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Anaerobic oxidation of methane (AOM) is a microbial process of importance in the global carbon cycle. AOM is predominantly mediated by anaerobic methanotrophic archaea (ANME), the physiology of which is still poorly understood. Here we present a new addition to the current physiological understanding of ANME by examining, for the first time, the biochemical and redox-active properties of the extracellular polymeric substances (EPS) of an ANME enrichment culture. Using a 'Candidatus Methanoperedens nitroreducens '- dominated methanotrophic consortium as the representative, we found it can produce an EPS matrix featuring a high protein-to-polysaccharide ratio of ∼8. Characterization of EPS using FTIR revealed the dominance of protein-associated amide I and amide II bands in the EPS. XPS characterization revealed the functional group of C-(O/N) from proteins accounted for 63.7% of total carbon. Heme-reactive staining and spectroscopic characterization confirmed the distribution of c -type cytochromes in this protein-dominated EPS, which potentially enabled its electroactive characteristic. Redox-active c -type cytochromes in EPS mediated the EET of ' Ca. M. nitroreducens' for the reduction of Ag+ to metallic Ag, which was confirmed by both ex-situ experiments with extracted soluble EPS and in-situ experiments with pristine EPS matrix surrounding cells. The formation of nanoparticles in the EPS matrix during in-situ extracellular Ag + reduction resulted in a relatively lower intracellular Ag distribution fraction, beneficial for alleviating the Ag toxicity to cells. The results of this study provide the first biochemical information on EPS of anaerobic methanotrophic consortia and a new insight into its physiological role in AOM process. • EPS produced by ' Ca. M. nitroreducens ' enrichment culture was characterized. • EPS of ' Ca. M. nitroreducens ' enrichment culture was dominated by protein. • EPS of ' Ca. M. nitroreducens ' enrichment culture was electroactive. • Heme proteins likely contribute to the redox property of EPS matrix. • Ag + can be reduced in EPS matrix to reduce the Ag access into ' Ca. M. nitroreducens' cells. [ABSTRACT FROM AUTHOR]

Biocompatible materials and biocarriers have attracted great attention in biological wastewater treatment owing to their excellent performance in improving pollutant removal. Graphene-based material, a biocarrier candidate, with excellent adsorbability and conductivity was increasingly applied in anaerobic digestion due to its exceptional potential in the adsorption and electron transfer process. Nevertheless, the green approach for the formation of bio-graphene complexes and their mechanism in dye removal is limited. The aim of this study is to investigate and assess the performance of biological graphene hydrogel (BGH) formed by Shewanella putrefaciens CN32 on the removal of methyl orange (MO) and methylene blue (MB). The results showed that the formation of BGH is determined by the physicochemical characteristics of graphene oxide, including sheet size, oxidation degree, and interlayer distance. BGHs significantly increased the removal efficiency of dyes in comparison to non-graphene samples, with a 24-h removal rate of MO and MB reaching 92.9% and 91%, respectively. The synergetic mechanism of BGH on the enhanced removal rate of organic dye can be ascribed to GO's ability in accelerating extracellular electron transfer and stimulating biodegradation pathways relating to c -type cytochromes, including MtrA and MtrC. These findings provided an understanding of the relationship between graphene-based nanomaterials and Shewanella , which facilitated their future application in environmental biotechnology. [Display omitted] • The reduction of GO by S. putrefaciens CN32 prompt BGH generation. • Sheet area and degree of defects of GO determine the BGH formation. • Bacteria-immobilized rGO hydrogel displayed a synergistic effect in bioremediation. • BGH increased dye removal efficiency by accelerating extracellular electron transfer. [ABSTRACT FROM AUTHOR]

This study examined the biotransformation of phytocannabinoids in human hepatocytes. The susceptibility of the tested compounds to transformations in hepatocytes exhibited the following hierarchy: cannabinol (CBN) > cannabigerol (CBG) > cannabichromene (CBC) > cannabidiol (CBD). Biotransformation included hydroxylation, oxidation to a carboxylic acid, dehydrogenation, hydrogenation, dehydration, loss/shortening of alkyl, glucuronidation and sulfation. CBN was primarily metabolized by oxidation of a methyl to a carboxylic acid group, while CBD, CBG and CBC were preferentially metabolized by direct glucuronidation. The study also screened for the activity of recombinant human cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs), which could catalyze the hydroxylation and glucuronidation of the tested compounds, respectively. We found that CBD was hydroxylated mainly by CYPs 2C8, 2C19, 2D6; CBN by 1A2, 2C9, 2C19 and 2D6; and CBG by 2B6, 2C9, 2C19 and 2D6. CBC exhibited higher susceptibility to CYP-mediated transformation than the other tested compounds, mainly with CYPs 1A2, 2B6, 2C8, 2C19, 2D6 and 3A4 being involved. Further, CBD was primarily glucuronidated by UGTs 1A3, 1A7, 1A8, 1A9 and 2B7; CBN by 1A7, 1A8, 1A9 and 2B7; CBG by 1A3, 1A7, 1A8, 1A9, 2B4, 2B7 and 2B17; and the glucuronidation of CBC was catalyzed by UGTs 1A1, 1A8, 1A9 and 2B7. • LC-MS study on biotransformation of phytocannabinoids in human hepatocytes. • MS-fragmentation patterns for CBD, CBN, CBC, CBG and their metabolites reported. • The study screened for the activity of recombinant human cytochromes P450 and UDP-glucuronosyltransferases. • The susceptibility to transformations in hepatocytes: CBN > CBG > CBC > CBD. [ABSTRACT FROM AUTHOR]

