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Representatives of the colorless sulfur bacteria of the genus Beggiatoa use reduced sulfur compounds in the processes of lithotrophic growth, which is accompanied by the storage of intracellular sulfur. However, it is still unknown how the transformation of intracellular sulfur occurs in Beggiatoa representatives. Annotation of the genome of Beggiatoa leptomitoformis D-402 did not identify any genes for the oxidation or reduction of elemental sulfur. By searching BLASTP, two putative persulfide dioxygenase (PDO) homologs were found in the genome of B. leptomitoformis . In some heterotrophic prokaryotes, PDO is involved in the oxidation of sulfane sulfur. According to HPLC-MS/MS, the revealed protein was reliably detected in a culture sample grown only in the presence of endogenous sulfur and CO 2 . The recombinant protein from B. leptomitoformis was active in the presence of glutathione persulfide. The crystal structure of recombinant PDO exhibited consistency with known structures of type I PDO. Thus, it was shown that B. leptomitoformis uses PDO to oxidize endogenous sulfur. Additionally, on the basis of HPLC-MS/MS, RT-qPCR, and the study of PDO reaction products, we predicted the interrelation of PDO and Sox-system function in the oxidation of endogenous sulfur in B. leptomitoformis and the connection of this process with energy metabolism.

Chitosan (CHT) is a deacylated derivative of chitin and improves growth and yield performance, activates defensive genes, and also induces stomatal closure in plants. Glutathione (GSH) has significant functions in the growth, development, defense systems, signaling, and gene expression. GSH negatively regulates abscisic acid-, methyl jasmonate-, and salicylic acid-induced stomatal closure. However, the negative regulation by GSH of CHT-induced stomatal closure is still unknown. Regulation of CHT-induced stomatal closure by GSH in guard cells was investigated using two GSH-deficient mutants, cad2-1 and chlorina 1-1 (ch1-1), and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). The cad2-1 and ch1-1 mutations and CDNB treatment enhanced CHT-induced stomatal closure. Treatment with glutathione monoethyl ester restored the GSH level in the guard cells of cad2-1 and ch1-1 and complemented the stomatal phenotype of the mutants. These results indicate that GSH negatively regulates CHT-induced stomatal closure in Arabidopsis thaliana., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC 50 ) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC 50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population., 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. Published by Elsevier B.V.)

Infectious diseases caused by trypanosomatids, including African trypanosomiasis (sleeping sickness), Chagas disease, and different forms of leishmaniasis, are Neglected Tropical Diseases affecting millions of people worldwide, mainly in vulnerable territories of tropical and subtropical areas. In general, current treatments against these diseases are old-fashioned, showing adverse effects and loss of efficacy due to misuse or overuse, thus leading to the emergence of resistance. For these reasons, searching for new antitrypanosomatid drugs has become an urgent necessity, and different metabolic pathways have been studied as potential drug targets against these parasites. Considering that trypanosomatids possess a unique redox pathway based on the trypanothione molecule absent in the mammalian host, the key enzymes involved in trypanothione metabolism, trypanothione reductase and trypanothione synthetase, have been studied in detail as druggable targets. In this review, we summarize some of the recent findings on the molecules inhibiting these two essential enzymes for Trypanosoma and Leishmania viability.

Vitamin B 12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B 12 is obtained from food and travels from the stomach, through the intestine, and into the bloodstream by three B 12 -transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor, and transcobalamin (TC), which all bind B 12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B 12 , Cbl in the aquo/hydroxocobalamin form can coordinate various nucleophiles, for example, GSH, giving rise to glutathionylcobalamin (GSCbl), a naturally occurring form of vitamin B 12 . Currently, there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B 12 transporter and GSCbl, which compared to aquo/hydroxocobalamin, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, haptocorrin and intrinsic factor are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We investigated the effects of glutathione trisulfide (GSSSG) on lipopolysaccharide (LPS)-induced inflammatory gene expression in immortalized ARPE-19, and primary human and mouse retinal pigment epithelial (RPE) cells. Sulfane sulfur molecules were significantly increased in GSSSG-treated ARPE-19 cells. GSSSG prevented the LPS-induced upregulation of interleukin ( IL )- 1β, IL-6 , and C-C motif chemokine ligand 2 ( CCL2 ) in ARPE-19/primary RPE cells. Moreover, GSSSG prevented the activation of the nuclear factor-kappa B p65 subunit, and promoted the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in LPS-treated ARPE-19 cells. ERK1/2 inhibition prevented the GSSSG-mediated inhibition of LPS-induced IL-6 and CCL2 upregulation. Additionally, ERK1/2 activation prevented the upregulation of these genes in the absence of GSSSG. Knockdown of HMOX1 or NRF2 , known as anti-oxidative genes, did not affect the activity of GSSSG in the context of LPS stimulation. These findings suggest that GSSSG attenuates LPS-induced inflammatory gene expression via ERK signaling hyperactivation, independently of the NRF2/HMOX1 pathway.

The abnormal accumulation of methylglyoxal (MG) leading to increased glycation of protein and DNA has emerged as an important metabolic stress, dicarbonyl stress, linked to aging, and disease. Increased MG glycation produces inactivation and misfolding of proteins, cell dysfunction, activation of the unfolded protein response, and related low-grade inflammation. Glycation of DNA and the spliceosome contribute to an antiproliferative and apoptotic response of high, cytotoxic levels of MG. Glyoxalase 1 (Glo1) of the glyoxalase system has a major role in the metabolism of MG. Small molecule inducers of Glo1, Glo1 inducers, have been developed to alleviate dicarbonyl stress as a prospective treatment for the prevention and early-stage reversal of type 2 diabetes and prevention of vascular complications of diabetes. The first clinical trial with the Glo1 inducer, trans -resveratrol and hesperetin combination (tRES-HESP)-a randomized, double-blind, placebo-controlled crossover phase 2A study for correction of insulin resistance in overweight and obese subjects, was completed successfully. tRES-HESP corrected insulin resistance, improved dysglycemia, and low-grade inflammation. Cell permeable Glo1 inhibitor prodrugs have been developed to induce severe dicarbonyl stress as a prospective treatment for cancer-particularly for high Glo1 expressing-related multidrug-resistant tumors. The prototype Glo1 inhibitor is prodrug S-p-bromobenzylglutathione cyclopentyl diester (BBGD). It has antitumor activity in vitro and in tumor-bearing mice in vivo. In the National Cancer Institute human tumor cell line screen, BBGD was most active against the glioblastoma SNB-19 cell line. Recently, potent antitumor activity was found in glioblastoma multiforme tumor-bearing mice. High Glo1 expression is a negative survival factor in chemotherapy of breast cancer where adjunct therapy with a Glo1 inhibitor may improve treatment outcomes. BBGD has not yet been evaluated clinically. Glycation by MG now appears to be a pathogenic process that may be pharmacologically manipulated for therapeutic outcomes of potentially important clinical impact.

