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Methanogens are a diverse group of Archaea that obligately couple energy conservation to the production of methane. Some methanogens encode alternate pathways for energy conservation, like anaerobic respiration, but the biochemical details of this process are unknown. We show that a multiheme c-type cytochrome called MmcA from Methanosarcina acetivorans is important for intracellular electron transport during methanogenesis and can also reduce extracellular electron acceptors like soluble Fe3+ and anthraquinone-2,6-disulfonate. Consistent with these observations, MmcA displays reversible redox features ranging from -100 to -450 mV versus SHE. Additionally, mutants lacking mmcA have significantly slower Fe3+ reduction rates. The mmcA locus is prevalent in members of the Order Methanosarcinales and is a part of a distinct clade of multiheme cytochromes that are closely related to octaheme tetrathionate reductases. Taken together, MmcA might act as an electron conduit that can potentially support a variety of energy conservation strategies that extend beyond methanogenesis.

A novel lipoformulation was developed by encapsulating cationic (S^C)-cyclometallated gold(III) complex [Au(dppta)(N2Py-PZ-dtc)]+ (AuPyPZ) in liposomes. The liposomal form of compound AuPyPZ has a bactericidal action similar to that of the free drug without any appreciable effect on the viability of mammalian cells. Furthermore, the nanoformulation reduces metalloantibiotic-induced inhibition of hERG and the inhibition of cytochromes, significantly decreasing the potential liabilities of the metallodrug. The obtained metalloantibiotic liposomal formulation shows high stability and suitable properties for drug delivery, representing an effective strategy to fight against drug-resistant bacteria. [ABSTRACT FROM AUTHOR]

Heme is a primordial macrocycle upon which most aerobic life on Earth depends. It is essential to the survival and health of nearly all cells, functioning as a prosthetic group for oxygen‐carrying proteins and enzymes involved in oxidation/reduction and electron transport reactions. Heme is essential for the function of numerous hemoproteins and has numerous other roles in the biochemistry of life. In mammals, heme is synthesised from glycine, succinyl‐CoA, and ferrous iron in a series of eight steps. The first and normally rate‐controlling step is catalysed by 5‐aminolevulinate synthase (ALAS), which has two forms: ALAS1 is the housekeeping form with highly variable expression, depending upon the supply of the end‐product heme, which acts to repress its activity; ALAS2 is the erythroid form, which is regulated chiefly by the adequacy of iron for erythroid haemoglobin synthesis. Abnormalities in the several enzymes of the heme synthetic pathway, most of which are inherited partial enzyme deficiencies, give rise to rare diseases called porphyrias. The existence and role of heme importers and exporters in mammals have been debated. Recent evidence established the presence of heme transporters. Such transporters are important for the transfer of heme from mitochondria, where the penultimate and ultimate steps of heme synthesis occur, and for the transfer of heme from cytoplasm to other cellular organelles. Several chaperones of heme and iron are known and important for cell health. Heme and iron, although promoters of oxidative stress and potentially toxic, are essential cofactors for cellular energy production and oxygenation. [ABSTRACT FROM AUTHOR]

Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR‐1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell‐cell click chemistry strategy. By introducing alkyne‐ or azide‐modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C‐type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell‐cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications. [ABSTRACT FROM AUTHOR]

Cupriavidus necator, despite lacking direct electron transfer capabilities, demonstrates efficient reduction of various redox mediators in oxygen‐free cultivation within bioelectrochemical systems. This study investigates the reduction site of ferricyanide through inhibition and expression rate analysis of oxygen and nitrate respiration chain complexes, comparing aerobic cultivation conditions with fructose as carbon and electron donor to autotrophic (CO2/H2/O2) and anodic cultivation conditions (fructose/anode). Azide inhibition identified cytochrome c oxidase as the primary complex facilitating electron transfer to ferricyanide, with a secondary role proposed for nitrite reductase NirS, demonstrating a 3.9±1.1‐fold higher expression when exposed to anodic conditions. The 2.9±0.6‐fold increase in the expression of the natural porin OmpA under anodic conditions implies its potential involvement in ferricyanide uptake. Additionally, chemically permeabilizing cell membranes with cetyltrimethylammonium bromide doubles ferricyanide reduction rates without an anode present, offering insights for optimizing redox mediation in C. necator based bioelectrochemical systems. This study opens up new possibilities for the targeted optimization of mediated electron transfer in C. necator and other organisms. [ABSTRACT FROM AUTHOR]

The liver plays a key role in the metabolism of lipoproteins, controlling both production and catabolism. To accelerate the development of new lipid‐lowering therapies in humans, it is essential to have a relevant in vitro study model available. The current hepatocyte‐like cells (HLCs) models derived from hiPSC can be used to model many genetically driven diseases but require further improvement to better recapitulate the complexity of liver functions. Here, we aimed to improve the maturation of HLCs using a three‐dimensional (3D) approach using Biomimesys®, a hyaluronic acid‐based hydroscaffold in which hiPSCs may directly form aggregates and differentiate toward a functional liver organoid model. After a 28‐day differentiation 3D protocol, we showed that many hepatic genes were upregulated in the 3D model (liver organoids) in comparison with the 2D model (HLCs). Liver organoids, grown on Biomimesys®, exhibited an autonomous cell organization, were composed of different cell types and displayed enhanced cytochromes P450 activities compared to HLCs. Regarding the functional capacities of these organoids, we showed that they were able to accumulate lipids (hepatic steatosis), internalize low‐density lipoprotein and secrete apolipoprotein B. Interestingly, we showed for the first time that this model was also able to produce apolipoprotein (a), the apolipoprotein (a) specific of Lp(a). This innovative hiPSC‐derived liver organoid model may serve as a relevant model for studying human lipopoprotein metabolism, including Lp(a). [ABSTRACT FROM AUTHOR]

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD-containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure-based engineering. The electron transfer kinetics of wild-type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped-flow spectrophotometry and structural effects were studied by small-angle X-ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway - its mutation reducing the interdomain electron transfer 10-fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer.

The interdomain electron transfer (IET) between the catalytic flavodehydrogenase domain and the electron-transferring cytochrome domain of cellobiose dehydrogenase (CDH) plays an essential role in biocatalysis, biosensors and biofuel cells, as well as in its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of CDH, which is hypothesised to limit IET in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair-distance distribution functions and Kratky plots, we show an increase in CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS-based multistate modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multistate modelling and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the IET governed by the movement of the CDH cytochrome domain.

