Visualization of an Endogenous Mitochondrial Azoreductase Activity under Normoxic Conditions Using a Naphthalimide Azo-Based Fluorogenic Probe.

In this paper, we demonstrate the existence of an endogenous mitochondrial azoreductase (AzoR) activity that can induce the cleavage of N═N double bonds of azobenzene compounds under normoxic conditions. To this end, 100% OFF-ON azo-based fluorogenic probes derived from 4-amino-1,8-naphthalimide fluorophores were synthesized and evaluated. The in vitro study conducted with other endogenous reducing agents of the cell, including reductases, demonstrated both the efficacy and the selectivity of the probe for AzoR. Confocal experiments with the probe revealed an AzoR activity in the mitochondria of living cells under normal oxygenation conditions, and we were able to demonstrate that this endogenous AzoR activity appears to be expressed at different levels across different cell lines. This discovery provides crucial information for our understanding of the biochemical processes occurring within the mitochondria. It thus contributes to a better understanding of its function, which is implicated in numerous pathologies.

Innovative Colorimetric NQO1 Detection Strategy via Substrate Competitive and Biomimetic Cascade Reactions with a Highly Active NADH Oxidase Mimic.

NAD(P)H: quinone oxidoreductase-1 (NQO1) plays critical roles in antioxidation and abnormally overexpresses in tumors. Developing a fast and sensitive method of monitoring NQO1 will greatly promote cancer diagnosis in clinical practice. This study introduces a transformative colorimetric detection strategy for NQO1, harnessing an innovative competitive substrate mechanism between NQO1 and a new NADH oxidase (NOX) mimic, cobalt-nitrogen-doped carbon nanozyme (CoNC). This method ingeniously exploits the differential consumption of NADH in the presence of NQO1 to modulate the generation of H2O2 from CoNC catalysis, which is then quantified through a secondary, peroxidase-mimetic cascade reaction involving Prussian blue (PB) nanoparticles. This dual-stage reaction framework not only enhances the sensitivity of NQO1 detection, achieving a limit of detection as low as 0.67 µg mL-1, but also enables the differentiation between cancerous and noncancerous cells by their enzymatic activity profiles. Moreover, CoNC exhibits exceptional catalytic efficiency, with a specific activity reaching 5.2 U mg-1, significantly outperforming existing NOX mimics. Beyond mere detection, CoNC serves a dual role, acting as both a robust mimic of cytochrome c reductase (Cyt c) and a cornerstone for enzymatic regeneration, thereby broadening the scope of its biological applications. This study not only marks a significant step forward in the bioanalytical application of nanozymes but also sets the stage for their expanded use in clinical diagnostics and therapeutic monitoring.

GRIM-19 deficiency promotes macrophage polarization to the M1 phenotype partly through glycolysis in unexplained recurrent spontaneous abortion†.

The occurrence of unexplained recurrent spontaneous abortion (URSA) is closely related to immune system disorders, however, the underlying mechanisms remain unclear. The purpose of this study was to investigate the expression of GRIM-19 in URSA and the possible pathogenesis of URSA according to macrophage polarization. Here, we showed that GRIM-19 was downregulated in the uterine decidual macrophages of patients with URSA and that GRIM-19 downregulation was accompanied by increased M1 macrophage polarization. Furthermore, the expression levels of glycolytic enzymes were substantially enhanced in the uterine decidual macrophages of URSA patients, and glycolysis in THP-1-derived macrophages was further enhanced by the downregulation of GRIM-19. Additionally, the increase of M1 macrophages resulting from the loss of GRIM-19 was significantly reversed in cells treated with 2-deoxy-D-glucose (2-DG, an inhibitor of glycolysis). To provide more direct evidence, GRIM-19 deficiency was shown to promote macrophage polarization to the M1 phenotype in GRIM-19+/- mouse uteri. Overall, our study provides evidence that GRIM-19 deficiency may play a role in regulating macrophage polarization in URSA, and that glycolysis may participate in this process.

Sequence similarity network analysis of drug- and dye-modifying azoreductase enzymes found in the human gut microbiome.