Extracellular glutathione (GSH) and oxidized glutathione (GSSG) can modulate the function of the extracellular calcium sensing receptor (CaSR). The CaSR has a binding pocket in the extracellular domain of CaSR large enough to bind either GSH or GSSG, as well as the naturally occurring oxidized derivative L-cysteine glutathione disulfide (CySSG) and the compound cysteinyl glutathione (CysGSH). Modeling the binding energies (ΔG) of CySSG and CysGSH to CaSR reveals that both cysteine derivatives may have greater affinities for CaSR than either GSH or GSSG. GSH, CySSG, and GSSG are found in circulation in mammals and, among the three, CySSG is more affected by HIV/AIDs and aging than either GSH or GSSG. The beta-carbon linkage of cysteine in CysGSH may model a new class of calcimimetics, exemplified by etelcalcetide. Circulating glutathionergic compounds, particularly CySSG, may mediate calcium-regulatory responses via receptor-binding to CaSR in a variety of organs, including parathyroids, kidneys, and bones. Receptor-mediated actions of glutathionergics may thus complement their roles in redox regulation and detoxification. The glutathionergic binding site(s) on CaSR are suggested to be a target for development of drugs that can be used in treating kidney and other diseases whose mechanisms involve CaSR dysregulation.

Redox signaling, wherein reactive and diffusible small molecules are channeled into specific messenger functions, is a critical component of signal transduction. A central principle of redox signaling is that the redox modulators are produced in a highly controlled fashion to specifically modify biotargets. Thiols serve as primary mediators of redox signaling as a function of the rich variety of adducts, which allows initiation of distinct cellular effects. Coupling the inherent reactivity of thiols with highly sensitive and selective chemical analysis protocols can facilitate identification of redox signaling agents, both in solution and in cultured cells. Here, we describe use of capillary zone electrophoresis to both identify and quantify sulfinamides, which are specific markers of the reaction of thiols with nitroxyl (HNO), a putative biologically relevant reactive nitrogen species., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Acrylamide (AA) is classified as a probable human carcinogen and is ubiquitous in foods processed at high temperatures. The carcinogenicity of AA has been attributed to its active metabolite, glycidamide (GA). Both AA and GA can spontaneously or enzymatically conjugate with glutathione (GSH) to form their corresponding GSH conjugates. Profiling AA-glutathione conjugate (AA-GSH) and GA-glutathione conjugates (2 isomers: GA2-GSH and GA3-GSH) in serum would better illustrate AA detoxification compared with urinary metabolite analysis. However, the lack of AA-, GA2, and GA3-GSH study remains a critical data gap. Our study aimed to investigate the toxicokinetics of AA-, GA2-and GA3-GSH in Sprague Dawley rats treated with 0.1 mg/kg, 1.0 mg/kg, or 5.0 mg/kg AA. Blood samples were collected for LC-MS/MS analysis of the GSH conjugate products. Within 24 h of treatment, we observed rapid formation, elimination, and linear kinetics of AA-, GA2-and GA3-GSH. The ∑GA-GSH AUC/AA-GSH AUC ratios were 0.14-0.29, similar to ∑GA/AA AUC in serum but different from ∑GA/AA-derived urinary mercapturic acids in rodents. Our analysis of AA- and GA-GSHs values represents direct detoxification of AA and GA in vivo. This study advances our understanding of sex and inter-species differences in AA detoxification and may refine the existing kinetic models for a more relevant risk extrapolation., (Copyright © 2021. Published by Elsevier B.V.)

Functions of selenium are diverse as antioxidant, anti-inflammation, increased immunity, reduced cancer incidence, blocking tumor invasion and metastasis, and further clinical application as treatment with radiation and chemotherapy. These functions of selenium are mostly related to oxidation and reduction mechanisms of selenium metabolites. Hydrogen selenide from selenite, and methylselenol (MSeH) from Se-methylselenocyteine (MSeC) and methylseleninicacid (MSeA) are the most reactive metabolites produced reactive oxygen species (ROS); furthermore, these metabolites may involve in oxidizing sulfhydryl groups, including glutathione. Selenite also reacted with glutathione and produces hydrogen selenide via selenodiglutathione (SeDG), which induces cytotoxicity as cell apoptosis, ROS production, DNA damage, and adenosine-methionine methylation in the cellular nucleus. However, a more pronounced effect was shown in the subsequent treatment of sodium selenite with chemotherapy and radiation therapy. High doses of sodium selenite were effective to increase radiation therapy and chemotherapy, and further to reduce radiation side effects and drug resistance. In our study, advanced cancer patients can tolerate until 5000 μg of sodium selenite in combination with radiation and chemotherapy since the half-life of sodium selenite may be relatively short, and, further, selenium may accumulates more in cancer cells than that of normal cells, which may be toxic to the cancer cells. Further clinical studies of high amount sodium selenite are required to treat advanced cancer patients.

Atomoxetine (ATX) is a neurological drug widely used for the treatment of attention deficit-hyperactivity disorder. Liver injury has been documented in patients administered ATX. The mechanism of ATX's toxic action is less clear. This study is aimed to characterize reactive metabolites of ATX in vitro and in vivo to assist our understanding of the mechanisms of ATX hepatotoxicity. A hydroxylated metabolite, along with an O -dealkylation metabolite, was found in ATX-supplemented rat liver microsome incubations. Additionally, two glutathione (GSH) conjugates and two N -acetylcysteine (NAC) conjugates were observed in rat liver microsome incubations containing ATX, NADPH, and GSH or NAC. The corresponding GSH conjugates and NAC conjugates were found in bile and urine of ATX-treated rats, respectively. Recombinant P450 enzyme incubation study demonstrated that CYP2D6 dominated the metabolic activation of ATX. The insights gained from this study may be of assistance to illuminate the mechanisms of ATX-induced hepatotoxicity.

Metallo-β-lactamases (MBLs) are class B β-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of β-lactam antibiotics. A previous study on BLEG-1 (formerly called Bleg1_2437), a hypothetical protein from Bacillus lehensis G1, revealed sequence similarity and activity to B3 subclass MBLs, despite its evolutionary divergence from these enzymes. Its relatedness to glyoxalase II (GLXII) raises the possibility of its enzymatic promiscuity and unique structural features compared to other MBLs and GLXIIs. This present study highlights that BLEG-1 possessed both MBL and GLXII activities with similar catalytic efficiencies. Its crystal structure revealed highly similar active site configuration to YcbL and GloB GLXIIs from Salmonella enterica , and L1 B3 MBL from Stenotrophomonas maltophilia . However, different from GLXIIs, BLEG-1 has an insertion of an active-site loop, forming a binding cavity similar to B3 MBL at the N-terminal region. We propose that BLEG-1 could possibly have evolved from GLXII and adopted MBL activity through this insertion.

