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Class II photolyases (PLs) are a distant subclade in the photolyase/cryptochrome superfamily, displaying a unique Trp-Tyr tetrad for photoreduction and exhibiting a lower quantum yield (QY) of DNA repair (49%) than class I photolyases (82%) [M. Zhang, L. Wang, S. Shu, A. Sancar, D. Zhong, Science 354, 209-213 (2016)]. Using layer-by-layer mutant design and femtosecond spectroscopy, we have successfully determined the rates of electron transfer and proton transfer, driving force, and reorganization energy for nine elementary steps involved in the initial photoreduction of class II Arabidopsis thaliana photolyase (AtPL), thereby constructing the photoreduction network specific to class II PLs. Several dynamic features have been revealed including a slow-rise (172 ps) and fast-decay (26 ps) kinetics between the excited lumiflavin and adenine groups within the flavin adenine dinucleotide cofactor, a slower electron transfer (ET) (22 ps) between the excited lumiflavin and the nearest Trp in the Trp triad (Wa) as compared to reported class I PL (0.8 ps), and a rapid deprotonation of the distal Trp in the Trp triad (Wc). Most strikingly, we captured a slightly energetically unfavorable ET step between Wa and the center Trp (Wb), as opposed to the decreasing reduction potential observed in class I PL that drives the electron flow unidirectionally. Such an energetically uphill ET step leads to a lower photoreduction quantum yield (~34%) in class II AtPL compared to that of class I PL (~45%), raising an important question on the evolutionary implications of various photoreduction networks in photolyases and cryptochromes. [ABSTRACT FROM AUTHOR]

A single-component flavin-dependent halogenase, AetF, has emerged as an attractive biocatalyst for catalyzing halogenation. However, its flavin chemistry remains unexplored and cannot be predicted due to its uniqueness in sequence and structure compared to other flavin-dependent monooxygenases. Here, we investigated the flavin reactions of AetF using transient kinetics. Our data revealed that NADP+ binding is required for formation of C4a-hydroperoxy flavin adenine dinucleotide (FAD) (FADC4aOOH), a key flavin-oxygen adduct required for generating a halogenating species. In the presence of NaBr without L-tryptophan, the flavin oxygen adduct intermediates [possibly FADC4aOOH and C4a-hydroxy FAD (FADC4aOH)] are highly stabilized (>4,000 s) before returning to the oxidized FAD state. In the presence of L-tryptophan, the rate of FADC4aOH dehydration to form oxidized FAD increased by ~825-fold. These data suggest that the presence of all substrates is required for speeding up AetF's catalytic cycle. Our findings underscore the adeptness of AetF in managing its reactivity through ligand control. Structural and tunnel analyses revealed that the binding of NADP+ and L-tryptophan induces changes in protein tunnels which may potentially link to the ligand-controlled mechanisms. Leveraging these catalytic insights, we employed light-induced flavin reduction and NADP+ stimulation to enable AetF halogenation of various compounds. Our findings demonstrate the mechanisms of precise control over flavin chemistry by AetF. These mechanistic insights may be useful for the biocatalytic development of single-component flavin-dependent halogenases. [ABSTRACT FROM AUTHOR]

Flavin-dependent halogenases (FDHs) are promising candidates for the sustainable production of halogenated organic molecules by biocatalysis. FDHs require only oxygen, halide and a fully reduced flavin adenine dinucleotide (FADH−) cofactor to generate the reactive HOX that diffuses 10 Å to the substrate binding pocket and enables regioselective oxidative halogenation. A key challenge for the application of FDHs is the regeneration of the FADH−. In vitro, FADH− can be regenerated by photoreduction of the oxidized FAD inside the protein using blue light, turning the halogenase into an inefficient artificial photoenzyme. We aimed to improve the photochemical properties of the tryptophan 5-halogenase PyrH from Streptomyces rugosporus by structure-guided mutagenesis. W279 and W281 of the conserved WxWxIP-motif close to FAD were exchanged against phenylalanine. Time-resolved UV-vis spectroscopy showed that the W281F exchange indeed increased the quantum yield of the one- and two-electron reduction, respectively. The cofactor binding affinity decreased slightly with dissociation constants rising from 31 to 74 μM, as examined by fluorescence anisotropy. FTIR difference spectroscopy demonstrated that the allosteric coupling between the FAD and substrate binding sites was mostly preserved. In contrast, the double mutant did not improve the yield further, while negatively affecting binding affinity and structural coupling. The distal W279F exchange was less effective in all parameters. Photoreductions were additionally delayed by a reversible inactive conformation. We conclude that there is a delicate balance to be considered for screening of FDHs for biocatalysis. Variant PyrH-W281F was found to be the most promising candidate for the application as artificial photoenzyme. [ABSTRACT FROM AUTHOR]

The rapid identification of pathogenic bacterial strains is becoming a challenging task as it causes many hospital associated infections. Many have reported on the use of fluorescence spectroscopy as an alternative to characterize bacteria. As bacteria possess intrinsic fluorophores, attempts were made to characterize eight strains using excitation emission matrix (EEM) measurements. From the results of parallel factor analysis (PARAFAC), four fluorophores, tryptophan, anthranilic acid, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide, were identified with varying distributions. The data obtained from PARAFAC analysis were subjected to hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA). This study demonstrates the potential of EEM technique to classify bacteria with 100% accuracy. [ABSTRACT FROM AUTHOR]

L-arginine oxidase (AROD, EC 1.4.3.25) is an oxidoreductase that catalyses the deamination of L-arginine, with flavin adenine dinucleotide (FAD) as a cofactor. Recently identified AROD from Pseudomonas sp. TPU 7192 (PT-AROD) demonstrates high selectivity for L-arginine. This enzyme is useful for accurate assays of L-arginine in biological samples. The structural characteristics of the FAD-dependent AROD, however, remain unknown. Here, we report the structure of PT-AROD at a resolution of 2.3 Å by cryo-electron microscopy. PT-AROD adopts an octameric structure with D4 symmetry, which is consistent with its molecular weight in solution, estimated by mass photometry. Comparative analysis of this structure with that determined using X-ray crystallography reveals open and closed forms of the lid-like loop at the entrance to the substrate pocket. Furthermore, mutation of Glu493, located at the substrate binding site, diminishes substrate selectivity, suggesting that this residue contributes significantly to the high selectivity of PT-AROD. [ABSTRACT FROM AUTHOR]

The article discusses the importance of understanding the biosynthetic pathways of plant natural products (PNPs) and the challenges faced in traditional methods due to the dispersed nature of biosynthetic genes in plants. Chemoproteomics, utilizing activity probes, has shown promise in rapidly identifying functional proteins involved in biosynthesis, such as steviol glycosides, chalcomoracin, and camptothecin. This approach offers advantages in directly targeting enzyme activity, enabling the discovery of key biosynthetic genes and facilitating their application in synthetic biology for large-scale production of medicinal plant natural products. Despite facing challenges like non-specific binding and probe design limitations, chemoproteomics is a valuable tool in advancing research on plant-derived natural products for pharmaceutical and industrial applications. [Extracted from the article]