Drug metabolism by human gut microbes is often exemplified by azo bond reduction in the anticolitic prodrug sulfasalazine. Azoreductase activity is often found in incubations with cell cultures or ex vivo gut microbiome samples and contributes to the xenobiotic metabolism of drugs and food additives. Applying metagenomic studies to personalized medicine requires knowledge of the genes responsible for sulfasalazine and other drug metabolism, and candidate genes and proteins for drug modifications are understudied. A representative gut-abundant azoreductase from Anaerotignum lactatifermentan DSM 14214 efficiently reduces sulfasalazine and another drug, phenazopyridine, but could not reduce all azo-bonded drugs in this class. We used enzyme kinetics to characterize this enzyme for its NADH-dependent reduction of these drugs and food additives and performed computational docking to provide the groundwork for understanding substrate specificity in this family. We performed an analysis of the Flavodoxin-like fold InterPro family (IPR003680) by computing a sequence similarity network to classify distinct subgroups of the family and then performed chemically-guided functional profiling to identify proteins that are abundant in the NIH Human Microbiome Project dataset. This strategy aims to reduce the number of unique azoreductases needed to characterize one protein family in the diverse set of potential drug- and dye-modifying activities found in the human gut microbiome.

Enzyme-Triggered Degradation of Supramolecularly Cross-Linked Polymersomes of Azobenzene-Based Polyurethane: Cell-Selective Anticancer Drug Release.

Enzyme-responsive self-assembled nanostructures for drug delivery applications have gained a lot of attention, as enzymes exhibit dysregulation in many disease-associated microenvironments. Azoreductase enzyme levels are strongly elevated in many tumor tissues; hence, here, we exploited the altered enzyme activity of the azoreductase enzyme and designed a main-chain azobenzene-based amphiphilic polyurethane, which self-assembles into a vesicular nanostructure and is programmed to disassemble in response to a specific enzyme, azoreductase, with the help of the nicotinamide adenine dinucleotide phosphate (NADPH) coenzyme in the hypoxic environment of solid tumors. The vesicular nanostructure sequesters, stabilizes the hydrophobic anticancer drug, and releases the drug in a controlled fashion in response to enzyme-triggered degradation of azo-bonds and disruption of vesicular assembly. The biological evaluation revealed tumor extracellular matrix pH-induced surface charge modulation, selective activated cellular uptake to azoreductase overexpressed lung cancer cells (A549), and the release of the anticancer drug followed by cell death. In contrast, the benign nature of the drug-loaded vesicular nanostructure toward normal cells (H9c2) suggested excellent cell specificity. We envision that the main-chain azobenzene-based polyurethane discussed in this manuscript could be considered as a possible selective chemotherapeutic cargo against the azoreductase overexpressed cancer cells while shielding the normal cells from off-target toxicity.

Genome-Scale Investigation of the Regulation of azoR Expression in Escherichia coli Using Computational Analysis and Transposon Mutagenesis.

Bacterial azoreductases are enzymes that catalyze the reduction of ingested or industrial azo dyes. Although azoreductase genes have been well identified and characterized, the regulation of their expression has not been systematically investigated. To determine how different factors affect the expression of azoR, we extracted and analyzed transcriptional data from the Gene Expression Omnibus (GEO) resource, then confirmed computational predictions by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results showed that azoR expression was lower with higher glucose concentration, agitation speed, and incubation temperature, but higher at higher culture densities. Co-expression and clustering analysis indicated ten genes with similar expression patterns to azoR: melA, tpx, yhbW, yciK, fdnG, fpr, nfsA, nfsB, rutF, and chrR (yieF). In parallel, constructing a random transposon library in E. coli K-12 and screening 4320 of its colonies for altered methyl red (MR)-decolorizing activity identified another set of seven genes potentially involved in azoR regulation. Among these genes, arsC, relA, plsY, and trmM were confirmed as potential azoR regulators based on the phenotypic decolorization activity of their transposon mutants, and the expression of arsC and relA was confirmed, by qRT-PCR, to significantly increase in E. coli K-12 in response to different MR concentrations. Finally, the significant decrease in azoR transcription upon transposon insertion in arsC and relA (as compared to its expression in wild-type E. coli) suggests their probable involvement in azoR regulation. In conclusion, combining in silico analysis and random transposon mutagenesis suggested a set of potential regulators of azoR in E. coli.

Expression of the alfalfa gene MsMDHAR in Arabidopsis thaliana increases water stress tolerance.