Low molecular weight thiols including trypanothione and glutathione play an important function in the cellular growth, maintenance and reduction of oxidative stress in Leishmania species. In particular, parasite specific trypanothione has been established as a prime target for new anti-leishmania drugs. Previous studies into the interaction of the front-line Sb(V) based anti-leishmanial drug meglumine antimoniate with glutathione, have demonstrated that a reduction pathway may be responsible for its effective and selective nature. The new suite of organometallic complexes, of general formula [MAr 3 (O 2 CR) 2 ] (M = Sb or Bi) have been shown to have potential as new selective drug candidates. However, their behaviour towards the critical thiols glutathione and trypanothione is still largely unknown. Using NMR spectroscopy and mass spectrometry we have examined the interaction of the analogous Sb(V) and Bi(V) organometallic complexes, [SbPh 3 (O 2 CCH 2 (C 6 H 4 CH 3 )) 2 ] S1 and [BiPh 3 (O 2 CCH 2 (C 6 H 4 CH 3 )) 2 ] B1, with the trifluoroacetate (TFA) salt of trypanothione and L-glutathione. In the presence of trypanothione or glutathione at the clinically relevant pH of 4-5 for Leishmania amastigotes, both complexes undergo facile and rapid reduction, with no discernible difference. However, at a higher pH (6-7), the complexes behave quite differently towards glutathione. The Bi(V) complex is again reduced rapidly but the Sb(V) complex undergoes slow reduction over 8 h (t 1/2  = 54 min.) These results give the first insights into why the highly oxidising Bi(V) complexes display low selectivity in their cytotoxicity towards leishmanial and mammalian cells, while the Sb(V) complexes show good selectivity., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

The main objectives of this study was to identify the effects of a relationship of hyper-concentration of Gamma-glutamyltransferase (γ-GTP) in follicle fluid (FF) on the levels of glutathione (GSH)/reactive oxygen species (ROS) in oocytes and subsequent embryo development in cattle with abnormal livers. Furthermore, we investigated the effect of supplementing in vitro maturation medium with glutathione ethyl ester (GSH-OEt) on the subsequent developmental potential of oocytes from such cattle. We used a control group of cattle (with normal livers) and a liver disorder (LD) group, in which the liver was diagnosed as being abnormal. In experiment 1, the LD group was divided to two subgroups according to the concentration of γ-GTP in FF: a low group (≤50 IU/L; the low LD group), and a high group (>50 IU/L: the high LD group). Cumulus oocyte-complexes (COCs) were matured and fertilized in vitro and then cultured to the blastocyst stage. The levels of GSH and ROS in the matured oocytes after IVM were then assessed in each group. On day 7 after fertilization, embryo cleavage and development were assessed. We found that the rate of development to the blastocyst stage was significantly lower in the high LD group than in the control group and the low LD group. The levels of GSH in matured oocytes were significantly lower in the high LD group than in the control group and low LD group. The levels of ROS in matured oocytes was significantly higher in the high LD group than in the control group and the low LD group. In experiment 2, COCs from cattle in the high LD group were matured in m-199 supplemented with 5 mM GSH-OEt, then IVF and IVC was performed for 7 days. The GSH levels were determined in some COCs after IVM. The supplementation of media with GSH-OEt during IVM increased the levels of GSH in mature oocytes and improved the rate of blastocyst development compared with the control group. In conclusion, GSH-OEt supplementation to media during IVM can improve the developmental potential of oocytes in liver-diseased cattle with high γ-GTP concentrations in the FF by increasing intracellular GSH synthesis and scavenging ROS., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest., (Copyright © 2021. Published by Elsevier Inc.)

Leishmaniasis is one of the most neglected diseases worldwide and is considered a serious public health issue. The current therapeutic options have several disadvantages that make the search for new therapeutics urgent. Gold compounds are emerging as promising candidates based on encouraging in vitro and limited in vivo results for several Au I and Au III complexes. The antiparasitic mechanisms of these molecules remain only partially understood. However, a few studies have proposed the trypanothione redox system as a target, similar to the mammalian thioredoxin system, pointed out as the main target for several gold compounds with significant antitumor activity. In this review, we present the current status of the investigation and design of gold compounds directed at treating leishmaniasis. In addition, we explore potential targets in Leishmania parasites beyond the trypanothione system, taking into account previous studies and structure modulation performed for gold-based compounds., (© 2021 Wiley-VCH GmbH.)

Proteins in the eye lens have negligible turnover and therefore progressively accumulate chemical modifications during aging. Carbonyls and oxidative stresses, which are intricately linked to one another, predominantly drive such modifications. Oxidative stress leads to the loss of glutathione (GSH) and ascorbate degradation; this in turn leads to the formation of highly reactive dicarbonyl compounds that react with proteins to form advanced glycation end products (AGEs). The formation of AGEs leads to the crosslinking and aggregation of proteins contributing to lens aging and cataract formation. To inhibit AGE formation, we developed a disulfide compound linking GSH diester and mercaptoethylguanidine, and we named it carboxitin. Bovine lens organ cultured with carboxitin showed higher levels of GSH and mercaptoethylguanidine in the lens nucleus. Carboxitin inhibited erythrulose-mediated mouse lens protein crosslinking, AGE formation and the formation of 3-deoxythreosone, a major ascorbate-derived AGE precursor in the human lens. Carboxitin inhibited the glycation-mediated increase in stiffness in organ-cultured mouse lenses measured using compressive mechanical strain. Delivery of carboxitin into the lens increases GSH levels, traps dicarbonyl compounds and inhibits AGE formation. These properties of carboxitin could be exploited to develop a therapy against the formation of AGEs and the increase in stiffness that causes presbyopia in aging lenses.

