The "Doorstop Pocket" In Thioredoxin Reductases─An Unexpected Druggable Regulator of the Catalytic Machinery.

Pyridine nucleotide-disulfide oxidoreductases are underexplored as drug targets, and thioredoxin reductases (TrxRs) stand out as compelling pharmacological targets. Selective TrxR inhibition is challenging primarily due to the reliance on covalent inhibition strategies. Recent studies identified a regulatory and druggable pocket in Schistosoma mansoni thioredoxin glutathione reductase (TGR), a TrxR-like enzyme, and an established drug target for schistosomiasis. This site is termed the "doorstop pocket" because compounds that bind there impede the movement of an aromatic side-chain necessary for the entry and exit of NADPH and NADP+ during enzymatic turnover. This discovery spearheaded the development of new TGR inhibitors with efficacies surpassing those of current schistosomiasis treatment. Targeting the "doorstop pocket" is a promising strategy, as the pocket is present in all members of the pyridine nucleotide-disulfide oxidoreductase family, opening new avenues for exploring therapeutic approaches in diseases where the importance of these enzymes is established, including cancer and inflammatory and infectious diseases.

Free Radical Production Induced by Nitroimidazole Compounds Lead to Cell Death in Leishmania infantum Amastigotes.

Leishmania infantum is the vector-borne trypanosomatid parasite causing visceral leishmaniasis in the Mediterranean basin. This neglected tropical disease is treated with a limited number of obsolete drugs that are not exempt from adverse effects and whose overuse has promoted the emergence of resistant pathogens. In the search for novel antitrypanosomatid molecules that help overcome these drawbacks, drug repurposing has emerged as a good strategy. Nitroaromatic compounds have been found in drug discovery campaigns as promising antileishmanial molecules. Fexinidazole (recently introduced for the treatment of stages 1 and 2 of African trypanosomiasis), and pretomanid, which share the nitroimidazole nitroaromatic structure, have provided antileishmanial activity in different studies. In this work, we have tested the in vitro efficacy of these two nitroimidazoles to validate our 384-well high-throughput screening (HTS) platform consisting of L. infantum parasites emitting the near-infrared fluorescent protein (iRFP) as a biomarker of cell viability. These molecules showed good efficacy in both axenic and intramacrophage amastigotes and were poorly cytotoxic in RAW 264.7 and HepG2 cultures. Fexinidazole and pretomanid induced the production of ROS in axenic amastigotes but were not able to inhibit trypanothione reductase (TryR), thus suggesting that these compounds may target thiol metabolism through a different mechanism of action.

Design and Synthesis of an Azo Reductase Responsive Flavonol-Indomethacin Hybrid Used for the Diagnosis and Treatment of Colitis.

Human intestinal bacteria are the primary producers of azo reductase, and the content of azo reductase is closely associated with various intestinal diseases, including ulcerative colitis (UC). The rapid detection of changes in azo reductase levels is crucial for diagnosing and promptly intervening in UC. In this study, a therapeutic agent, FAI, specifically targeting UC, was designed and synthesized. This agent was developed by linking the anti-inflammatory drug indomethacin to flavonols with antioxidant activity via an azo bond (off-on). Breakage of the azo bond breaks results in the release of both fluorophores and drugs, achieving targeted tracing and integrated treatment effects. In vivo and in vitro fluorescence imaging experiments were used to demonstrate the potential of FAI in the diagnosis of UC, together with synergistic therapeutic effects through the release of both fluorophores and anti-inflammatory agents. Therefore, this diagnostic agent shows promise as a potential tool for diagnosing and treating UC.

The compartment-specific manipulation of the NAD+/NADH ratio affects the metabolome and the function of glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive type of cancer in the brain and has an inferior prognosis because of the lack of suitable medicine, largely due to its tremendous invasion. GBM has selfish metabolic pathways to promote migration, invasion, and proliferation compared to normal cells. Among various metabolic pathways, NAD (nicotinamide adenine dinucleotide) is essential in generating ATP and is used as a resource for cancer cells. LbNOX (Lactobacillus brevis NADH oxidase) is an enzyme that can directly manipulate the NAD+/NADH ratio. In this study, we found that an increased NAD+/NADH ratio by LbNOX or mitoLbNOX reduced intracellular glutamate and calcium responses and reduced invasion capacity in GBM. However, the invasion was not affected in GBM by rotenone, an ETC (Electron Transport Chain) complex I inhibitor, or nicotinamide riboside, a NAD+ precursor, suggesting that the crucial factor is the NAD+/NADH ratio rather than the absolute quantity of ATP or NAD+ for the invasion of GBM. To develop a more accurate and effective GBM treatment, our findings highlight the importance of developing a new medicine that targets the regulation of the NAD+/NADH ratio, given the current lack of effective treatment options for this brain cancer.

