Feature-Based Molecular Networking (FBMN) on the Global Natural Products Social Molecular Networking (GNPS) platform offers a transformative framework for visualizing drug-drug interaction (DDI)-driven metabolic reprogramming that conventional analytical approaches fail to capture. Here, we demonstrate its application as a novel strategy to resolve complex metabolic alterations arising from DDI-induced toxicity. Using FBMN-integrated MS2 mapping, we established a proof-of-concept workflow to elucidate large-scale metabolic transitions and pathway rewiring. As a model system, co-administration of methamphetamine (MA) and ethanol (EtOH)─a combination known to potentiate toxicity─was analyzed through liquid chromatography-quadrupole time-of-flight mass spectrometry. Merging MS2 spectra across multiple collision energies revealed diagnostic fragment ions that enabled network connectivity and comprehensive annotation of 21 MA-related features, including glucuronide conjugates and in-source fragments. FBMN visualization uncovered an EtOH-driven metabolic shift, characterized by suppression of benzene ring hydroxylation and the emergence of N-acetylamphetamine and novel glucuronidated metabolites. Collectively, these findings establish GNPS-FBMN as a powerful and generalizable analytical strategy for delineating DDI-associated metabolic reprogramming, providing mechanistic insight into the metabolic basis of toxicity arising from drug combinations.

Neuroinflammation, driven by the activation of immune cells such as microglia, is tightly linked to metabolic reprogramming, thereby emerging as a key therapeutic target. This study investigated the potential of laminarin, a natural β-glucan polysaccharide, to ameliorate immunometabolic dysfunction in lipopolysaccharide (LPS)-stimulated microglia. Laminarin potently suppressed the production of pro-inflammatory cytokines, reduced oxidative stress, and ameliorated metabolic dysfunction through the activation of sirtuin 1 (SIRT1), which was identified as a key molecular target. Molecular docking simulations predicted a high-affinity binding of its representative unit, laminarihexaose, to the SIRT1 allosteric activation site, leading to its effective activation. In LPS-challenged microglia, laminarin's ability to enhance cellular NAD+ levels via the NAD+ salvage pathway also contributed to SIRT1 activation. Furthermore, SIRT1 activation was linked to laminarin's capacity to promote the phosphorylation of AMP-activated protein kinase (AMPK), suggesting a potential positive feedback loop that reinforces the integrity of the SIRT1-AMPK axis. Laminarin prevented LPS-induced abnormal flux of the TCA cycle by regulating the related genes and the concentration of intermediates such as aconitic acid and 2-hydroxyglutaric acid. Laminarin's efficacy also extended to suppressing the compensatory glycolytic switch and ameliorating mitochondrial dysfunction by restoring the mitochondrial membrane potential and regulating genes involved in mitochondrial respiration. Collectively, these results suggest that laminarin ameliorates microglial inflammation and metabolic dysregulation by activating the SIRT1-AMPK axis to restore metabolic homeostasis. These findings position laminarin as a promising therapeutic candidate for neuroinflammatory disorders by targeting the crucial link between immunity and metabolism.

Menthol, a prevalent additive in tobacco and electronic cigarette products, has been reported to interact with nicotine at both neuropharmacological and behavioral levels. Although menthol alone is not reinforcing, converging evidence suggests that it may modulate the pharmacological and behavioral effects of nicotine through nicotinic acetylcholine receptor (nAChR) mechanisms. Using an inhalation-based rodent model that mimics human exposure to electronic nicotine delivery systems, we examined how menthol aerosol influences nicotine-induced hypothermia, discriminative stimulus effects, and the potential involvement of α4β2* nAChRs in male rats. Plasma nicotine, cotinine, and trans-3'-hydroxycotinine (3'-OH-cotinine) concentrations were also measured to assess pharmacokinetic interactions. Menthol aerosol prolonged and potentiated nicotine-induced hypothermia, and this effect was attenuated by the α4β2* receptor antagonist dihydro-β-erythroidine (DHβE). In drug discrimination (DD) assays, coinhalation of menthol enhanced nicotine-appropriate responding, suggesting facilitation of nicotine's interoceptive stimulus properties. These effects were also attenuated by DHβE, consistent with involvement of α4β2* nAChR-mediated mechanisms. In addition, the behavioral effects were accompanied by alterations in nicotine pharmacokinetics, including increased levels of nicotine metabolites. Collectively, these findings suggest that menthol modulates nicotine's pharmacological and behavioral effects, potentially through α4β2* nAChR-dependent mechanisms. By prolonging nicotine's physiological and interoceptive effects, menthol may increase the salience of nicotine-related cues during inhalation exposure. These interactions could contribute to enhanced abuse liability associated with menthol-containing nicotine products, although further studies are required to establish causal mechanisms. This study provides mechanistic insight into menthol-nicotine interactions and highlights considerations relevant to mentholated e-cigarette products.

