Bacterial catabolism of acetovanillone, a lignin-derived compound.

Bacterial catabolic pathways have considerable potential as industrial biocatalysts for the valorization of lignin, a major component of plant-derived biomass. Here, we describe a pathway responsible for the catabolism of acetovanillone, a major component of several industrial lignin streams. Rhodococcus rhodochrous GD02 was previously isolated for growth on acetovanillone. A high-quality genome sequence of GD02 was generated. Transcriptomic analyses revealed a cluster of eight genes up-regulated during growth on acetovanillone and 4-hydroxyacetophenone, as well as a two-gene cluster up-regulated during growth on acetophenone. Bioinformatic analyses predicted that the hydroxyphenylethanone (Hpe) pathway proceeds via phosphorylation and carboxylation, before ß-elimination yields vanillate from acetovanillone or 4-hydroxybenzoate from 4-hydroxyacetophenone. Consistent with this prediction, the kinase, HpeHI, phosphorylated acetovanillone and 4-hydroxyacetophenone. Furthermore, HpeCBA, a biotin-dependent enzyme, catalyzed the ATP-dependent carboxylation of 4-phospho-acetovanillone but not acetovanillone. The carboxylase's specificity for 4-phospho-acetophenone (kcat/KM = 34 ± 2 mM-1 s-1) was approximately an order of magnitude higher than for 4-phospho-acetovanillone. HpeD catalyzed the efficient dephosphorylation of the carboxylated products. GD02 grew on a preparation of pine lignin produced by oxidative catalytic fractionation, depleting all of the acetovanillone, vanillin, and vanillate. Genomic and metagenomic searches indicated that the Hpe pathway occurs in a relatively small number of bacteria. This study facilitates the design of bacterial strains for biocatalytic applications by identifying a pathway for the degradation of acetovanillone.

Hepatoprotective effect of acetovanillone against methotrexate hepatotoxicity: Role of Keap-1/Nrf2/ARE, IL6/STAT-3, and NF-κB/AP-1 signaling pathways.

This study targeted to examine the protective effects of acetovanillone (AV) against methotrexate (MTX)-induced hepatotoxicity. Thirty-two rats were allocated into four groups of eight animals; Group 1: Normal; Group 2: administered AV (100 ml/kg; P.O.) for 10 days; Group 3: challenged with MTX (20 mg/kg, i.p; single dose); Group 4: administered AV 5 days before and 5 days after MTX. For the first time, this study affords evidence for AV's hepatoprotective effects on MTX-induced hepatotoxicity. The underlined mechanisms behind its hepatic protection include counteracting MTX-induced oxidative injury via down-regulation of NADPH oxidase and up-regulation of Nrf2/ARE, SIRT1, PPARγ, and cytoglobin signals. Additionally, AV attenuated hepatic inflammation through down-regulation of IL-6/STAT-3 and NF-κB/AP-1 signaling. Network pharmacology analysis exhibited a high enrichment score between the interacting proteins and strongly suggested the intricate and essential role of the target proteins regulating MTX-induced oxidative damage and inflammatory perturbation. Besides, AV increased the in vitro cytotoxic activity of MTX toward PC-3, HeLa, and K562 cancer cell lines. On the whole, our investigation suggested that AV might be regarded as a promising adjuvant for the amelioration of MTX hepatotoxicity and/or increased its in vitro antitumor efficacy, and it could be used in patients receiving MTX.

Streptomyces tunisiensis DSM 42037 mediated bioconversion of ferulic acid released from barley bran.

Streptomyces tunisiensis DSM 42037 exhibited growth capacity on a minimum medium containing 1% barley bran. This peculiar strain released 83.5% of total ferulic acid present in barley bran after 5 days of incubation and the highest amount of released ferulic acid (19 mg/L) was observed on the 3rd day of incubation. The concentrated supernatant of S. tunisiensis also released ferulic acid from the parietal arabinoxylan complex of barley bran. This strain was able to convert the free ferulic acid into 4-vinyl guaiacol (14 mg/L) and acetovanillone (12 mg/L) at molar yield of 97% and 83% respectively. The biotransformation products were successively purified by preparative thin layer and silica gel column chromatography followed by HPLC and identified by 1H nuclear magnetic resonance. Streptomyces tunisiensis DSM 42037 could have potential applications in the food, pharmaceutical and cosmetic industries thanks to its ability in biotransforming ferulic acid into 4-vinyl guaiacol and acetovanillone.