The article discusses lethal capecitabine toxicity in patients with complete dihydropyrimidine dehydrogenase deficiency due to ultra-rare DPYD variants. It emphasizes the importance of preemptive genetic analysis of DPYD to reduce fluoropyrimidine-related adverse events and highlights the need for comprehensive genotyping and phenotyping strategies for identifying patients at risk of severe toxicity. The study presents two cases of patients with complete DPYD deficiency due to ultra-rare DPYD variants and lethal capecitabine toxicity, underscoring the challenges in predicting severe toxicity in patients with rare DPYD variants. The research advocates for a combined genotyping/phenotyping approach to enhance the safety of fluoropyrimidine-based therapy, even in patients with complete DPYD deficiency. [Extracted from the article]

Significance: Cellular metabolic dynamics can occur within milliseconds, yet there are no optimal tools to spatially and temporally capture these events. Autofluorescence imaging can provide metabolic information on the cellular level due to the intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD). Aim: Our goal is to build and evaluate a widefield microscope optimized for rapid autofluorescence imaging of metabolic changes in cells. Approach: A widefield, fluorescence microscope was assembled from an inverted microscope base, an light-emitting diode (LED) for excitation, and an image splitter for simultaneous but separate imaging of two bandwidths of emission (451/106 and 560/94 nm) on a single scientific complementary metal-oxide-semiconductor (sCMOS) camera. MCF-7 cells and primary murine hippocampal neurons were metabolically perturbed using cyanide and imaged to optimize illumination and camera exposure. To capture a rapid change in metabolism, MCF-7 cells were starved for 1 h and imaged while reintroduced to glucose. Results: Significant differences in the optical redox ratio (ORR) and intensity of NAD(P)H divided by the summed intensities of NAD(P)H and FAD were quantified for cyanide-treated neurons and MCF-7 cells at illumination powers above 0.30 mW and camera exposures as low as 5 ms; however, low illumination and camera exposures hindered the ability to identify subcellular features. Minimal photobleaching was quantified for 30 s of continuous imaging for illuminations at 4.14 mW and below. Using the optimized illumination power of 4.14 mW and an exposure of 10 ms, continuous autofluorescence imaging of starved MCF-7 cells demonstrated a rapid, yet heterogeneous, increase in the ORR of cells upon exposure to glucose. Conclusions: Ultimately, this widefield autofluorescence imaging system allowed for dynamic imaging and quantification of cellular metabolism at 99.6 Hz. [ABSTRACT FROM AUTHOR]

Purpose: Drug photodegradation is a matter of great concern because it can result in potency loss and adverse side effects. This study examines the light-induced degradation of dacarbazine catalyzed by vitamin B2 and flavin adenine dinucleotide (FAD) under light-emitting diode (LED) or fluorescent light irradiation. Methods: Dacarbazine was irradiated with LED (405 nm) or fluorescent light in the presence of various equivalents of vitamin B2 or FAD. The photodegradation of the drug in D2O was monitored by 1H nuclear magnetic resonance spectroscopy. Results: Dacarbazine dissolved in D2O decomposed in the presence of vitamin B2 or FAD under irradiation with LED or fluorescent light. The decomposition products were 2-azahypoxanthine 2, which has previously been observed after light irradiation in the absence of vitamin B2, and 1H-imidazole-5-carboxamide 6, a new product formed in the presence of vitamin B2. Irradiation with LED light was more effective than irradiation with fluorescent light in degrading dacarbazine. Conclusion: Vitamin B2 and FAD induced dacarbazine photodegradation. Thus, the interfusion of vitamin B2 or FAD under excessive light exposure should be avoided during the intravenous administration of dacarbazine. [ABSTRACT FROM AUTHOR]

By integrating enzymatic catalysis with photocatalysis, photoenzymatic catalysis emerges as a powerful strategy to enhance enzyme catalytic capabilities and provide superior stereocontrol in reactions involving reactive intermediates. Repurposing naturally occurring enzymes using visible light is among the most active directions of photoenzymatic catalysis. This Minireview focuses on a cutting‐edge strategy in this direction, namely single‐electron‐oxidation‐triggered non‐natural biotransformations catalyzed by photoexcited enzymes. These straightforward transformations feature a unique radical mechanism initiated by single‐electron oxidation, achieving redox‐neutral non‐natural C−C, C−O, and C−S bond formation, and expanding the chemical toolbox of enzymes. By highlighting recent advances in this field and emphasizing their catalytic mechanisms and synthetic potential, innovative approaches for photobiomanufacturing are anticipated. [ABSTRACT FROM AUTHOR]

The article discusses a case of lethal 5-Fluorouracil toxicity in a patient carrying the DPYD c.704G>A variant. This variant is associated with increased risk of severe toxicity to fluorouracil, a common treatment for colorectal carcinoma. The patient, of South Asian descent, experienced severe adverse effects to standard doses of fluorouracil, leading to multiorgan failure and ultimately death. The study highlights the limitations of targeted genotyping in identifying rare deleterious variants, especially in populations with non-European ancestry, and emphasizes the need for further research in this area to guide dose adjustments and improve treatment outcomes. [Extracted from the article]

The heterotrimeric flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase derived from Burkholderia cepacia (BcGDH) has many exceptional features for its use in glucose sensing—including that this enzyme is capable of direct electron transfer with an electrode in its heterotrimeric configuration. However, this enzyme's high catalytic activity towards not only glucose but also galactose presents an engineering challenge. To increase the substrate specificity of this enzyme, it must be engineered to reduce its activity towards galactose while maintaining its activity towards glucose. To aid in these mutagenesis studies, the crystal structure composed of BcGDH's small subunit and catalytic subunit (BcGDHγα), in complex with D -glucono-1,5-lactone was elucidated and used to construct the three-dimensional model for targeted, site-directed mutagenesis. BcGDHγα was then mutated at three different residues, glycine 322, asparagine 474 and asparagine 475. The single mutations that showed the greatest glucose selectivity were combined to create the resulting mutant, α-G322Q-N474S-N475S. The α-G322Q-N474S-N475S mutant and BcGDHγα wild type were then characterized with dye-mediated dehydrogenase activity assays to determine their kinetic parameters. The α-G322Q-N474S-N475S mutant showed more than a 2-fold increase in V max towards glucose and this mutant showed a lower activity towards galactose in the physiological range (5 mM) of 4.19 U mg−1, as compared to the wild type, 86.6 U mg−1. This resulting increase in specificity lead to an 81.7 gal/glc % activity for the wild type while the α-G322Q-N474S-N475S mutant had just 10.9 gal/glc % activity at 5 mM. While the BcGDHγα wild type has high specificity towards galactose, our engineering α-G322Q-N474S-N475S mutant showed concentration dependent response to glucose and was not affected by galactose. • X-ray crystal structure of catalytic subunit of DET-type GDH in complex with 1,5-Glucono-δ-lactone was elucidated. • Three residues, G322, N474 and N475, were solicited to be responsible for enzyme substrate specificity. • G322Q/N474S/N475S mutant improved specificity toward glucose by decreasing activity toward galactose. • Structural model provided the impact of mutations, confirming the narrowed substrate binding pocket in the mutant. [ABSTRACT FROM AUTHOR]

