Electrophilic Compounds Research Articles

The potentials of Calotropis procera against filarial elephantiasis: an in-silico approach.

Lymphatic filariasis is one of the major diseases that belong to the category of neglected tropical illness. Filarial nematodes are the cause of the disease and are transmitted to humans via blood-feeding arthropod vectors. Drugs such as Albendazole, Ivermectin and diethylcarbamazine are administered either individually or in combination to overcome the progress of the lymphatic filariasis. These drugs have some minor side effects like temporary hair loss, dizziness, nausea etc. The filarial parasites have multifunctional proteins including the Glutathione-s-transferase (GST) enzyme. This study aims at the identification of a natural molecule that has the potential to bind with the GST enzyme, which plays a major role in detoxification of endogenous electrophilic compounds. Thus the binding interrupts the detoxification process within the filarial parasite, Brugia malayi. A medicinal plant Calotropis procera, owing to its anthelmintic properties was searched for the presence of potential phytocompounds. The phytocompounds were docked against the homology modeled GST enzyme using the MOE software. The results were screened and analyzed based on the Lipinski rule of 5. N-octanoate was the phytocompound obtained based on molecular docking, subjected to molecular dynamics. These results require further in vitro and in vivo validation to consider n-octanoate as a potential drug candidate for lymphatic filariasis treatment.

Effects of Substitution on Cytotoxicity of Diphenyl Ditelluride in Cultured Vascular Endothelial Cells

Among organic–inorganic hybrid molecules consisting of organic structure(s) and metal(s), only few studies are available on the cytotoxicity of nucleophilic molecules. In the present study, we investigated the cytotoxicity of a nucleophilic organotellurium compound, diphenyl ditelluride (DPDTe), using a cell culture system. DPDTe exhibited strong cytotoxicity against vascular endothelial cells and fibroblasts along with high intracellular accumulation but showed no cytotoxicity and had less accumulation in vascular smooth muscle cells and renal epithelial cells. The cytotoxicity of DPDTe decreased when intramolecular tellurium atoms were replaced with selenium or sulfur atoms. Electronic state analysis revealed that the electron density between tellurium atoms in DPDTe was much lower than those between selenium atoms of diphenyl diselenide and sulfur atoms of diphenyl disulfide. Moreover, diphenyl telluride did not accumulate and exhibit cytotoxicity. The cytotoxicity of DPDTe was also affected by substitution. p-Dimethoxy-DPDTe showed higher cytotoxicity, but p-dichloro-DPDTe and p-methyl-DPDTe showed lower cytotoxicity than that of DPDTe. The subcellular distribution of the compounds revealed that the compounds with stronger cytotoxicity showed higher accumulation rates in the mitochondria. Our findings suggest that the electronic state of tellurium atoms in DPDTe play an important role in accumulation and distribution of DPDTe in cultured cells. The present study supports the hypothesis that nucleophilic organometallic compounds, as well as electrophilic organometallic compounds, exhibit cytotoxicity by particular mechanisms.

Glutathione S-Transferases in Marine Copepods

The glutathione S-transferase (GST) is a complex family of phase II detoxification enzymes, known for their ability to catalyze the conjugation of the reduced form of glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds for detoxification purposes. In marine environments, copepods are constantly exposed to multiple exogenous stressors, thus their capability of detoxification is key for survival. Full identification of the GST family in copepods has been limited only to few species. As for insects, the GST family includes a wide range of genes that, based on their cellular localization, can be divided in three classes: cytosolic, microsomal, and mitochondrial. The role of GSTs might have class-specific features, thus understanding the nature of the GST family has become crucial. This paper covers information of the GST activity in marine copepods based on studies investigating gene expression, protein content, and enzymatic activity. Using published literature and mining new publicly available transcriptomes, we characterized the multiplicity of the GST family in copepods from different orders and families, highlighting the possible role of these genes as biomarker for ocean health status monitoring.