Nanaerobes are a newly described class of microorganisms that use a unique cytochrome bd oxidase to achieve nanaerobic respiration at

Background. Side effects of endocrine therapy with tamoxifen (TAM) reduce the quality of life of patients with breast cancer (BC) and adversely affect treatment compliance. Against the background of treatment of BC with TAM, the risk of developing endometrial hyperplasia (EH) increases. At the moment, algorithms for monitoring patients receiving TAM by an obstetrician-gynecologist have not been developed and approved. There are also divergent opinions of experts regarding the diagnosis and management of patients in the detection of EH. This article presents the results of the final stage of the study in 2017–2022 – a survey study. The purpose of this study was to assess adherence to endocrine therapy and satisfaction with observation by a gynecologist of women with BC after 5 years of observation and their relationship with the identified early associated polymorphisms of the CYP2D6, CYP3A5, CYP2C9 and ABCB1 genes. Materials and methods. 54 patients with BC, out of 120 who had previously passed the first pharmacogenetic stage of the study, were interviewed with specially designed questionnaire. Due to the small sample size, delta percentages (∆%) were used in each comparison group with a difference threshold of 5%. To assess associations with the studied genetic polymorphisms, previously obtained significant associations of adverse drug reactions with pharmacogenetics in the same patients were used. Results. The prevalence of all adverse drug reactions (ADRs), with the exception of EH, was higher in the group of patients who canceled TAM. Over 5 years of follow-up, 57.4% of patients were regularly observed by an obstetrician-gynecologist, 42.59% of patients visited a gynecologist less than once a year. Of all the respondents, 53.7% of patients are satisfied with the regularity and quality of dispensary observation by an oncologist, 33.33% of patients are satisfied with the regularity and quality of dispensary observation by an obstetrician-gynecologist. All studied ADRs (EH, hot flashes, asthenia, bone pain and dyspepsia) associated with various genetic polymorphisms, prevailed in the group of patients who stopped taking TAM due to drug intolerance (∆%: 25.72, 6.97, 4.81, 6.97 and 24.52 respectively) compared with the TAM-group. Conclusion. The results obtained confirm the role of the studied genetic polymorphisms in the development of ADRs of TAM, but only systemic (hot flashes, asthenia, bone pain and dyspepsia). With regard to endometrial hyperplasia, no significant differences were obtained, which requires more extensive studies.

Borgs are huge extrachromosomal elements (ECE) of anaerobic methane-consuming "Candidatus Methanoperedens" archaea. Here, we used nanopore sequencing to validate published complete genomes curated from short reads and to reconstruct new genomes. 13 complete and four near-complete linear genomes share 40 genes that define a largely syntenous genome backbone. We use these conserved genes to identify new Borgs from peatland soil and to delineate Borg phylogeny, revealing two major clades. Remarkably, Borg genes encoding nanowire-like electron-transferring cytochromes and cell surface proteins are more highly expressed than those of host Methanoperedens, indicating that Borgs augment the Methanoperedens activity in situ. We reconstructed the first complete 4.00 Mbp genome for a Methanoperedens that is inferred to be a Borg host and predicted its methylation motifs, which differ from pervasive TC and CC methylation motifs of the Borgs. Thus, methylation may enable Methanoperedens to distinguish their genomes from those of Borgs. Very high Borg to Methanoperedens ratios and structural predictions suggest that Borgs may be capable of encapsulation. The findings clearly define Borgs as a distinct class of ECE with shared genomic signatures, establish their diversification from a common ancestor with genetic inheritance, and raise the possibility of periodic existence outside of host cells. Borgs are huge extrachromosomal elements associated with anaerobic methane-consuming archaea. Here, the authors use nanopore sequencing to identify new Borgs from peatland soil and their hosts, uncover their DNA modifications and activities, and elucidate the evolutionary relationships of Borgs. [ABSTRACT FROM AUTHOR]

Chiral methyl mandelates are useful synthons in organic transformation and pharmaceutical synthesis. Green synthesis of these valuable compounds by direct C–H activating oxidative hydroxylation has attracted keen interest. Described herein is achieving the stereoselective and efficient bio-hydroxylation of methyl 2-phenylacetates to the chiral methyl mandelates by directed evolution of the cytochrome P450DA hydroxylase. In the present study, a new colorimetric high-throughput screening assay was successfully developed based on a dualenzyme cascade for the engineering of the P450DA's hydroxylation activity. Several beneficial variants with enhanced bio-hydroxylation activity were created by combining random mutagenesis and site-saturated/directed mutagenesis strategies. Whole-cell bio-hydroxylation of various methyl 2-phenylacetates using the best septupletmutant P450DA-11 yielded the corresponding chiral methyl mandelates in up to 92% isolated yields and >99% ee. [ABSTRACT FROM AUTHOR]

As compounds of natural origin enter human body, it is necessary to investigate their possible interactions with the metabolism of drugs and xenobiotics in general, namely with the cytochrome P450 (CYP) system. Phytic acid (myo-inositol hexaphosphoric acid, IP6) is mainly present in plants but is also an endogenous compound present in mammalian cells and tissues. It has been shown to exhibit protective effect in many pathological conditions. For this paper, its interaction with CYPs was studied using human liver microsomes, primary human hepatocytes, the HepG2 cell line, and molecular docking. Docking experiments and absorption spectra demonstrated the weak ability of IP6 to interact in the heme active site of CYP1A. Molecular docking suggested that IP6 preferentially binds to the protein surface, whereas binding to the active site of CYP1A2 was found to be less probable. Subsequently, we investigated the ability of IP6 to modulate the metabolism of xenobiotics for both the mRNA expression and enzymatic activity of CYP1A enzymes. Our findings revealed that IP6 can slightly modulate the mRNA levels and enzyme activity of CYP1A. However, thanks to the relatively weak interactions of IP6 with CYPs, the chances of the mechanisms of clinically important drug–drug interactions involving IP6 are low. [ABSTRACT FROM AUTHOR]

Human cytochrome P450 2F1, as well as its ortholog 2F3 in domestic goat, is considered to be a rather unusual enzyme. The only type of reaction it catalyzes has been described: the conversion of skatole, a product of anaerobic tryptophan metabolism, into a pulmonary toxin. Endogenous substrates of CYP2F are unknown, and although more than 30 years have passed since the discovery of the enzyme, its biological role remains unclear. We hypothesized that the physiological functions of CYP2F can be specifically implemented in the brain, remaining previously unnoticed owing to the high compartmentalization of the organ. Using open data, we studied the covariation of the expression of CYP2F1 and genes for behavioral traits: in the human brain, as well as the polymorphism of their orthologs and CYP2F3 in 180 populations of domestic goats (Capra hircus). Two SNPs were found in the CYP2F3 gene, one of which had pronounced traces of selection, and the frequency of homozygotes increased with geographic distance from the center of domestication. Expression of CYP2F1 mRNA in the human brain also had regional specificity. In both species, factor analysis revealed the relationship between CYP2F1/3 and a number of genes regulating behavior: the serotonin transporter SLC6A4 and its receptor HTR2A3, the ABCB1 transporter, the purine receptor P2RX7, the GABA receptor GABRA4, the circadian rhythm regulator PER3, and T-cadherin CDH13. Thus, analysis of the genomic data of the domestic goat and human transcriptomic data revealed the evolutionary and functional relationships of CYP2F cytochromes and neurochemical systems for regulating behavior. This evidence of the cerebral function of the enzyme is indirect, since it is based on correlation analysis, but indicates the promise of further search in this direction. [ABSTRACT FROM AUTHOR]

