Melatonin has demonstrated antitumor activity in various cancer models, but its effects may depend on the therapeutic dose and cellular environment. We found that melatonin at millimolar concentrations significantly downregulated transglutaminase 2 (TGM2), histone serotonylation (H3K4me3Q5ser), and YTHDF1/m6A (N6-methyladenosine) in a colorectal cancer model. Cross-interference between TGM2 and YTHDF1 supports a link between these regulatory processes, while molecular docking, pharmacological interventions, and co-immunoprecipitation experiments suggest the existence of a signaling pathway involving melatonin and the HTR2B receptor. A combined analysis of CHIP-seq and meRIP-seq identified salt-induced kinase 1 (SIK1) as a potential downstream target, and SIK1 interference affected the proliferation and migration of colorectal cancer. Collectively, these findings support a working model in which melatonin-induced tumor suppression is associated with coordinated changes in TGM2/H3K4me3Q5ser and YTHDF1-associated m6A regulation, with SIK1 emerging as a potential convergent downstream effector.

Hyperpigmentation is a dermatological condition characterized by excessive melanin production, resulting in skin darkening. Intrinsic and extrinsic mediators, including ultraviolet (UV) radiation, hormonal changes, inflammation, and genetic predisposition, contribute to hyperpigmentation pathogenesis. The current review thoroughly examines the biological processes involved in hyperpigmentation, with particular emphasis on its signaling pathways, particularly the cyclic adenosine monophosphate (cAMP), wingless-related integration site (Wnt)/β-catenin, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and mitogen-activated protein kinase (MAPK), thereby providing mechanistic insights that may facilitate the development of effective treatments. The review also evaluates the safety, toxicity, and therapeutic efficacy of TYR inhibitors, arbutin, azelaic acid, ellagic acid, kojic acid, resveratrol, and the melanosome inhibitor niacinamide, for managing hyperpigmentation. Additionally, the review summarizes the in vitro and in vivo studies of these agents and other emerging plant extracts. Recent advances in the use of plant extracts for hyperpigmentation have shown promising efficacy; nevertheless, their translation into clinical and commercial applications remains limited. This limited translation might be attributed to the challenges related to standardization and the paucity of clinical evidence regarding long-term safety. Moreover, ongoing studies on natural anti-hyperpigmentation agents have highlighted the critical role of patent analyses in the advancement of biomedical research. This review provides a strategic direction for researchers to assess the safety, efficacy, and toxicity profiles of natural anti-hyperpigmentation agents.

The Coronary Vasomotor Disorders International Study group (COVADIS) developed the diagnostic criteria for vasospastic angina and microvascular angina more than 8 years ago. This article critically reappraises these criteria (Latin: quid fecisti - what has been done) and proposes the strategies for their advancement (Latin: Quo Vadis - where are you are going?). Although the criteria have some limitations, they remain relevant considering their multifaceted clinical perspective including the presence of symptoms, myocardial ischaemia and objective evidence of coronary vasomotor dysfunction. To improve the criteria, the following strategic directions (Quo Vadis) are proposed, (1) optimising invasive functional coronary angiography assessment - the benchmark diagnostic investigation for coronary vasomotor disorders, (2) diagnosing mixed macrovascular and microvascular coronary vasomotor disorders, (3) advancing non-invasive coronary vasomotor assessment methods, (4) linking coronary vasomotor disorder criteria to clinical outcomes, (5) evaluating coronary vasomotor disorders in the presence of obstructive coronary artery disease, and (6) global partnering of coronary vasomotor disorder initiatives in national networks and patient forums. These strategies will improve the diagnostic approaches to coronary vasomotor disorders thereby facilitating the evolution of the COVADIS Diagnostic Criteria and thus management of these elusive disorders.

