5-Hydroxytryptophan (5-HTP) is a functional precursor of serotonin; however, achieving sustained 5-HTP production in vivo by engineered microbes remains challenging. To address this, we developed a genome-integrated engineered probiotic system enabling gut-local biosynthesis of 5-HTP. Escherichia coli Nissle 1917 (EcN), a clinically validated probiotic with an established safety profile for intestinal application, was selected as the chassis. Constitutive expression was employed to eliminate inducer dependence, while genomic integration was used to enhance genetic stability and avoid reliance on antibiotic selection for pathway maintenance. In addition, BH4 biosynthesis and regeneration modules were incorporated to address the limited endogenous BH4 availability in the gut environment. Different administration strategies were systematically evaluated to characterize in vivo performance. Notably, our results showed that the hTPH2 hydroxylation module, together with the BH4 biosynthesis and regeneration system, supported 5-HTP biosynthesis in EcN. The engineered EcN strain achieved a 5-HTP titer of 863.2 mg/L, the highest level reported in EcN-based systems. Under non-selective conditions, EcN[Pconst-5-HTP] carrying the chromosomally integrated pathway maintained stable 5-HTP production in vitro and retained 5-HTP-producing capacity after intestinal passage, whereas plasmid loss in non-integrated constructs resulted in a pronounced decrease in production. In vivo analysis showed that single-dose administration induced a transient elevation in plasma 5-HTP, whereas repeated dosing generated reproducible, time-dependent fluctuations in fecal bacterial abundance and gut-local biosynthetic activity. Treatment with the engineered probiotic further improved behavioral phenotypes and modulated brain 5-HT-related metabolites in a reserpine-induced mouse model. Together, these findings provide a translational framework linking in vitro 5-HTP production by engineered probiotics to their in vivo functional performance.

Tilia americana var. mexicana is a medicinal species whose reported pharmacological effects primarily target the central nervous system, including anxiolytic, antidepressant, and anticonvulsant activities, attributed in part to flavonoids such as tiliroside (8). In this study, 14 compounds were isolated and identified from the methanolic extract (Ta-MeOH) and evaluated by molecular docking against the monoamine oxidases MAO-A and MAO-B, enzymes implicated in Parkinson's disease (PD) due to their involvement in dopamine metabolism. Chemical analysis identified six terpenes: α- and β-amyrine (1-2), β-sitosterol (3), stigmasterol (4), ursolic acid (5), and β-sitosterol glucoside (6); the novel diglycosylated monoterpene 4α-terpineol sambubioside (7), characterized through its hexaacetate derivative (7a); the flavonoids tiliroside (8) and rutin (9); sucrose (10); and four phenolic compounds: scopoletin (11), caffeic acid (12), coumaric acid (13), and chlorogenic acid (14). Molecular docking against monoamine oxidases A and B (MAO-A and MAO-B) was used as a computational strategy to prioritize isolated metabolites for biological evaluation. β-Sitosterol glucoside (6), 4α-terpineol sambubioside (7), tiliroside (8) and rutin (9) exhibited the most favorable docking scores toward MAO-B, with predicted binding energies of -10.8733, -11.1434, -12.16986 and -12.9474 kcal/mol, respectively. Considering the docking results together with phytochemical and experimental criteria, (6) and (8) (1 mg/kg) were prioritized for biological evaluation, together with Ta-MeOH extract (100 mg/kg) and selected fractions (25 mg/kg), in a reserpine-induced mouse model of parkinsonism using L-DOPA (150 mg/kg) as a positive control. In the open field test, spontaneous locomotor activity was assessed by total crossings and expressed as AUC. Ta-MeOH significantly increased locomotor activity compared with the reserpine-treated group (AUC: 1281.8 vs. 551.7, respectively; p < 0.05). Tiliroside (8) also increased locomotor activity (AUC: 858). In the Rota-Rod test, fine motor coordination was assessed by latency to fall and expressed as AUC. Ta-MeOH showed the greatest recovery, with AUC values of 431, 89, and 34 at 4, 10, and 20 rpm, respectively. In addition, treatments derived from T. americana attenuated behavioral alterations induced by reserpine in the Irwin test. Overall, this study demonstrates that integrating phytochemical isolation, molecular docking, and in vivo pharmacological evaluation provides a useful strategy for prioritizing bioactive metabolites from T. americana. These findings support further pharmacological investigation of this medicinal species and its isolated metabolites, while additional studies are required to establish their molecular targets and mechanisms of action.