The aberrant redox regulation and anti-oxidative defense is one of the main causes of age-induced decline in oocytes quality and embryo development in mammals. The present study aimed to elucidate the effect of glutathione ethyl ester (GSH-OEt), a cell-permeable glutathione (GSH) donor, on the developmental competence of oocytes in cows with advanced reproductive age. Oocytes were collected from cows aged 30-50 months or >120 months, which were defined as young or aged, respectively, and subjected to in vitro maturation (IVM) in the presence of 5 mM of GSH-OEt. In aged cows, the GSH level in follicular fluid was lower, and the intracellular levels of reactive oxygen species (ROS) in post-IVM oocytes was higher than those in young cows. GSH-OEt supplementation during IVM reduced the ROS contents of oocyte in aged cows but not in young cows. GSH-OEt treatment promoted the meiotic progression and increased the proportion of oocytes with mature cytoplasm containing evenly dispersed cortical granules in aged cows. After in vitro fertilization, the normal fertilization and development to the blastocyst stage were enhanced by GSH-OEt in aged cows to levels comparable to those in young cows. Further, oocyte maturation in the presence of GSH-OEt increased the proportion of diploid blastocyst in aged cows. In contrast, GSH-OEt failed to enhance the oocyte maturation, fertilization, and embryo development in young cows. Taken together, the exogenous supplementation of GSH-OEt during IVM modulated the age-related oxidative damage of bovine oocytes and improved the developmental competence of oocytes in aged cows. Oocytes presented a distinct response to GSH-OEt treatment depending on the donor age. GSH-OEt supplementation during IVM could be of practical value through the efficiency improvement of chromosomally normal embryo production in aged cows., Competing Interests: Declaration of competing interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the contents of the paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Enzymatic conjugation of glutathione (GSH) to trichloroethylene (TCE) followed by catabolism to the corresponding cysteine-conjugate, S-(dichlorovinyl)-L-cysteine (DCVC), and subsequent bioactivation by renal cysteine conjugate beta-lyases is considered to play an important role in the nephrotoxic effects observed in TCE-exposed rat and human. In this study, it is shown for the first time that three regioisomers of GSH-conjugates of TCE are formed by rat and human liver fractions, namely S-(1,2-trans-dichlorovinyl)-glutathione (1,2-trans-DCVG), S-(1,2-cis-dichlorovinyl)-glutathione (1,2-cis-DCVG) and S-(2,2-dichlorovinyl)-glutathione (2,2-DCVG). In incubations of TCE with rat liver fractions their amounts decreased in order of 1,2-cis-DCVG > 1,2-trans-DCVG > 2,2-DCVG. Human liver cytosol showed a more than 10-fold lower activity of GSH-conjugation, with amounts of regioisomers decreasing in order 2,2-DCVG > 1,2-trans-DCVG > 1,2-cis-DCVG. Incubations with recombinant human GSTs suggest that GSTA1-1 and GSTA2-2 play the most important role in human liver cytosol. GSTP1-1, which produces regioisomers in order 1,2-trans-DCVG > 2,2-cis-DCVG > 1,2-cis-DCVG, is likely to contribute to extrahepatic GSH-conjugation of TCE. Analysis of the products formed by a beta-lyase mimetic model showed that both 1,2-trans-DCVC and 1,2-cis-DCVC are converted to reactive products that form cross-links between the model nucleophile 4-(4-nitrobenzyl)-pyridine (NBP) and thiol-species. No NBP-alkylation was observed with 2,2-DCVC corresponding to its low cytotoxicity and mutagenicity. The lower activity of GSH-conjugation of TCE by human liver fractions, in combination with the lower fraction of potential nephrotoxic and mutagenic 1,2-DCVG-isomers, suggest that humans are at much lower risk for TCE-associated nephrotoxic effects than rats., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Rosacea is a very common, chronic inflammatory disease characterized by flushing, erythema and inflammatory lesions. Increased oxidative stress plays a relevant pathogenetic role in Rosacea. Intracellular Glutathione (GSH) is the main scavenger protective mechanism against increased oxidative stress. An altered GSH metabolism in Rosacea has been described. GSH-C4 is a modified GSH molecule characterized by a better intracellular bioavailability and longer half-life. A daily cream (E-AR) containing GSH-C4 (0.1%) with beta-Glycyrrhetic (0.5%) and azelaic acids (10%), with an SPF of 30, is available., Aim: In a pilot, prospective, two-center, assessor-blinded study we evaluate the efficacy and the tolerability of E-AR cream in subjects with mild to moderate Rosacea treated for 8 weeks., Patients and Methods: The main outcomes were the Investigator Global Assessment (IGA) 7-point score (from 0, completely clear; to 6, severe) and the clinical and instrumental erythema severity score (ESS) (from 0 to 4) evaluated in a blinded fashion (randomly coded photographs) at baseline, after 4 (only clinical) and 8 weeks (clinical and instrumental). VISIA evaluation for erythema and lesion counts and ANTERA 3D analysis for skin haemoglobin concentration (a parameter associated with inflammation) were also performed at the same time points. Analysis of primary outcomes was performed on an intention-to-treat basis. Tolerability was evaluated at week 4 and 8 recording spontaneously reported side effects., Results: Thirty subjects (22 women and 8 men; mean age 38 years) were enrolled after their written informed consent. Twenty-six (87%) subjects completed the study phases. Four subjects stopped prematurely the trial due to low skin tolerability (n=3) or lost to follow-up (n=1). At baseline, mean (SD) IGA score was 2.6 (0.9). At week 4, IGA score decreased (NS) to 2.3 (1.2). IGA score decreased significantly (p=0.0001) at week 8 to 1.2 (1) (mean difference 1.3; 95% CI of the difference from 0.9 to 1.7) in comparison with the baseline. The inflammatory mean (SD) lesion count, evaluated clinically, were 5.1(2.5) at baseline, 2.8 (1.9) at week 4, and 1.9 (1.7) at week 8 (P=0.0001; ANOVA Test), representing a 63% reduction. This reduction was confirmed by inflammatory lesions count performed on VISIA pictures (from 4.5 at baseline to 1.7 lesions at week 8). Similar evolution was observed for the clinical and instrumental ESS with a reduction of 56% (clinical) and 48% (VISIA), respectively, at week 8 in comparison with the baseline. ANTERA 3D photographs confirmed the positive evolution observed clinically with a significant reduction (-24%) in hemoglobin content: from 1.88 at baseline to 1.44 at week 8., Conclusion: This new GSH-C4, beta-glycyrrethic and azelaic acids cream has shown to be efficacious in mild to moderate rosacea subjects. Local tolerability is in line with other anti-rosacea treatments., (© 2020 Wiley Periodicals LLC.)

There is an urgent need to develop novel compounds that prevent the deleterious effects of opioids such as fentanyl on minute ventilation while, if possible, preserving the analgesic actions of the opioids. We report that L-glutathione ethyl ester (GSHee) may be such a novel compound. In this study, we measured tail flick latency (TFL), arterial blood gas (ABG) chemistry, Alveolar-arterial gradient, and ventilatory parameters by whole body plethysmography to determine the responses elicited by bolus injections of fentanyl (75 μg/kg, IV) in male adult Sprague-Dawley rats that had received a bolus injection of GSHee (100 μmol/kg, IV) 15 min previously. GSHee given alone had minimal effects on TFL, ABG chemistry and A-a gradient whereas it elicited changes in some ventilatory parameters such as an increase in breathing frequency. In vehicle-treated rats, fentanyl elicited (1) an increase in TFL, (2) decreases in pH, pO 2 and sO 2 and increases in pCO 2 (all indicative of ventilatory depression), (3) an increase in Alveolar-arterial gradient (indicative of a mismatch in ventilation-perfusion in the lungs), and (4) changes in ventilatory parameters such as a reduction in tidal volume, that were indicative of pronounced ventilatory depression. In GSHee-pretreated rats, fentanyl elicited a more prolonged analgesia, relatively minor changes in ABG chemistry and Alveolar-arterial gradient, and a substantially milder depression of ventilation. GSHee may represent an effective member of a novel class of thiolester drugs that are able to prevent the ventilatory depressant effects elicited by powerful opioids such as fentanyl and their deleterious effects on gas-exchange in the lungs without compromising opioid analgesia.

We herein propose an easy-to-prepare mini-chemosensor array constructed by two-types of off-the-shelf coumarin dyes for simultaneous classification and quantification of sulfur-containing amino acids (SCAAs) (i.e., l-cysteine, l-cystine, l-homocysteine, glutathione, and glutathione disulfide). The detection mechanism of SCAAs relied on a coordination-based sensor array (CBSA) utilizing the competitive binding among the coumarin dye, a Zn 2+ ion and SCAAs. The reversible feature of the coordination bond of the coumarin-Zn 2+ resulted in UV/vis spectral shifts and fluorescence enhancement in response to the analytes, offering the multianalyte detection in high accuracy. Furthermore, a mixture of glutathione and l-cysteine was successfully quantified in a sample containing human blood serum. This study would be an important example of the optical chemosensor array toward the rapid and accurate detection of biomarkers.