Dry age-related macular degeneration (AMD) is the leading cause of central vision loss among the elderly, yet no curative treatment exists. While exudative AMD can be managed with anti-vascular endothelial growth factor (VEGF) therapy, dry AMD-accounting for more than 85% of cases-progresses insidiously from drusen accumulation to geographic atrophy (GA). Although the recent U.S. Food and Drug Administration (FDA) approvals of pegcetacoplan and avacincaptad pegol represent major milestones, their therapeutic effects remain modest. This review provides an integrated overview of the molecular and cellular mechanisms underlying dry AMD, highlighting key pathogenic pathways involving oxidative stress, lipid dysregulation, complement activation, mitochondrial impairment, and RPE-specific bisretinoid lipofuscin accumulation. We further summarize mechanistic mouse models that replicate these pathological processes and discuss how each model contributes to understanding the disease. Finally, we review current and emerging therapeutic strategies-including complement inhibitors, visual cycle modulators, and mitochondrial-protective approaches-and outline future directions for translational research. Collectively, this review synthesizes mechanistic insights, disease models, and therapeutic innovation to support the development of targeted and stage-specific interventions for dry AMD.

Methamphetamine (MA) is a psychostimulant with high potential for abuse and neurotoxicity, and overdose or concurrent use with ethanol (EtOH) has been associated with increased hospitalizations and mortality. This study investigated the pharmacokinetic and metabolic interactions resulting from concomitant exposure to MA (1, 4, and 10 mg/kg, intraperitoneal [i.p.]) and EtOH (2 g/kg, 30 % v/v, i.p.) in a rat model by assessing dose-dependent behavioral responses and EtOH-induced potentiation of MA-mediated neurotoxicity. Pharmacokinetic analysis revealed that EtOH co-administration increased the maximum plasma concentration, half-life, and area under the plasma concentration-time curve of MA while decreasing its volume of distribution and total clearance. Notably, EtOH co-administration reduced the hydroxylation of MA and enhanced its demethylation, potentially contributing to elevated toxicity and addictive potential. Time-course metabolic profiling of amino acids and polyamines showed that EtOH-induced potentiation of stereotypic behaviors correlated with a significant increase in plasma spermidine and spermine levels, suggesting a temporal association between behavioral and metabolomic alterations. At the time of peak behavioral abnormalities, extensive metabolic perturbations were observed following MA and EtOH co-administration. Receiver operating characteristic curve and network analyses identified two polyamines (spermidine and spermine) and two bile acids (glycocholic acid and taurocholic acid) as key metabolites associated with the MA-EtOH interaction, implicationg their roles in MA-EtOH intoxication. These findings reveal previously uncharacterized pharmacometabolic pathways and behavioral manifestations resulting from MA and EtOH co-exposure, providing novel mechanistic insights into the pathophysiology of MA-EtOH co-intoxication.

gamma-Hydroxybutyric acid (GHB) is a central nervous system depressant with high addiction risk, causing oxidative stress and neurotoxicity. Urinary nucleoside metabolomics studies in single- and multiple- administration rats have not been performed. Most nucleoside levels were higher in the single-administered group than the multiple-administered group. Multivariate analysis identified cytidine, pseudouridine, uridine, N4-acetylcysteine, adenosine, and 5'-deoxy-5'-methylthioadenosine as significant for group discrimination in principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). These metabolites performed well in receiver operating characteristic (ROC) analysis, suggesting they could be potential biomarkers for GHB exposure and addiction.