Abstract Cupriavidus necator, despite lacking direct electron transfer capabilities, demonstrates efficient reduction of various redox mediators in oxygen‐free cultivation within bioelectrochemical systems. This study investigates the reduction site of ferricyanide through inhibition and expression rate analysis of oxygen and nitrate respiration chain complexes, comparing aerobic cultivation conditions with fructose as carbon and electron donor to autotrophic (CO2/H2/O2) and anodic cultivation conditions (fructose/anode). Azide inhibition identified cytochrome c oxidase as the primary complex facilitating electron transfer to ferricyanide, with a secondary role proposed for nitrite reductase NirS, demonstrating a 3.9±1.1‐fold higher expression when exposed to anodic conditions. The 2.9±0.6‐fold increase in the expression of the natural porin OmpA under anodic conditions implies its potential involvement in ferricyanide uptake. Additionally, chemically permeabilizing cell membranes with cetyltrimethylammonium bromide doubles ferricyanide reduction rates without an anode present, offering insights for optimizing redox mediation in C. necator based bioelectrochemical systems. This study opens up new possibilities for the targeted optimization of mediated electron transfer in C. necator and other organisms.

Heme-copper oxygen reductases are membrane-bound oligomeric complexes that are integral to prokaryotic and eukaryotic aerobic respiratory chains. Biogenesis of these enzymes is complex and requires coordinated assembly of the subunits and their cofactors. Some of the components are involved in the acquisition and integration of different heme and copper (Cu) cofactors into these terminal oxygen reductases. As such, MFS-type transporters of the CalT family (e.g., CcoA) are required for Cu import and heme-CuB center biogenesis of the cbb3-type cytochrome c oxidases (cbb3-Cox). However, functionally homologous Cu transporters for similar heme-Cu containing bo3-type quinol oxidases (bo3-Qox) are unknown. Despite the occurrence of multiple MFS-type transporters, orthologs of CcoA are absent in bacteria like Escherichia coli that contain bo3-Qox. In this work, we identified a subset of uncharacterized MFS transporters, based on the presence of putative metal-binding residues, as likely candidates for the missing Cu transporter. Using a genetic approach, we tested whether these transporters are involved in the biogenesis of E. coli bo3-Qox. When respiratory growth is dependent on bo3-Qox, because of deletion of the bd-type Qox enzymes, three candidate genes, yhjE, ydiM, and yfcJ, were found to be critical for E. coli growth. Radioactive metal uptake assays showed that ΔydiM has a slower 64Cu uptake, whereas ΔyhjE accumulates reduced 55Fe in the cell, while no similar uptake defect is associated with ΔycfJ. Phylogenomic analyses suggest plausible roles for the YhjE, YdiM, and YfcJ transporters, and overall findings illustrate the diverse roles that the MFS-type transporters play in cellular metal homeostasis and production of active heme-Cu oxygen reductases.

A novel photosensitized living biohybrid was fabricated by inward-to-outward assembly of amine-functionalized carbon dots (NCDs) and polydopamine (PDA) to Shewanella oneidensis MR-1 and applied for high-efficiency, microbial-photoreduction of Cr(VI). Within a 72 h test period, biohybrids achieved a pronounced catalytic reduction capacity (100%) for 100 mg/L Cr(VI) under visible illumination, greatly surpassing the poor capacity (only 2.5%) displayed by the wild strain under dark conditions. Modular configurations of NCDs and PDA afforded biohybrids with a large electron flux by harvesting extracellular photoelectrons generated from illuminated NCDs and increasing reducing equivalents released from an enlarged intracellular NADH/NAD+ pool. Further, increased production of intracellular c-type cytochromes and extracellular flavins resulting from the modular configuration enhanced the biohybrid electron transport ability. The enhancement of electron transport was also attributed to more conductive conduits at NCDs-PDA junction interfaces. Moreover, because NCDs are highly reductive, the enhanced Cr(VI) reduction was also attributed to direct reduction by the NCDs and the direct Cr(VI) reduction by sterile NCDs-assembled biohybrid was up to 20% in the dark. Overall, a highly efficient strategy for removal/transformation of Cr(VI) by using NCD-assembled photosensitized biohybrids was proposed in this work, which greatly exceeded the performance of Cr(VI)-remediation strategies based on conventional microbial technologies.

Anaerobic methane oxidation exerts a key control on greenhouse gas emissions1, yet factors that modulate the activity of microorganisms performing this function remain poorly understood. Here we discovered extraordinarily large, diverse DNA sequences that primarily encode hypothetical proteins through studying groundwater, sediments and wetland soil where methane production and oxidation occur. Four curated, complete genomes are linear, up to approximately 1 Mb in length and share genome organization, including replichore structure, long inverted terminal repeats and genome-wide unique perfect tandem direct repeats that are intergenic or generate amino acid repeats. We infer that these are highly divergent archaeal extrachromosomal elements with a distinct evolutionary origin. Gene sequence similarity, phylogeny and local divergence of sequence composition indicate that many of their genes were assimilated from methane-oxidizing Methanoperedens archaea. We refer to these elements as 'Borgs'. We identified at least 19 different Borg types coexisting with Methanoperedens spp. in four distinct ecosystems. Borgs provide methane-oxidizing Methanoperedens archaea access to genes encoding proteins involved in redox reactions and energy conservation (for example, clusters of multihaem cytochromes and methyl coenzyme M reductase). These data suggest that Borgs might have previously unrecognized roles in the metabolism of this group of archaea, which are known to modulate greenhouse gas emissions, but further studies are now needed to establish their functional relevance.

