Many disease-associated genetic variants likely exert context-dependent regulatory effects that are underrepresented in healthy, steady-state adult tissues. To investigate gene regulation across diverse environmental and cellular contexts, we exposed heterogeneous differentiating cultures from 51 human induced pluripotent stem cell lines to nicotine, caffeine, or ethanol and profiled 1.4 million cells by single-cell RNA sequencing. We identified hundreds of response expression quantitative trait loci (eQTLs) across diverse cell types, as well as dynamic regulatory effects that vary along differentiation trajectories. In contrast to baseline steady-state eQTLs and similar to disease risk loci, response eQTLs are enriched in distal enhancers and regulate genes under strong selective constraint, contain complex regulatory landscapes, and display diverse biological functions. Response eQTLs also coincide with disease-associated loci not explained by baseline steady-state eQTLs. These findings indicate that interpreting disease-associated loci will benefit from studying gene-by-environment interactions across diverse biological contexts.

Changes in cytoplasmic free Mg[2+] concentration ([Mg[2+]]cyto) might play a key role in regulating skeletal muscle function. Mag-indo-1, a fluorescence dye that specifically binds to Mg[2+], can be used both to detect and buffer [Mg[2+]]cyto. In this study, we hypothesized that buffering the fatigue-induced elevations in [Mg[2+]]cyto would mitigate fatigue by preserving sarcoplasmic reticulum (SR) Ca[2+] release. Intact fibers were isolated from the flexor digitorum brevis muscle, and loaded with varying concentration of Mag-indo-1-AM. The fibers were repeatedly stimulated at 70 Hz for 300 msec every 2 sec for 100 contractions (fatiguing stimulation: FS). To examine the relationship between fatigue and Mg[2+] buffering, we compared the force produced during the final contraction of FS with the amount of Mag-indo-1 loading before FS (Fatigue-Mg[2+] buffer relationship). Fatigue-Mg[2+] buffer relationship exhibited an inverted U-shaped curve: fatigue resistance improved when [Mag-indo-1] increased up to an optimal level, but declined when [Mag-indo-1] became excessive. Therefore, the fibers were classified into three groups: underloaded, optimal-loaded and overloaded groups. Caffeine treatment immediately after FS significantly enhanced force recovery in the underloaded group, reaching a level comparable to that of caffeine-treated, optimal-loaded group. Furthermore, mitochondrial coupled respiration was impaired, and proton leak was enhanced in the presence of nM level Mag-indo-1. These results suggest that elevated [Mg[2+]]cyto contributes to muscle fatigue due to impairments of SR Ca[2+] release. Conversely, excessive buffering of [Mg[2+]]cyto may also exacerbate muscle fatigue, indicating that a proper [Mg[2+]]cyto balance is critical for maintaining fatigue resistance.

Tea (Camellia sinensis) quality is determined by key metabolites including catechins, caffeine, theanine, and volatiles, yet their biosynthesis and spatial distribution at the cellular level remain poorly understood. Here, we constructed a cell-type-resolved spatial multi-omics atlas of tea leaves from C. sinensis var. sinensis 'Jinxuan' and C. sinensis var. assamica 'Yinghong No. 9' by integrating single-nucleus RNA sequencing (snRNA-seq), physically isolated cell-type multi-omics, and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Multi-omics analyses identified mesophyll cells as the major site of catechin biosynthesis, particularly for terminal esterification steps. Theanine biosynthesis was inferred to be predominantly associated with mesophyll cells but accumulated in both mesophyll and vascular bundle cells, consistent with the distribution of its precursors, glutamate and glutamine. Caffeine biosynthesis and accumulation were mainly associated with mesophyll cells, whereas other alkaloids showed vascular-associated accumulation patterns. Benzenoid and fatty acid-derived volatiles showed broader cellular distributions, whereas monoterpene biosynthesis was mainly associated with mesophyll and epidermal cells and their glycosylated derivatives preferentially accumulated in vascular tissues. Further comparison of the two cultivars showed that linalool and geraniol displayed distinct cultivar-dependent accumulation patterns associated with cell density, synthase expression, and enzyme catalytic properties. Overall, key flavor-related metabolites are mainly synthesized in mesophyll cells, whereas vascular bundle cells may contribute to their accumulation and redistribution. These findings reveal the cellular organization of tea flavor metabolism and provide a cellular and molecular framework for future evaluation of tea germplasm with desirable flavor traits.