Gamma-hydroxybutyric acid (GHB) is a potent central nervous system depressant produced during gammaaminobutyric acid metabolism. Although it has therapeutic uses in conditions such as narcolepsy and alcohol withdrawal, GHB is often misused in drug-facilitated crimes. Monitoring its use is challenging owing to the difficulty in distinguishing endogenous from exogenous GHB and its rapid metabolism. Previous metabolomic studies have identified potential biomarkers for GHB exposure; however, controversies and limited detection windows have hindered their forensic and clinical utility. This study aimed to identify novel biomarkers of GHB intoxication by analyzing urinary metabolic alterations in rats administered a sedation-inducing dose (600 mg/ kg) of GHB. We used both targeted and non-targeted metabolomics using liquid chromatography-tandem mass spectrometry to assess metabolic alterations. Targeted analysis demonstrated significant increases in all identified GHB biomarker metabolite levels following administration. Through principal component analysis, nontargeted metabolomics revealed a clear separation between the control and GHB-treated groups, indicating disturbances in tryptophan metabolism. Notably, 4-guanidinobutyric acid (GBA) emerged as a novel biomarker for GHB intoxication, with urinary levels peaking at 3 h post-administration before declining. These findings highlight GHB-induced metabolic perturbations and indicate that GBA may serve as a valuable biomarker for the bioanalytical detection of GHB intoxication, with potential applications in forensic and clinical settings.

Substance use disorders, particularly drug addiction, are complex neurophysiological conditions characterized by cycles of compulsive drug use, withdrawal symptoms, and relapses. Methamphetamine (MA) addiction evolves through repeated exposure, altering brain circuits related to reward and neuroplasticity. The need for reliable biomarkers to diagnose and monitor MA addiction has become increasingly critical in clinical practice. In this study, we explored the time-dependent transcriptomic changes in the rat striatum immediately after short-term abstinence following MA self-administration. Using a rat model, we conducted RNA sequencing to analyze the transcriptomic alterations in the striatum immediately after the self-administration and short-term abstinence phases (12- and 24-h post-MA). Through protein–protein interaction (PPI) network analysis and gene expression pattern assessment, we identified key genes that demonstrated significant expression changes. These genes were strongly linked to reward mechanisms, synaptic plasticity, and memory processes, suggesting a role in mediating MA-associated behaviors. Understanding the expression dynamics of these genes provides valuable insights into the molecular mechanisms underlying MA addiction and offers a foundation for developing diagnostic tools and therapeutic strategies targeting addiction-related neural adaptations.

Amino acids and polyamines play essential roles in physiological processes, such as protein synthesis, neurotransmission, and cell growth, and are emerging as potential biomarkers for diseases including cancer and diabetes. The accurate quantification of these compounds in biological sample is challenging, particularly due to matrix effects during liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This study aimed to mitigate the matrix effects by employing a mixed-mode internal standard (IS) strategy that uses both isotopederivatized ISs (Method 1, with analyte standards derivatized using an isotopic reagent, BzCl-d5) and isotopic standards (Method 2, where isotopic analyte standards are derivatized with BzCl). A novel method was developed for the simultaneous quantitative analysis of 21 amino acids and three polyamines in rat urine and plasma samples using LC-MS/MS with benzoyl chloride derivatization. To improve analytical accuracy, the two IS preparation techniques were explored and the initial results showed poor parallelism for several analytes using Method 1. However, significant improvements were observed with Method 2, highlighting the impact of the IS strategy on reducing the matrix effects and improving quantification accuracy. By combining both approaches, we successfully achieved accurate quantification of the target compounds in biological matrices. This methodology offers a powerful tool for investigating metabolic alterations in diseases, enhancing our understanding of disease pathology and aiding in biomarker identification.