This article explores the potential of cytochromes P450 (P450s) as biocatalysts for oxidation reactions. Despite their potential, P450s have not been extensively used in research and industry due to challenges. The authors present a case study on the iron-catalyzed Wacker-type oxidation of olefins using ambient air as the sole oxidant, which has shown promising results for the functionalization of complex molecules. The text also discusses other strategies for the oxidation of organic compounds to produce aryl aldehydes, highlighting their selectivity and compatibility with complex molecules. The article concludes by discussing recent advances in catalysis inspired by P450 enzymes, which have the potential to accelerate the development of versatile synthetic catalysts with applications in drug synthesis and bioremediation. [Extracted from the article]

Variants in the gene encoding human cytochrome c (CYCS) cause mild autosomal dominant thrombocytopenia. Despite high sequence conservation between mouse and human cytochrome c, this phenotype is not recapitulated in mice for the sole mutant (G41S) that has been investigated. The effect of the G41S mutation on the in vitro activities of cytochrome c is also not conserved between human and mouse. Peroxidase activity is increased in both mouse and human G41S variants, whereas apoptosome activation is increased for human G41S cytochrome c but decreased for mouse G41S cytochrome c. These apoptotic activities of cytochrome c are regulated at least in part by conformational dynamics of the main chain. Here we use computational and in vitro approaches to understand why the impact of the G41S mutation differs between mouse and human cytochromes c. The G41S mutation increases the inherent entropy and main chain mobility of human but not mouse cytochrome c. Exclusively in human G41S cytochrome c this is accompanied by a decrease in occupancy of H-bonds between protein and heme during simulations. These data demonstrate that binding of cytochrome c to Apaf-1 to trigger apoptosome formation, but not the peroxidase activity of cytochrome c, is enhanced by increased mobility of the native protein conformation. [ABSTRACT FROM AUTHOR]

BACKGROUND: The mechanisms by which salt increases blood pressure in people with salt sensitivity remain unclear. Our previous studies found that high sodium enters antigen-presenting cells (APCs) via the epithelial sodium channel and leads to the production of isolevuglandins and hypertension. In the current mechanistic clinical study, we hypothesized that epithelial sodium channel–dependent isolevuglandin-adduct formation in APCs is regulated by epoxyeicosatrienoic acids (EETs) and leads to salt-sensitive hypertension in humans. METHODS: Salt sensitivity was assessed in 19 hypertensive subjects using an inpatient salt loading and depletion protocol. Isolevuglandin-adduct accumulation in APCs was analyzed using flow cytometry. Gene expression in APCs was analyzed using cellular indexing of transcriptomes and epitopes by sequencing analysis of blood mononuclear cells. Plasma and urine EETs were measured using liquid chromatography–mass spectrometry. RESULTS: Baseline isolevuglandin+ APCs correlated with higher salt-sensitivity index. Isolevuglandin+ APCs significantly decreased from salt loading to depletion with an increasing salt-sensitivity index. We observed that human APCs express the epithelial sodium channel δ subunit, SGK1 (salt-sensing kinase serum/glucocorticoid kinase 1), and cytochrome P450 2S1. We found a direct correlation between baseline urinary 14,15 EET and salt-sensitivity index, whereas changes in urinary 14,15 EET negatively correlated with isolevuglandin+ monocytes from salt loading to depletion. Coincubation with 14,15 EET inhibited high-salt–induced increase in isolevuglandin+ APC. CONCLUSIONS: Isolevuglandin formation in APCs responds to acute changes in salt intake in salt-sensitive but not salt-resistant people with hypertension, and this may be regulated by renal 14,15 EET. Baseline levels of isolevuglandin+ APCs or urinary 14,15 EET may provide diagnostic tools for salt sensitivity without a protocol of salt loading. [ABSTRACT FROM AUTHOR]

Growth hormone (GH) modifies liver gene transcription in a sexually dimorphic manner to meet liver metabolic demands related to sex; thus, GH dysregulation leads to sex-biased hepatic disease. We dissected the steps of the GH regulatory cascade modifying GH-dependent genes involved in metabolism, focusing on the male-predominant genes Lcn13 , Asns , and Cyp7b1 , and the female-predominant genes Hao2 , Pgc1a , Hamp2 , Cyp2a4 , and Cyp2b9. We explored mRNA expression in 2 settings: (i) intact liver GH receptor (GHR) but altered GH and insulin-like growth factor 1 (IGF1) levels (NeuroDrd2KO, HiGH, aHepIGF1kd, and STAT5bCA mouse lines); and (ii) liver loss of GHR, with or without STAT5b reconstitution (aHepGHRkd, and aHepGHRkd + STAT5bCA). Lcn13 was downregulated in males in most models, while Asns and Cyp7b1 were decreased in males by low GH levels or action, or constant GH levels, but unexpectedly upregulated in both sexes by the loss of liver Igf1 or constitutive Stat5b expression. Hao , Cyp2a4 , and Cyp2b9 were generally decreased in female mice with low GH levels or action (NeuroDrd2KO and/or aHepGHRkd mice) and increased in HiGH females, while in contrast, Pgc1a was increased in female NeuroDrd2KO but decreased in STAT5bCA and aHepIGF1kd females. Bioinformatic analysis of RNAseq from aHepGHRkd livers stressed the greater impact of GHR loss on wide gene expression in males and highlighted that GH modifies almost completely different gene signatures in each sex. Concordantly, we show that altering different steps of the GH cascade in the liver modified liver expression of Lcn13 , Asns , Cyp7b1 , Hao2 , Hamp2 , Pgc1a , Cyp2a4 , and Cyp2b9 in a sex- and gene-specific manner. [ABSTRACT FROM AUTHOR]

Paclitaxel is an anticancer therapeutic produced by the yew tree. Over the last two decades, a significant bottleneck in the reconstitution of early paclitaxel biosynthesis has been the propensity of heterologously expressed pathway cytochromes P450, including taxadiene 5α-hydroxylase (T5αH), to form multiple products. Here, we structurally characterize four new products of T5αH, many of which appear to be over-oxidation of the primary mono-oxidized products. By tuning the promoter strength for T5αH expression in Nicotiana plants, we observe decreased levels of these proposed byproducts with a concomitant increase in the accumulation of taxadien-5α-ol, the paclitaxel precursor, by three-fold. This enables the reconstitution of a six step biosynthetic pathway, which we further show may function as a metabolic network. Our result demonstrates that six previously characterized Taxus genes can coordinatively produce key paclitaxel intermediates and serves as a crucial platform for the discovery of the remaining biosynthetic genes. Paclitaxel is an important anticancer drug whose biosynthetic pathway reconstruction is hindered by the propensity of heterologously expressed pathway cytochromes P450, including taxadiene 5α-hydroxylase (T5αH), to form multiple products. Here, the authors tune the promoter strength for T5αH expression in Nicotiana plants to increase the levels of paclitaxel precursor taxadien-5α-ol by three-fold and reconstitute the six step early biosynthetic pathway of paclitaxel. [ABSTRACT FROM AUTHOR]

Aim: Cytochrome P450 is a hepatic enzyme system responsible for drug metabolism that comprises different iso-enzymes. The CYP2D6 enzyme, which composes 2-4% of all cytochrome enzymes, is responsible for 25% of the metabolisms of the drugs used in clinics. The genetic polymorphisms of this enzyme can change the efficiency and toxicity of the drugs used. In this study, the retrospective evaluation of CYP2D6 gene polymorphisms that influence enzyme activity in the Turkish population is targeted. Materials and Methods: From the 192 patients sent from psychiatry, medical genetics, and neurology polyclinics to our laboratory, we determined CYP2D6 enzyme gene polymorphisms by multiplex polymerase chain reaction (PCR) and multiplex allele-specific primer extension (ASPE). Results: Of all the cases, 82.29% were determined to be extensive, 12.5% intermediate, 2.08% poor, and 3.13% ultra-rapid metabolizers. In our population, the most frequent alleles were *1 (37.63%), *2 (24.75%), *41 (15.15%), *4 (9.85%), and *10 (4.29%), respectively. Conclusion: Compared to previous studies conducted on Turkish society, the percentage of extensive metabolizers was higher in the current examined population. In contrast, the percentages of poor and ultra-rapid metabolizers were lower. In addition, the *41 allele, which has not been studied before in our population, that follows the decrease in enzyme function was detected as the most frequent allele in this study. The fact that the percentage of the cases in which changes were observed was 17.1% will help determine the CYP2D6 gene mutations in the pre-treatment cases. [ABSTRACT FROM AUTHOR]