The abnormal structure of tumor vascular seriously hinders the delivery and deep penetration of drug in tumor therapy. Herein, an integrated and tumor microenvironment (TME)-responsive nanocarrier is designed, which can dilate vessle and improve the drug penetration by in situ releasing nitric oxide (NO). Briefly, S-nitroso-glutathione (GSNO) and curcumin (Cur) were encapsulatd into the Cu-doped zeolite imidazole framework-8 (Cu-ZIF-8) and modified with hyaluronic acid. The nanocarrier degradation in the weakly acidic of TME releases Cu2+, then deplete overexpressed intratumourally glutathione and transformed into Cu+, thus disrupting the balance between nicotinamide adenine dinucleotide phosphate and flavin adenine dinucleotide (NADPH/FAD) during the metabolism homeostasis of tumor. The Cu+ can generate highly toxic hydroxyl radical through the Fenton-like reaction, enhancing the chemodynamic therapeutic effect. In addition, Cu+ also decomposes GSNO to release NO by ionic reduction, leading to vasodilation and increased vascular permeability, significantly promoting the deep penetration of Cur in tumor. Afterwards, the orderly operation of cell cycle is disrupted and arrested in the S-phase to induce tumor cell apoptosis. Deep-hypothermia potentiated 2D/3D fluorescence imaging demonstrated nanocarrier regulated endogenous metabolism homeostasis of tumor. The cascade-catalysed multifunctional nanocarrier provides an approach to treat orthotopic tumor. [ABSTRACT FROM AUTHOR]

The increasing incidence of neurodegenerative and other diseases is considered to involve an excessive production of reactive oxygen species (ROS). Water supplies are often characterized by excessive organic waste that is decomposed by bacteria, using dissolved oxygen, leading to oxygen depletion. The potassium content of these waters may also affect negatively the mitochondrial metabolism and cellular ROS formation. This work focused on characterizing mitochondrial autofluorescence changes, with flavoprotein origin, and fluorescence ROS signals measured using the 2',7'-dichlorodihydrofluorescein diacetate indicator H2DCFDA. All signals were evoked by hypoxia or by the depolarizing agent KCl (20 mM), at the hippocampal mossy fiber synapses of CA3 area. It was observed that both hypoxia and KCl-induced depolarization elicited a small rise in the autofluorescence and ROS changes. The hypoxia-induced signals were maintained upon normal reoxygenation, but of those evoked by KCl, the autofluorescence signals recovered during washout, while the ROS changes were irreversible. [ABSTRACT FROM AUTHOR]

Synthetic lethality, involving the simultaneous deactivation of two genes, disrupts cellular functions or induces cell death. This study examines its role in cancer, focusing on focal adhesion kinase and Neurofibromin 2. Inhibiting FAK, crucial for synthetic lethality with NF2/Merlin, offers significant cancer treatment potential. No FAK inhibitor has been clinically approved, underscoring the need for new, effective inhibitors. The small-molecule FAK inhibitors identified in this study show promise, with SP docking, IFD, QPLD, and MD simulations revealing intricate interactions. Based on the comprehensive analysis, the MM/GBSA scores from SP docking for amprenavir, bosutinib, ferric derisomaltose, flavin adenine dinucleotide, lactulose, and tafluprost were determined as −72.81, −71.84, −76.70, −69.09, −74.86, and −65.77 kcal/mol, respectively. The MMGBSA results following IFD docking MD identified the top-performing compounds with scores of −84.0518, −75.2591, −71.8943, −84.2638, −56.3019, and −75.3873 kcal/mol, respectively. The MMGBSA results from QPLD docking MD identified the leading compounds with scores of −77.8486, −69.5773, −71.9171, N/A, −62.5716, and −66.8067 kcal/mol, respectively. In conclusion, based on the MMGBSA scores obtained using the three docking methods and the 100 ns MD simulations, and considering the combined evaluation of these methods, amprenavir, ferric derisomaltose, and bosutinib are proposed as the most promising candidates. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a metabolic disorder. Discovering the metabolic products involved in the development of AD may help not only in the early detection and prevention of AD but also in understanding its pathogenesis and treatment. This study investigated the causal association between the latest large-scale plasma metabolites (1091 metabolites and 309 metabolite ratios) and AD. Through the application of Mendelian randomization analysis methods such as inverse-variance weighted (IVW), MR-Egger, and weighted median models, 66 metabolites and metabolite ratios were identified as potentially having a causal association with AD, with 13 showing significant causal associations. During the replication validation phase, six metabolites and metabolite ratios were confirmed for their roles in AD: N-lactoyl tyrosine, argininate, and the adenosine 5'-monophosphate to flavin adenine dinucleotide ratio were found to exhibit protective effects against AD. In contrast, ergothioneine, piperine, and 1,7-dimethyluric acid were identified as contributing to an increased risk of AD. Among them, argininate showed a significant effect against AD. Replication and sensitivity analyses confirmed the robustness of these findings. Metabolic pathway analysis linked "Vitamin B6 metabolism" to AD risk. No genetic correlations were found, but colocalization analysis indicated potential AD risk elevation through top SNPs in APOE and PSEN2 genes. This provides novel insights into AD's etiology from a metabolomic viewpoint, suggesting both protective and risk metabolites. [ABSTRACT FROM AUTHOR]

Optical imaging of metabolism can provide key information about health and disease progression in cells and tissues; however, current methods have lacked gold-standard information about histological structure. Conversely, histology and virtual histology methods have lacked metabolic contrast. Here, we present metabolic light absorption, scattering, and emission (MetaLASE) microscopy, which rapidly provides a virtual histology and optical metabolic readout simultaneously. Hematoxylin-like nucleic contrast and eosin-like cytoplasmic contrast are obtained using photoacoustic remote sensing and ultraviolet reflectance microscopy, respectively. The same ultraviolet source excites endogenous Nicotinamide adenine dinucleotide (phosphate), flavin adenine dinucleotide, and collagen autofluorescence, providing a map of optical redox ratios to visualize metabolic variations including in areas of invasive carcinoma. Benign chronic inflammation and glands also are seen to exhibit hypermetabolism. MetaLASE microscopy offers promise for future applications in intraoperative margin analysis and in research applications where greater insights into metabolic activity could be correlated with cell and tissue types. [ABSTRACT FROM AUTHOR]

Significance: Autofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications. Aim: We present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells. Approach: We use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids. Results: The use of a multispectral detector in combination with an excitation at a single wavelength of 750 nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins. Conclusions: The approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750 nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids. [ABSTRACT FROM AUTHOR]

Mitochondria are commonly recognized as the powerhouses of the cell, primarily responsible for energy production through oxidative phosphorylation. Alongside this vital function, they also play crucial roles in regulating calcium signaling, maintaining membrane potential, and modulating apoptosis. Their involvement in various cellular pathways becomes particularly evident during oogenesis and embryogenesis, where mitochondrial quantity, morphology, and distribution are tightly controlled. The efficiency of the mitochondrial network is maintained through multiple quality control mechanisms that are essential for reproductive success. These include mitochondrial unfolded protein response, mitochondrial dynamics, and mitophagy. Not surprisingly, mitochondrial dysfunction has been implicated in infertility and ovarian aging, prompting investigation into mitochondria as diagnostic and therapeutic targets in assisted reproduction. To date, mitochondrial DNA copy number in oocytes, cumulus cells, and trophectoderm biopsies, and fluorescent lifetime imaging microscopy-based assessment of NADH and flavin adenine dinucleotide content have been explored as potential predictors of embryo competence, yielding limited success. Despite challenges in the clinical application of mitochondrial diagnostic strategies, these enigmatic organelles have a significant impact on reproduction, and their potential role as diagnostic targets in assisted reproduction is likely to remain an active area of investigation in the foreseeable future. [ABSTRACT FROM AUTHOR]