Thioredoxin Reductase Inhibition for Cancer Therapy.

The cytosolic selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1), and to some extent mitochondrial TrxR2 (TXNRD2), can be inhibited by a wide range of electrophilic compounds. Many such compounds also yield cytotoxicity toward cancer cells in culture or in mouse models, and most compounds are likely to irreversibly modify the easily accessible selenocysteine residue in TrxR1, thereby inhibiting its normal activity to reduce cytosolic thioredoxin (Trx1, TXN) and other substrates of the enzyme. This leads to an oxidative challenge. In some cases, the inhibited forms of TrxR1 are not catalytically inert and are instead converted to prooxidant NADPH oxidases, named SecTRAPs, thus further aggravating the oxidative stress, particularly in cells expressing higher levels of the enzyme. In this review, the possible molecular and cellular consequences of these effects are discussed in relation to cancer therapy, with a focus on outstanding questions that should be addressed if targeted TrxR1 inhibition is to be further developed for therapeutic use.

Recent advances in chemical derivatization-based chromatography-mass spectrometry methods for analysis of aldehyde biomarkers

人体接触环境中的化学污染物会导致多种疾病,包括癌症、糖尿病、心血管疾病、神经退行性疾病(阿尔茨海默症、帕金森病等)等。作为一类具有高反应活性的亲电化合物,醛类(包括外源性醛类或环境污染物暴露后产生的内源性醛类)可与人体中多种重要生物分子形成共价修饰产物而产生毒害作用。暴露组研究自2005年被首次提出以来一直是一个前沿热门领域,暴露组研究可绘制生物标志物与疾病风险之间的复杂关系,因此,所有生物标志物的可测量的和特征性的变化共同构成了暴露组研究的关键基础。醛类是化学暴露组的主要成分之一。由于醛类化合物自身物理化学性质和样品大量基质干扰存在,对它们进行分析和表征特别困难。醛类化合物的分析检测方法主要有传感分析法、电化学法、荧光成像、色谱法、质谱法、色谱-质谱联用法等。基于色谱-质谱的分析技术已成为化学暴露组研究的主要方法之一。化学衍生化,特别是稳定同位素标记衍生化(亦称化学同位素标记)结合液相色谱-质谱(LC-MS)技术能够解决靶向和非靶向代谢组和暴露组分析工作中的诸多问题。化学衍生化联合色谱-质谱的分析策略是复杂体系中醛类精准分析非常重要的解决方案之一。特别是近5年,基于化学衍生化的色谱-质谱分析方法开发与应用已成为醛类分析方法中的热点和亮点。该文主要总结与评述了近5年基于化学衍生化的气相色谱-质谱(GC-MS)和LC-MS最新进展,重点关注生物基质(血液、尿液、唾液、生物组织等)中醛类暴露标志物的分析方法进展。通过探讨标记小分子醛的各种衍生试剂、定性/定量分析方法及应用价值,评述醛类暴露标志物不同分析方法的优缺点以及未来发展趋势,为暴露组学、代谢组学、脂质组学的整合发展和环境生态健康研究提供一定的帮助。为了阐明外源性和内源性醛类化合物在生理和病理事件中所起的复杂作用,需要大力改进研究醛组学(aldehydome)的分析表征技术和工具。随着更先进的质谱仪的研发和使用,以及高效色谱分离和不断进步的生物信息学手段,并同时伴随着单细胞分析、质谱成像的兴起,未来的醛类暴露组分析方法会具有更高的灵敏度、更高的分析通量,更有希望筛选鉴定未知醛类化合物并发现新的暴露组生物标志物。

Assessment and characterization of tomato lipophilic electrophiles and their potential contribution to nutraceutical properties via SKN-1/Nrf2 signaling activation

