid metabolite, M407, implying that the covalent binding may trigger the immune-mediated hepatotoxicity. There was no evidence, however, that such a thiazoline acid metabolite was formed in vivo. In the present study, we administered a single oral dose of VG to male Sprague-Dawley rats, and detected M407 in plasma. The sum of urinary and fecal excretions of M407 reached approximately 2% of the dose 48 hours postdosing. Using bile duct-cannulated rats, we demonstrated that M407 was secreted into bile as a glucuronide, designated as M583. Another newly identified thiazoline metabolite of VG, the cysteinylglycine conjugate M464, was detected in urine, feces, and bile. The formation of M464 was confirmed by in vitro incubation of VG with glutathione even in the absence of metabolic enzymes. A glutathione adduct against the nitrile moiety M611 was also detected in vitro but not in vivo. In summary, we found three new thiazoline-containing thiol adduct metabolites in VG-administered rats. Nonenzymatic covalent binding of VG would likely occur in humans, and it may be relevant to predicting adverse reactions., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

The balance between free oxygen radicals and antioxidant defense systems is usually assessed by an antioxidant capacity assay. A rapid and sensitive antioxidant capacity assay is described here. It is making use of NaYF 4 :Yb/Er@NaYF 4 core-shell upconversion nanoparticles (UCNPs) and potassium permanganate (KMnO 4 ). In this strategy, added KMnO 4 reduces the green (540 nm) emission of the UCNPs (under 980 nm photoexcitation) due to an inner filter effect. The antioxidants cysteine, ascorbic acid and glutathione (GSH) reduce the intense purple color of KMnO 4 because it is reduced to Mn(II) ion. Hence, the green upconversion fluorescence is restored after the addition of antioxidants. Figures of merit for this assay (for the case of GSH) include a detection limit of 3.3 μM, a detection range that extends from 10 μM to 2.5 mM, and an assay time of a few seconds. The assay was applied to the evaluation of antioxidant capacity in human plasma samples spiked with GSH and gave satisfactory repeatability and specificity. Graphical abstract Schematic presentation of a fluorometric assay based on inner filter effect (IFE) between upconversion nanoparticles (UCNPs) and potassium permanganate (KMnO 4 ) for the determination of antioxidant capacity in human plasma.

In this work, we report a sensitive and selective turn-on fluorescence detection of cysteine with glutathione protected silver nanoclusters (GSH-Ag NCs). The glutathione stabilized silver nanoclusters were synthesized by the boiling water method. When excited at 380 nm, the GSH-Ag NCs exhibited a weak emission at about 680 nm, which could be enhanced by cysteine. The proposed method allows evaluation of cysteine in the range of 2-3000 μM with a detection limit of 0.51 μM. The recoveries were found to be 95.07%-101.38% when detecting cysteine contents in fetal bovine serum samples. In addition, we also discussed the possible mechanism for the fluorescence enhancement of GSH-Ag NCs by addition of cysteine. It might be the formation of cysteine and glutathione co-capped Ag NCs. This work reported a fluorimetric method for the assay of cysteine and provided a strategy for the synthesis of dual ligand-protected Ag nanoclusters.

A turn-on fluorometric method is described for selective and sensitive detection of cysteine (Cys). Gold nanoclusters (Au NCs) capped with glutathione (GSH) are used as a fluorescent probe. If Ce 3+ ion are present, they will bind to the carboxy groups of the GSH-capped Au NC. This results in aggregation-induced emission enhancement (AIEE), best measured at excitation/emission wavelengths of 360/575 nm. On addition of Cys, which has less steric hindrance compared with GSH and higher affinity for Ce 3+ , it will bind to Ce 3+ through the carboxyl group and link with Au NCs via Au-S bond. Hence, the AIEE is increased and Cys can be quantified via this effect with a linear response in the 0.4-120 μmol L -1 Cys concentration range and a detection limit of 0.15 μmol L -1 . Graphical abstract Schematic presentation of cysteine detection via the Ce 3+ -triggered aggregation of glutathione capped gold nanoclusters which leads to increased yellow fluorescence.