The fossil record demonstrates the preservation of porphyrins (e.g., heme) in organic sediments and the fossilized remains of animals. These molecules are essential components in modern metabolic processes, such as electron transport (cytochromes) and oxygen transport (hemoglobin), and likely originated before the emergence of life. The integration and adaptation of porphyrins and structurally similar molecules (e.g., chlorophylls) are key aspects in the evolution of energy production (i.e., aerobic respiration and photosynthesis) and complex life (i.e., eukaryotes and multicellularity). Here, we discuss the evolution and functional diversity of heme-bound hemoglobin proteins in vertebrates, along with the preservation of these molecules in the fossil record. By elucidating the pivotal role of these molecules in the evolution of life, this review lays the groundwork necessary to explore hemoglobin as a means to investigate the paleobiology of extinct taxa, including non-avian dinosaurs. [ABSTRACT FROM AUTHOR]

This study assessed the effect of tubeimoside I on CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 to reveal the potential of tubeimoside I to induce drug-drug interaction. The evaluation of cytochromes P450 enzyme (CYP) activity was performed in pooled human liver microsomes with probing substrates of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Typical inhibitors were employed as positive controls and the effect of 0, 2.5, 5, 10, 25, 50, and 100 μM tubeimoside I was investigated. The activity of CYP2D6, 2E1, and 3A4 was significantly inhibited by tubeimoside I with the IC50 values of 10.34, 11.58, and 9.74 μM, respectively. The inhibition of CYP2D6 and 2E1 was competitive with the Ki value of 5.66 and 5.29 μM, respectively. While the inhibition of CYP3A4 was non-competitive with the Ki value of 4.87 μM. Moreover, the inhibition of CYP3A4 was time-dependent with the KI and Kinact values of 0.635 μM−1 and 0.0373 min−1, respectively. Tubeimoside I served as a competitive inhibitor of CYP2D6 and 2E1 exerting weak inhibition and a non-competitive inhibitor of CYP3A4 exerting moderate inhibition. [ABSTRACT FROM AUTHOR]

Cytochrome P450 3A4 and 2D6 (EC 1.14.13.97 and 1.14.14.1; CYP3A4 and 2D6) are heme-containing enzymes that catalyze the oxidation of a wide number of xenobiotic and drug substrates and thus broadly impact human biology and pharmacologic therapies. Although their activities are directly proportional to their heme contents, little is known about the cellular heme delivery and insertion processes that enable their maturation to functional form. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into CYP3A4 and 2D6 after they were transiently expressed in HEK293T and GlyA CHO cells or when naturally expressed in HEPG2 cells in response to rifampicin, and also investigated their associations with GAPDH and Hsp90 in cells. The results indicate that GAPDH and its heme binding function is involved in delivery of mitochondria-generated heme to apo-CYP3A4 and 2D6, and that cell chaperone Hsp90 is additionally involved in driving their heme insertions. Uncovering how cells allocate heme to CYP3A4 and 2D6 provides new insight on their maturation processes and how this may help to regulate their functions in health and disease. [ABSTRACT FROM AUTHOR]

Background: Neonatal opioid withdrawal syndrome (NOWS), arises due to increased opioid use during pregnancy. Cytochrome P450 (CYP) enzymes play a pivotal role in metabolizing a wide range of substances in the human body, including opioids, other drugs, toxins, and endogenous compounds. The association between CYP gene methylation and opioid effects is unexplored and it could offer promising insights. Objective: To investigate the impact of prenatal opioid exposure on disrupted CYPs in infants and their anticipated long-term clinical implications. Study Design: DNA methylation levels of CYP genes were analyzed in a cohort of 96 placental tissues using Illumina Infinium MethylationEPIC (850 k) BeadChips. This involved three groups of placental tissues: 32 from mothers with infants exposed to opioids prenatally requiring pharmacologic treatment for NOWS, 32 from mothers with prenatally opioid-exposed infants not needing NOWS treatment, and 32 from unexposed control mothers. Results: The study identified 20 significantly differentially methylated CpG sites associated with 17 distinct CYP genes, with 14 CpGs showing reduced methylation across 14 genes (CYP19A1, CYP1A2, CYP4V2, CYP1B1, CYP24A1, CYP26B1, CYP26C1, CYP2C18, CYP2C9, CYP2U1, CYP39A1, CYP2R1, CYP4Z1, CYP2D7P1 and), while 8 exhibited hypermethylation (CYP51A1, CYP26B1, CYP2R1, CYP2U1, CYP4X1, CYP1A2, CYP2W1, and CYP4V2). Genes such as CYP1A2, CYP26B1, CYP2R1, CYP2U1, and CYP4V2 exhibited both increased and decreased methylation. These genes are crucial for metabolizing eicosanoids, fatty acids, drugs, and diverse substances. Conclusion: The study identified profound methylation changes in multiple CYP genes in the placental tissues relevant to NOWS. This suggests that disruption of DNA methylation patterns in CYP transcripts might play a role in NOWS and may serve as valuable biomarkers, suggesting a future pathway for personalized treatment. Further research is needed to confirm these findings and explore their potential for diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Sanghuangporus, also known as "Sanghuang" in China, is a well-known genus of traditional Chinese medicinal macrofungi. To make more effective use of Sanghuangporus resources, we completed the first genome assembly and annotation of a monokaryon strain of S. weigelae in the present study. A 33.96-Mb genome sequence was assembled as 13 contigs, leading to prediction of 9377 protein-coding genes. Phylogenetic and average nucleotide identity analyses indicated that the S. weigelae genome is closely related to those of other Sanghuangporus species in evolutionary tree, which clustered in one clade. Collinearity analysis revealed a high level of collinearity of S. weigelae with S. baumii, S. vaninii, and S. sanghuang. Biosynthesis pathways potentially involved in medicinal properties, including terpenoid and polysaccharide synthesis, were identified in S. weigelae, while polysaccharides were identified as the main medicinal metabolites in S. weigelae, with flavonoids more important in Sanghuangporus than other medicinal mushroom groups. Genes encoding 332 carbohydrate-active enzymes were identified in the S. weigelae genome, including major glycoside hydrolases and glycosyltransferases predicted, revealing the robust lignocellulose degradation capacity of S. weigelae. Further, 130 genes, clustered in seven classes were annotated to encode cytochromes P450 in the S. weigelae genome. Overall, our results reveal the remarkably medicinal capacity of S. weigelae and provide new insights that will inform the study of evolution and medicinal application of S. weigelae. The data are a reference resource for the formulation of scientific and rational ecological protection policies for Sanghuangporus species. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup. [ABSTRACT FROM AUTHOR]