Phytochemical electrophiles are drawing significant attention due to their properties to modulate signaling pathways related to cellular homeostasis. The aim of this study was to develop new tools to examine the electrophilic activity in food and predict their beneficial effects on health. We developed a spectrophotometric assay based on the nitrobenzenethiol (NBT) reactivity, as a thiol-reactive nucleophile, to screen electrophiles in tomato fruits. The method is robust, simple, inexpensive, and could be applied to other types of food. We quantified the electrophile activity in a tomato collection and associated this activity with the pigment composition. Thus, we identified lycopene, β- and γ-carotenes, 16 by-products of carotenoid oxidation and 18 unknown compounds as NBT-reactive by HPLC-MS/MS. The potential benefits of NBT-reactive compounds on health were evaluated in the in vivo model of C. elegans where they activated the SKN-1/Nrf2 pathway, evidencing the ability of electrophilic compounds to induce a biological response.

Proteomic Analysis Reveals IRAK4 As a Therapeutic Target in Chronic Lymphocytic Leukemia

Dimethylfumarate (DMF) and other electrophilic compounds are highly toxic to CLL cells in vitro and in xenograft animal models [Wu et al., 2010; Wu et al., 2016]. To study the mechanism of action, competitive isotopic tandem Orthogonal Proteolysis Activity Based Protein Profiling (isoTOP ABPP) - a chemical proteomic platform, which utilizes broad-spectrum cysteine reactive probes, - was applied to quantitatively map DMF protein targets in whole proteomes. Cells from 10 CLL patients were treated with 30 μM DMF for 4h followed by an isoTOP ABPP sample preparation and analysis workflow. Based on reproducible high isoTOP ABPP ratios across multiple biological replicates, cysteine 13 of interleukin-1 receptor-associated kinase 4 (IRAK4) was identified as a prominent target of DMF in CLL patient cells.IRAK4 plays a critical role in Toll-like receptor (TLR) signal transduction. Engagement of IRAK4 with the signaling adaptor protein MyD88 in TLRs activates downstream NF-κB and type-I IFN pathways. Approximately 15-20% of CLL patients have dominant activating mutations of MyD88, and nearly all patients with the CLL-related disease Waldenstrom's Macroglobulinemia have such mutations. Hence, we sought to evaluate known inhibitors of IRAK4 kinase activity for their effect on CLL cells. Multiple IRAK4 inhibitors are currently in clinical development, primarily for the treatment of autoimmune conditions. We used PF06650833, a specific inhibitor of IRAK4, previously tested in phase 1 trials for patients with systemic lupus erythematosus, to evaluate its effect on CLL cells viability. In vitro , PF06650833 reduced CLL cell viability in a dose-dependent manner. Reduction in IRAK4 and IκBα protein levels were also observed in samples treated with at least 15 µM PF06650833 by Western blot analysis. In vivo studies provide a better opportunity to evaluate the effect of this drug in blocking microenvironment signaling. We utilized a murine xenograft model in which primary CLL cells are transplanted into the peritoneum of Rag2/gamma chain immunodeficient mice. Each mouse received a single intraperitoneal injection of 1-10 mg/kg PF06650833 one day after transplantation. Cells were recovered 6 days after the treatment and analyzed by flow cytometry, showing remarkable reduction in CLL survival in a dose-dependent manner.In conclusion, unbiased proteomics analysis of a non-specific drug, DMF, identified IRAK4 as a target in cells from CLL patients. While the exact mode of action for DMF and PF06650833 might differ (disruption of higher IRAK4 complexes, inhibition of kinase dependent signaling or other), our results suggest that IRAK4 represents a potential druggable target in the management of patients with CLL. [Display omitted] DisclosuresChoi:Gilead, Genentech, Abbvie, and PCYC: Speakers Bureau; AbbVie: Other: Institutional research funding; AbbVie, Genentech, and PCYC: Consultancy.

Local Temperature as a Chemical Reactivity Descriptor.