In Parkinson's disease (PD), the limitations in disease-modifying therapies have broadened the concept of neuroprotection beyond targeting specific pathogenic pathways to include the strengthening of motor reserve and resilience through modifiable lifestyle factors. This review synthesizes current evidence on lifestyle medicine for PD, focusing on its potential contribution to neuroprotection, symptom control, and implementation in Asian contexts. We first discuss physical activity and exercise, including community-based and technology-assisted approaches, as interventions that improve motor and non-motor outcomes and may have long-term disease-modifying potential. We then review nutrition from prevention-oriented, genetic, and disease-stage-specific perspectives, including dietary patterns, caffeine and tea exposure, and nutritional vulnerability in established PD. Sleep health and stress management are considered as interconnected determinants of daily function, quality of life (QoL), and resilience. We further discuss integrated lifestyle medicine as a multidomain care model that combines core lifestyle pillars with personalized, sustainable, and culturally responsive implementation. Future directions may include rigorous validation, practical implementation models, and digitally enabled monitoring tailored to diverse Asian healthcare systems. Although definitive disease-modifying effects remain unproven, integrated lifestyle medicine may offer a practical foundation for translating accumulating lifestyle evidence into more rigorous and scalable neuroprotective strategies for PD.

Coffee pulp (CP) and coffee husk (CH) are abundant agro-industrial residues with potential for biomethane production through anaerobic digestion (AD), but their contrasting characteristics impose different conversion constraints. This review critically examines AD of CP and CH, focusing on feedstock characteristics, inhibition, pretreatment, process enhancement, and scale-up. CP is characterised by high moisture, a relatively accessible biodegradable fraction, acidity, soluble phenolics and caffeine, and susceptibility to rapid acidification, whereas CH is drier and more structurally recalcitrant, with hydrolysis and lignocellulosic accessibility as major limitations. Phenolic, caffeine, tannin, furanic, and pretreatment-derived inhibitors are examined in relation to their concentration-dependent and combined effects on anaerobic microbial activity. Physical, thermal, hydrothermal, chemical, biological, and hybrid pretreatments are compared, with emphasis on trade-offs among substrate accessibility, methane recovery, inhibitor formation, and energy and chemical requirements. Process-enhancement strategies, including adsorbents and conductive materials, trace-element supplementation, co-digestion, and semi-continuous, continuous, and two-stage digestion, are evaluated using quantitative evidence where available. Energy balance, techno-economic and life-cycle performance, feedstock logistics, regional applicability, system configuration, and scale-up are also considered. CP is primarily suited to strategies targeting inhibitor control, nutrient and buffering management, and process stability, whereas CH requires greater emphasis on structural deconstruction and hydrolysis. Future research should prioritise residue-specific optimisation, long-term reactor validation, integrated techno-economic and life-cycle assessment, and pilot-scale demonstration. A constraint-driven pathway linking feedstock characterisation, constraint identification, targeted intervention, long-term validation, and scale-up is proposed for sustainable coffee-residue biomethane production.

Green tea contains bioactive components with varying polarities. Single deep eutectic solvents (DESs) struggle to achieve simultaneous extraction of different polarity targets. This study developed a temperature-responsive biphasic DES (TRBDES) system for the selective extraction of six bioactive substances with distinct polarities from green tea. The system exhibited reversible biphasic-monophasic transition via temperature modulation. Under optimal conditions, the total extraction efficiency reached 158.24 ± 6.30 mg/g, significantly higher than conventional methods. Hydrophilic (e.g., EGCG) and hydrophobic (e.g., chlorophyll) components were almost completely partitioned into the lower and upper phases, respectively, while caffeine was distributed between both phases. Notably, the TRBDES extracts exhibited superior antioxidant activity (4.4-fold increase over ethanol extraction) and low melting points, confirming structural formation and functionality. Density functional theory calculations elucidated the interaction mechanisms. Demonstrating environmental friendliness, operational simplicity, and strong cell fiber structure disruption capability, this system provides a novel strategy for multi-polarity bioactive substance extraction.