Endogenous tricarboxylic acid (TCA) cycle metabolites are critical biomarkers of metabolic perturbation; however, their accurate quantification is complicated by the absence of analyte-free matrices, variable endogenous backgrounds, and matrix effects. We developed and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for 10 TCA metabolites across rat urine (Method 1), rat plasma (Method 2), and human hepatic organoid medium (Method 3). Water was used to prepare calibrators for each matrix, and the validity of this approach was rigorously evaluated with assessments of parallelism and matrix effects. Validation demonstrated selectivity, sensitivity, accuracy, and precision with acceptable linearity across the calibration ranges in all matrices. The developed methods were applied to both in vivo and in vitro models of ethanol exposure in rats (4 g/kg ethanol, 40% v/v, oral gavage at 12 h intervals for 3 days) and human hepatic organoids (200 mM ethanol for 24 h). The analyses revealed that ethanol induced distinct matrix-specific perturbations of the TCA cycle. In rats, the disruption pattern was consistent with a bottleneck in specific TCA-related enzyme activities, whereas in human hepatic organoids, the alterations highlighted impaired energy metabolism and compromised hepatic function. These findings highlight the importance of accurate quantification of TCA cycle metabolites in both target organs and peripheral fluids. Matrix-specific LC-MS/MS methods enable reliable quantification of endogenous TCA cycle metabolites and support mechanistic interpretation of ethanol-related metabolic dysregulation.

Tumor-associated macrophages (TAMs) constitute 50-80% of stromal cells in most solid tumors with high mortality and poor prognosis. Tumor-infiltrating dendritic cells (TIDCs) and TAMs are key components mediating immune responses within the tumor microenvironment (TME). Considering their refractory properties, simultaneous remodeling of TAMs and TIDCs is a potential strategy of boosting tumor immunity and restoring immunosurveillance. In this study, mannose-decorated poly(lactic-co-glycolic acid) nanoparticles loading with R848 (Man-pD-PLGA-NP@R848) were prepared to dually target TAMs and TIDCs for efficient tumor immunotherapy. The three-dimensional (3D) cell culture model can simulate tumor growth as influenced by the TME and its 3D structural arrangement. Consequently, cancer spheroids enriched with tumor-associated macrophages (TAMs) were fabricated to assess the therapeutic effectiveness of Man-pD-PLGA-NP@R848. In the TME, Man-pD-PLGA-NP@R848 targeted both TAMs and TIDCs in a mannose receptor-mediated manner. Subsequently, Man-pD-PLGA-NP@R848 released R848 to activate Toll-like receptors 7 and 8, following dual-reprograming of TIDCs and TAMs. Man-pD-PLGA-NP@R848 could uniquely reprogram TAMs into antitumoral phenotypes, decrease angiogenesis, reprogram the immunosuppressive TME from "cold tumor" into "hot tumor", with high CD4+ and CD8+ T cell infiltration, and consequently hinder tumor development in B16F10 tumor-bearing mice. Therefore, dual-reprograming of TIDCs and TAMs with the Man-pD-PLGA-NP@R848 is a promising cancer immunotherapy strategy.

Neurological disorders, encompassing conditions such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS), pose a significant global health challenge, affecting millions worldwide. With an aging population and increased life expectancy, the prevalence of these disorders is escalating rapidly, leading to substantial economic burdens exceeding trillions of dollars annually. Animal models play a crucial role in understanding the underlying mechanisms of these disorders and developing effective treatments. Various species, including rodents, non-human primates, and fruit flies, are utilized to replicate specific aspects of human neurological conditions. However, selecting the ideal animal model requires careful consideration of its proximity to human disease conditions and its ability to mimic disease pathobiology and pharmacological responses. An Animal Model Quality Assessment (AMQA) tool has been developed to facilitate this selection process, focusing on assessing models based on their similarity to human conditions and disease pathobiology. Therefore, integrating intrinsic and extrinsic factors linked to the disease into the study's objectives aids in constructing a biological information matrix for comparing disease progression between the animal model and human disease. Ultimately, selecting an ideal animal disease model depends on its predictive, face, and construct validity, ensuring relevance and reliability in translational research efforts.