Antioxidants protect against diabetes by improving glucose homeostasis in mouse models of inducible insulin resistance and obesity.

AIMS/HYPOTHESIS: In the context of diabetes, the health benefit of antioxidant treatment has been widely debated. In this study, we investigated the effect of antioxidant treatment during the development of insulin resistance and hyperphagia in obesity and partial lipodystrophy. METHODS: We studied the role of antioxidants in the regulation of insulin resistance using the tamoxifen-inducible fat-specific insulin receptor knockout (iFIRKO) mouse model, which allowed us to analyse the antioxidant's effect in a time-resolved manner. In addition, leptin-deficient ob/ob mice were used as a hyperphagic, chronically obese and diabetic mouse model to validate the beneficial effect of antioxidants on metabolism. RESULTS: Acute induction of insulin receptor knockout in adipocytes changed the substrate preference to fat before induction of a diabetic phenotype including hyperinsulinaemia and hyperglycaemia. In healthy chow-fed animals as well as in morbidly obese mice, this diabetic phase could be reversed within a few weeks. Furthermore, after the induction of insulin receptor knockout in mature adipocytes, iFIRKO mice were protected from subsequent obesity development through high-fat diet feeding. By genetic tracing we show that the persistent fat mass loss in mice after insulin receptor knockout in adipocytes is not caused by the depletion of adipocytes. Treatment of iFIRKO mice with antioxidants postponed and reduced hyperglycaemia by increasing insulin sensitivity. In ob/ob mice, antioxidants rescued both hyperglycaemia and hyperphagia. CONCLUSIONS/INTERPRETATION: We conclude that fat mass reduction through insulin resistance in adipocytes is not reversible. Furthermore, it seems unlikely that adipocytes undergo apoptosis during the process of extreme lipolysis, as a consequence of insulin resistance. Antioxidants have a beneficial health effect not only during the acute phase of diabetes development, but also in a temporary fashion once chronic obesity and diabetes have been established.

The protective effect of apocynin on testicular ischemia-reperfusion injury.

PURPOSE: We investigated the protective effect of the NADPH oxidase inhibitor apocynin on testicular damage induced by ischemia-reperfusion injury in rats. MATERIALS AND METHODS: A total of 32 rats were randomly divided into 4 groups. Controls underwent left scrotal exploration only. The 3 groups with ischemia-reperfusion underwent 4-hour torsion followed by 1-hour detorsion. The ischemia-reperfusion only group underwent left testicular torsion and detorsion. The ischemia-reperfusion plus saline group underwent left testicular torsion, received 10 ml/kg saline intraperitoneally at minute 210 of ischemia and then underwent detorsion. The ischemia-reperfusion plus apocynin group underwent left testicular torsion, received 20 mg/kg apocynin intraperitoneally at minute 210 of ischemia and then underwent detorsion. We determined histopathological findings and performed specific biochemical analyses. RESULTS: In the ischemia-reperfusion only and the ischemia-reperfusion plus saline groups malondialdehyde, total oxidative capacity and the oxidative stress index were significantly higher. Superoxide dismutase, catalase, glutathione peroxidase and glutathione were significantly lower. Apocynin significantly decreased malondialdehyde, total oxidative capacity and the oxidative stress index, and significantly increased superoxide dismutase and catalase. There was a significantly increase in the number of giant, degenerated and desquamated cells in the ischemia-reperfusion group. Apocynin significantly improved these histological alterations. CONCLUSIONS: These histopathological and biochemical findings show the beneficial effects of apocynin on testicular ischemia-reperfusion injury.

Molecular imaging reveals rapid reduction of endothelial activation in early atherosclerosis with apocynin independent of antioxidative properties.