The impact of Flavin adenine dinucleotide (FAD) on sulfate-reducing bacteria (SRB) corrosion of a pipeline welded joint (WJ) was investigated under anaerobic condition in this paper. The results showed that the thickness of the corrosion product on heat affected zone (HAZ) was lower than that on base metal (BM) and welded zone (WZ), and the FAD addition enhanced the development of the protruding microbial tubercles on the WJ. The local corrosion degrees of the BM and WZ coupons were significantly higher than that of the HAZ coupon. Besides, the FAD addition simultaneously promoted local corrosion of all three zones of the WJ in the SRB inoculated environment, and the promotion role was much more pronounced on the WZ coupons. The selective promotion effect of FAD on SRB corrosion in the WJ was attributed to the special structure of the WZ, the selected SRB attachment and the FAD/FADH2 redox feedback cycle. [ABSTRACT FROM AUTHOR]

Ovarian cancer (OC) is a deadly disease with a high mortality rate, primarily due to its often asymptomatic nature in early stages and the absence of effective screening methods. Conventional imaging methods have several drawbacks, such as difficulty in detecting small tumors, challenges in differentiating between benign and malignant tumors, dangerous radiation exposure, high cost, low sensitivity, and low specificity in detecting early-stage malignancies. On the other hand, 2D nanomaterials-based electrochemical biosensors present novel opportunities for early detection of ovarian cancer biomarkers, leveraging their unique properties such as high surface-to-volume ratio and excellent electrical conductivity. These materials enhance sensor sensitivity and selectivity, enabling the detection of biomarkers at ultra-low concentrations, thus improving diagnostic accuracy. Their compatibility with miniaturization further facilitates the development of portable, point-of-care devices, revolutionizing early screening efforts in ovarian cancer detection. This review paper overviews recent advancements in OC biomarker detection using 2D nanomaterials-based electrochemical biosensors. A detailed discussion is presented on the electrochemical detection of carbohydrate antigen 125 (CA125), mucin 1 (MUC1), and human epididymis protein 4 (HE4) biomarkers regarding sensing materials, sensing approach, analytical technique, and performance characteristics like limit of detection and linear range. Other OC biomarkers such as SKOV3, breast cancer gene 1 (BRCA1), stress-induced phosphoprotein 1 (STIP1), and flavin adenine dinucleotide are also briefly discussed. Finally, the review discusses the difficulties and future outlooks of diagnosing OC via an electrochemical sensing approach. The present review article emphasizes the application of 2D nanomaterials in the early diagnosis of ovarian cancer through an electrochemical sensing approach. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus infections present a significant threat to the global healthcare system. The increasing resistance to existing antibiotics and their limited efficacy underscores the urgent need to identify new antibacterial agents with low toxicity to effectively combat various S. aureus infections. Hence, in this study, we have screened T-muurolol for possible interactions with several S. aureus-specific bacterial proteins to establish its potential as an alternative antibacterial agent. Based on its binding affinity and interactions with amino acids, T-muurolol was identified as a potential inhibitor of S. aureus lipase, dihydrofolate reductase, penicillin-binding protein 2a, D-Ala:D-Ala ligase, and ribosome protection proteins tetracycline resistance determinant (RPP TetM), which indicates its potentiality against S. aureus and its multi-drug-resistant strains. Also, T-muurolol exhibited good antioxidant and anti-inflammatory activity by showing strong binding interactions with flavin adenine dinucleotide (FAD)-dependent nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase, and cyclooxygenase-2. Consequently, molecular dynamics (MD) simulation and recalculating binding free energies elucidated its binding interaction stability with targeted proteins. Furthermore, quantum chemical structure analysis based on density functional theory (DFT) depicted a higher energy gap between the highest occupied molecular orbital and lowest unoccupied molecular orbital (EHOMO-LUMO) with a lower chemical potential index, and moderate electrophilicity suggests its chemical hardness and stability and less polarizability and reactivity. Additionally, pharmacological parameters based on ADMET, Lipinski's rules, and bioactivity score validated it as a promising drug candidate with high activity toward ion channel modulators, nuclear receptor ligands, and enzyme inhibitors. In conclusion, the current findings suggest T-muurolol as a promising alternative antibacterial agent that might be a potential phytochemical-based drug against S. aureus. This study also suggests further clinical research before human application. [ABSTRACT FROM AUTHOR]

Enzymes play a fundamental role in cellular metabolism. A wide range of enzymes require the presence of complementary coenzymes and cofactors to function properly. While coenzymes are believed to have been part of the last universal ancestor (LUCA) or have been present even earlier, the syntheses of crucial coenzymes like the redox‐active coenzymes flavin adenine dinucleotide (FAD) or nicotinamide adenine dinucleotide (NAD+) remain challenging. Here, we present a pathway to NAD+ under prebiotic conditions starting with ammonia, cyanoacetaldehyde, prop‐2‐ynal and sugar‐forming precursors, yielding in situ the nicotinamide riboside. Regioselective phosphorylation and water stable light activated adenosine monophosphate derivatives allow for topographically and irradiation‐controlled formation of NAD+. Our findings indicate that NAD+, a coenzyme vital to life, can be formed non‐enzymatically from simple organic feedstock molecules via photocatalytic activation under prebiotically plausible early Earth conditions in a continuous process under aqueous conditions. [ABSTRACT FROM AUTHOR]

The development of noninvasive approaches to precisely control neural activity in mammals is highly desirable. Here, we used the ion channel transient receptor potential ankyrin-repeat 1 (TRPA1) as a proof of principle, demonstrating remote near-infrared (NIR) activation of endogenous neuronal channels in mice through an engineered nanoagonist. This achievement enables specific neurostimulation in nongenetically modified mice. Initially, target-based screening identified flavins as photopharmacological agonists, allowing for the photoactivation of TRPA1 in sensory neurons upon ultraviolet A/blue light illumination. Subsequently, upconversion nanoparticles (UCNPs) were customized with an emission spectrum aligned to flavin absorption and conjugated with flavin adenine dinucleotide, creating a nanoagonist capable of NIR activation of TRPA1. Following the intrathecal injection of the nanoagonist, noninvasive NIR stimulation allows precise bidirectional control of nociception in mice through remote activation of spinal TRPA1. This study demonstrates a noninvasive NIR neurostimulation method with the potential for adaptation to various endogenous ion channels and neural processes by combining photochemical toolboxes with customized UCNPs. [ABSTRACT FROM AUTHOR]

Introduction: Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters, RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), which are involved in different redox reactions, including the energetic metabolism processes occurring in mitochondria. To date, human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons, which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far, no attention has been dedicated to astrocytes. Results and discussion: Here, we demonstrate that in vitro differentiation of astrocytes, which guarantee trophic and metabolic support to neurons, from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons, RTD2 does not compromise the morpho-functional features of astrocytes. [ABSTRACT FROM AUTHOR]