Polystyrene nanoplastics (NPs) may affect macrophage function, but the relationships between inflammatory responses and mitochondrial alterations remain unclear. We combined exploratory analysis of public zebrafish intestinal single-cell RNA-sequencing data with transcriptomic, metabolomic, and experimental studies in RAW264.7 cells and primary mouse macrophages. In RAW264.7 cells, NPs increased reactive oxygen species (ROS), nitric oxide (NO), and the secretion of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). ROS and cytokine responses occurred at concentrations below those causing significant cytotoxicity in this cell line. A complex I-associated activity assay detected changes at lower NP concentrations than assays of cellular adenosine triphosphate (ATP) and the NAD+/NADH ratio. MitoSOX and JC-1 measurements further indicated changes in mitochondrial oxidant-sensitive fluorescence and membrane-potential-associated signals. Primary mouse macrophages showed concentration- and time-dependent responses, including increased TNF-α secretion. NPs exposure was also associated with reduced CPEB4 and INO80B protein abundance. CPEB4 knockdown increased ROS and reduced ATP at a later time point, whereas cellular thermal shift assay showed altered CPEB4 thermal behavior after NPs exposure. These findings identify inflammatory and mitochondrial responses associated with NPs exposure and implicate CPEB4 as a candidate contributor. However, they do not establish direct binding of intact NPs to CPEB4 or a causal role for CPEB4 or INO80B.

Ochratoxin A (OTA) contamination severely threatens global food safety. Herein, a ratiometric fluorescent immunoassay method was established for trace OTA detection based on an alkaline phosphatase (ALP)-catalyzed Eu3+ coordination polymer (Eu-CP). The Eu-CP probe is self-assembled from ciprofloxacin, adenosine monophosphate, and Eu3+. Upon activation by the Mg2+ cofactor, ALP drives the dephosphorylation and subsequent structural disassembly of the Eu-CP framework, which synchronously regulates dual-emission fluorescent signals. In indirect competitive reaction, OTA competes with bovine serum albumin-OTA for limited antibodies, and the bound ALP-labeled secondary antibody is inversely correlated with OTA concentration. Based on the ratiometric fluorescence signal (F618/F418), the proposed method exhibited a reliable linear range of 0.4883-62.5 ng/mL (R2 = 0.9909) and a limit of detection as low as 0.0273 ng/mL. Recoveries in corn, oat and rice were 97.07%-119.11% with inter-batch CV < 10%. This study provides a simple and sensitive method for trace OTA detection.

FAD-dependent glucose dehydrogenase (FAD-GDH) is a promising glucose sensing enzymes owing to its excellent thermostability and substrate specificity. To produce recombinant FAD-GDH with higher enzymatic activity by Pichia pastoris to meet commercial demands, three complementary strategies were proposed. First, the OST signal peptide, which outperformed ADV and MSB, replaced α-MF to yield much more efficient secretion, enhancing enzyme activity and protein yield up to 201.4% and 214.0% compared to the levels obtained with the original secretion signal, respectively, in shake-flask cultures. Second, to address the low specific activity of FAD-GDH, mixed feeding with riboflavin, adenosine, and yeast extract during induction period of fermentation had improved enzyme activity and specific activity to196.6% and 161.4%, respectively, while maintaining cell viability, compared with the control without additional feeding. Third, to further enhance specific activity of FAD-GDH, endogenous FAD synthase was ectopically overexpressed under the control of either the GAP or AOX1 promoter via CRISPR-Cas9-mediated genomic integration. The strain with AOX1-drivenFAD synthase (WCG-AOX1-FAD1) exhibited the most significant improvements, with enzyme activity of 1023.46 ± 95.85 U/mL (239.4% of WCG-OST) and specific activity of 886.31 ± 25.42 U/mg (233.4% of WCG-OST), both representing relatively high levels among similar studies. The synergistic application of these three strategies provides a reference for the industrial production of various cofactor-dependent enzymes.

DEAD-box RNA helicases are widespread ATP-driven enzymes that reshape RNA structures and RNA-protein complexes during many steps of RNA metabolism. In bacteria, DEAD-box RNA helicases support cellular growth and responses to stress by contributing to ribosome biogenesis, RNA degradation, and translation initiation. Growing evidence indicates that several DEAD-box RNA helicases can also form liquid-liquid phase-separated condensates that concentrate RNA and partner proteins. This behavior adds a new layer of control to RNA regulation, although the molecular factors that promote or tune liquid-liquid phase separation (LLPS) are still unclear. Here, we describe an in vitro microscopy-based assay to characterize the LLPS behavior of bacterial DEAD-box RNA helicases. The protocol includes step-by-step procedures to evaluate condensate formation, salt sensitivity and dissolution, RNA dependency and specificity, and ATP effects. We further provide detailed instructions for quantitative analysis of microscope images, including size and assessment of protein enrichment within condensates. Altogether, this workflow enables systematic dissection of key physicochemical parameters governing DEAD-box helicase LLPS and provides a standardised framework for comparative studies across bacterial DEAD-box proteins or variants.