OBJECTIVE: Antioxidative drugs continue to be developed for the treatment of atherosclerosis. Apocynin is an nicotinamide adenine dinucleotide phosphate oxidase inhibitor with anti-inflammatory properties. We used contrast-enhanced ultrasound molecular imaging to assess whether short-term apocynin therapy in atherosclerosis reduces vascular oxidative stress and endothelial activation APPROACH AND RESULTS: Genetically modified mice with early atherosclerosis were studied at baseline and after 7 days of therapy with apocynin (4 mg/kg per day IP) or saline. Contrast-enhanced ultrasound molecular imaging of the aorta was performed with microbubbles targeted to vascular cell adhesion molecule 1 (VCAM-1; MB(V)), to platelet glycoprotein Ibα (MB(Pl)), and control microbubbles (MB(Ctr)). Aortic vascular cell adhesion molecule 1 was measured using Western blot. Aortic reactive oxygen species generation was measured using a lucigenin assay. Hydroethidine oxidation was used to assess aortic superoxide generation. Baseline signal for MBV (1.3 ± 0.3 AU) and MB(Pl )(1.5 ± 0.5 AU) was higher than for MBCtr (0.5 ± 0.2 AU; P<0.01). In saline-treated animals, signal did not significantly change for any microbubble agent, whereas short-term apocynin significantly (P<0.05) reduced vascular cell adhesion molecule 1 and platelet signal (MBV: 0.3 ± 0.1; MBPl: 0.4 ± 0.1; MBCtr: 0.3 ± 0.2 AU; P=0.6 between agents). Apocynin reduced aortic vascular cell adhesion molecule 1 expression by 50% (P<0.05). However, apocynin therapy did not reduce reactive oxygen species content, superoxide generation, or macrophage content. CONCLUSIONS: Short-term treatment with apocynin in atherosclerosis reduces endothelial cell adhesion molecule expression. This change in endothelial phenotype can be detected by molecular imaging before any measurable decrease in macrophage content and is not associated with a detectable change in oxidative burden.

Development of a rapid assay for ß-etherase activity using a novel chromogenic substrate.

Biocatalytic processes play a crucial role in the valorization of lignin; therefore, methods enabling the monitoring of enzymes such as ß-etherases, capable of breaking ß-O-4 aryl-ether bonds, are of significant biotechnological interest. A novel method for quantifying ß-etherase activity was developed based on the ß-ester bond formation between a chromophore and acetovainillone. The chromogenic substrate ß-(ρ-nitrophenoxy)-α-acetovanillone (PNPAV), was chemically synthesized. Kintetic monitoring of ρ-nitrophenolate release at 410 nm over 10 min, using recombinant LigF from Sphingobium sp SYK-6, LigF-AB and LigE-AB from Althererytrobacter sp B11, yielded enzimatic activities of 404. 3 mU/mg, 72 mU/mg, and 50 mU/mg, respectively. This method is applicable in a pH range of 7.0-9.0, with a sensitivity of up to 50 ng of enzyme, exhibiting no interference with lipolytic, glycolytic, proteolytic, and oxidoreductase enzymes.

Supplementation of Mito TEMPO and acetovanillone in semen extender improves freezability of buffalo spermatozoa.

BACKGROUND: Oxidative stress is one of the leading factors responsible for poor post-thaw semen quality because of overproduction of reactive oxygen species (ROS) over neutralizing antioxidants present in semen. Mainly two ROS generation sites are present in spermatozoa, that is, mitochondria and plasma membrane. Therefore, the idea of targeting these specific sites for minimization of ROS production with the compounds having known mechanism of actions was built up as a core for this research. OBJECTIVE: Present study was done to investigate the effects of Mito TEMPO and acetovanillone individually and in combination on freezability of buffalo spermatozoa. MATERIALS AND METHODS: For the experiment, semen extender was supplemented with Mito TEMPO (50 µM), acetovanillone (50 µM), and a combination of Mito TEMPO + acetovanillone (50 µM+ 50 µM), designated as Group II, Group III, and Group IV, respectively. Control group without any supplementation was designated as Group I. A total of 24 ejaculates with individual progressive motility (IPM) of ≥70% were selected for the study. After final dilution, filling-sealing of straws, equilibration, and freezing were done as per the standard procedure. Semen samples were evaluated for IPM, plasma membrane integrity, lipid peroxidation, total antioxidant capacity (TAC), and cholesterol to phospholipids (C/P) ratio at both fresh and post-thaw stages. Evaluation of ROS, mitochondrial membrane potential (MMP), capacitation status (CTC assay), and in vitro fertility potential were conducted only on frozen-thawed samples. RESULTS: The addition of Mito TEMPO (50 µM) and acetovanillone (50 µM) individually and in combination significantly (p < 0.05) improved post-thaw semen quality in terms of IPM, plasma membrane integrity, TAC, cholesterol content, C/P ratio, MMP, Chlortetracycline (CTC)-Full (F) pattern, and zona binding ability of buffalo spermatozoa, while significantly (p < 0.05) reduced ROS production, lipid peroxidation, and capacitation like changes as compared to the control group. DISCUSSION: As Mito TEMPO acts as an SOD mimetic and also detoxifies ferrous iron at the mitochondria level, it aids in neutralization of excessive ROS production and minimizes oxidative stress-related damages that enhances the antioxidant potential of sperm mitochondria. Earlier studies also indicated improved post-thaw semen quality in 50 µM supplemented group. The improvement observed in acetovanillone (50 µM) group might be because of inhibition of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as this enzyme activation by various physical/chemical inducers during cryopreservation process leads to activation of CatSper channel resulting in calcium influx, premature capacitation, and acrosomal reaction like changes through activation of adenylate cyclase and cAMP/PKA-mediated tyrosine phosphorylation of sperm proteins. Acetovanillone also prevents NADPH oxidase-mediated inhibition of glutathione reductase activity, which has a vital role in protecting the structural and functional integrity of sperm plasma membrane. CONCLUSION: Results indicated beneficial effects of supplementation of Mito TEMPO and acetovanillone on sperm freezability and individual supplementation was as efficient as the combination group for sustaining post-thaw semen quality.