Using the electron density and its associated quantities in a molecular system to quantify chemical reactivity in density functional theory is of considerable recent interest. Local temperature based on the kinetic energy density is an intrinsic property of a molecular system, which can be employed for this purpose. In this work, we explore such a possibility. To this end, we examine the local behavior of local temperature with a few choices of the kinetic energy density, apply it to determine regioselectivity of nucleophilic and electrophilic compounds, and then investigate its performance in appreciating reactions along the intrinsic reaction pathway for exothermic, endothermic, and thermoneutral transformations. Our results confirm that local temperature can be used as an effective descriptor of molecular reactivity.

Novel 4-Hydroxybenzyl Adducts in Human Hemoglobin: Structures and Mechanisms of Formation.

Humans are exposed to large numbers of electrophiles from their diet, the environment, and endogenous physiological processes. Adducts formed at the N-terminal valine of hemoglobin are often used as biomarkers of human exposure to electrophilic compounds. We previously reported the formation of hemoglobin N-terminal valine adducts (added mass, 106.042 Da) in the blood of human smokers and nonsmokers and identified their structure as 4-hydroxybenzyl-Val. In the present work, mass spectrometry-based proteomics was utilized to identify additional sites for 4-hydroxybenzyl adduct formation at internal nucleophilic amino acid side chains within hemoglobin. Hemoglobin isolated from human blood was treated with para-quinone methide (para-QM) followed by global nanoLC-MS/MS and targeted nanoLC-MS/MS to identify amino acid residues containing the 4-hydroxybenzyl modification. Our experiments revealed the formation of 4-hydroxybenzyl adducts at the αHis20, αTyr24, αTyr42, αHis45, βSer72, βThr84, βThr87, βSer89, βHis92, βCys93, βCys112, βThr123, and βHis143 residues (in addition to N-terminal valine) through characteristic MS/MS spectra. These amino acid side chains had variable reactivity toward para-QM with αHis45, αTyr42, βCys93, βHis92, and βSer72 forming the largest numbers of adducts upon exposure to para-QM. Two additional mechanisms for formation of 4-hydroxybenzyl adducts in humans were investigated: exposure to 4-hydroxybenzaldehyde (4-HBA) followed by reduction and UV-mediated reactions of hemoglobin with tyrosine. Exposure of hemoglobin to a 5-fold molar excess of 4-HBA followed by reduction with sodium cyanoborohydride produced 4-hydroxybenzyl adducts at several amino acid side chains of which αHis20, αTyr24, αTyr42, αHis45, βSer44, βThr84, and βHis92 were verified in targeted mass spectrometry experiments. Similarly, exposure of human blood to ultraviolet radiation produced 4-hydroxybenzyl adducts at αHis20, αTyr24, αTyr42, αHis45, βSer44, βThr84, and βSer89. Overall, our results reveal that 4-hydroxybenzyl adducts form at multiple nucleophilic sites of hemoglobin and that para-QM is the most likely source of these adducts in humans.

The maximum carbonyl ratio (MCR) as a new index for the structural classification of secondary organic aerosol components.

Organic aerosols (OA) account for a large fraction of atmospheric fine particulate matter and thus are affecting climate and public health. Elucidation of the chemical composition of OA is the key for addressing the role of ambient fine particles at the atmosphere-biosphere interface and mass spectrometry is the main method to achieve this goal. High-resolution mass spectrometry (HRMS) is on its way to becoming one of the most prominent analytical techniques, also for the analysis of atmospheric aerosols. The combination of high mass resolution and accurate mass determination allows the elemental compositions of numerous compounds to be easily elucidated. Here a new parameter for the improved classification of OA is introduced - the maximum carbonyl ratio (MCR) - which is directly derived from the molecular composition and is particularly suitable for the identification and characterization of secondary organic aerosols (SOA). The concept is exemplified by the analysis of ambient OA samples from two measurement sites (Hyytiälä, Finland; Beijing, China) and of laboratory-generated SOA based on ultrahigh-performance liquid chromatography (UHPLC) coupled to Orbitrap MS. To interpret the results, MCR-Van Krevelen (VK) diagrams are generated for the different OA samples and the individual compounds are categorized into specific areas in the diagrams. The results show that the MCR index is a valuable parameter for representing atmospheric SOA components in composition and structure-dependent visualization tools such as VK diagrams. The MCR index is suggested as a tool for a better characterization of the sources and the processing of atmospheric OA components based on HRMS data. Since the MCR contains information on the concentration of highly electrophilic organic compounds in particulate matter (PM) as well as on the concentration of organic (hydro)peroxides, the MCR could be a promising metric for identifying health-related particulate matter parameters by HRMS.