The ascorbate-glutathione pathway plays an essential role in the physiology of vascular plants, particularly in their response to environmental stresses. This pathway is responsible for regulating the cellular redox state, which is critical for maintaining cell function and survival under adverse conditions. To study the involvement of the alfalfa monodehydroascorbate reductase (MsMDHAR) in water stress processes, Arabidopsis thaliana plants constitutively expressing the sequence encoding MsMDHAR were developed. Transgenic events with low and high MsMDHAR expression and ascorbate levels were selected for further analysis of drought and waterlogging tolerance. Under water stress, Arabidopsis transgenic plants generated higher biomass, produced more seeds, and had larger roots than wild type ones. This higher tolerance was associated with increased production of waxes and chlorophyll a at the basal level, greater stomatal opening and stability in regulating the relative water content and reduced H2O2 accumulation under stress conditions in transgenic plants. Overall, these results show that MsMDHAR is involved in plant tolerance to abiotic stresses. The data presented here also emphasises the potential of the MsMDHAR enzyme as a plant breeding tool to improve water stress tolerance.

Sustained release of 5-aminosalicylic acid from azoreductase-responsive polymeric prodrugs for prolonged colon-targeted colitis therapy.

Ulcerative colitis (UC) is a challenging inflammatory gastrointestinal disorder, whose therapies encounter limitations in overcoming insufficient colonic retention and rapid systemic clearance. In this study, we report an innovative polymeric prodrug nanoformulation for targeted UC treatment through sustained 5-aminosalicylic acid (5-ASA) delivery. Amphiphilic polymer-based 13.5 nm micelles were engineered to incorporate azo-linked 5-ASA prodrug motifs, enabling cleavage via colonic azoreductases. In vitro, micelles exhibited excellent stability under gastric/intestinal conditions while demonstrating controlled 5-ASA release over 24 h in colonic fluids. Orally administered micelles revealed prolonged 24-h retention and a high accumulation within inflamed murine colonic tissue. At an approximately 60% dose reduction from those most advanced recent studies, the platform halted DSS colitis progression and outperformed standard 5-ASA therapy through a 77-97% suppression of inflammatory markers. Histological analysis confirmed intact colon morphology and restored barrier protein expression. This integrated prodrug nanoformulation addresses limitations in colon-targeted UC therapy through localized bioactivation and tailored pharmacokinetics, suggesting the potential of nanotechnology-guided precision delivery to transform disease management.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

In Silico Comparison of Bioactive Compounds Characterized from Azadirachta indica with an FDA-Approved Drug against Schistosomal Agents: New Insight into Schistosomiasis Treatment.

The burden of human schistosomiasis, a known but neglected tropical disease in Sub-Saharan Africa, has been worrisome in recent years. It is becoming increasingly difficult to tackle schistosomiasis with praziquantel, a drug known to be effective against all Schistosoma species, due to reports of reduced efficacy and resistance. Therefore, this study seeks to investigate the antischistosomal potential of phytochemicals from Azadirachta indica against proteins that have been implicated as druggable targets for the treatment of schistosomiasis using computational techniques. In this study, sixty-three (63) previously isolated and characterized phytochemicals from A. indica were identified from the literature and retrieved from the PubChem database. In silico screening was conducted to assess the inhibitory potential of these phytochemicals against three receptors (Schistosoma mansoni Thioredoxin glutathione reductase, dihydroorotate dehydrogenase, and Arginase) that may serve as therapeutic targets for schistosomiasis treatment. Molecular docking, ADMET prediction, ligand interaction, MMGBSA, and molecular dynamics simulation of the hit compounds were conducted using the Schrodinger molecular drug discovery suite. The results show that Andrographolide possesses a satisfactory pharmacokinetic profile, does not violate the Lipinski rule of five, binds with favourable affinity with the receptors, and interacts with key amino acids at the active site. Importantly, its interaction with dihydroorotate dehydrogenase, an enzyme responsible for the catalysis of the de novo pyrimidine nucleotide biosynthetic pathway rate-limiting step, shows a glide score and MMGBSA of -10.19 and -45.75 Kcal/mol, respectively. In addition, the MD simulation shows its stability at the active site of the receptor. Overall, this study revealed that Andrographolide from Azadirachta indica could serve as a potential lead compound for the development of an anti-schistosomal drug.

Targeting Trypanothione Metabolism in Trypanosomatids.

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.

Fragment Merging, Growing, and Linking Identify New Trypanothione Reductase Inhibitors for Leishmaniasis.