Benthodytes occidentpalauta sp. nov. was collected from the Kyushu-Palau Ridge at a depth of 5 481 m in 2021. This new species is characterized by a gelatinous body wall, violet skin, six pairs of dorsal papillae, and a rough mid-ventral surface without tube feet. The dorsal deposits are rod-shaped and tripartite. Two types of papillae deposits as crosses with four arms with central bipartite apophyses. Ventral deposits are rods. Tentacle ossicles are rod-shaped with end protrusions. Gonad deposits are rod-shaped, tripartite, and cross-shaped. The phylogenetic analyses based on cytochrome oxidase subunit 1 (COI) and 16S individually and a concatenated dataset of COI and 16S genes of this species support that B. occidentpalauta sp. nov. belongs to Benthodytes. [ABSTRACT FROM AUTHOR]

c-Type cytochromes (cyt c) are proteins that undergo post-translational modification to covalently bind heme, which allows them to facilitate redox reactions in electron transport chains across all domains of life. Genomic evidence suggests that cyt c are involved in electron transfer processes among the Archaea, especially in members that produce or consume the potent greenhouse gas methane. However, neither the maturation machinery for cyt c in Archaea nor their role in methane metabolism has ever been functionally characterized. Here, we have used CRISPR-Cas9 genome editing tools to map a distinct pathway for cyt c biogenesis in the model methanogenic archaeon Methanosarcina acetivorans, and have also identified substrate-specific functional roles for cyt c during methanogenesis. Although the cyt c maturation machinery from M. acetivorans is universally conserved in the Archaea, our evolutionary analyses indicate that different clades of Archaea acquired this machinery through multiple independent horizontal gene transfer events from different groups of Bacteria. Overall, we demonstrate the convergent evolution of a novel Archaea-specific cyt c maturation machinery and its physiological role during methanogenesis, a process which contributes substantially to global methane emissions.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large class of secondary metabolites that have garnered scientific attention due to their complex scaffolds with potential roles in medicine, agriculture, and chemical ecology. RiPPs derive from the cleavage of ribosomally synthesized proteins and additional modifications, catalyzed by various enzymes to alter the peptide backbone or side chains. Of these enzymes, cytochromes P450 (P450s) are a superfamily of heme-thiolate proteins involved in many metabolic pathways, including RiPP biosyntheses. In this review, we focus our discussion on P450 involved in RiPP pathways and the unique chemical transformations they mediate. Previous studies have revealed a wealth of P450s distributed across all domains of life. While the number of characterized P450s involved in RiPP biosyntheses is relatively small, they catalyze various enzymatic reactions such as C–C or C–N bond formation. Formation of some RiPPs is catalyzed by more than one P450, enabling structural diversity. With the continuous improvement of the bioinformatic tools for RiPP prediction and advancement in synthetic biology techniques, it is expected that further cytochrome P450-mediated RiPP biosynthetic pathways will be discovered. Summary The presence of genes encoding P450s in gene clusters for ribosomally synthesized and post-translationally modified peptides expand structural and functional diversity of these secondary metabolites, and here, we review the current state of this knowledge. [ABSTRACT FROM AUTHOR]

Electroactive microorganisms (EAMs) could utilize extracellular electron transfer (EET) pathways to exchange electrons and energy with their external surroundings. Conductive cytochrome proteins and nanowires play crucial roles in controlling electron transfer rate from cytosol to extracellular electrode. Many previous studies elucidated how the c‐type cytochrome proteins and conductive nanowires are synthesized, assembled, and engineered to manipulate the EET rate, and quantified the kinetic processes of electron generation and EET. Here, we firstly overview the electron transfer pathways of EAMs and quantify the kinetic parameters that dictating intracellular electron production and EET. Secondly, we systematically review the structure, conductivity mechanisms, and engineering strategies to manipulate conductive cytochromes and nanowire in EAMs. Lastly, we outlook potential directions for future research in cytochromes and conductive nanowires for enhanced electron transfer. This article reviews the quantitative kinetics of intracellular electron production and EET, and the contribution of engineered c‐type cytochromes and conductive nanowire in enhancing the EET rate, which lay the foundation for enhancing electron transfer capacity of EAMs. [ABSTRACT FROM AUTHOR]

Cytochrome c552 from Thermus thermophilus is one of the hot topics for creating smart biomaterials as it possesses remarkable stability, is tolerant to multiple mutations and has therefore been recently reported for a number of functionalizations upon substitution of the original prosthetic group with an artificial prosthetic group. However, all of the substitutions were driven by the coordination through the axial ligands followed by complete reconstitution with a metal–porphyrin complex. This limits the scope of the cytochrome c for incorporating a metal-less non-natural heme species that could improve the versatility of cytochrome c for a new generation of engineered cytochrome proteins for further enhancement in their functionalities such as biocatalysts. In this connection, a new variant of Cytochrome c (rC552 C14A) from Thermus thermophilus was reported, where an easy approach to remove the original prosthetic group was achieved, followed by the incorporation of a number of metal-PPIX derivatives that ultimately led to the formation of artificial c-type cytochromes through covalent bonding. The apo-cytochrome was found to be thermally tolerant and to possess a distinctive overall structure as that of the wild type, as was evident from the corresponding CD spectra, which ultimately encouraged reconstitution with a metal-less protoporphyrin derivative for better understanding the role of axial ligands in the reconstitution process. Successful reconstitution was achieved, resulting in a new type of Cytochrome b-type artificial protein without the metal in its active site, indicating the non-involvement of the axial ligand. In order to prove the non-involvement of the axial ligand, a subsequent double mutant (C14A/M69A) was constructed, replacing the methionine at 69 position with non-coordinating alanine residue. Accordingly, the apo-C14A/M69A was prepared and found to be extremely stable as the earlier mutants and the WT showed no signs of denaturation, even at the elevated temperature of 98°C. Subsequently, heme b was successfully incorporated into the apo-C14A/M69A, which demonstrated itself as a highly thermally tolerant protein scaffold for incorporating a metal-less artificial prosthetic group in the absence of the axial ligand. Further improvement in the reconstitution process is achieved by replacing the methionine at 69 position with phenyl alanine (C14A/M69F mutant), resulting in further stabilization of heme species, possibly through non-covalent π–interactions, as corroborated by molecular docking. [ABSTRACT FROM AUTHOR]