Molecular Docking-Based Virtual Screening of FDA-Approved Drugs Using Trypanothione Reductase Identified New Trypanocidal Agents.

American trypanosomiasis or Chagas disease, caused by Trypanosoma cruzi (T. cruzi), affects approximately 6-7 million people worldwide. However, its pharmacological treatment causes several uncomfortable side effects, causing patients' treatment abandonment. Therefore, there is a need for new and better treatments. In this work, the molecular docking of nine hundred twenty-four FDA-approved drugs on three different sites of trypanothione reductase of T. cruzi (TcTR) was carried out to find potential trypanocidal agents. Finally, biological evaluations in vitro and in vivo were conducted with the selected FDA-approved drugs. Digoxin, alendronate, flucytosine, and dihydroergotamine showed better trypanocidal activity than the reference drugs benznidazole and nifurtimox in the in vitro evaluation against the trypomastigotes form. Further, these FDA-approved drugs were able to reduce 20-50% parasitemia in a short time in an in vivo model, although with less efficiency than benznidazole. Therefore, the results suggest a combined therapy of repurposed and canonical drugs against T. cruzi infection.

Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem.

2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.

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.

Identification of Novel Antileishmanial Chemotypes By High-Throughput Virtual and In Vitro Screening.

BACKGROUND: Leishmaniasis is a deadly protozoan parasitic disease and a significant health problem in underdeveloped and developing countries. The global spread of the parasite, coupled with the emergence of drug resistance and severe side effects associated with existing treatments, has necessitated the identification of new and potential drugs. OBJECTIVE: This study aimed to identify promising compounds for the treatment of leishmaniasis by targeting two essential enzymes of Leishmania donovani: trypanothione reductase (Try-R) and trypanothione synthetase (Try-S). METHODS: High-throughput virtual and in vitro screening of in-house and commercial databases was conducted. A pharmacophore model with seven features was developed and validated using the Guner-Henery method. The pharmacophore-based virtual screening yielded 690 hits, which were further filtered through Lipinski's rule, ADMET analysis, and molecular docking against Try-R and Try-S. Molecular dynamics studies were performed on selected compounds, and in vitro experiments were conducted to evaluate their activity against the promastigote and amastigote forms of L. donovani. RESULTS: The virtual screening and subsequent analysis identified 33 promising compounds. Molecular dynamics studies of two compounds (comp-1 and comp-2) demonstrated stable binding interactions with the target enzymes and high affinity. In vitro experiments revealed that 13 compounds exhibited moderate activity against both the promastigote (IC50, 41 µM-76 µM) and the amastigote (IC50, 44 µM-72 µM) forms of L. donovani. Compounds 1 and 2 showed the highest percent inhibition and the lowest IC50 values. CONCLUSION: The identified compounds demonstrated significant inhibitory activity against Leishmania donovani and stable interactions with target enzymes. These findings suggest that the compounds could serve as promising leads for developing new treatments for leishmaniasis.

Assessing proteases and enzymes of the trypanothione system in subpopulations of Leishmania (Viannia) braziliensis Thor strain during macrophage infection.

BACKGROUND: Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22. OBJECTIVES: Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations. METHODS: Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study. FINDINGS: Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes' expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03. MAIN CONCLUSIONS: The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells.

Pt-Cluster-Embedded Metal-Organic Frameworks-Derived Fe@C as Dual-Enzyme Mimics for NADH Detection in Serum.