Supercritical fluid extraction coupled on-line with supercritical fluid chromatography creates a powerful multidimensional analysis platform, but its adoption is limited by the resource-heavy process of method development. This research presents a surrogate optimization approach as a better alternative to traditional design of experiments and response surface methodology. Unlike fixed experimental designs, the surrogate model iteratively updates the response surface after each run, facilitating effective global optimization through continuous exploration and exploitation. The study adjusts both SFE- and SFC-specific variables-such as back-pressure regulation and split flow-to reduce peak FWHM and enhance extraction efficiency. Analytes, specifically reserpine, vigabatrin, hydromorphone, and hydrocodone, covering diverse physicochemical profiles, were optimized sequentially, guided by a molecular similarity metric, to broaden the design space. The surrogate model identified critical factors, including modifier concentration, chromatography pressure, dynamic extraction time, and flow rate, with adjusted coefficient of determination (R[2]) values reaching 0.97 as more data was collected and the model was refined. Response surface and parallel coordinate analyses showed that hydrocodone, hydromorphone, and vigabatrin favored high modifier concentrations, while reserpine had a distinct optimum more sensitive to static extraction time. Sensitivity analysis over 62.5-1000 ng mL[-1] yielded detection limits/quantification limits of 6.9/23.0 and 2.4/8.1 ng mL[-1] for hydrocodone and reserpine, respectively. Sequential optimization enhanced sensitivity by 15%, demonstrating the scalability and transferability of the surrogate optimization framework for developing SFE-SFC methods.

Terrestrial jumping is a typical evolutionarily conserved 'survival' fish behavior. Zebrafish (Danio rerio) are widely used as a model organism in CNS research and drug discovery. Here, we explore the potential utility of the terrestrial jumping assay to characterize behavioral effects of a wide range of pharmacological and other experimental manipulations in adult zebrafish. For this, zebrafish were individually placed in a large glass tank and their jumping behavioral profile was assessed for 60 s by video-recording and subsequent off-line analyses of escape-like jumps and immobility episodes in a slow mode. Treatment groups including acute exposure to ethanol, arecoline, caffeine, alarm pheromone and live predator (Nandus nandus), as well as chronic exposure to neuroactive drugs fluoxetine, reserpine, cuprizone and rotenone. Overall, the assay shows high sensitivity to various experimental manipulations, supporting the utility of the terrestrial jumping assay as a fast, low-cost and single-trial behavioral paradigm to assess both motor phenotypes and affective-like responses in zebrafish.

This paper proposes a plasma modification-trichlorosilane impregnation composite process for fabricating paper microfluidic channels, which is applied to paper spray mass spectrometry detection of prohibited drugs in serum samples from health supplement addition. Plasma modification plays a dual core role: precisely constructing hydrophilic microchannels on silane-impregnated filter paper and stripping surface residual impurities and ash to optimize physicochemical properties. The plasma-modified microchannel paper spray ionization system (PM-PSI) significantly reduces sample loss during migration versus conventional paper spray ionization (PSI), thus enhancing the detection signal of target analytes in blood samples. Combined with filter paper's matrix effect mitigation and plasma-induced surface optimization, PM-PSI exhibits better detection efficiency than traditional electrospray ionization (ESI) for direct blood sample analysis. Performance validation with venlafaxine showed that PM-PSI increased the signal intensity by 3-5 times and reduced the relative standard deviation (RSD) by 5-6 times compared with PSI at 50-500 ng/mL, and the signal intensity was 10-30 times higher than that of ESI. Quantitative analysis of four typical prohibited drugs (venlafaxine, reserpine, risperidone, sildenafil) revealed their linear range of 1-1000 ng/mL with a significantly improved coefficient of determination (R[2]) versus PSI. Moreover, PM-PSI coupled with a domestic miniaturized mass spectrometer successfully detected the parent ions of the four drugs at 300 ng/mL, confirming its suitability for complex sample analysis of miniaturized mass spectrometers and providing a technical solution for rapid on-site testing.