Trypanothione reductase (TR) is a suitable target for drug discovery approaches against leishmaniasis, although the identification of potent inhibitors is still challenging. Herein, we harnessed a fragment-based drug discovery (FBDD) strategy to develop new TR inhibitors. Previous crystallographic screening identified fragments 1-3, which provided ideal starting points for a medicinal chemistry campaign. In silico investigations revealed critical hotspots in the TR binding site, guiding our structure- and ligand-based structure-actvity relationship (SAR) exploration that yielded fragment-derived compounds 4-14. A trend of improvement in Leishmania infantum TR inhibition was detected along the optimization and confirmed by the crystal structures of 9, 10, and 14 in complex with Trypanosoma brucei TR. Compound 10 showed the best TR inhibitory profile (Ki = 0.2 µM), whereas 9 was the best one in terms of in vitro and ex vivo activity. Although further fine-tuning is needed to improve selectivity, we demonstrated the potentiality of FBDD on a classic but difficult target for leishmaniasis.

Design of a cofactor self-sufficient whole-cell biocatalyst for enzymatic asymmetric reduction via engineered metabolic pathways and multi-enzyme cascade.

NAD(P)H-dependent oxidoreductases are crucial biocatalysts for synthesizing chiral compounds. Yet, the industrial implementation of enzymatic redox reactions is often hampered by an insufficient supply of expensive nicotinamide cofactors. Here, a cofactor self-sufficient whole-cell biocatalyst was developed for the enzymatic asymmetric reduction of 2-oxo-4-[(hydroxy)(-methyl)phosphinyl] butyric acid (PPO) to L-phosphinothricin (L-PPT). The endogenous NADP+ pool was significantly enhanced by regulating Preiss-Handler pathway toward NAD(H) synthesis and, in the meantime, introducing NAD kinase to phosphorylate NAD(H) toward NADP+. The intracellular NADP(H) concentration displayed a 2.97-fold increase with the strategy compared with the wild-type strain. Furthermore, a recombinant multi-enzyme cascade biocatalytic system was constructed based on the Escherichia coli chassis. In order to balance multi-enzyme co-expression levels, the strategy of modulating rate-limiting enzyme PmGluDH by RBS strengths regulation successfully increased the catalytic efficiency of PPO conversion. Finally, the cofactor self-sufficient whole-cell biocatalyst effectively converted 300 mM PPO to L-PPT in 2 h without the need to add exogenous cofactors, resulting in a 2.3-fold increase in PPO conversion (%) from 43% to 100%, with a high space-time yield of 706.2 g L-1 d-1 and 99.9% ee. Overall, this work demonstrates a technological example for constructing a cofactor self-sufficient system for NADPH-dependent redox biocatalysis.

GRIM19 deficiency aggravates metabolic disorder and ovarian dysfunction in PCOS.

CONTEXT: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women. Retinoid-interferon-induced mortality 19 (GRIM19) is a functional component of mitochondrial complex I that plays a role in cellular energy metabolism. However, the role of GRIM19 in the pathogenesis of PCOS is still unclear. OBJECTIVE: To investigate the role of GRIM19 in the pathogenesis of PCOS. DESIGN: We first measured the expression of GRIM19 in human granulosa cells (hGCs) from patients with and without PCOS (n = 16 per group), and then established a PCOS mouse model with WT and Grim19+/- mice for in vivo experiments. Glucose uptake-related genes RAC1 and GLUT4 and energy metabolism levels in KGN cells were examined in vitro by knocking down GRIM19 in the cell lines. Additionally, ovulation-related genes such as p-ERK1/2, HAS2, and PTX3 were also studied to determine their expression levels. RESULTS: GRIM19 expression was reduced in hGCs of PCOS patients, which was negatively correlated with BMI and serum testosterone level. Grim19+/- mice with PCOS exhibited a markedly anovulatory phenotype and disturbed glycolipid metabolism. In vitro experiments, GRIM19 deficiency inhibited the RAC1/GLUT4 pathway, reducing insulin-stimulated glucose uptake in KGN cells. Moreover, GRIM19 deficiency induced mitochondrial dysfunction, defective glucose metabolism, and apoptosis. In addition, GRIM19 deficiency suppressed the expression of ovulation-related genes in KGN cells, which was regulated by dihydrotestosterone mediated androgen receptor. CONCLUSIONS: GRIM19 deficiency may mediate ovulation and glucose metabolism disorders in PCOS patients. Our results suggest that GRIM19 may be a new target for diagnosis and treatment.

Revealing the degrading-possibility of methyl red by two azoreductases of Anoxybacillus sp. PDR2 based on molecular docking.