Objective: Two phase I studies of darinaparsin including Japanese and Korean patients with relapsed/refractory peripheral T-cell lymphoma were performed to evaluate its safety (primary purpose), efficacy and pharmacokinetic profile (ClinicalTrials.gov: NCT01435863 and NCT01689220)., Methods: Patients received intravenous darinaparsin for 5 consecutive days at 200 mg/m2/day in 4-week cycles, 300 mg/m2/day in 4-week cycles or 300 mg/m2/day in 3-week cycles., Results: Seventeen Japanese and 6 Korean patients were enrolled and treated. Drug-related adverse events developed in 18 patients (78%). Dose-limiting toxicity, grade 3 hepatic dysfunction, was reported on Day 15 of cycle 1 in 1 Japanese patient who received 300 mg/m2/day. The most common drug-related, grade ≥ 3 adverse events were lymphopenia (9%), neutropenia (9%) and thrombocytopenia (9%). No deaths occurred. In 14 evaluable patients, 1 and 3 patients had complete response and partial response, respectively. The plasma concentration-time profiles of arsenic, a surrogate marker for darinaparsin, were similar between Japanese and Korean patients. No significant difference was found in its pharmacokinetic profile., Conclusions: These data indicate the good tolerability and potential efficacy of darinaparsin in patients with relapsed/refractory peripheral T-cell lymphoma. Darinaparsin 300 mg/m2/day for 5 consecutive days in 3-week cycles is the recommended regimen for phase II study., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sulfur mustard (SM), a chemical warfare agent, is a strong alkylating compound that readily reacts with numerous biomolecules. The goal of the present work was to define and validate new biomarkers of exposure to SM that could be easily accessible in urine or plasma. Because investigations using SM are prohibited by the Organisation for the Prohibition of Chemical Weapons, we worked with 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM. We developed an ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) approach to the conjugate of CEES to glutathione and two of its metabolites: the cysteine and the N-acetylcysteine conjugates. The N7-guanine adduct of CEES (N7Gua-CEES) was also targeted. After synthesizing the specific biomarkers, a solid-phase extraction protocol and a UHPLC-MS/MS method with isotopic dilution were optimized. We were able to quantify N7Gua-CEES in the DNA of HaCaT keratinocytes and of explants of human skin exposed to CEES. N7Gua-CEES was also detected in the culture medium of these two models, together with the glutathione and the cysteine conjugates. In contrast, the N-acetylcysteine conjugate was not detected. The method was then applied to plasma from mice cutaneously exposed to CEES. All four markers could be detected. Our present results thus validate both the analytical technique and the biological relevance of new, easily quantifiable biomarkers of exposure to CEES. Because CEES behaves very similar to SM, the results are promising for application to this toxic of interest.

The role of post-transcriptional RNA modification is of growing interest. One example is the addition of non-templated uridine residues to the 3' end of transcripts. In mammalian systems, uridylation is integral to cell cycle control of histone mRNA levels. This regulatory mechanism is dependent on the nonsense-mediated decay (NMD) component, Upf1, which promotes histone mRNA uridylation and degradation in response to the arrest of DNA synthesis. We have identified a similar system in Aspergillus nidulans, where Upf1 is required for the regulation of histone mRNA levels. However, other NMD components are also implicated, distinguishing it from the mammalian system. As in human cells, 3' uridylation of histone mRNA is induced upon replication arrest. Disruption of this 3' tagging has a significant but limited effect on histone transcript regulation, consistent with multiple mechanisms acting to regulate mRNA levels. Interestingly, 3' end degraded transcripts are also subject to re-adenylation. Both mRNA pyrimidine tagging and re-adenylation are dependent on the same terminal-nucleotidyltransferases, CutA, and CutB, and we show this is consistent with the in vitro activities of both enzymes. Based on these data we argue that mRNA 3' tagging has diverse and distinct roles associated with transcript degradation, functionality and regulation., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

A novel analytical method was developed for the quantification of glutathione hydropersulfide (G-SSH) in biological samples by high-performance liquid chromatography (HPLC) with post-column derivatization. G-SSH was treated with iodoacetamide as an alkylating agent for 5 min at 37 °C, and the resultant acetamide-labeled G-SSH (G-SS-acetamide) was subjected to HPLC. After separation on a reversed-phase column, G-SS-acetamide was quantified by detection using a post-column reaction with orthophthalaldehyde under alkaline conditions. The standard G-SS-acetamide was synthesized through the S-S exchange reaction between oxidized glutathione and 2-mercaptoacetamide. It should be noted that some types of alkylating agents, including N-ethylmaleimide and monobromobimane, cleave the polysulfide chains of polysulfides that consist of glutathione, resulting in the production of alkylated G-SSHs. We confirmed that iodoacetamide did not enhance the cleavage of acetamide-labeled glutathione trihydropersulfide (G-SSS-acetamide). The lowest quantification limit was estimated to be 25 nM for G-SS-acetamide. This method can be useful for studying the dynamics of sulfane sulfur in glutathione-containing matrices., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The formation of a persulfide group (-SSH) on cysteine residues has gained attention as a reversible posttranslational modification contributing to protein regulation or protection. The widely distributed 3-mercaptopyruvate sulfurtransferases (MSTs) are implicated in the generation of persulfidated molecules and H 2 S biogenesis through transfer of a sulfane sulfur atom from a suitable donor to an acceptor. Arabidopsis has two MSTs, named STR1 and STR2, but they are poorly characterized. To learn more about these enzymes, we conducted a series of biochemical experiments including a variety of possible reducing systems. Our kinetic studies, which used a combination of sulfur donors and acceptors revealed that both MSTs use 3-mercaptopyruvate efficiently as a sulfur donor while thioredoxins, glutathione, and glutaredoxins all served as high-affinity sulfane sulfur acceptors. Using the redox-sensitive GFP (roGFP2) as a model acceptor protein, we showed that the persulfide-forming MSTs catalyze roGFP2 oxidation and more generally trans-persulfidation reactions. However, a preferential interaction with the thioredoxin system and glutathione was observed in case of competition between these sulfur acceptors. Moreover, we observed that MSTs are sensitive to overoxidation but are protected from an irreversible inactivation by their persulfide intermediate and subsequent reactivation by thioredoxins or glutathione. This work provides significant insights into Arabidopsis STR1 and STR2 catalytic properties and more specifically emphasizes the interaction with cellular reducing systems for the generation of H 2 S and glutathione persulfide and reactivation of an oxidatively modified form., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the content of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: COVID-19 is a life-threatening novel corona viral infection to our civilization and spreading rapidly. Tremendousefforts have been made by the researchers to search for a drug to control SARS-CoV-2., Methods: Here, a series of arsenical derivatives were optimized and analyzed with in silico study to search the inhibitor of RNA dependent RNA polymerase (RdRp), the major replication factor of SARS-CoV-2. All the optimized derivatives were blindly docked with RdRp of SARS-CoV-2 using iGEMDOCK v2.1., Results: Based on the lower idock score in the catalytic pocket of RdRp, darinaparsin (-82.52 kcal/- mol) was revealed to be the most effective among them. Darinaparsin strongly binds with both Nsp9 replicase protein (-8.77 kcal/mol) and Nsp15 endoribonuclease (-8.3 kcal/mol) of SARS-- CoV-2 as confirmed from the AutoDock analysis. During infection, the ssRNA of SARS-CoV-2 is translated into large polyproteins forming viral replication complex by specific proteases like 3CL protease and papain protease. This is also another target to control the virus infection where darinaparsin also performs the inhibitory role to proteases of 3CL protease (-7.69 kcal/mol) and papain protease (-8.43 kcal/mol)., Conclusion: In the host cell, the furin protease serves as a gateway to the viral entry and darinaparsin docked with furin protease, which revealed a strong binding affinity. Thus, screening of potential arsenic drugs would help in providing the fast in-vitro to in-vivo analysis towards the development of therapeutics against SARS-CoV-2., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Glutathione S-transferase P (GSTP) is a component of a complex series of pathways that provide cellular redox homeostasis. It is an abundant protein in certain tumors and is over-expressed in cancer drug resistance. It has diverse cellular functions that include, thiolase activities with small electrophilic agents or susceptible cysteine residues on the protein to mediate S-glutathionylation, and chaperone binding with select protein kinases. Preclinical and clinical testing of a nanomolar inhibitor of GSTP, TLK199 (Telintra; Ezatiostat) has indicated a role for the enzyme in hematopoiesis and utility for the drug in the treatment of patients with myelodysplastic syndrome.