Bacterial Transformation of Aromatic Monomers in Softwood Black Liquor.

The valorization of lignin, a major component of plant-derived biomass, is essential to sustainable biorefining. We identified the major monoaromatic compounds present in black liquor, a lignin-rich stream generated in the kraft pulping process, and investigated their bacterial transformation. Among tested solvents, acetone extracted the greatest amount of monoaromatic compounds from softwood black liquor, with guaiacol, vanillin, and acetovanillone, in an approximately 4:3:2 ratio, constituting ~90% of the total extracted monoaromatic content. 4-Ethanol guaiacol, vanillate, and 4-propanol guaiacol were also present. Bacterial strains that grew on minimal media supplemented with the BL extracts at 1mM total aromatic compounds included Pseudomonas putida KT2442, Sphingobium sp. SYK-6, and Rhodococcus rhodochrous EP4. By contrast, the extracts inhibited the growth of Rhodococcus jostii RHA1 and Rhodococcus opacus PD630, strains extensively studied for lignin valorization. Of the strains that grew on the extracts, only R. rhodochrous GD01 and GD02, isolated for their ability to grow on acetovanillone, depleted the major extracted monoaromatics. Genomic analyses revealed that EP4, GD01, and GD02 share an average nucleotide identity (ANI) of 98% and that GD01 and GD02 harbor a predicted three-component carboxylase not present in EP4. A representative carboxylase gene was upregulated ~100-fold during growth of GD02 on a mixture of the BL monoaromatics, consistent with the involvement of the enzyme in acetovanillone catabolism. More generally, quantitative RT-PCR indicated that GD02 catabolizes the BL compounds in a convergent manner via the ß-ketoadipate pathway. Overall, these studies help define the catabolic capabilities of potential biocatalytic strains, describe new isolates able to catabolize the major monoaromatic components of BL, including acetovanillone, and facilitate the design of biocatalysts to valorize under-utilized components of industrial lignin streams.

Role of Two Sets of RND-Type Multidrug Efflux Pump Transporter Genes, mexAB-oprM and mexEF-oprN, in Virulence of Pseudomonas syringae pv. tabaci 6605.

Pseudomonas syringae pv. tabaci 6605 has two multidrug resistance (MDR) efflux pump transporters, MexAB-OprM and MexEF-OprN. To understand the role of these MDR efflux pumps in virulence, we generated deletion mutants, ∆mexB, ∆mexF, and ∆mexB∆mexF, and investigated their sensitivity to plant-derived antimicrobial compounds, antibiotics, and virulence. Growth inhibition assays with KB soft agar plate showed that growth of the wild-type (WT) was inhibited by 5 µl of 1 M catechol and 1 M coumarin but not by other plant-derived potential antimicrobial compounds tested including phytoalexins. The sensitivity to these compounds tended to increase in ∆mexB and ∆mexB∆mexF mutants. The ∆mexB∆mexF mutant was also sensitive to 2 M acetovanillone. The mexAB-oprM was constitutively expressed, and activated in the ∆mexF and ∆mexB∆mexF mutant strains. The swarming and swimming motilities were impaired in ∆mexF and ∆mexB∆mexF mutants. The flood inoculation test indicated that bacterial populations in all mutant strains were significantly lower than that of WT, although all mutants and WT caused similar disease symptoms. These results indicate that MexAB-OprM extrudes plant-derived catechol, acetovanillone, or coumarin, and contributes to bacterial virulence. Furthermore, MexAB-OprM and MexEF-OprN complemented each other's functions to some extent.