Background: Vitamins have a great impact on metabolis. Aims: To determine the role of 1,25(OH)2D On Cytochromes CYP27A1 and CYP27B1 in Periodontitis. Material and Method: The investigation was carried out on 45 participants of ages within the range of (30-45 years) who were attending the private dental clinics. Diagnosis of chronic periodontitis was established depending on dental history, clinical examinations (periodontal indices). All participants were examined by the same dentist. They were classified into three groups: Group 1 (control negative): (15) participants with normal serum vitamin D3 level and with pocket depth =3 mm, good oral health and normal periodontal tissues and no previous history of periodontal diseases. Group 2 (control positive): (15) participants with normal serum vitamin D3 level and periodontitis with pocket depth =5 mm, they received placebo medication orally, Group3(treatment): (15) participants with vitamin D3 deficiency (below 30 IU), and periodontitis with pocket depth ≥5 mm, they received oral Vitamin D3 fast acting liquid soft gel capsule 2000 IU/day for 3 months. 3 blood samples were taken from each participant at 0,45,90 days, for research examinations. CYP27A1, CYP27B1 serum levels was measured for each sample in three groups by ELISA kit. Result: there was a highly significant reduction in CYP27A1 serum level in the treatment group at the ninety days of the study while there was no significant elevation CYP27B1 serum level in all groups during 45,90 days of the study. Conclusion: The present study suggested that the 1,25(OH)2D has effects on serum levels of both Cytochromes CYP27A1 and CYP27B1 and this was associated with periodontitis. [ABSTRACT FROM AUTHOR]

We evaluate cryoEM and crystal structures of two molecular machines that traffick heme and attach it to cytochrome c (cyt c), the second activity performed by a cyt c synthase. These integral membrane proteins, CcsBA and CcmF/H, both covalently attach heme to cyt c, but carry it out via different mechanisms. A CcsB-CcsA complex transports heme through a channel to its external active site, where it forms two thioethers between reduced (Fe+2) heme and CysXxxXxxCysHis in cyt c. The active site is formed by a periplasmic WWD sequence and two histidines (P-His1 and P-His2). We evaluate each proposed functional domain in CcsBA cryoEM densities, exploring their presence in other CcsB-CcsA proteins from a wide distribution of organisms (e.g., from Gram positive to Gram negative bacteria to chloroplasts.) Two conserved pockets, for the first and second cysteines of CXXCH, explain stereochemical heme attachment. In addition to other universal features, a conserved periplasmic beta stranded structure, called the beta cap, protects the active site when external heme is not present. Analysis of CcmF/H, here called an oxidoreductase and cyt c synthase, addresses mechanisms of heme access and attachment. We provide evidence that CcmF/H receives Fe+3 heme from holoCcmE via a periplasmic entry point in CcmF, whereby heme is inserted directly into a conserved WWD/P-His domain from above. Evidence suggests that CcmF acts as a heme reductase, reducing holoCcmE (to Fe+2) through a transmembrane electron transfer conduit, which initiates a complicated series of events at the active site. [ABSTRACT FROM AUTHOR]

Atrazine, as an herbicide, is used widely worldwide. Because of its prolonged persistence in the environment and accumulation in the body, atrazine exposure is a potential threat to human health. The present study evaluated the possible protective effects of zinc oxide nanoparticles and vitamin C against atrazine-induced hepatotoxicity in rats. Atrazine administered to rats orally at a dose of 300 mg/kg for 21 days caused liver oxidative stress as it increased malondialdehyde (MDA) formation and decreased reduced glutathione (GSH) contents. Atrazine induced inflammation accompanied by apoptosis via upregulation of hepatic gene expression levels of NF-κB, TNF-α, BAX, and caspase-3 and downregulation of Bcl-2 gene expression levels. Additionally, it disturbed the metabolic activities of cytochrome P450 as it downregulated hepatic gene expression levels of CYP1A1, CYP1B1, CYP2E1. The liver function biomarkers were greatly affected upon atrazine administration, and the serum levels of AST and ALT were significantly increased, while BWG%, albumin, globulins, and total proteins levels were markedly decreased. As a result of the above-mentioned influences of atrazine, histopathological changes in liver tissue were recorded in our findings. The administration of zinc oxide nanoparticles or vitamin C orally at a dose of 10 mg/kg and 200 mg/kg, respectively, for 30 days prior and along with atrazine, could significantly ameliorate the oxidative stress, inflammation, and apoptosis induced by atrazine and regulated the hepatic cytochrome P450 activities. Furthermore, they improved liver function biomarkers and histopathology. In conclusion, our results revealed that zinc oxide nanoparticles and vitamin C supplementations could effectively protect against atrazine-induced hepatotoxicity. [ABSTRACT FROM AUTHOR]

Anaerobic digestion is a valuable strategy for environmentally-friendly and sustainable disposal of kitchen waste. However, anaerobic digestion of kitchen waste (KWAD) is prone to acidification, which can cause poor reaction outcomes. Utilising conductive materials can ameliorate hydrogen, acid and ammonia inhibition during KWAD, ultimately enhancing methane production. This technique is widely applied to increase methane yield from anaerobic digestion. An analysis of conductive materials to promote methanogenesis in KWAD indicates that applying such materials can enhance methane production, alleviate acid and ammonia inhibition, enrich functional microorganisms, and replace conductive pili and c-type cytochromes for direct interspecies electron transfer. Additionally, metagenomics has shown that conductive materials can increase the expression of functional genes on microbial metabolism, ultimately improving the activity of key enzymes in the methanogenic process. Finally, it is recommended that the combination of macrogenomics, macrotranscriptomics and macroproteomics should be used to investigate KWAD's mechanism and to anticipate future engineering applications that reduce treatment expenses and ensure environmentally responsible disposal. [ABSTRACT FROM AUTHOR]

Geobacter sulfurreducens mediates extracellular electron transfer (EET) reactions with different substrates, such as solid-phase Fe(III)-containing minerals, anodes and the cells of Geobacter metallireducens. To compare their roles in EET, the pilA-N, omcE, omcS, omcT and omcZ genes of G. sulfurreducens were systematically deleted. All mutants showed impaired and varied ability to form biofilms on nonconductive surface. Deletion of omcE also impaired bacterial ability to reduce ferrihydrite, but its impacts on the ability for anode reduction and the co-culture of G. metallireducens-G. sulfurreducens were minimal. The mutant without omcS showed diminished ability to reduce ferrihydrite and to form the co-culture, but was able to regain its ability to reduce anodes. Deletion of omcT, omcZ or pilA-N alone impaired bacterial ability to reduce ferrihydrite and anodes and to form the co-culture. Deletion of all tested genes abolished bacterial ability to reduce ferrihydrite and anodes. Triple-deletion of all omcS, omcT and omcZ abolished the ability of G. sulfurreducens to co-culture with G. metallireducens. However, deletion of only omcZ or pilA-N or both omcS and omcT abolished the ability of G. sulfurreducens without hydrogenase gene hybL to co-culture with G. metallireducens, which show their indispensable roles in direct electron transfer from G. metallireducens to G. sulfurreducens. Thus, the roles of pilA-N, omcE, omcS, omcT and omcZ for G. sulfurreducens in EET vary substantially, which also suggest that possession of PilA-N and multiple cytochromes of different structures enables G. sulfurreducens to mediate EET reactions efficiently with substrates of different properties. [ABSTRACT FROM AUTHOR]