Persistent neurochemical and biological disturbances resulting from repeated cycles of drug reward, withdrawal, and relapse contribute to drug dependence. Methamphetamine (MA) is a psychostimulant with substantial abuse potential and neurotoxic effects, primarily affecting monoamine neurotransmitter systems in the brain. In this study, we aimed to explore the progression of drug dependence in rat models of MA self-administration, extinction, and reinstatement through targeted and non-targeted metabolomics analyses. Metabolic profiles were examined in rat plasma during the following phases: after 16 days of MA self-administration (Group M); after 16 days of self-administration followed by 14 days of extinction (Group MS); and after self-administration and extinction followed by a reinstatement injection of MA (Group MSM). Each group of MA self-administration, extinction, and reinstatement induces distinct changes in the metabolic pathways, particularly those related to the TCA cycle, arginine and proline metabolism, and arginine biosynthesis. Additionally, the downregulation of glycerophospholipids and sphingomyelins in Group MSM suggests their potential role in MA reinstatement. These alterations may signify the progressive deterioration of these metabolic pathways, possibly contributing to drug dependence following repeated cycles of drug reward, withdrawal, and relapse. These results provide valuable insights into the metabolic changes associated with MA use at various stages, potentially facilitating the discovery of early diagnostic biomarkers and therapeutic targets for MA use disorders.

Electric fields affect the activity of neurons and brain circuits, yet how this happens at the cellular level remains enigmatic. Lack of understanding of how to stimulate the brain to promote or suppress specific activity significantly limits basic research and clinical applications. Here, we study how electric fields impact subthreshold and spiking properties of major cortical neuronal classes. We find that neurons in the rodent and human cortex exhibit strong, cell-class-dependent entrainment that depends on stimulation frequency. Excitatory pyramidal neurons, with their slower spike rate, entrain to both slow and fast electric fields, while inhibitory classes like Pvalb and Sst (with their fast spiking) predominantly phase-lock to fast fields. We show that this spike-field entrainment is the result of two effects: non-specific membrane polarization occurring across classes and class-specific excitability properties. Importantly, these properties are present across cortical areas and species. These findings allow for the design of selective and class-specific neuromodulation.

The dynamic equilibrium between acetylation and deacetylation is vital for cellular homeostasis. Parkinson’s disease (PD), a neurodegenerative disorder marked by α-synuclein (α-syn) accumulation and dopaminergic neuron loss in the substantia nigra, is associated with a disruption of this balance. Therefore, correcting this imbalance with histone deacetylase (HDAC) inhibitors represents a promising treatment strategy for PD. CAY10603 (CAY) is a potent and selective HDAC6 inhibitor. However, because of its poor water solubility and short biological half-life, it faces clinical limitations. Herein, we engineered lactoferrin-decorated CAY-loaded poly(lactic-co-glycolic acid) nanoparticles (denoted as PLGA@CAY@Lf NPs) to effectively counter methamphetamine (Meth)-induced PD. PLGA@CAY@Lf NPs showed enhanced blood–brain barrier crossing and significant brain accumulation. Notably, CAY released from PLGA@CAY@Lf NPs restored the disrupted acetylation balance in PD, resulting in neuroprotection by reversing mitochondrial dysfunction, suppressing reactive oxygen species, and inhibiting α-syn accumulation. Additionally, PLGA@CAY@Lf NPs treatment normalized dopamine and tyrosine hydroxylase levels, reduced neuroinflammation, and improved behavioral impairments. These findings underscore the potential of PLGA@CAY@Lf NPs in treating Meth-induced PD and suggest that an innovative HDAC6-inhibitor-based strategy can be used to treat PD.

Novel psychoactive substances (NPSs) are new psychotropic drugs designed to evade substance regulatory policies. 25E-NBOMe (2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine) has recently been identified as an NPS, and its recreational misuse has been reported to be rapidly increasing. However, the psychopharmacological effects and mechanisms of 25E-NBOMe have not been studied. We examined the abuse potential of 25E-NBOMe using the conditioned place preference in male mice and self-administration paradigms in male rats. Additionally, immunoblot assay, enzyme-linked immunosorbent assay, and microdialysis were used to determine the molecular effects of 25E-NBOMe in the nucleus accumbens (NAc). Our data demonstrated that 25E-NBOMe induces conditioned place preference, and the dopaminergic signaling in the NAc mediates these. Following 25E-NBOMe administration, expression of dopamine transporter and dopamine D1 receptor (D1DR) were enhanced in the NAc of male mice, and NAc dopamine levels were reduced in both male mice and rats. Induction of intracellular dopaminergic pathways, DARPP32, and phosphorylation of CREB in the NAc of male mice was also observed. Significantly, pharmacological blockade of D1DR or chemogenetic inhibition of D1DR-expressing medium spiny neurons in the NAc attenuated 25E-NBOMe-induced conditioned place preference in male mice. We also examined the hallucinogenic properties of 25E-NBOMe using the head twitch response test in male mice and found that this behavior was mediated by serotonin 2A receptor activity. Our findings demonstrate that D1DR signaling may govern the addictive potential of 25E-NBOMe. Moreover, our study provides new insights into the potential mechanisms of substance use disorder and the improvement of controlled substance management.