This article provides an analysis of Oomycete Nudix effectors, which are proteins secreted by phytopathogens to aid in the colonization of host plants. The researchers conducted a phylogenetic analysis and examined the crystal structure of the P. sojae effector Avr3b. They found that oomycete Nudix effectors have distinct structural features and functions compared to fungal and bacterial Nudix effectors. The study also revealed that Avr3b and other oomycete Nudix effectors have mRNA decapping activity, suggesting their involvement in modulating host immune responses. Further research is needed to fully understand the role of these effectors in promoting pathogen infection and their impact on host immunity. [Extracted from the article]

Objective Adrenal cortisol production occurs through a biosynthetic pathway which depend on NADH and NADPH for energy supply. The mitochondrial respiratory chain and the reactive oxygen species (ROS) detoxification system are therefore important for steroidogenesis. Mitochondrial dysfunction leading to oxidative stress has been implicated in the pathogenesis of several adrenal conditions. Nonetheless, only very few patients with variants in one gene of the ROS detoxification system, Thioredoxin Reductase 2 (TXNRD2), have been described with variable phenotypes. Design Clinical, genetic, structural, and functional characterization of a novel, biallelic TXNRD2 splice variant. Methods On human biomaterial, we performed whole exome sequencing to identify and RNA analysis to characterize the specific TXNRD2 splice variant. Amino acid conservation analysis and protein structure modeling were performed in silico. Using patient's fibroblast-derived human induced pluripotent stem cells, we generated adrenal-like cells (iALC) to study the impact of wild-type (WT) and mutant TXNRD2 on adrenal steroidogenesis and ROS production. Results The patient had a complex phenotype of primary adrenal insufficiency (PAI), combined with genital, ophthalmological, and neurological features. He carried a homozygous splice variant c.1348-1G > T in TXNRD2 which leads to a shorter protein lacking the C-terminus and thereby affecting homodimerization and flavin adenine dinucleotide binding. Patient-derived iALC showed a loss of cortisol production with overall diminished adrenal steroidogenesis, while ROS production was significantly increased. Conclusion Lack of TXNRD2 activity for mitochondrial ROS detoxification affects adrenal steroidogenesis and predominantly cortisol production. [ABSTRACT FROM AUTHOR]

Even with 100% certainty of a complete cure for breast cancer (BC), there is still a long way to go toward more efficient treatment because it requires sensitive and timely detection and accurate pre/post-clinical characterizations. Despite the availability of advanced diagnostic tools, many cancer patients lack access to efficient diagnostics that are both highly reliable and affordable. The fluorescence-based optical technique aims to make another significant leap forward in improving patient safety. It offers a convenient operation that reduces healthcare costs compared to visual examination tools (VETs). The primary and metastatic stages of BC consider different cancerous cell lines (MDAs), meaning the highest number of cells in this research (up to 300,000) represents the metastatic stages of BC, and 50,000 represents the primary level of BC. Developments have been studied based on fluorescence-enhanced photodynamic characterizations. The ability to characterize the fluorescence caused by MDA with 50,000 cells compared to the dominant radiation of MDA with 300,000 cells is emphatic proof of the high potential of fluorescence technique in timely BC detections, specifically before it spreads to the axillary lymph nodes. The specific cell numbers of 50,000 and 300,000 were chosen arbitrarily based on the cultivation of common biological limitations. Comparing the outcomes between 50,000 and 300,000 cells allows for evaluating the fluorescence technique's diagnostic capability across various stages of breast cancer. This assessment provides valuable insights into the effectiveness of the fluorescence-based characterizing approach in detecting cancerous cells at different stages of the disease. Here, we have assessed fluorescence's spectral shift and intensity difference as a diagnostic approach to distinguish between cancerous and normal breast cells. This study also presents a two-way structure of the 5-aminolevulinic acid (5-ALA) prodrug and Fluorescein Sodium (FS) effect in BC cell characterization from the perspective of photodynamical procedures and the detection side. 5-ALA induces an accumulation of protoporphyrin IX (PpIX) photosensitizer through a biosynthetic pathway, leading to red radiation of fluorescence measurements depending on different factors, such as temperature, incubation time, added glucose of the culturing medium, as well as photosynthesis processes. The presence and progression of breast cancer can be indicated by elevated levels of Reactive Oxygen Species (ROS), associated with the production of PpIX in cells following the administration of 5-ALA. In addition, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) fluorophores are recognized as the main factors for fluorescence emissions at around 420–580 nm emission intervals. Considering the MDA's high metastatic potential, the impact of 5-ALA on MDA's cellular morphology and viability has been investigated. The molecular fluorophores are the primary probes to MDA's cellular photodynamic considerations, allowing this widespread pre/post-clinical approach. The fluorescence signal reduction due to decreased cell viability and increased MDA's cellular death rate after 24 h of the 5-ALA-induced staining corresponds to the changes in lipid metabolism enzymes of MDAs cultured at different doses, which could be known as a cell death inducer function. Furthermore, statistical concerns have been studied using PCA multivariate component analysis to differentiate MDA cell lines administrated by 5-ALA. [ABSTRACT FROM AUTHOR]

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology. [ABSTRACT FROM AUTHOR]

Formate oxidase (FOx), which contains 8-formyl flavin adenine dinucleotide (FAD), exhibits a distinct advantage in utilizing ambient oxygen molecules for the oxidation of formic acid compared to other glucose-methanol-choline (GMC) oxidoreductase enzymes that contain only the standard FAD cofactor. The FOx-mediated conversion of FAD to 8-formyl FAD results in an approximate 10-fold increase in formate oxidase activity. However, the mechanistic details underlying the autocatalytic formation of 8-formyl FAD are still not well understood, which impedes further utilization of FOx. In this study, we employ molecular dynamics simulation, QM/MM umbrella sampling simulation, enzyme activity assay, site-directed mutagenesis, and spectroscopic analysis to elucidate the oxidation mechanism of FAD to 8-formyl FAD. Our results reveal that a catalytic water molecule, rather than any catalytic amino acids, serves as a general base to deprotonate the C8 methyl group on FAD, thus facilitating the formation of a quinone-methide tautomer intermediate. An oxygen molecule subsequently oxidizes this intermediate, resulting in a C8 methyl hydroperoxide anion that is protonated and dissociated to form OHC-RP and OH−. During the oxidation of FAD to 8-formyl FAD, the energy barrier for the rate-limiting step is calculated to be 22.8 kcal/mol, which corresponds to the required 14-hour transformation time observed experimentally. Further, the elucidated oxidation mechanism reveals that the autocatalytic formation of 8-formyl FAD depends on the proximal arginine and serine residues, R87 and S94, respectively. Enzymatic activity assay validates that the mutation of R87 to lysine reduces the kcat value to 75% of the wild-type, while the mutation to histidine results in a complete loss of activity. Similarly, the mutant S94I also leads to the deactivation of enzyme. This dependency arises because the nucleophilic OH− group and the quinone-methide tautomer intermediate are stabilized through the noncovalent interaction provided by R87 and S94. These findings not only explain the mechanistic details of each reaction step but also clarify the functional role of R87 and S94 during the oxidative maturation of 8-formyl FAD, thereby providing crucial theoretical support for the development of novel flavoenzymes with enhanced redox properties. [ABSTRACT FROM AUTHOR]