Electroactive microbial cells have evolved unique extracellular electron transfer to conduct the reactions via redox outer-membrane (OM) proteins. However, the electron transfer mechanism at the interface of OM proteins and nanomaterial remains unclear. In this study, the mechanism for the electron transfer at biological/inorganic interface is investigated by integrating molecular modeling with electrochemical and spectroscopic measurements. For this purpose, a model system composed of OmcA, a typical OM protein, and the hexagonal tungsten trioxide (h-WO3) with good biocompatibility is selected. The interfacial electron transfer is dependent mainly on the special molecular configuration of OmcA and the microenvironment of the solvent exposed active center. Also, the apparent electron transfer rate can be tuned by site-directed mutagenesis at the axial ligand of the active center. Furthermore, the equilibrium state of the OmcA/h-WO3 systems suggests that their attachment is attributed to the limited number of residues. The electrochemical analysis of OmcA and its variants reveals that the wild type exhibits the fastest electron transfer rate, and the transient absorption spectroscopy further shows that the axial histidine plays an important role in the interfacial electron transfer process. This study provides a useful approach to promote the site-directed mutagenesis and nanomaterial design for bioelectrocatalytic applications. [ABSTRACT FROM AUTHOR]

The recent reclassification of the strict anaerobe Geobacter sulfurreducens bacterium as aerotolerant brought attention for oxidative stress protection pathways. Although the electron transfer pathways for oxygen detoxification are not well established, evidence was obtained for the formation of a redox complex between the periplasmic triheme cytochrome PpcA and the diheme cytochrome peroxidase MacA. In the latter, the reduction of the high-potential heme triggers a conformational change that displaces the axial histidine of the low-potential heme with peroxidase activity. More recently, a possible involvement of the triheme periplasmic cytochrome family (PpcA-E) in the protection from oxidative stress in G. sulfurreducens was suggested. To evaluate this hypothesis, we investigated the electron transfer reaction and the biomolecular interaction between each PpcA-E cytochrome and MacA. Using a newly developed method that relies on the different NMR spectral signatures of the heme proteins, we directly monitored the electron transfer reaction from reduced PpcA-E cytochromes to oxidized MacA. The results obtained showed a complete electron transfer from the cytochromes to the high-potential heme of MacA. This highlights PpcA-E cytochromes' efficient role in providing the necessary reducing power to mitigate oxidative stress situations, hence contributing to a better knowledge of oxidative stress protection pathways in G. sulfurreducens. [ABSTRACT FROM AUTHOR]

Cytochromes P450 are a unique family of enzymes found in all Kingdoms of living organisms (animals, bacteria, plants, fungi, and archaea), whose main function is biotransformation of exogenous and endogenous compounds. The review discusses approaches to enhancing the efficiency of electrocatalysis by cytochromes P450 for their use in biotechnology and design of biosensors and describes main methods in the development of reconstituted and electrochemical catalytic systems based on the biochemical mechanism of cytochromes P450, as well as and modern trends for their practical application. [ABSTRACT FROM AUTHOR]

N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates. There are only limited data on the genotoxicity of nitrosamines in human cell systems. In this study, we used metabolically competent human HepaRG cells, whose metabolic capability is comparable to that of primary human hepatocytes, to evaluate the genotoxicity of eight nitrosamines [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. Under the conditions we used to culture HepaRG cells, three-dimensional (3D) spheroids possessed higher levels of CYP activity compared to 2D monolayer cells; thus the genotoxicity of the eight nitrosamines was investigated using 3D HepaRG spheroids in addition to more conventional 2D cultures. Genotoxicity was assessed as DNA damage using the high-throughput CometChip assay and as aneugenicity/clastogenicity in the flow-cytometry-based micronucleus (MN) assay. Following a 24-h treatment, all the nitrosamines induced DNA damage in 3D spheroids, while only three nitrosamines, NDBA, NDEA, and NDMA, produced positive responses in 2D HepaRG cells. In addition, these three nitrosamines also caused significant increases in MN frequency in both 2D and 3D HepaRG models, while NMBA and NMPA were positive only in the 3D HepaRG MN assay. Overall, our results indicate that HepaRG spheroids may provide a sensitive, human-based cell system for evaluating the genotoxicity of nitrosamines. [ABSTRACT FROM AUTHOR]

Nivasorexant, a selective orexin‐1‐receptor antagonist, has recently been assessed in the treatment of humans with binge‐eating disorder. Herein, the inhibitory potential of nivasorexant on cytochromes P450 (CYPs) 2C9, 2C19, and 3A4 was evaluated. Human liver microsomes/recombinant CYP enzymes were evaluated in vitro. In vivo, a single‐center, open‐label, fixed‐sequence study was performed in healthy adults to explore the effect of 100 mg nivasorexant administered twice daily (b.i.d.) on the pharmacokinetics (PK) of flurbiprofen (50 mg, CYP2C9), omeprazole (20 mg, CYP2C19), midazolam (2 mg, CYP3A4) making use of a cocktail approach. Plasma PK sampling was performed over 24 h on Day 1 (Cocktail alone), 8 (Cocktail + nivasorexant), and 15 (Cocktail + nivasorexant at steady state). Genotyping of subjects' CYPs was performed while safety and tolerability were also assessed. In vitro, nivasorexant inhibited CYP2C9, 2C19, and 3A4 in competitive inhibition assays with IC50 values of 8.6, 1.6, and 19–44 μM, respectively, while showing a significant time‐dependent CYP2C19 inhibition. In 22 subjects, exposure to flurbiprofen, omeprazole, and midazolam was generally higher during concomitant single‐ (i.e., area under the plasma concentration–time curve [AUC] ratio increased by 1.04‐, 2.05‐, and 1.56‐fold, respectively) and repeated‐dose (i.e., AUC ratio increased by 1.47‐, 6.84‐, and 3.71‐fold, respectively) nivasorexant administration compared with the cocktail substrates administered alone. The most frequently reported adverse event was somnolence. According to regulatory guidance, nivasorexant is classified as a moderate CYP2C19 and weak CYP3A4 inhibitor after 1 day and as a weak CYP2C9, strong CYP2C19, and moderate CYP3A4 inhibitor after 8 days of 100 mg b.i.d. administration. Clinicaltrials.gov ID: NCT05254548. [ABSTRACT FROM AUTHOR]