Cancer immunotherapy is a groundbreaking strategy that has revolutionized the field of oncology compared to other therapeutic strategies, such as surgery, chemotherapy, or radiotherapy. However, cancer complexity, tumor heterogeneity, and immune escape have become the main hurdles to the clinical application of immunotherapy. Moreover, conventional immunotherapies cause many harmful side effects owing to hyperreactivity in patients, long treatment durations and expensive cost. Nanotechnology is considered a transformative approach that enhances the potency of immunotherapy by capitalizing on the superior physicochemical properties of nanocarriers, creating highly targeted tissue delivery systems. These advantageous features include a substantial specific surface area, which enhances the interaction with the immune system. In addition, the capability to finely modify surface chemistry enables the achievement of controlled and sustained release properties. These advances have significantly increased the potential of immunotherapy, making it more powerful than ever before. In this review, we introduce recent nanocarriers for application in cancer immunotherapy based on strategies that target different main immune cells, including T cells, dendritic cells, natural killer cells, and tumor-associated macrophages. We also provide an overview of the role and significance of nanotechnology in cancer immunotherapy.

Betaine-homocysteine S-methyltransferase (BHMT) is one of the most abundant proteins in the liver and regulates homocysteine metabolism. However, the molecular mechanisms underlying Bhmt transcription have not yet been elucidated. This study aimed to assess the molecular mechanisms underlying Bhmt transcription and the effect of BHMT deficiency on metabolic functions in the liver mediated by liver receptor homolog-1 (LRH-1). During fasting, both Bhmt and Lrh-1 expression increased in the liver of Lrh-1f/f mice; however, Bhmt expression was decreased in LRH-1 liver specific knockout mice. Promoter activity analysis confirmed that LRH-1 binds to a specific site in the Bhmt promoter region. LRH-1 deficiency was associated with elevated production of reactive oxygen species (ROS), lipid peroxidation, and mitochondrial stress in hepatocytes, contributing to hepatic triglyceride (TG) accumulation. In conclusion, this study suggests that the absence of an LRH-1-mediated decrease in Bhmt expression promotes TG accumulation by increasing ROS levels and inducing mitochondrial stress. Therefore, LRH-1 deficiency not only leads to excess ROS production and mitochondrial stress in hepatocytes, but also disrupts the methionine cycle. Understanding these regulatory pathways may pave the way for novel therapeutic interventions against metabolic disorders associated with hepatic lipid accumulation.

Age-related macular degeneration (AMD) severely affects central vision due to progressive macular degeneration and its staggering prevalence is rising globally, especially in the elderly population above 55 years. Increased oxidative stress with aging is considered an important contributor to AMD pathogenesis despite multifaceted risk factors including genetic predisposition and environmental agents. Wet AMD can be managed with routine intra-vitreal injection of angiogenesis inhibitors, but no satisfactory medicine has been approved for the successful management of the dry form. The toxic carbonyls due to photo-oxidative degradation of accumulated bisretinoids within lysosomes initiate a series of events including protein adduct formation, impaired autophagy flux, complement activation, and chronic inflammation, which is implicated in dry AMD. Therapy based on antioxidants has been extensively studied for its promising effect in reducing the impact of oxidative stress. This paper reviews the dry AMD pathogenesis, delineates the effectiveness of dietary and nutrition supplements in clinical studies, and explores pre-clinical studies of antioxidant molecules, extracts, and formulations with their mechanistic insights.