Chagas Disease, African sleeping sickness, and leishmaniasis are neglected diseases caused by pathogenic trypanosomatid parasites, which have a considerable impact on morbidity and mortality in poor countries. The available drugs used as treatment have high toxicity, limited access, and can cause parasite drug resistance. Long-term treatments, added to their high toxicity, result in patients that give up therapy. Trypanosomatids presents a unique trypanothione based redox system, which is responsible for maintaining the redox balance. Therefore, inhibition of these essential and exclusive parasite's metabolic pathways, absent from the mammalian host, could lead to the development of more efficient and safe drugs. The system contains different redox cascades, where trypanothione and tryparedoxins play together a central role in transferring reduced power to different enzymes, such as 2-Cys peroxiredoxins, non-selenium glutathione peroxidases, ascorbate peroxidases, glutaredoxins and methionine sulfoxide reductases, through NADPH as a source of electrons. There is sufficient evidence that this complex system is essential for parasite survival and infection. In this review, we explore what is known in terms of essentiality, kinetic and structural data, and the development of inhibitors of enzymes from this trypanothione-based redox system. The recent advances and limitations in the development of lead inhibitory compounds targeting these enzymes have been discussed. The combination of molecular biology, bioinformatics, genomics, and structural biology is fundamental since the knowledge of unique features of the trypanothione-dependent system will provide tools for rational drug design in order to develop better treatments for these diseases., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Selenium (Se) shows biologically ambivalent characteristics in animals. It is an essential element but becomes severely toxic when the amount ingested exceeds the adequate intake level. Animals must be able to metabolize the various selenocompounds in meat, fish and vegetables to utilize Se for selenoprotein synthesis. It is known that the biological, nutritional, and toxicological effects of Se are strongly dependent on its chemical form. First, we evaluated the nutritional availability of nine naturally occurring Se compounds, or the so-called bioselenocompounds, in vivo. Second, we evaluated that gut microflora might contributes to the Se nutritional availability. Se is mainly excreted into urine. However, a substantial amount of Se was secreted into bile although Se was hardly detected in feces. Third, we evaluated the biological significance of biliary secretion of Se in terms of mineral nutrition. Finally, we discussed the entire Se metabolism in gut contributing to Se homeostasis in animal.

Leishmania and Trypanosoma parasites are responsible for the challenging neglected tropical diseases leishmaniases, Chagas disease, and human African trypanosomiasis, which account for up to 40,000 deaths annually mainly in developing countries. Current chemotherapy relies on drugs with significant limitations in efficacy and safety, prompting the urgent need to explore innovative approaches to improve the drug discovery pipeline. The unique trypanothione-based redox pathway, which is absent in human hosts, is vital for all trypanosomatids and offers valuable opportunities to guide the rational development of specific, broad-spectrum and innovative anti-trypanosomatid agents. Major efforts focused on the key metabolic enzymes trypanothione synthetase-amidase and trypanothione reductase, whose inhibition should affect the entire pathway and, finally, parasite survival. Herein, we will report and comment on the most recent studies in the search for enzyme inhibitors, underlining the promising opportunities that have emerged so far to drive the exploration of future successful therapeutic approaches., (© 2020 Wiley-VCH GmbH.)

Busulfan is an alkylating agent used in chemotherapy conditioning regimens prior to hematopoietic stem cell transplantation (HSCT). However, its administration is associated with a great risk of adverse toxicities, which have been historically attributed to busulfan's mechanism of non-specific DNA alkylation. A phase II generated metabolite of busulfan, EdAG (γ-glutamyldehydroalanylglycine), is a dehydroalanine analog of glutathione (GSH) with an electrophilic moiety, suggesting it may bind to proteins and disrupt biological function. However, EdAG's reactions with common cellular thiols such as glutathione (GSH) and l-cysteine are understudied, along with possible inhibition of glutathionylation-dependent enzymes (with active site cysteine residues). We established a physiologically-relevant in vitro model to readily measure thiol loss over time. Using this model, we compared the apparent rates of thiol depletion in the presence of EdAG or arecoline, a toxic constituent of the areca (betel) nut and known GSH depletor. Simulated kinetic modeling revealed that the mean (±SE) alpha (α) second order rate constants describing GSH and l-cysteine depletion in the presence of EdAG were 0.00522 (0.00845) μM -1 ∙min -1 and 0.0207 (0.00721) μM -1 ∙min -1 , respectively; in the presence of arecoline, the apparent rates of depletion were 0.0619 (0.009) μM -1 ∙min -1 and 0.2834 (0.0637) μM -1 ∙min -1 for GSH and l-cysteine, respectively. Under these experimental conditions, we conclude that EdAG was a weaker electrophile than arecoline. Arecoline and EdAG both depleted apparent l-cysteine concentrations to a much greater extent than GSH, approximately 4.58-fold and 3.97-fold change greater, respectively. EdAG modestly inhibited (∼20%) the human thioredoxin-1 (hTrx-1) catalyzed reduction of insulin with a mean IC 50 of 93 μM [95% CI: 78.6-110 μM). In summary, EdAG's ability to spontaneously react with endogenous thiols and inhibit hTrx-1 are potentially biochemically relevant in humans. These findings continue to support the growing concept that EdAG, an underrecognized phase II metabolite of busulfan, plays a role in untoward cellular toxicities during busulfan pharmacotherapy., 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 © 2020 Elsevier Inc. All rights reserved.)

Trypanothione synthetase (TryS) produces N 1 ,N 8 -bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised N 5 -substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC 50 against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC 50 0.5-10 µM) and selectivity (20-35) against the clinically relevant stage of Leishmania braziliensis (mucocutaneous leishmaniasis) and L. infantum (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different Leishmania species.

Persulfides (RSSH/RSS - ) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H 2 S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/GSS - ), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H 2 S. A p K a of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through S N 2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS - presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called α effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the α effect correlated with the Brønsted nucleophilic factor, β nuc , for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the p K a of a biological persulfide and the first examination of the α effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Benchoam et al.)

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.