A long-wavelength fluorescent probe NR-CY was developed for simultaneous identification of cysteine/glutathione and sulphide by combining the derivative of Nile red with 7-nitrobenzofurazan. The response of NR-CY to thiols is regulated by intramolecular charge transfer and photoinduced electron transfer mechanisms. For sulphide at 560 nm, cysteine at 475 nm and glutathione at 425 nm, different absorbance increases can be observed. NR-CY can detect cysteine at fluorescence emission 543 nm and distinguish sulphide from other analytes by kinetic experiments at 636 nm. The probe showed a rapid response to these thiols (cysteine was 90 s and sulphide was 30 s). In addition, NR-CY has been successfully applied to live MCF-7 cell imaging., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Grx1, a cytosolic thiol-disulfide oxidoreductase, actively maintains cellular redox homeostasis using glutathione substrates (reduced, GSH, and oxidized, GSSG). Here, the crystallization of reduced Grx1 from the yeast Saccharomyces cerevisiae (yGrx1) in space group P2 1 2 1 2 1 and its structure solution and refinement to 1.22 Å resolution are reported. To study the structure-function relationship of yeast Grx1, the crystal structure of reduced yGrx1 was compared with the existing structures of the oxidized and glutathionylated forms. These comparisons revealed structural differences in the conformations of residues neighbouring the Cys27-Cys30 active site which accompany alterations in the redox status of the protein.

Interaction of platinum-based drugs with proteins containing sulphur amino acids is usually argued as one of the major reasons for the observed resistance to these drugs, mainly due to the deactivation of the native compounds by very efficient thiolation processes in the organism. In this work, we have investigated the detailed thermodynamics and kinetics of reaction between cisplatin cis-[PtCl2(NH3)2] and its major hydrolysed forms (monohydroxocisplatin cis-[PtCl(OH)(NH3)2] and monoaquacisplatin cis-[PtCl(H2O)(NH3)2]+) with various thiolates (methanethiolate, cysteine and glutathione) and methionine. We have used a demanding quantum chemistry approach at the MP2 and DFT levels of theory to determine the Gibbs free energies and the barrier of reactions of the most possible reaction paths. The substitution of the four ligands of the complexes studied here (Cl-, OH-, H2O and NH3) can either proceed by direct thiolations or bidentations. Our Raman spectroscopy measurements show that only two thiolations actually occur, although four are possible in principle. The reason could lie in the bidentation reactions eventually taking place after each thiolation, which is backed up by our computational results. The observed lability scale of the ligands under thiolate exposure was found to be in the following order H2O > Cl- ≈ NH3(trans) > NH3(cis) > OH-, the difference between ammine ligands being induced by a significant trans-labilization by thiolates. Finally, the S,N bidentation is shown to be preferred with respect to the S,O one.

After evidence of the cysteinylated precursors of 3-sulfanyl-4-methylpentan-1-ol (Cys-26) and 3-sulfanylhexan-1-ol (Cys-23) in hop, S-glutathione precursors (G-23 and G-26) were recently discovered in different dual-purpose hop varieties. Because free 3-sulfanylpentan-1-ol (21) has also been detected in hop, the present work aimed to identify its potential precursors. The compounds S-3-(1-hydroxylpentyl)cysteine (Cys-21) and S-3-(1-hydroxylpentyl)glutathione (G-21) were first synthesized and characterized by nuclear magnetic resonance and high-resolution mass spectrometry. High-performance liquid chromatography-positive electrospray ionization-tandem mass spectrometry evidenced both for the first time in hop. Both S conjugates were further quantitated in six hop samples: the well-known Saaz, Amarillo, Citra, Hallertau Blanc, Nelson Sauvin, and Polaris. Similar to G-23, G-21 appeared ubiquitous to all varieties. Of all of the samples investigated here, Citra (harvest 2017) emerged as the richest in G-21, with 18 mg/kg of dry matter. Cys-21 was found in all samples at a much lower concentration (up to 0.2 mg/kg of dry matter in Polaris, harvest 2017). Model media spiked with Cys-21 or G-21 allowed for the confirmation that brewing yeast is able to release free compound 21 from them.

Biological thiols (biothiols), such as glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), play a vital role in the process of reversible redox reactions in physiological systems. In this work, flow cytometry-based fluorescent sensor is for the first time developed for the detection of biothiols in a fluorescence "turn on" manner. The probe which we name "Polystyrene/Quantum Dots/Gold Nanoparticles" or (PS/QDs/Au) is constructed by immobilizing QDs onto the surface of PS microbeads to obtain fluorescent microbeads. The probe (PS/QDs/Au) is constructed by immobilizing QDs onto the surface of PS microbeads to obtain fluorescent microbeads, followed by gold NPs absorption through electrostatic interaction to quench their fluorescence. In the presence of biothiols, the fluorescence of our probe can be restored in less than 5 min, and the detection limits for GSH, Cys and Hcy are 0.5 μM, 0.1 μM and 0.3 μM, respectively. Most importantly, the fluorescence signal of each of our probe microbeads can be collected individually by flow cytometry, realizing single microbead-based biothiols detection for the first time. Moreover, the probe is successfully applied to imaging of intracellular biothiols in A549 cells, demonstrating its potential in biological application., (Copyright © 2018 Elsevier B.V. All rights reserved.)