Electrophilic and Drug-Induced Stimulation of NOTCH3 N-terminal Fragment Oligomerization in Cerebrovascular Pathology.

Small vessel disease is a prevalent age-related condition linked to increased risk of dementia and stroke. We investigate the most commonly inherited form, CADASIL, caused by cysteine-involving mutations in NOTCH3. Recent studies highlight accumulation of NOTCH3 N-terminal fragmentation product (NTF) in disease. In vitro, NTF is capable of both spontaneous and catecholamine-enhanced cysteine-mediated oligomerization. Despite well-characterized genetic influence on CADASIL, environmental effects, including medication usage, on disease remain unclear. We studied effects of assorted electrophilic compounds and drugs on NTF oligomerization by SDS-PAGE and dynamic light scattering. We then examined direct proton pump inhibitor-NTF binding with antibodies designed against proton pump inhibitor-labeled proteins and mass spectrometry. Finally, we used monoclonal NTF antibodies with Proximity Ligation Assay to identify NTF oligomers in 3 CADASIL and 2 age-matched control brains. We identified enhancement of NTF oligomerization by two electrophilic cysteine-modifying compounds, N-ethylmaleimide and iodoacetamide, and an electrophilic compound capable of oxidizing cysteines, ferric chloride. Electrophilic clinical drugs (fenoldopam, omeprazole, tenatoprazole, lansoprazole, and rabeprazole) also promoted oligomerization, and we identified direct omeprazole-NTF and tenatoprazole-NTF complexes. Additionally, we provide novel evidence of NTF multimers in human CADASIL brains. A broad array of electrophilic chemicals, including clinically relevant drugs, influences oligomerization of a pathological CADASIL protein, providing mechanistic insight into disease protein oligomerization. We posit that environmental influences, which may include usage of electrophilic drugs, may affect CADASIL presentations.

Sudden infant death syndrome: deletions of glutathione-S-transferase genes M1 and T1 and tobacco smoke exposure

In developed countries, sudden infant death syndrome (SIDS) is the leading cause of death in infants in their first year of life. The risk of SIDS is increased if parents smoked during pregnancy and in presence of the child. Glutathione S-transferases (GSTs) catalyse the conjugation of glutathione with electrophilic compounds and toxins, making them less reactive and easier to excrete. As a gene dose effect was observed for GSTM1 and GSTT1, the aim of this study was to investigate whether there is a connection between homozygous or heterozygous gene deletions of GSTM1 or GSTT1 and the occurrence of SIDS. We found that heterozygous deletion of GSTM1 occurred significantly more frequently in the SIDS case group compared to the control group. A homozygous deletion of GSMT1 was slightly more frequently in the control group. A homozygous gene deletion of GSTT1 showed no significant difference between the SIDS group and the control group. We also found that in the SIDS group, the number of victims that were exposed to cigarette smoke was significantly higher than the number of victims without cigarette smoke exposure and that the mean lifetime of children whose mothers smoked was shorter in comparison with non-smoking mothers. In SIDS cases with homozygous gene deletions of GSTM1, the median life span of children with tobacco smoke exposure was 60 days shorter than without smoke exposure. In conclusion, the absence of these two genes is not the only trigger for SIDS but could be a critical aspect of SIDS aetiology, particularly in SIDS cases with smoking parents.