Dihydro-nicotinamide adenine dinucleotide (NADH) detection is crucial since it is a vital coenzyme in organism metabolism. Compared to the traditional method based on natural NADH oxidase (NOX), nanozymes with multienzyme-like activity can catalyze multistage reactions in a singular setup, simplifying detection processes and enhancing sensitivity. In this study, an innovative NADH detection method was developed using iron-doped carbon (Fe@C) nanozyme synthesized from metal-organic frameworks with in situ reduced Pt clusters. This nanozyme composite (Pt/Fe@C) demonstrated dual NOX and peroxidase-like characteristics, significantly enhancing the catalytic efficiency and enabling NADH conversion to NAD+ and H2O2 with subsequent detection. The collaborative research involving both experimental and theoretical simulations has uncovered the catalytic process and the cooperative effect of Fe and Pt atoms, leading to enhanced oxygen adsorption and activation, as well as a decrease in the energy barrier of the key step in the H2O2 decomposition process. These findings indicate that the catalytic performance of Pt/Fe@C in NOX-like and POD-like reactions can be significantly improved. The colorimetric sensor detects NADH with a limit of detection as low as 0.4 nM, signifying a breakthrough in enzyme-mimicking nanozyme technology for precise NADH measurement.

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.

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.

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.

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.

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.

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.

Antioxidant, anti-amylase, anti-lipase, and efficiency of Satureja fatty acid on the anti-inflammatory parameters in lipopolysaccharide-stimulated macrophage through Nrf2/NF-kB/NADH oxidase pathway.

Satureja is an aromatic plant that is used for flavoring, perfume, and food manufacturing due to its pleasant essential oil. Modern medicine research revealed several biological activities of Satureja essential oil, including antifungal, antibacterial, antiviral, antioxidant, anticancer, and anti-inflammatory. However, the functional properties of Satureja fatty acid have not been explored. This study examined the fatty acid profile, lipid nutritional quality, antioxidant, anti-amylase, and anti-lipase capacities of Satureja. The efficiency of Satureja fatty acid on the anti-oxidative and anti-inflammatory parameters in LPS-induced macrophage through the Nrf2/NF-kB/NADH oxidase pathway was examined. The whole lipid extract was prepared with chloroform/methanol/water solution. Fatty acids methyl ester from whole lipid extract were prepared with methanol/sulfuric acid reagent. The fatty acid profile was analyzed using gas chromatography-mass spectrometry. Total antioxidant was determined by ABTS decolorization. Lipase and amylase activities were determined by monitoring the decomposition of p-nitrophenyl butyrate and starch. The macrophage cell line was grown in DMEM media in the presence of fatty acid. The hydrogen peroxide production in treated cells was monitored using the FOX reagent. NADH oxidase activity was measured by monitoring NADH breakdown. The expression of NOX, NF-kB, and NRF2, were tested in the treated cells by real-time PCR. The main components of the Satureja fatty acid were linolenic acid (24.67-37.32%), palmitic acid (10.65-20.29%), linoleic acid (8.31-13.39%), oleic acid (4.42-14.35%), stearic acid (2.76-8.77%) and palmitoleic acid (1.77-4.95%). Given the nutritional quality, omega-3 PUFA (23.58-37.32%), SFA (21.53-26.70%), omega-6 PUFA (10.86-16.14%), omega-9 MUFA (4.42-14.35%), and omega-7 MUFA (1.77-4.95%) comprise the majority of fatty acids. Satureja fatty acid has a promising unsaturation index (120.77-164.27), PUFA/MUFA (2.07-6.41), hypocholesterolemic index (2.44-3.47), health-promoting index (2.03-2.42), PUFA/SFA (1.37-1.94), nutritive value index (0.53-1.71), MUFA/SFA (0.30-0.80) omega-6/omega-3 (0.34-0.65), atherogenicity index (0.41-0.49), and thrombogenicity index (0.17-0.27). Satureja fatty acid displayed strong antioxidant capacity (with IC50 ranging from 354 to 428 µg/mL), anti-lipase capacity (with IC50 ranging from 354 to 428 µg/mL), and anti-amylase capacity (with IC50 ranging from 370 to 390 µg/mL). LPS induced the expression of NOX, NRF2, and NF-kB and the synthesis of hydrogen peroxide in macrophage cells. In LPS-stimulated macrophages, Satureja fatty acid reduced NOX expression, hydrogen peroxide, and NF-kB expression and increased NRF2 at 0.04 mg/mL. In conclusion, Satureja fatty acids have potent antioxidant, anti-amylase, anti-lipase, and anti-inflammatory activities. The mechanisms in lowering oxidative stress markers depended on down-regulating superoxide-producing enzymes at gene and protein levels. Satureja polyunsaturated omega-3 fatty acids could be recommended for healthy products combined with dietary therapy to treat obesity, diabetes, and oxidative stress.

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.

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.