Azo dyes, as the most widely used synthetic dyes, are considered to be one of the culprits of water resources and environmental pollution. Anoxybacillus sp. PDR2 is a thermophilic bacterium with the ability to degrade azo dyes, whose genome contains two genes encoding azoreductases (named AzoPDR2-1 and AzoPDR2-2). In this study, through response surface methodology (RSM), when the initial pH, inoculation volume and Mg2+ addition amount were 7.18, 10.72% and 0.1 g/L respectively, the decolorization rate of methyl red (MR) (200 mg/L) could reach its maximum (98.8%). The metabolites after biodegradation were detected by UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and liquid chromatography mass spectrometry (LC-MS/MS), indicating that MR was successfully decomposed into 4-aminobenzoic acid and other small substrates. In homologous modeling, it was found that both azoreductases were flavin-dependent azoreductases, and belonged to the α/ß structure, using the Rossmann fold. In their docking results with the cofactor flavin mononucleotide (FMN), FMN bound to the surface of the protein dimer. Nicotinamide adenine dinucleotide (NADH) was superimposed on the plane of the pyrazine ring between FMN and the activity pocket of protein. Besides, both azoreductase complexes (azoreductase-FMN-NADH) exhibited a substrate preference for MR. Asn104 and Tyr74 played an important role in the combination of the azoreductase AzoPDR2-1 complex and the azoreductase AzoPDR2-2 complex with MR, respectively. This provided assistance for studying the mechanism of azoreductase biodegradation of azo dyes in thermophilic bacteria.

Cloning and characterization of FMN-dependent azoreductases from textile industry effluent identified through metagenomic sequencing.

Azo dyes, when released untreated in the environment, cause detrimental effects on flora and fauna. Azoreductases are enzymes capable of cleaving commercially used azo dyes, sometimes in less toxic by-products which can be further degraded via synergistic microbial cometabolism. In this study, azoreductases encoded by FMN1 and FMN2 genes were screened from metagenome shotgun sequences generated from the samples of textile dye industries' effluents, cloned, expressed, and evaluated for their azo dye decolorization efficacy. At pH 7 and 45°C temperature, both recombinant enzymes FMN1 and FMN2 were able to decolorize methyl red at 20 and 100 ppm concentrations, respectively. FMN2 was found to be more efficient in decolorization/degradation of methyl red than FMN1. This study offers valuable insights into the possible application of azoreductases to reduce the environmental damage caused by azo dyes, with the hope of contributing to sustainable and eco-friendly practices for the environment management. This enzymatic approach offers a promising solution for the bioremediation of textile industrial effluents. However, the study acknowledges the need for further process optimization to enhance the efficacy of these enzymes in large-scale applications.Implications: The study underscores the environmental hazards associated with untreated release of azo dyes into the environment and emphasizes the potential of azoreductases, specifically those encoded by FMN1 and FMN2 genes, to mitigate the detrimental effects. The study emphasizes the ongoing commitment to refining and advancing the enzymatic approach for the bioremediation of azo dye-containing effluents, marking a positive stride toward more sustainable industrial practices.

Unveiling potent inhibitors for schistosomiasis through ligand-based drug design, molecular docking, molecular dynamics simulations and pharmacokinetics predictions.

Schistosomiasis is a neglected tropical disease which imposes a considerable and enduring impact on affected regions, leading to persistent morbidity, hindering child development, diminishing productivity, and imposing economic burdens. Due to the emergence of drug resistance and limited management options, there is need to develop additional effective inhibitors for schistosomiasis. In view of this, quantitative structure-activity relationship studies, molecular docking, molecular dynamics simulations, drug-likeness and pharmacokinetics predictions were applied to 39 Schistosoma mansoni Thioredoxin Glutathione Reductase (SmTGR) inhibitors. The chosen QSAR model demonstrated robust statistical parameters, including an R2 of 0.798, R2adj of 0.767, Q2cv of 0.681, LOF of 0.930, R2test of 0.776, and cR2p of 0.746, confirming its reliability. The most active derivative (compound 40) was identified as a lead candidate for the development of new potential non-covalent inhibitors through ligand-based design. Subsequently, 12 novel compounds (40a-40l) were designed with enhanced anti-schistosomiasis activity and binding affinity. Molecular docking studies revealed strong and stable interactions, including hydrogen bonding, between the designed compounds and the target receptor. Molecular dynamics simulations over 100 nanoseconds and MM-PBSA free binding energy (ΔGbind) calculations validated the stability of the two best-designed molecules. Furthermore, drug-likeness and pharmacokinetics prediction analyses affirmed the potential of these designed compounds, suggesting their promise as innovative agents for the treatment of schistosomiasis.

Water-accommodated fractions of crude oil and its mixture with chemical dispersant impairs oxidase stress and energy metabolism disorders in Oryzias melastigma embryos.