In-depth understanding of molecular mechanisms of aldehyde toxicity to engineer robust Saccharomyces cerevisiae.

Aldehydes are ubiquitous electrophilic compounds that ferment microorganisms including Saccharomyces cerevisiae encounter during the fermentation processes to produce food, fuels, chemicals, and pharmaceuticals. Aldehydes pose severe toxicity to the growth and metabolism of the S. cerevisiae through a variety of toxic molecular mechanisms, predominantly via damaging macromolecules and hampering the production of targeted compounds. Compounds with aldehyde functional groups are far more toxic to S. cerevisiae than all other functional classes, and toxic potency depends on physicochemical characteristics of aldehydes. The yeast synthetic biology community established a design-build-test-learn framework to develop S. cerevisiae cell factories to valorize the sustainable and renewable biomass, including the lignin-derived substrates. However, thermochemically pretreated biomass-derived substrate streams contain diverse aldehydes (e.g., glycolaldehyde and furfural), and biological conversions routes of lignocellulosic compounds consist of toxic aldehyde intermediates (e.g., formaldehyde and methylglyoxal), and some of the high-value targeted products have aldehyde functional group (e.g., vanillin and benzaldehyde). Numerous studies comprehensively characterized both single and additive effects of aldehyde toxicity via systems biology investigations, and novel molecular approaches have been discovered to overcome the aldehyde toxicity. Based on those novel approaches, researchers successfully developed synthetic yeast cell factories to convert lignocellulosic substrates to valuable products, including aldehyde compounds. In this mini-review, we highlight the salient relationship of physicochemical characteristics and molecular toxicity of aldehydes, the molecular detoxification and macromolecules protection mechanisms of aldehydes, and the advances of engineering robust S. cerevisiae against complex mixtures of aldehyde inhibitors. KEY POINTS: • We reviewed structure-activity relationships of aldehyde toxicity on S. cerevisiae. • Two-tier protection mechanisms to alleviate aldehyde toxicity are presented. • We highlighted the strategies to overcome the synergistic toxicity of aldehydes.

The analysis of GSTA1 promoter genetic and functional diversity of human populations

GSTA1 encodes a member of a family of enzymes that function to add glutathione to target electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins, and products of oxidative stress. GSTA1 has several functional SNPs within its promoter region that are responsible for a change in its expression by altering promoter function. This study aims to investigate distributions of GSTA1 promoter haplotypes across different human populations and to assess their impact on the expression of GSTA1. PHASE 2.1.1 was used to infer haplotypes and diplotypes of six GSTA1 promoter SNPs on 2501 individuals from 26 populations classified by the 1000 Genomes Project into five super-populations that included Africa (N = 660), America (N = 347), East Asia (N = 504), Europe (N = 502), and South Asia (N = 488). We used pairwise FST analysis to compare sub-populations and luciferase reporter assay (LRA) to evaluate the impact of each SNP on activation of transcription and interaction with other SNPs. The distributions of GSTA1 promoter haplotypes and diplotypes were significantly different among the different human populations. Three new promoter haplotypes were found in the African super-population. LRA demonstrated that SNPs at -52 and -69 has the most impact on GSTA1 expression, however other SNPs have a significant impact on transcriptional activity. Based on LRA, a new model of cis-elements interaction is presented. Due to the significant differences in GSTA1 diplotype population frequencies, future pharmacogenomics or disease-related studies would benefit from the inclusion of the complete GSTA1 promoter haplotype based on the newly proposed metabolic grouping derived from the LRA results.