Single-molecule manipulation methods are useful techniques to probe the interactions of proteins and nucleic acid structures. Here, we describe magnetic tweezers-based single-molecule investigation of the binding of helicases to G-quadruplex structures and their ATP-dependent unwinding activity, using DHX36 (also known as RHAU and G4R1) helicase and both DNA and RNA G-quadruplex structures as examples. We specifically emphasize the principle and method to probe the interactions between DHX36 and the DNA G-quadruplex in different intermediate states during an ATPase cycle of DHX36, based on detecting the DHX36-induced changes in the lifetime of the DNA and RNA G-quadruplex under tension. The principle of the measurement can be broadly extended to the studies of other DNA or RNA G-quadruplex helicases.

Helicases are versatile enzymes. Superfamily 2 (SF2) is the largest and most diverse group of helicases. SF2 helicases are involved in fundamental processes dealing with nucleic acids in the three domains of life and can be specific to DNA, RNA, or DNA:RNA hybrids. They are known to catalyze unwinding reactions in vitro or disrupt duplexes in vivo. More recently, it has emerged that they can also anneal single strands to form duplexes. Here, we describe biochemical assays used to investigate the activities of the Ski2-like helicases ASH-Ski2 and Hel308, as well as the Lhr-like helicase Lhr2 from Thermococcales, an order of hyperthermophilic archaea. Unwinding and strand-annealing assays using a range of substrates revealed that they are RNA-, DNA-, or RNA:DNA-specific helicases, respectively.

Cellular RNAs depend on proteins for efficient folding to specific functional structures and for transitions between functional structures. This dependence arises from intrinsic properties of RNA structure. Specifically, RNAs possess stable local structure, largely in the form of helices, and there are abundant opportunities for RNAs to form alternative helices and tertiary contacts and therefore to populate alternative structures. Proteins with RNA chaperone activity, either ATP-dependent or ATP-independent, can promote structural transitions by interacting with single-stranded RNA (ssRNA) to compete away partner interactions and then release ssRNA so that it can form new interactions. In this chapter, we review the basic properties of RNA and the proteins that function as chaperones and remodelers. We then use these properties as a foundation to explore key points for the design and interpretation of experiments that probe RNA rearrangements and their acceleration by proteins.

The nucleoside diphosphates linked to moiety-X (Nudix) hydrolases are a superfamily of proteins defined by the characteristic Nudix box sequence, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. The Nudix hydrolase associated with CRISPR-Cas-associated adenosine deaminases (CAAD) from Limisphaera ngatamarikiensis (LngNH) mitigates CAAD-induced toxicity by hydrolyzing ITP to IMP and pyrophosphate. Here, we determined crystal structures of LngNH in the apo form and in complex with ITP using catalytic mutants and validated key interactions by site-directed mutagenesis. The hypoxanthine of ITP is deeply buried in a hydrophobic pocket and engages a distinct hydrogen-bond network, whereas the triphosphate tail is coordinated by conserved residues and divalent metal ions. Together, these findings provide a structural framework for CAAD-linked nucleotide sanitization and suggest that CRISPR-associated Nudix hydrolases function as local self-protection modules that limit toxicity from noncanonical inosine nucleotides.IMPORTANCENucleotide quality-control enzymes protect cells from harmful metabolic by-products. Here, we characterize a CRISPR-associated nucleoside diphosphates linked to moiety-X (Nudix) hydrolase that removes ITP, a toxic noncanonical nucleotide generated during antiviral defense. Biochemical and structural analyses show how this enzyme specifically recognizes and hydrolyzes ITP while avoiding related cellular nucleotides. Our findings suggest that CRISPR-associated nucleotide deaminase systems require "self-protection" mechanisms to prevent damage from their own antiviral activities. These results expand our understanding of how bacteria maintain nucleotide homeostasis during immune responses and reveal an additional regulatory layer within CRISPR-associated defense systems. The structural framework reported here also provides a foundation for future studies of nucleotide sanitization enzymes in microbial physiology and antiviral immunity.