Terrestrial organisms are likely to face hypoxic stress during natural disasters such as floods or landslides, which can lead to inevitable hypoxic conditions for those commonly residing within soil. Pardosa pseudoannulata often inhabits soil crevices and has been extensively studied, yet research on its response to hypoxic stress remains unclear. Therefore, we investigated the adaptive strategies of Pardosa pseudoannulata under hypoxic stress using metabolomics and transcriptomics approaches. The results indicated that under hypoxic stress, metabolites related to energy and antioxidants such as ATP, D-glucose 6-phosphate, flavin adenine dinucleotide (FAD), and reduced L-glutathione were significantly differentially expressed. Pathways such as the citric acid (TCA) cycle and oxidative phosphorylation were significantly enriched. Transcriptome analysis and related assessments also revealed a significant enrichment of pathways associated with energy metabolism, suggesting that Pardosa pseudoannulata primarily copes with hypoxic environments by modulating energy metabolism and antioxidant-related substances. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes. Methods: Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm). Results: We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M− 1.S− 1. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2. Since schistosomes are damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M− 1.S− 1. Conclusions: The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) is a dynamic and complex microenvironment that modulates cell behavior and cell fate. Changes in ECM composition and architecture have been correlated with development, differentiation, and disease progression in various pathologies, including breast cancer [1]. Studies have shown that aligned fibers drive a pro-metastatic microenvironment, promoting the transformation of mammary epithelial cells into invasive ductal carcinoma via the epithelial-to-mesenchymal transition (EMT) [2]. The impact of ECM orientation on breast cancer metabolism, however, is largely unknown. Here, we employ two non-invasive imaging techniques, fluorescence-lifetime imaging microscopy (FLIM) and intensity-based multiphoton microscopy, to assess the metabolic states of cancer cells cultured on ECM-mimicking nanofibers in a random and aligned orientation. By tracking the changes in the intrinsic fluorescence of nicotinamide adenine dinucleotide and flavin adenine dinucleotide, as well as expression levels of metastatic markers, we reveal how ECM fiber orientation alters cancer metabolism and EMT progression. Our study indicates that aligned cellular microenvironments play a key role in promoting metastatic phenotypes of breast cancer as evidenced by a more glycolytic metabolic signature on nanofiber scaffolds of aligned orientation compared to scaffolds of random orientation. This finding is particularly relevant for subsets of breast cancer marked by high levels of collagen remodeling (e.g. pregnancy associated breast cancer), and may serve as a platform for predicting clinical outcomes within these subsets [3–6]. [ABSTRACT FROM AUTHOR]

Significance: To enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM). Aim: Using murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death. Approach: We use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays. Results: Through a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected. Conclusions: These data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments. [ABSTRACT FROM AUTHOR]

Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple β-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation. [ABSTRACT FROM AUTHOR]

The Mediterranean diet well known for its beneficial health effects, including mood enhancement, is characterised by the relatively high consumption of extra virgin olive oil (EVOO), which is rich in bioactive phenolic compounds. Over 200 phenolic compounds have been associated with Olea europaea, and of these, only a relatively small fraction have been characterised. Utilising the OliveNetTM library, phenolic compounds were investigated as potential inhibitors of the epigenetic modifier lysine-specific demethylase 1 (LSD1). Furthermore, the compounds were screened for inhibition of the structurally similar monoamine oxidases (MAOs) which are directly implicated in the pathophysiology of depression. Molecular docking highlighted that olive phenolics interact with the active site of LSD1 and MAOs. Protein–peptide docking was also performed to evaluate the interaction of the histone H3 peptide with LSD1, in the presence of ligands bound to the substrate-binding cavity. To validate the in silico studies, the inhibitory activity of phenolic compounds was compared to the clinically approved inhibitor tranylcypromine. Our findings indicate that olive phenolics inhibit LSD1 and the MAOs in vitro. Using a cell culture model system with corticosteroid-stimulated human BJ fibroblast cells, the results demonstrate the attenuation of dexamethasone- and hydrocortisone-induced MAO activity by phenolic compounds. The findings were further corroborated using human embryonic stem cell (hESC)-derived neurons stimulated with all-trans retinoic acid. Overall, the results indicate the inhibition of flavin adenine dinucleotide (FAD)-dependent amine oxidases by olive phenolics. More generally, our findings further support at least a partial mechanism accounting for the antidepressant effects associated with EVOO and the Mediterranean diet. [ABSTRACT FROM AUTHOR]

Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP+ or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD+, NADP+, flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors. • Uracil-thymine dehydrogenase was found to be a heteromer consisting of three different subunits. • Uracil-thymine dehydrogenase catalyzed oxidation of uracil with artificial electron acceptors. • The activity of uracil-thymine dehydrogenase was enhanced by cerium ions. [ABSTRACT FROM AUTHOR]

ABSTRACT Flavin adenine dinucleotide (FAD) is an essential cofactor for numerous flavoenzymes present in all living organisms. The biosynthesis of FAD from riboflavin involves two sequential reactions catalyzed by riboflavin kinase and flavin adenine dinucleotide synthase (FADS). Entamoeba histolytica, the protozoan parasite responsible for amebiasis, apparently lacks a gene encoding FADS that share similarity with bacterial and eukaryotic canonical FADS, yet it can synthesize FAD. In this study, we have identified the gene responsible for FADS and thoroughly characterized physiological and biochemical properties of FADS from E. histolytica. Phylogenetic analysis revealed that the gene was likely laterally transferred from archaea. The kinetic properties of recombinant EhFADS were consistent with the notion that EhFADS is of archaeal origin, exhibiting KM and kcat values similar to those of the arachaeal enzyme while significantly differing from the human counterpart. Repression of gene expression of EhFADS by epigenetic gene silencing caused substantial reduction in FAD levels and parasite growth, underscoring the importance of EhFADS for the parasite. Furthermore, we demonstrated that EhFADS gene silencing reduced thioredoxin reductase activity, which requires FAD as a cofactor and makes the ameba more susceptible to metronidazole. In summary, this study unveils unique evolutionary and biochemical features of EhFADS and underscores its significance as a promising drug target in combating human amebiasis.IMPORTANCEFAD is important for all forms of life, yet its role and metabolism are still poorly studied in E. histolytica, the protozoan parasite causing human amebiasis. Our study uncovers the evolutionary unique key enzyme, archaeal-type FADS for FAD biosynthesis from E. histolytica for the first time. Additionally, we showed the essentiality of this enzyme for parasite survival, highlighting its potential as target for drug development against E. histolytica infections.