The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d -lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35–40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d -lactate plus [14C]- l -lactate, the radioactive label was found in methane, CO 2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d -lactate oxidation was coupled to O 2 -consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d -lactate as carbon source and showed membrane-bound d -lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d -lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd : methanophenazine (quinol) oxidoreductase. Biomass generation and O 2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen. [Display omitted] • Air-adapted Methanosarcina acetivorans can grow in presence of acetate + lactate. • Lactate was oxidized in the presence of O 2 , producing methane, glycogen and biomass. • D-iLDH and cyt bd oxidase transcripts increased in cells showing high O 2 -consumption. • Respiration linked to lactate oxidation was sensitive to HQNO and partially to azide. • Lactate oxidation potentially through cytochromes, consumed the otherwise toxic O 2. [ABSTRACT FROM AUTHOR]

Rhodococcus globerulus is a metabolically active organism that has been shown to utilise eucalypt oil as its sole source of carbon and energy. This oil includes 1,8-cineole, p -cymene and limonene. Two identified and characterised cytochromes P450 (P450s) from this organism initiate the biodegradation of the monoterpenes 1,8-cineole (CYP176A1) and p -cymene (CYP108N12). Extensive characterisation has been completed for CYP176A1 and it has been successfully reconstituted with its immediate redox partner, cindoxin, and E. coli flavodoxin reductase. Two putative redox partner genes are encoded in the same operon as CYP108N12 and here the isolation, expression, purification, and characterisation of its specific [2Fe–2S] ferredoxin redox partner, cymredoxin is presented. Reconstitution of CYP108N12 with cymredoxin in place of putidaredoxin, a [2Fe–2S] redox partner of another P450, improves both the rate of electron transfer (from 13 ± 2 to 70 ± 1 μM NADH/min/ μ M CYP108N12) and the efficiency of NADH utilisation (the so-called coupling efficiency increases from 13% to 90%). Cymredoxin improves the catalytic ability of CYP108N12 in vitro. Aldehyde oxidation products of the previously identified substrates p -cymene (4-isopropylbenzaldehyde) and limonene (perillaldehyde) were observed in addition to major hydroxylation products 4-isopropylbenzyl alcohol and perillyl alcohol respectively. These further oxidation products had not previously been seen with putidaredoxin supported oxidation. Furthermore, when supported by cymredoxin CYP108N12 is able to oxidise a wider range of substrates than previously reported. These include o -xylene, α-terpineol, (−)-carveol and thymol yielding o -tolylmethanol, 7-hydroxyterpineol, (4 R)-7-hydroxycarveol and 5-hydroxymethyl-2-isopropylphenol, respectively. Cymredoxin is also capable of supporting CYP108A1 (P450 terp) and CYP176A1 activity, allowing them to catalyse the hydroxylation of their native substrates α-terpineol to 7-hydroxyterpineol and 1,8-cineole to 6β-hydroxycineole respectively. These results indicate that cymredoxin not only improves the catalytic capability of CYP108N12 but can also support the activity of other P450s and prove useful for their characterisation. [Display omitted] • Cloned, expressed and purified cymredoxin from Rhodococcus globerulus. • Cymredoxin, a [2Fe–2S] ferredoxin, is the native redox partner of CYP108N12. • CYP108N12 oxidation of p- cymene faster and more efficient with cymredoxin. • Substrate range of CYP108N12 extended. • Catalytic activity of other bacterial P450s supported by this ferredoxin. [ABSTRACT FROM AUTHOR]