Docking-based virtual screening studies aiming at the covalent inhibition of SARS-CoV-2 MPro by targeting the cysteine 145

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 which has infected millions of people worldwide. The main protease of SARS-CoV-2 (MPro) has been recognized as a key target for the development of antiviral compounds. Taking advantage of the X-ray crystal complex with reversible covalent inhibitors interacting with the catalytic cysteine 145 (Cys145), we explored flexible docking studies to select alternative compounds able to target this residue as covalent inhibitors. First, docking studies of three known electrophilic compounds led to results consistent with co-crystallized data validating the method for SARS-CoV-2 MPro covalent inhibition. Then, libraries of soft electrophiles (overall 41 757 compounds) were submitted to docking-based virtual screening resulting in the identification of 17 molecules having their electrophilic group close to the Cys145 residue. We also investigated flexible docking studies of a focused approved covalent drugs library including 32 compounds with various electrophilic functional groups. Among them, the calculations resulted in the identification of four compounds, namely dimethylfumarate, fosfomycin, ibrutinib and saxagliptin, able first, to bind to the active site of the protein and second, to form a covalent bond with the catalytic cysteine.

Heuristics in Role of Human Glutathione S-transferase Mu 1 as Nitric Oxide Carrier and its Engineered Variants for Enhanced Activity.

Human glutathione S-transferases (hGSTs) are phase-II detoxification enzymes that catalyze the conjugation of electrophilic compounds and glutathione. Anomalous excess production of NO in the cellular environment under diseased or stressed condition results in lethal effects to the cell. Studies have reported that the evolution of tyrosine-based GSTs as a defense mechanism by the cell to mitigate Nitric Oxide (NO) toxicity. The dual role of hGSTP1 as NO carrier and scavenger is a prelude for the research forthwith. A plausible role of hGSTM1 as NO carrier is considered. Being a prominent cellular messenger and secondary metabolite, excess production of NO is lethal to the cell. Moreover, hGSTM1 polymorphisms lead to diminished catalytic activity that promotes a diseased state. Hence, it is compelling to generate hGSTM1 mutants that have more catalytic efficacy compared to Wild Type (WT). hGSTM1 mutants with enhanced efficiency were generated using in silico and in vitro Site-Directed Mutagenesis (SDM). WT and mutant proteins were overexpressed and purified using affinity chromatography. The catalytic activity and binding efficiency of WT and mutant proteins towards CDNB (1-chloro-2, 4-dinitrobenzene) & NO were determined. NO assay reveals the probable interaction of WT hGSTM1 with NO. In silico, SDM studies provided E129K and Q109K mutants with superior NO binding efficiency as compared to WT. The catalytic activity (GST and NO assays) of the mutants corroborate the in silico results. WT hGSTM1 is recognized as a positive NO carrier. The novel mutant enzymes E129K and Q109K are inferred to possess superior NO carrying capacity.

SigB-regulated antioxidant functions in gram-positive bacteria.

Oxidative stress can have lethal consequences if organisms do not respond and remediate the damage to DNA, proteins and lipids. Bacterial species respond to oxidative stress by activating transcriptional profiles that include biochemical functions to reduce oxidized cellular components, regenerate pools of reducing molecules, and detoxify harmful metabolites. Interestingly, the general stress response in Gram positive bacteria controlled by SigB is induced by oxidative stress from reactive oxygen and electrophilic species. The upregulation of SigB regulated genes during exposure to electrophilic and oxidative compounds suggests SigB contributes directly to the adaptations required for oxidative stress survival. A subset of the functions of SigB regulated genes can be categorized with antioxidant biochemical activities, such as redoxins, reductases and dehydrogenases, including regulation of low molecular weight thiols, yet their exact cellular role is not fully understood. Here, we present an overview of the predicted antioxidant biochemical functions regulated by SigB, with potential for biomedical research given the prevalence of oxidative stress during bacterial infection, as well as during industrial applications of large-scale production of compounds by microbes.