S-adenosyl-L-methionine is an endogenous molecule with hepato-protective properties linked to redox regulation and methylation. Here, the potential therapeutic value of SAMe was tested in 17 patients with PBC, a cholestatic disease with autoimmune phenomena targeting small bile ducts. Nine patients responded to SAMe (SAMe responders) with increased serum protein S-glutathionylation. That posttranslational protein modification was associated with reduction of serum anti-mitochondrial autoantibodies (AMA-M2) titers and improvement of liver biochemistry. Clinically, SAMe responders were younger at diagnosis, had longer duration of the disease and lower level of serum S-glutathionylated proteins at entry. SAMe treatment was associated with negative correlation between protein S-glutathionylation and TNFα. Furthermore, AMA-M2 titers correlated positively with INFγ and FGF-19 while negatively with TGFβ. Additionally, cirrhotic PBC livers showed reduced levels of glutathionylated proteins, glutaredoxine-1 (Grx-1) and GSH synthase (GS). The effect of SAMe was also analyzed in vitro. In human cholangiocytes overexpressing miR-506, which induces PBC-like features, SAMe increased total protein S-glutathionylation and the level of γ-glutamylcysteine ligase (GCLC), whereas reduced Grx-1 level. Moreover, SAMe protected primary human cholangiocytes against mitochondrial oxidative stress induced by tBHQ (tert-Butylhydroquinone) via raising the level of Nrf2 and HO-1. Finally, SAMe reduced apoptosis (cleaved-caspase3) and PDC-E2 (antigen responsible of the AMA-M2) induced experimentally by glycochenodeoxycholic acid (GCDC). These data suggest that SAMe may inhibit autoimmune events in patients with PBC via its antioxidant and S-glutathionylation properties. These findings provide new insights into the molecular events promoting progression of PBC and suggest potential therapeutic application of SAMe in PBC., 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 © 2020 Elsevier B.V. All rights reserved.)

We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 μM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 μM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 μM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 μM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This study aimed to examine whether the addition of glutathione ethyl ester (GSH-OEt) to the in vitro maturation (IVM) medium would improve the resilience of bovine oocytes to withstand vitrification. The effects of GSH-OEt on spindle morphology, levels of reactive oxygen species (ROS), mitochondrial activity and distribution, and embryo developmental potential were assessed together with the expression of genes with a role in apoptosis ( BAX, BCL2 ), oxidative-stress pathways ( GPX1, SOD1 ), water channels ( AQP3 ), implantation ( IFN-τ ) and gap junctions ( CX43 ) in oocytes and their derived blastocysts. Vitrification gave rise to abnormal spindle microtubule configurations and elevated ROS levels. Supplementation of IVM medium with GSH-OEt before vitrification preserved mitochondrial distribution pattern and diminished both cytoplasmic and mitochondrial ROS contents and percentages of embryos developing beyond the 8-cell stage were similar to those recorded in fresh non-vitrified oocytes. Although not significantly different from control vitrified oocytes, vitrified oocytes after GSH-OEt treatment gave rise to similar day 8-blastocyst and hatching rates to fresh non-vitrified oocytes. No effects of GSH-OEt supplementation were noted on the targeted gene expression of oocytes and derived blastocysts, with the exception of GPX1 , AQP3 and CX43 in derived blastocysts. The addition of GSH-OEt to the IVM medium before vitrification may be beneficial for embryo development presumably as the consequence of additional anti-oxidant protection during IVM.

Despite early treatment with antimycobacterial combination therapy, drug resistance continues to emerge. Maintenance of redox homeostasis is essential for Mycobacterium avium (M. avium) survival and growth. The aim of the present study was to investigate the antimycobacterial activity of two pro-glutathione (pro-GSH) drugs that are able to induce redox stress in M. avium and to modulate cytokine production by macrophages. Hence, we investigated two molecules shown to possess antiviral and immunomodulatory properties: C4-GSH, an N-butanoyl GSH derivative; and I-152, a prodrug of N-acetyl-cysteine (NAC) and β-mercaptoethylamine (MEA). Both molecules showed activity against replicating M. avium, both in the cell-free model and inside macrophages. Moreover, they were even more effective in reducing the viability of bacteria that had been kept in water for 7 days, proving to be active both against replicating and non-replicating bacteria. By regulating the macrophage redox state, I-152 modulated cytokine production. In particular, higher levels of interferon-gamma (IFN-γ), interleukin 1 beta (IL-1β), IL-18 and IL-12, which are known to be crucial for the control of intracellular pathogens, were found after I-152 treatment. Our results show that C4-GSH and I-152, by inducing perturbation of redox equilibrium, exert bacteriostatic and bactericidal activity against M. avium. Moreover, I-152 can boost the host response by inducing the production of cytokines that serve as key regulators of the Th1 response., (Copyright © 2020. Published by Elsevier Ltd.)

The transcriptome of eukaryotic cells is constantly monitored for errors to avoid the production of undesired protein variants. The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway degrades aberrant mRNAs, but also functions in the regulation of transcript abundance in response to changed physiological states. Here, we describe a zebrafish mutant of upf1 , encoding the central component of the NMD machinery. Fish homozygous for the upf1 t 20450 allele (Y163X) survive until day 10 after fertilization, presenting with impaired T cell development as one of the most conspicuous features of the mutant phenotype. Analysis of differentially expressed genes identified dysregulation of the pre-mRNA splicing pathway, accompanied by perturbed autoregulation of canonical splicing activators (SRSF) and repressors (HNRNP). In upf1 -deficient mutants, NMD-susceptible transcripts of ribosomal proteins that are known for their role as noncanonical splicing regulators were greatly increased, most notably, rpl10a When the levels of NMD-susceptible rpl10a transcripts were artificially increased in zebrafish larvae, T cell development was significantly impaired, suggesting that perturbed autoregulation of rpl10a splicing contributes to failing T cell development in upf1 deficiency. Our results identify an extraribosomal tissue-specific function to rpl10a in the immune system, and thus exemplify the advantages of the zebrafish model to study the effects of upf1 -deficiency in the context of a vertebrate organism., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Aldose reductase (AKR1B1), the first enzyme in the polyol pathway, is likely involved in the onset of diabetic complications. Differential inhibition of AKR1B1 has been proposed to counteract the damaging effects linked to the activity of the enzyme while preserving its detoxifying ability. Here, we show that epigallocatechin gallate (EGCG), one of the most representative catechins present in green tea, acts as a differential inhibitor of human recombinant AKR1B1. A kinetic analysis of EGCG, and of its components, gallic acid (GA) and epigallocatechin (EGC) as inhibitors of the reduction of L-idose, 4-hydroxy2,3-nonenal (HNE), and 3-glutathionyl l-4-dihydroxynonanal (GSHNE) revealed for the compounds a different model of inhibition toward the different substrates. While EGCG preferentially inhibited L-idose and GSHNE reduction with respect to HNE, gallic acid, which was still active in inhibiting the reduction of the sugar, was less active in inhibiting HNE and GSHNE reduction. EGC was found to be less efficient as an inhibitor of AKR1B1 and devoid of any differential inhibitory action. A computational study defined different interactive modes for the three substrates on the AKR1B1 active site and suggested a rationale for the observed differential inhibition. A chromatographic fractionation of an alcoholic green tea extract revealed that, besides EGCG and GA, other components may exhibit the differential inhibition of AKR1B1., Competing Interests: The authors declare no conflict of interest. The sponsor had no role in the design, execution, interpretation, or writing of the study.