Electroactive Geobacter bacteria can perform extracellular electron transfer and present a wide metabolic versatility. These bacteria reduce organic, toxic and radioactive compounds, and produce electric current while interacting with electrodes, making them interesting targets for numerous biotechnological applications. Their global electrochemical responses rely on an efficient interface between the inside and the cell's exterior, which is driven by the highly abundant periplasmic triheme PpcA-family cytochromes. The functional features of these cytochromes have been studied in G. sulfurreducens and G. metallireducens , and although they share a high degree of structural homology and sequence identity, their properties are quite distinct. In this work, the heme axial ligand geometries and the magnetic properties of PpcF from G. metallireducens were determined. The data obtained constitute important constraints for the determination of its solution structure in the oxidized state and indicate that the (i) heme core architecture; (ii) axial ligands geometries and (iii) magnetic properties of the cytochrome are conserved compared to the other members of the PpcA-families. Furthermore, the results also indicate that the heme arrangement is crucial to maintain an intrinsic regulation of the protein's redox properties and hence its electron transfer efficiency and functionality. • Geobacter are electroactive bacteria with proven biotechnological potential. • The cytochrome PpcF is important for electron transfer in G. metallireducens. • The heme core architecture and magnetic properties of PpcF were determined. • These features are conserved in the PpcA-family cytochromes from Geobacter. • The heme core arrangement broadens the redox range for electron transfer. [ABSTRACT FROM AUTHOR]

Alisertib (MLN8237), a novel Aurora A kinase inhibitor, is currently being clinically tested in late-phase trials for the therapy of various malignancies. In the present work, we describe alisertib's potential to perpetrate pharmacokinetic drug-drug interactions (DDIs) and/or to act as an antagonist of multidrug resistance (MDR). In accumulation assays, alisertib potently inhibited ABCC1 transporter, but not ABCB1 or ABCG2. The results of molecular modeling suggested a bifunctional mechanism for interaction on ABCC1. In addition, alisertib was characterized as a low- to moderate-affinity inhibitor of recombinant CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 isoenzymes, but without potential clinical relevance. Drug combination studies revealed the capability of alisertib to synergistically antagonize ABCC1-mediated resistance to daunorubicin. Although alisertib exhibited substrate characteristics toward ABCB1 transporter in monolayer transport assays, comparative proliferation studies showed lack of its MDR-victim behavior in cells overexpressing ABCB1 as well as ABCG2 and ABCC1. Lastly, alisertib did not affect the expression of ABCC1 , ABCG2 , ABCB1 transporters and CYP1A2, CYP3A4 , CYP2B6 isozymes on mRNA level in various systemic and tumoral models. In conclusion, our study suggests that alisertib is a drug candidate with negligible potential for perpetrating systemic pharmacokinetic DDIs on ABCB1, ABCG2 and cytochromes P450. In addition, we introduce alisertib as an effective dual-activity chemosensitizer whose MDR-antagonistic capacities are not impaired by efflux or effect on MDR phenotype. Our in vitro findings provide important pieces of information for clinicians when introducing alisertib into the clinical area. • Alisertib inhibits ABCC1, but not ABCB1 or ABCG2 drug efflux transporters. • Alisertib inhibits several cytochrome P450 isoforms but without clinical relevance. • Alisertib reverses ABCC1-mediated drug resistance. • ABC drug efflux transporters do not establish resistance to alisertib. • Alisertib does not affect the gene expression of examined enzymes or transporters. [ABSTRACT FROM AUTHOR]

We investigated the correspondence between drug metabolism routes and the composition of the P450 ensemble in human liver microsomes (HLM). As a probe, we used Coumarin 152 (C152), a fluorogenic substrate metabolized by multiple P450 species. Studying the substrate-saturation profiles (SSP) in seven pooled HLM preparations, we sought to correlate them with the P450 pool's composition characterized by targeted proteomics. This analysis, complemented with the assays with specific inhibitors of CYP3A4 and CYP2C19, the primary C152 metabolizers, demonstrated a significant contrast between different HLM samples. To unveil the source of these differences, we implemented Principal Component Analysis (PCA) of the SSP series obtained with HLM samples with a known composition of the P450 pool. Our analysis revealed that the parameters of C152 metabolism are primarily determined by the content of CYP2A6, CYP2B6, CYP2C8, CYP2E1, and CYP3A5 of those only CYP2B6 and CYP3A5 can metabolize C152. To validate this finding, we studied the effect of enriching HLM with CYP2A6, CYP2E1, and CYP3A5. The incorporation of CYP3A5 into HLM decreases the rate of C152 metabolism while increasing the role of CYP2B6 in its turnover. In contrast, incorporation of CYP2A6 and CYP2E1 reroutes the C152 demethylation towards some P450 enzyme with a moderate affinity to the substrate, most likely CYP3A4. Our results reveal a sharp non-additivity of the individual P450 properties and suggest a pivotal role of P450–P450 interactions in determining drug metabolism routes. This study demonstrates the high potential of our new PCA-based approach in unveiling functional interrelationships between different P450 species. [ABSTRACT FROM AUTHOR]