The article discusses the field of magnetobiology, which studies the non-thermal effects of magnetic fields on biological systems. The literature in this field is often plagued by conflicting claims and methodological shortcomings, leading to an unbalanced body of research. The article highlights the potential of the radical pair mechanism (RPM) in understanding the effects of magnetic interactions in living systems. It specifically focuses on the RPM's role in the magnetic compass sense of migratory songbirds and its potential applications in other areas of magnetobiology. The article also mentions the importance of realistic models and experimental optimization in studying radical pair reactions. [Extracted from the article]

Bacillus subtilis ferredoxin:NADP+ oxidoreductase (BsFNR) is a thioredoxin reductase-type FNR whose redox properties and reactivity with nonphysiological electron acceptors have been scarcely characterized. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of the flavin adenine dinucleotide (FAD) cofactor was estimated to be −0.240 V. Photoreduction using 5-deazaflavin mononucleotide (5-deazaFMN) as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.024 V. We examined the mechanisms of the reduction of several different groups of non-physiological electron acceptors catalyzed by BsFNR. The reactivity of quinones and aromatic N-oxides toward BsFNR increased when increasing their single-electron reduction midpoint redox potentials. The reactivity of nitroaromatic compounds was lower due to their lower electron self-exchange rate, but it exhibited the same trend. A mixed single- and two-electron reduction reaction was characteristic of quinones, whereas reactions involving nitroaromatics proceeded exclusively via the one-electron reduction reaction. The oxidation of FADH• to FAD is the rate-limiting step during the oxidation of fully reduced FAD. The calculated electron transfer distances in the reaction with nitroaromatics were close to those of other FNRs including the plant-type enzymes, thus demonstrating their similar active site accessibility to low-molecular-weight oxidants despite the fundamental differences in their structures. [ABSTRACT FROM AUTHOR]

D‐2‐hydroxyglutarate dehydrogenase (D2HGDH) is a mitochondrial enzyme containing flavin adenine dinucleotide FAD, existing as a dimer, and it facilitates the specific oxidation of D‐2HG to 2‐oxoglutarate (2‐OG), which is a key intermediate in the tricarboxylic acid (TCA) cycle. A Genome‐wide expression analysis (GWEA) has indicated an association between GhD2HGDH and flowering time. To further explore the role of GhD2HGDH, we performed a comprehensive investigation encompassing phenotyping, physiology, metabolomics, and transcriptomics in Arabidopsis thaliana plants overexpressing GhD2HGDH. Transcriptomic and qRT‐PCR data exhibited heightened expression of GhD2HGDH in upland cotton flowers. Additionally, early‐maturing cotton exhibited higher expression of GhD2HGDH across all tissues than delayed‐maturing cotton. Subcellular localization confirmed its presence in the mitochondria. Overexpression of GhD2HGDH in Arabidopsis resulted in early flowering. Using virus‐induced gene silencing (VIGS), we investigated the impact of GhD2HGDH on flowering in both early‐ and delayed‐maturing cotton plants. Manipulation of GhD2HGDH expression levels led to changes in photosynthetic pigment and gas exchange attributes. GhD2HGDH responded to gibberellin (GA3) hormone treatment, influencing the expression of GA biosynthesis genes and repressing DELLA genes. Protein interaction studies, including yeast two‐hybrid, luciferase complementation (LUC), and GST pull‐down assays, confirmed the interaction between GhD2HGDH and GhSOX (Sulfite oxidase). The metabolomics analysis demonstrated GhD2HGDH's modulation of the TCA cycle through alterations in various metabolite levels. Transcriptome data revealed that GhD2HGDH overexpression triggers early flowering by modulating the GA3 and photoperiodic pathways of the flowering core factor genes. Taken together, GhD2HGDH positively regulates the network of genes associated with early flowering pathways. [ABSTRACT FROM AUTHOR]

The ongoing expansion of fifth-generation (5G) and future sixth-generation (6G) mobile communications is expected to result in widespread human exposure to millimeter-wave (mmWave) radiation globally. Given the short penetration depth of mmWaves and their high absorption by the skin, it is imperative to investigate the potential effects of 5G radiation not only in terms of temperature increase but also at the cellular level. To understand the biological mechanisms of mmWave effects, accurate methods for assessing mmWave absorption in the skin are crucial. In this study, we use fluorescence lifetime imaging microscopy (FLIM) to explore these effects. Employing a mmWave exposure system operating at 26 gigahertz (GHz), porcine skin is irradiated for varying durations (5, 10, 20, and 30 min). We investigate changes in tissue temperature and the autofluorescence of flavin adenine dinucleotide (FAD). Our findings suggest that operating our mmWave exposure systems at the configured power level of 26 GHz is unlikely to cause damage to FADs, even after a 30 min exposure duration. [ABSTRACT FROM AUTHOR]

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site‐Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non‐canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH• and the nitroxide. The DEER data revealed a salt concentration‐dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before. [ABSTRACT FROM AUTHOR]

The presence of high chromium (Cr) levels induces the buildup of reactive oxygen species (ROS), resulting in hindered plant development. Riboflavin (vitamin B2) is produced by plants, fungi, and microbes. It serves as a precursor to the coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which play a crucial role in cellular metabolism. The objective of this work was to clarify the underlying mechanisms by which riboflavin alleviates Cr stress in Praecitrullus fistulosus L. Further, the role of riboflavin in growth, ions homeostasis, methylglyoxal detoxification, and antioxidant defense mechanism are not well documented in plants under Cr toxicity. We found greater biomass and minimal production of ROS in plants pretreated with riboflavin under Cr stress. Results manifested a clear abridge in growth, chlorophyll content, and nutrient uptake in Indian squash plants exposed to Cr stress. Findings displayed that Cr stress visibly enhanced oxidative injury reflected as higher malondialdehyde (MDA), hydrogen peroxide (H2O2), superoxide radical (O2•‒), methylglyoxal (MG) levels alongside vivid lipoxygenase activity. Riboflavin strengthened antioxidant system, enhanced osmolyte production and improved membrane integrity. Riboflavin diminished Cr accumulation in aerial parts that led to improved nutrient acquisition. Taken together, riboflavin abridged Cr phytotoxic effects by improving redox balance because plants treated with riboflavin had strong antioxidant system that carried out effective ROS detoxification. Riboflavin protected membrane integrity that, in turn, improved nutrient uptake in plants. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Does fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging assessment of human blastocysts prior to frozen transfer correlate with pregnancy outcomes? SUMMARY ANSWER FLIM failed to distinguish consistent patterns in mitochondrial metabolism between blastocysts leading to pregnancy compared to those that did not. WHAT IS KNOWN ALREADY FLIM measurements provide quantitative information on NAD(P)H and flavin adenine dinucleotide (FAD+) concentrations. The metabolism of embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. STUDY DESIGN, SIZE, DURATION This was a pilot trial enrolling 121 IVF couples who consented to have their frozen blastocyst measured using non-invasive metabolic imaging. After being warmed, 105 couples' good-quality blastocysts underwent a 6-min scan in a controlled temperature and gas environment. FLIM-assessed blastocysts were then transferred without any intervention in management. PARTICIPANTS/MATERIALS, SETTING, METHODS Eight metabolic parameters were obtained from each blastocyst (4 for NAD(P)H and 4 for FAD): short and long fluorescence lifetime, fluorescence intensity, and fraction of the molecule engaged with enzyme. The redox ratio (intensity of NAD(P)H)/(intensity of FAD) was also calculated. FLIM data were combined with known metadata and analyzed to quantify the ability of metabolic imaging to differentiate embryos that resulted in pregnancy from embryos that did not. De-identified discarded aneuploid human embryos (n = 158) were also measured to quantify correlations with ploidy status and other factors. Statistical comparisons were performed using logistic regression and receiver operating characteristic (ROC) curves with 5-fold cross-validation averaged over 100 repeats with random sampling. AUC values were used to quantify the ability to distinguish between classes. MAIN RESULTS AND THE ROLE OF CHANCE No metabolic imaging parameters showed significant differences between good-quality blastocysts resulting in pregnancy versus those that did not. A logistic regression using metabolic data and metadata produced an ROC AUC of 0.58. In contrast, robust AUCs were obtained when classifying other factors such as comparison of Day 5 (n = 64) versus Day 6 (n = 41) blastocysts (AUC = 0.78), inner cell mass versus trophectoderm (n = 105: AUC = 0.88) and aneuploid (n = 158) versus euploid and positive pregnancy embryos (n = 108) (AUC = 0.82). LIMITATIONS, REASONS FOR CAUTION The study protocol did not select which embryo to transfer and the cohort of 105 included blastocysts were all high quality. The study was also limited in number of participants and study sites. Increased power and performing the trial in more sites may have provided a stronger conclusion regarding the merits of the use of FLIM clinically. WIDER IMPLICATIONS OF THE FINDINGS FLIM failed to distinguish consistent patterns in mitochondrial metabolism between good-quality blastocysts leading to pregnancy compared to those that did not. Blastocyst ploidy status was, however, highly distinguishable. In addition, embryo regions and embryo day were consistently revealed by FLIM. While metabolic imaging detects mitochondrial metabolic features in human blastocysts, this pilot trial indicates it does not have the potential to serve as an effective embryo viability detection tool. This may be because mitochondrial metabolism plays an alternative role post-implantation. STUDY FUNDING/COMPETING INTEREST(S) This study was sponsored by Optiva Fertility, Inc. Boston IVF contributed to the clinical site and services. Becker Hickl, GmbH, provided the FLIM system on loan. T.S. was the founder and held stock in Optiva Fertility, Inc. and D.S. and E.S. had options with Optiva Fertility, Inc. during this study. TRIAL REGISTRATION NUMBER The study was approved by WCG Connexus IRB (Study Number 1298156). [ABSTRACT FROM AUTHOR]

Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCE Pseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate’s side chain. Structural analysis of the refined HspB crystal structure, combined with state-ofthe- art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development. [ABSTRACT FROM AUTHOR]

Lysine‐specific demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD) dependent monoamine oxidase (MAO) that erases the mono‐, and dimethylation of histone 3 lysine 4 (H3K4), resulting in the suppression of target gene transcriptions. Besides, it can also demethylate some nonhistone substrates to regulate their biological functions. As reported, LSD1 is widely upregulated and plays a key role in several kinds of cancers, pharmacological or genetic ablation of LSD1 in cancer cells suppresses cell aggressiveness by several distinct mechanisms. Therefore, numerous LSD1 inhibitors, including covalent and noncovalent, have been developed and several of them have entered clinical trials. Herein, we systemically reviewed and discussed the biological function of LSD1 in tumors, lymphocytes as well as LSD1‐targeting inhibitors in clinical trials, hoping to benefit the field of LSD1 and its inhibitors. [ABSTRACT FROM AUTHOR]

Ferroptosis is a recently discovered form of regulated cell death characterized by its distinct dependence on iron and the peroxidation of lipids within cellular membranes. Ferroptosis plays a crucial role in physiological and pathological situations and has attracted the attention of numerous scientists. Ferroptosis suppressive protein 1 (FSP1) is one of the main regulators that negatively regulates ferroptosis through the GPX4‐independent FSP1–CoQ10–NAD(P)H axis and is a potential therapeutic target for ferroptosis‐related diseases. However, the crystal structure of FSP1 has not been resolved, which hinders the development of therapeutic strategies targeting FSP1. To unravel this puzzle, we purified the human FSP1 (hFSP1) protein using the baculovirus eukaryotic cell expression system and solved its crystal structure at a resolution of 1.75 Å. Furthermore, we evaluated the oxidoreductase activity of hFSP1 with NADH as the substrate and identified E156 as the key amino acid in maintaining hFSP1 activity. Interestingly, our results indicated that hFSP1 exists and functions in a monomeric state. Mutagenesis analysis revealed the critical role of the C‐terminal domain in the binding of substrate. These findings significantly enhance our understanding of the functional mechanism of FSP1 and provide a precise model for further drug development. [ABSTRACT FROM AUTHOR]

A study published in the journal Cell Discovery has identified biallelic variants in the COASY gene as a new genetic cause of riboflavin-responsive lipid storage myopathy (RR-LSM). RR-LSM is a subtype of lipid metabolic disorders characterized by muscle weakness, excessive accumulation of lipid droplets in muscle fibers, and a dramatic response to riboflavin. The study conducted whole-exome sequencing on 28 RR-LSM patients and found that 16 of them carried biallelic COASY variants. Cellular and Drosophila models were used to demonstrate the impact of COASY variants on lipid metabolism and CoA biosynthesis. The study also showed that riboflavin treatment could stabilize the mutant COASY protein and potentially offer therapeutic benefits for COASY-related RR-LSM. [Extracted from the article]

Background: Poor oocyte quality remains one of the major challenges for polycystic ovary syndrome (PCOS) patients during in vitro fertilization (IVF) treatment. Granulosa cells (GCs) in PCOS display altered functions and could cause an unfavorable microenvironment for oocyte growth and maturation. Ferroptosis is a new form of programmed cell death, but its role in PCOS has been largely unclarified. Methods: Ferroptosis-related differentially expressed genes (DEGs) of GCs in women with PCOS were identified by bioinformatic analyses of GSE155489 and GSE168404 datasets. Functional enrichment analyses were conducted using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Core ferroptosis-related genes were further screened by random forest, and evaluated for diagnostic value by receiver operating characteristic curve analyses. Gene expression was validated by real-time quantitative polymerase chain reaction of collected GC samples, and analyzed for association with oocyte quality. In addition, gene regulatory network was constructed based on predicted RNA interactions and transcription factors, while potential therapeutic compounds were screened through molecular docking with crystallographic protein structures. Results: A total of 14 ferroptosis-related DEGs were identified. These DEGs were mainly enriched in reactive oxygen species metabolic process, mitochondrial outer membrane, antioxidant activity as well as ferroptosis and adipocytokine signaling pathways. Eight core ferroptosis-related genes (ATF3, BNIP3, DDIT4, LPIN1, NOS2, NQO1, SLC2A1 and SLC2A6) were further selected in random forest model, which showed high diagnostic performance for PCOS. Seven of them were validated in GC samples, and five were found to be significantly and positively correlated with one or more oocyte quality parameters in PCOS patients, including oocyte retrieval rate, mature oocyte rate, normal fertilization rate, and good-quality embryo rate. Gene regulatory network revealed JUN and HMGA1 as two important transcription factors, while dicoumarol and flavin adenine dinucleotide were predicted as small molecules with therapeutic potential. Conclusions: This is the first comprehensive report to study the differential expression of ferroptosis-related genes in GCs of PCOS and their clinical relevance with oocyte quality. Our findings could provide novel insights on the potential role of GC ferroptosis in PCOS pathogenesis, diagnosis, and targeted treatment. [ABSTRACT FROM AUTHOR]