Ultrasmall gold nanoparticles (AuNPs) are an emerging class of nanomaterials exhibiting distinctive physicochemical, molecular, and in vivo properties. Recently, we showed that ultrasmall AuNPs encompassing a zwitterionic glutathione monoethyl ester surface coating (AuGSH zwt ) were highly resistant to aggregation and serum protein interactions. Herein, we performed a new set of biointeraction studies to gain a more fundamental understanding into the behavior of both pristine and peptide-functionalized AuGSH zwt in complex media. Using the model Strep-tag peptide (WSHPQFEK) as an integrated functional group, we established that AuGSH zwt could be conjugated with increasing numbers of Strep-tags by simple ligand exchange, which provides a generic approach for AuGSH zwt functionalization. It was found that the strep-tagged AuGSH zwt particles were highly resistant to nonspecific protein interactions and retained their targeting capability in biological fluid, displaying efficient binding to Streptactin receptors in nearly undiluted serum. However, AuGSH zwt functionalized with multiple Strep-tags displayed somewhat lower resistance to protein interactions and lower levels of binding to Streptactin than monofunctionalized AuGSH zwt under given conditions. These results underscore the need for optimizing ligand density onto the surface of ultrasmall AuNPs for improved performance. Collectively, our findings support ultrasmall AuGSH zwt as an attractive platform for engineering functional, protein-mimetic nanostructures capable of specific protein recognition within the complex biological milieu.

Background: High glutaminase (GLS;EC3.5.1.2) activity is an important pathophysiological phenomenon in tumorigenesis and metabolic disease. Insight into the metabolic consequences of high GLS activity contributes to the understanding of the pathophysiology of both oncogenic pathways and inborn errors of glutamate metabolism. Glutaminase catalyzes the conversion of glutamine into glutamate, thereby interconnecting many metabolic pathways., Methods: We developed a HEK293-based cell-model that enables tuning of GLS activity by combining the expression of a hypermorphic GLS variant with incremental GLS inhibition. The metabolic consequences of increasing GLS activity were studied by metabolic profiling using Direct-Infusion High-Resolution Mass-Spectrometry (DI-HRMS)., Results and Conclusions: Of 12,437 detected features [m/z], 109 features corresponding to endogenously relevant metabolites were significantly affected by high GLS activity. As expected, these included strongly decreased glutamine and increased glutamate levels. Additionally, increased levels of tricarboxylic acid (TCA) intermediates with a truncation of the TCA cycle at the level of citrate were detected as well as increased metabolites of transamination reactions, proline and ornithine synthesis and GABA metabolism. Levels of asparagine and nucleotide metabolites showed the same dependence on GLS activity as glutamine. Of the nucleotides, especially metabolites of the pyrimidine thymine metabolism were negatively impacted by high GLS activity, which is remarkable since their synthesis depend both on aspartate (product of glutamate) and glutamine levels. Metabolites of the glutathione synthesizing γ-glutamyl-cycle were either decreased or unaffected., General Significance: By providing a metabolic fingerprint of increasing GLS activity, this study shows the large impact of high glutaminase activity on the cellular metabolome., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feedforward mechanisms of regulation. We have identified a PTM that is derived from the glycolytic by-product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D-lactate. We have identified the non-enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a "LactoylLys" modification on proteins. GLO2 knockout cells have elevated LGSH and a consequent marked increase in LactoylLys. Using an alkyne-tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg-like conditions., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glyoxalase I (GlxI) is a member of the glyoxalase system, which is important in cell detoxification and converts hemithioacetals of methylglyoxal (a cytotoxic byproduct of sugar metabolism that may react with DNA or proteins and introduce nucleic acid strand breaks, elevated mutation frequencies, and structural or functional changes of the proteins) and glutathione into d-lactate. GlxI accepts both the S and R enantiomers of hemithioacetal, but converts them to only the S -d enantiomer of lactoylglutathione. Interestingly, the enzyme shows this unusual specificity with a rather symmetric active site (a Zn ion coordinated to two glutamate residues; Glu-99 and Glu-172), making the investigation of its reaction mechanism challenging. Herein, we have performed a series of combined quantum mechanics and molecular mechanics calculations to study the reaction mechanism of GlxI. The substrate can bind to the enzyme in two different modes, depending on the direction of its alcoholic proton (H2; toward Glu-99 or Glu-172). Our results show that the S substrate can react only if H2 is directed toward Glu-99 and the R substrate only if H2 is directed toward Glu-172. In both cases, the reactions lead to the experimentally observed S -d enantiomer of the product. In addition, the results do not show any low-energy paths to the wrong enantiomer of the product from neither the S nor the R substrate. Previous studies have presented several opposing mechanisms for the conversion of R and S enantiomers of the substrate to the correct enantiomer of the product. Our results confirm one of them for the S substrate, but propose a new one for the R substrate.

Although selenium (Se) is mainly excreted in urine, it has been reported that an unknown Se metabolite is excreted in bile. When we administered selenomethionine (SeMet), selenocyanate or selenite to rats, a common biliary selenometabolite was detected 10 min after administration. The amount of the selenometabolite originating from SeMet was less than that originating from the two inorganic Se compounds, selenocyanate and selenite, suggesting that the transformation from the methylated organic selenocompound, i.e., SeMet, was less efficient than that from the inorganic Se compounds. The common biliary selenometabolite was concretely identified as selenodiglutathione (GSSeSG) by two types of mass spectrometry, i.e., LC-inductively coupled mass spectrometry (ICP-MS) and LC-ESI-Q/TOF. The bile-drained rats had lower urinary Se levels than the sham-operated rats. In addition, the Se amounts in urine plus bile of the bile-drained rats were comparable to the Se amount in the urine of the sham-operated rats. These results suggest that the biliary selenometabolite, GSSeSG, was reabsorbed in the gut and finally excreted in urine. Enterohepatic circulation occurs to maintain Se status in the body.

Leishmania protozoans are the causative agent of leishmaniasis, a neglected tropical disease consisting of three major clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis, and mucocutaneous leishmaniasis. VL is caused by Leishmania donovani in East Africa and the Indian subcontinent and by Leishmania infantum in Europe, North Africa, and Latin America, and causes an estimated 60,000 deaths per year. Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against leishmaniasis. This enzyme is fundamental for parasite survival in the human host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize the hydrogen peroxide produced by host macrophages during infection. Recently, we solved the X-ray structure of TR in complex with the diaryl sulfide compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine), which impairs the parasite defense against the reactive oxygen species by inhibiting TR with high efficiency. The compound binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466', Cys57 and Cys52, thereby inhibiting the trypanothione binding. On the basis of the RDS 777-TR complex, we synthesized structurally related diaryl sulfide analogs as TR inhibitors able to compete for trypanothione binding to the enzyme and to kill the promastigote in the micromolar range. One of the most active among these compounds (RDS 562) was able to reduce the trypanothione concentration in cell of about 33% via TR inhibition. RDS 562 inhibits selectively Leishmania TR, while it does not inhibit the human homolog glutathione reductase.