Edaravone and Acetovanillone Upregulate Nrf2 and PI3K/Akt/mTOR Signaling and Prevent Cyclophosphamide Cardiotoxicity in Rats.

INTRODUCTION: Cyclophosphamide (CP) causes redox imbalance and its use is associated with marked cardiotoxicity that limits its clinical applications. The present study investigated the protective effects of acetovanillone (AV) and edaravone (ED) against CP-induced oxidative stress and cardiac damage, emphasizing the role of PI3K/Akt/mTOR and Nrf2 signaling. MATERIALS AND METHODS: Rats received either AV (100 mg/kg) or ED (20 mg/kg) orally for 10 days and CP (200 mg/kg) on day 7. At day 11, the rats were sacrificed, and samples were collected for analysis. RESULTS: AV and ED ameliorated serum troponin I, CK-MB, LDH, AST and ALP, and prevented cardiac histological alterations in CP-intoxicated rats. Both treatments decreased cardiac lipid peroxidation and enhanced GSH, SOD and cytoglobin in CP-induced rats. AV and ED downregulated Keap1, whereas increased the expression of PI3K, Akt, mTOR and Nrf2 in the heart of rats received CP. Additionally, the binding modes of AV and ED to Keap1 were pinpointed in silico using molecular docking simulations. CONCLUSION: AV and ED prevent CP cardiotoxicity by attenuating oxidative stress and tissue injury, and modulating cytoglobin, and PI3K/Akt/mTOR and Keap1/Nrf2 signaling. Therefore, AV and ED may represent promising agents that can prevent cardiac injury in patients receiving CP.

Effect of innovative technology using staves and micro-oxygenation on the odorant and sensory profile of aged wine spirit.

The aged wine spirit is a beverage with great aromatic complexity. Their volatile compounds with odorant power coming from the distillate and from the wood used in its ageing, and the interactions that take place in the process, enhanced by oxygen, all contribute to this complexity. Due to time and cost inherent of ageing wine spirits in wooden barrels, research has sought to develop more sustainable alternatives to do it. In this context, the present study compares, the effect of traditional (wooden barrel) and alternative system (stainless steel tank with dipped staves and micro-oxygenation), on the odorant and sensory profile of a wine spirit, using Limousin oak and chestnut wood, after 12 months of ageing. The results suggest that the ageing process is accelerated by the alternative ageing technology and the chestnut wood, and the corresponding wine spirits presented characteristics of greater sensory evolution and strong wood compounds extraction.

The dynamics of apoplast phenolics in tobacco leaves following inoculation with bacteria.

This study demonstrates that the accumulation of apoplastic phenolics is stimulated in planta in response to bacterial inoculation. Past studies have shown that levels of extracellular phenolics are elicited in plant cell suspensions in response to bacteria, and that tomato plants infected with viroids showed changes in apoplastic phenolics. The method described here monitored changes in apoplastic phenolics in tobacco leaves following bacterial inoculation of the same tissue. Inoculation with a saprophyte, Pseudomonas fluorescens, which does not cause visible symptoms or physical damage, was used to elicit phenolics and examine the effects of variable parameters on phenolic composition. Location of the inoculation on the leaf, position, or developmental age of the leaf on the plant, and inoculum concentration were standardized for further experiments. The patterns of phenolic change in the apoplast were compared for tobacco inoculated with P. syringae pathovars, pv. syringae, which causes a resistant HR reaction within 15 h, and pv. tabaci, which causes a susceptible reaction with delayed visible symptoms. Both pathogens elicited lower increased levels of acetosyringone compared to the saprophyte, P. fluorescens but had greatly increased levels of the chlorogenic acid derivatives. The latter metabolites appear to have come from the intracellular stores, which could indicate a weakening of the apoplast/symplast barrier. This unexpected aspect will require further study of intracellular phenolics.