In this study, marine medaka (Oryzias melastigma) embryos were exposed to different concentrations of water-accommodated fractions (WAFs) and chemically enhanced water-accommodated fractions (CEWAFs) of Oman crude oil for 14 d by semi-static exposure methods. The effects on growth and development and energy metabolism process were evaluated. Results showed that embryo survival and hatchability were decreased in a dose-dependent manner with an increase in the concentration of petroleum hydrocarbon compounds, whereas the malformation exhibited a dose-dependent increase. Compared to the control, the adenosine triphosphate (ATP) content and Na+-K+-ATPase (NKA) activities of embryos exposed to both WAFs and CEWAFs were reduced, while intracellular reactive oxygen species (ROS) levels and NADH oxidase (NOX) activities were increased. Our study demonstrated that exposure to crude oil dispersed by chemical dispersant affected the growth and development of marine medaka embryos, caused oxidative stress while produced a series of malformations in the body and dysregulation in energy metabolism. In comparison, the toxic effects of chemically dispersed crude oil might be more severe than the oil itself in the equivalent diluted concentration treatment solution. These would provide more valuable and reliable reference data for the use of chemical dispersants in oil spills.

Identification of host antioxidant effectors as thioridazine targets: Impact on cardiomyocytes infection and Trypanosoma cruzi-induced acute myocarditis.

Phenothiazines inhibit antioxidant enzymes in trypanosomatids. However, potential interferences with host cell antioxidant defenses are central concerns in using these drugs to treat Trypanosoma cruzi-induced infectious myocarditis. Thus, the interaction of thioridazine (TDZ) with T. cruzi and cardiomyocytes antioxidant enzymes, and its impact on cardiomyocytes and cardiac infection was investigated in vitro and in vivo. Cardiomyocytes and trypomastigotes in culture, and mice treated with TDZ and benznidazole (Bz, reference antiparasitic drug) were submitted to microstructural, biochemical and molecular analyses. TDZ was more cytotoxic and less selective against T. cruzi than Bz in vitro. TDZ-pretreated cardiomyocytes developed increased infection rate, reactive oxygen species (ROS) production, lipid and protein oxidation; similar catalase (CAT) and superoxide dismutase (SOD) activity, and reduced glutathione's (peroxidase - GPx, S-transferase - GST, and reductase - GR) activity than infected untreated cells. TDZ attenuated trypanothione reductase activity in T. cruzi, and protein antioxidant capacity in cardiomyocytes, making these cells more susceptible to H2O2-based oxidative challenge. In vivo, TDZ potentiated heart parasitism, total ROS production, myocarditis, lipid and protein oxidation; as well as reduced GPx, GR, and GST activities compared to untreated mice. Benznidazole decreased heart parasitism, total ROS production, heart inflammation, lipid and protein oxidation in T. cruzi-infected mice. Our findings indicate that TDZ simultaneously interact with enzymatic antioxidant targets in cardiomyocytes and T. cruzi, potentiating the infection by inducing antioxidant fragility and increasing cardiomyocytes and heart susceptibility to parasitism, inflammation and oxidative damage.

Protein N-myristoylation plays a critical role in the mitochondrial localization of human mitochondrial complex I accessory subunit NDUFB7.

The present study examined human N-myristoylated proteins that specifically localize to mitochondria among the 1,705 human genes listed in MitoProteome, a mitochondrial protein database. We herein employed a strategy utilizing cellular metabolic labeling with a bioorthogonal myristic acid analog in transfected COS-1 cells established in our previous studies. Four proteins, DMAC1, HCCS, NDUFB7, and PLGRKT, were identified as N-myristoylated proteins that specifically localize to mitochondria. Among these proteins, DMAC1 and NDUFB7 play critical roles in the assembly of complex I of the mitochondrial respiratory chain. DMAC1 functions as an assembly factor, and NDUFB7 is an accessory subunit of complex I. An analysis of the intracellular localization of non-myristoylatable G2A mutants revealed that protein N-myristoylation occurring on NDUFB7 was important for the mitochondrial localization of this protein. Furthermore, an analysis of the role of the CHCH domain in NDUFB7 using Cys to Ser mutants revealed that it was essential for the mitochondrial localization of NDUFB7. Therefore, the present results showed that NDUFB7, a vital component of human mitochondrial complex I, was N-myristoylated, and protein N-myrisotylation and the CHCH domain were both indispensable for the specific targeting and localization of NDUFB7 to mitochondria.