Activation of Nrf2 by Electrophiles Is Largely Independent of the Selenium Status of HepG2 Cells

Selenoenzymes, whose activity depends on adequate selenium (Se) supply, and phase II enzymes, encoded by target genes of nuclear factor erythroid 2-related factor 2 (Nrf2), take part in governing cellular redox homeostasis. Their interplay is still not entirely understood. Here, we exposed HepG2 hepatoma cells cultured under Se-deficient, Se-adequate, or Se-supranutritional conditions to the Nrf2 activators sulforaphane, cardamonin, or diethyl maleate. Nrf2 protein levels and intracellular localization were determined by immunoblotting, and mRNA levels of Nrf2 target genes and selenoproteins were assessed by qRT-PCR. Exposure to electrophiles resulted in rapid induction of Nrf2 and its enrichment in the nucleus, independent of the cellular Se status. All three electrophilic compounds caused an enhanced expression of Nrf2 target genes, although with differences regarding extent and time course of their induction. Whereas Se status did not significantly affect mRNA levels of the Nrf2 target genes, gene expression of selenoproteins with a low position in the cellular “selenoprotein hierarchy”, such as glutathione peroxidase 1 (GPX1) or selenoprotein W (SELENOW), was elevated under Se-supplemented conditions, as compared to cells held in Se-deficient media. In conclusion, no major effect of Se status on Nrf2 signalling was observed in HepG2 cells.

Cysteamine assay for the evaluation of bioactive electrophiles

Covalent modifications of thiol and amine groups may control the function of proteins involved in the regulatory and signaling pathways of the cell. In this study, we developed a simple cysteamine assay which can be used to study the reactivity of electrophilic compounds towards primary amine and thiol groups in an aqueous environment. The detection principle is based on the electrochemical, photometrical and mass spectrometric analyses of cysteamine (2-aminoethanethiol) as the molecular probe. This technique is useful for studying the reaction kinetics of electrophiles with thiol (SH) and amino (NH2) groups. The decrease in analytical responses of cysteamine was monitored to evaluate the reactivity of three electrophilic activators of the Nrf2 pathway, which mediates the cellular stress response. The SH-reactivity under cell-free conditions of the tested electrophiles decreased in the following order: 4-hydroxy-2-nonenal ≥ nitro-oleic acid > sulforaphane. However, as shown in RAW264.7 cells, the tested compounds activated Nrf2-dependent gene expression in the opposite order: sulforaphane > nitro-oleic acid ≥ 4-hydroxy-2-nonenal. Although other factors in addition to chemical reactivity play a role in biological systems, we conclude that this cysteamine assay is a useful tool for screening potentially bioactive electrophiles and for studying their reactivity at a molecular level.

Superior performance of ZnCoOx/peroxymonosulfate system for organic pollutants removal by enhancing singlet oxygen generation: The effect of oxygen vacancies

Combination of sulfate radical (SO4−) and singlet oxygen (1O2) could enhance the removal of organic pollutants in PMS activation because of the high-selective oxidation of electrophilic compounds by 1O2. Co oxides are highly active towards PMS activation, but improving the generation of 1O2 in PMS activation with Co oxides as catalysts remains a great challenge. Herein, the Zn doped Co oxides (ZnCoOx) was prepared by calcining Zn doped ZIF-67 in air. The oxygen vacancies content of ZnCoOx could be adjusted by controlling Zn doping level and the presence of oxygen vacancies leads to the generation of 1O2 in ZnCoOx/PMS system. The optimized ZnCoOx-2 presents the kinetic constant of 0.538 min−1 for Rhodamine B removal, which is 14.2 times higher than that of undoped sample and 38.4 times higher than that of commercial Co3O4. 1O2 and SO4− exist in ZnCoOx/PMS system while 1O2 plays the dominant role for pollutants removal. The oxygen vacancies of ZnCoOx are demonstrated to be responsible for the generation of 1O2 and the mechanism is further investigated. This study provided an efficient catalyst to combine the advantages of 1O2 and SO4− for pollutants removal by PMS activation, and the results could open a new avenue for development of heterogeneous catalysts for PMS activation.