Monoaminergic signaling controls many aspects of human physiology, including autonomic responses, movement, and emotion. The vesicular monoamine transporters (VMAT1 and VMAT2) sequester monoamines into synaptic vesicles within the central and peripheral nervous system. Dysregulation of VMAT function contributes to neuropsychiatric and neurodegenerative disorders, including Parkinson's disease, depression, and psychostimulant substance abuse. Clinically, VMATs are targeted by the inhibitors tetrabenazine and reserpine, used to treat hyperkinetic movement disorders and hypertension, respectively, while amphetamines exploit VMAT2-mediated dopamine efflux to treat ADHD. Emerging VMAT2-selective compounds such as lobeline derivatives and GZ-11610 attenuate psychostimulant reinforcement. Tricyclic antidepressants upregulate VMAT2 function and rescue disease-causing variants, highlighting VMAT2 as a promising therapeutic target. Despite their importance, the molecular mechanisms driving proton-coupled transport, inhibition, and psychostimulant action remain poorly understood. This review summarizes current biochemical, structural, and functional insights into VMATs, highlighting proton coupling, transport kinetics, inhibition mechanisms, unresolved questions, and future research directions.

The present study investigated the therapeutic potential of quercetin nanoparticles (QNPs), administered alone or in combination with levodopa/carbidopa (L/C), against reserpine-induced neurochemical alterations and motor dysfunction in a rat model of Parkinson's disease (PD). Animals were randomly assigned to five experimental groups: a control group, a reserpine-induced PD group, and PD groups treated with QNPs, L/C, or their combination. Motor performance was assessed using the open-field and grid traction tests. Reserpine administration induced marked motor impairments accompanied by significant reductions in dopamine (DA), serotonin (5-HT), and norepinephrine (NE) levels in both the striatum and midbrain. These neurochemical deficits were associated with elevated monoamine oxidase (MAO) activity, increased lipid peroxidation (malondialdehyde, MDA), enhanced acetylcholinesterase (AChE) activity, and increased nitric oxide (NO) levels in both brain regions. Additionally, reduced glutathione (GSH) levels were decreased in the midbrain but elevated in the striatum. Treatment with QNPs significantly restored monoamine levels and normalized MAO and AChE activities. Moreover, QNPs markedly attenuated oxidative stress and improved motor performance in reserpine-treated rats. Similarly, L/C administration effectively reversed monoamine depletion, normalized enzymatic activities, and mitigated oxidative stress. Notably, combined treatment with QNPs and L/C produced effects comparable to those observed with either treatment alone, suggesting a lack of additive or synergistic interaction. These findings demonstrate that QNPs exert a significant neuroprotective effect against reserpine-induced motor dysfunction and neurochemical disturbances, supporting their potential as a therapeutic strategy for Parkinsonian pathology.

Nociplastic pain results from altered nociceptive processing without apparent tissue damage. Effective treatment remains challenging due to limited biomarker availability and poor response to conventional analgesics. Transient receptor potential ankyrin 1 (TRPA1) is a promising molecular target because of its role in pain hypersensitivity and neuroinflammatory signaling. Preclinical studies indicate that TRPA1 antagonists reduce nociception in various nociplastic pain models. However, their therapeutic significance has not been systematically evaluated. This systematic review and meta-analysis summarize preclinical evidence regarding TRPA1 antagonists as modulators of nociplastic pain hypersensitivity. The protocol was registered in PROSPERO (CRD42025629227). A systematic search of PubMed, Scopus, and Web of Science identified studies published from 2011 to 2024. Twelve in vivo rodent studies met the inclusion criteria, assessing TRPA1 antagonists in migraine-like nociception, chronic post-ischemia pain (CPIP), and reserpine-induced fibromyalgia models. Mechanical allodynia was the primary outcome. Data were pooled using a random-effects model, and heterogeneity was quantified using the I[2]. Study quality ranged from low to moderate, with recent publications revealing improved reporting standards and reduced risk of bias. Meta-analysis showed that TRPA1 antagonists produced consistent antinociceptive effects across nociplastic pain models, reducing mechanical and cold allodynia, with high-quality evidence for CPIP and migraine models. Consistent effects were observed across doses, administration routes, and antagonist types. No publication bias was detected, and sensitivity analyses validated the robustness of the findings. Despite variability in study quality, methodological limitations, and incomplete behavioral characterization, the evidence supports TRPA1 as an advantageous therapeutic target for prospective translational research.