Background/Objectives: Origanum majorana L. has reported antioxidant and other bioactive properties, but its effects on excitability and intracellular Ca[2+] handling in excitable cells remain poorly defined. We investigated how a hydroalcoholic marjoram extract modulates membrane ion currents, cytosolic Ca[2+] signaling, and MTT-reducing activity in bovine chromaffin cells and primary rat cortical neurons. Methods: A pressurized liquid O. majorana extract (ethanol:water, 70:30) was chemically characterized by HPLC-PAD. Whole-cell patch-clamp and current-clamp recordings were used to assess voltage-dependent Ca[2+], Na[+], and K[+] currents and membrane excitability in bovine chromaffin cells. Fluo-4-AM measurements were used to evaluate Ca[2+] responses to K[+], caffeine, and histamine. MTT assays assessed changes in MTT-reducing activity under veratridine-induced Ca[2+] overload and oligomycin/rotenone-induced mitochondrial stress in bovine chromaffin cells and cortical neurons prepared from embryonic day 18 Sprague-Dawley rats. For inferential statistics, replicate cells or wells from the same primary culture were averaged and the independent culture was used as the biological unit. Results: Culture-level analysis confirmed significant reductions in voltage-dependent Ca[2+] and Na[+] currents at 1.5 µg/mL (1 µL/mL) marjoram extract, together with significant overall effects on the putative Ca[2+]-dependent and voltage-dependent K[+] current components. The extract depolarized the resting membrane potential and markedly suppressed repetitive action-potential firing. Mean K[+]-evoked Fluo-4 responses were reduced by approximately 20%, although this effect did not reach statistical significance at the culture level. Caffeine-evoked Ca[2+] responses were higher in the presence of 0.015-1.5 µg/mL (0.01-1 µL/mL) marjoram extract, but these differences did not remain significant after correction for multiple comparisons. Histamine-evoked responses were unchanged. In MTT assays, significant preservation of MTT-reducing activity was limited to selected conditions, mainly at lower extract concentrations in bovine chromaffin cells, whereas the neuronal comparisons did not remain significant after multiplicity correction. Because extract-only, concentration-matched vehicle, cell-free extract/MTT interference controls, and an independent orthogonal assay of cell number or membrane integrity were not included, the MTT findings are reported strictly as changes in MTT-reducing activity and not as direct evidence of cytoprotection or neuroprotection. Conclusions:O. majorana extract modulates membrane excitability through effects on multiple plasma-membrane ion currents. The higher mean caffeine-evoked Ca[2+] responses are compatible with altered Ca[2+] mobilization from ryanodine-sensitive intracellular stores, but they do not demonstrate direct activation or sensitization of RyR receptors. The MTT findings do not establish cytoprotection or neuroprotection. These findings are correlative, and a causal contribution of ryanodine-sensitive intracellular stores to the preservation of MTT-reducing cellular metabolic activity remains to be established.

Background: Caffeine is a widely consumed psychostimulant that may affect anxiety-like, aggression-like, and social behaviors, potentially in association with oxidative imbalance. Objectives: This study evaluated the behavioral and biochemical effects of acute caffeine exposure in adult zebrafish and investigated the potential protective effects of N-acetylcysteine (NAC). Methods: Sixty adult wild-type AB zebrafish (Danio rerio), approximately 9-10 months old, were assigned to six groups (n = 10/group): control, caffeine (25 or 60 mg/L), NAC (1 mg/L), and NAC combined with either caffeine concentration. Behavioral effects were assessed using the Social Preference Test, Mirror-Biting Test, and Novel Tank Test. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were measured as oxidative stress-related markers. Results: Caffeine increased locomotor activity and induced anxiety-like and aggression-like behavioral alterations, including increased bottom-dwelling, thigmotaxis, and mirror-directed behavior, while reducing social preference. Caffeine significantly increased SOD activity at 25 mg/L (p = 0.010) and 60 mg/L (p = 0.008), and GPx activity at 25 mg/L (p = 0.008) and 60 mg/L (p = 0.006), compared with controls. NAC attenuated caffeine-induced hyperlocomotion and several anxiety-like and aggression-like alterations and modulated antioxidant enzyme responses, but did not fully restore social preference. Conclusions: Acute caffeine exposure produces behavioral alterations and changes in antioxidant enzyme activity in adult zebrafish. NAC partially attenuates these effects, particularly hyperlocomotion, anxiety-like behavior, and aggression-like responses, while showing more limited effects on caffeine-induced social alterations.