Metabolites最新文献

Background/objectives: Caffeic acid is a hydroxycinnamic acid that has a wide range of applications in the medical field. The synthesis of caffeic acid using microbial fermentation technology is an environmentally friendly method.

Methods: By engaging various enzymes, specifically 4-hydroxyphenylacetate 3-monooxygenase (HpaB), sourced from diverse bacterial strains, we successfully engineered a functional version of this enzyme within Escherichia coli, enabling the production of caffeic acid. In addition to the two common tyrosine ammonia lyases (TAL) and HpaC, different combinations of HpaB demonstrated varying abilities in converting the substrate L-tyrosine into the desired product, caffeic acid.

Results: Under shake-flask culture conditions, the highest yield of caffeic acid was achieved with an enzyme mixture containing HpaB from Escherichia coli, reaching 75.88 mg/L. Enhancing the activity of the rate-limiting enzyme through engineering could potentially increase caffeic acid titer. This study aims to conduct a semi-rational design of HpaB through structure-based approaches to screen for mutants that can enhance the production of caffeic acid. Initially, the predicted three-dimensional structure of HpaB was generated using AlphaFold2, and subsequent analysis was conducted to pinpoint the critical mutation sites within the substrate-binding pocket. Five key amino acid residues (R113, Y117, H155, S210 and Y461) located in the vicinity of the flavin adenine dinucleotide binding domain in HpaB from Escherichia coli could be instrumental in modulating enzyme activity. Subsequently, the mutant S210G/Y117A was obtained by iterative saturation mutagenesis, which increased the titer of caffeic acid by 1.68-fold. The caffeic acid titer was further improved to 2335.48 mg/L in a 5 L fermenter. The findings show that the yield of caffeic acid was significantly enhanced through the integration of semi-rational design and fermentation process optimization.

Cardiovascular diseases (CVDs) have emerged as a common health problem. However, despite their prevalence, little progress has been made in their treatment. In recent years, neurotrophic factors (NTFs) have been discovered to exert cardioprotective functions for CVDs. NTFs can modulate vascular integrity, myocardial remodeling, angiogenesis, and autonomic regulation, playing the roles of maintaining cardiovascular homeostasis and influencing disease progression. Under pathological conditions, the supplement of NTFs can induce substantial adaptations to mitigate adverse cardiac responses. Several NTFs have been investigated in this regard. This review briefly elaborates on present insights into the expression, signaling pathways, and regulatory effects of NTFs on the development of CVDs, and also discusses emerging therapeutic strategies based on NTFs, ranging from exercise to advanced modalities including stem cell therapy, gene transfer, recombinant protein therapy and NTF mimetics, among which the mimetics and exercise interventions emerge as the most promising avenues for clinical translation.

Background: Advances in genomic technologies combined with tandem mass newborn screening have enabled early detection and management of several common inborn errors of metabolism. Fructose-1,6-bisphosphatase deficiency, an autosomal recessive treatable disorder reported in around 150 patients worldwide, remains underdiagnosed despite an excellent prognosis with early detection. Although common in highly consanguineous populations, diagnosis is often delayed due to the non-specific clinical and biochemical profile. Methods: This report explores the diagnostic pathway using first-tier next-generation sequencing of three novel cases of fructose-1,6-bisphosphatase deficiency in a tertiary care center in Lebanon. Results: Two patients were diagnosed with first-tier exome sequencing within one month of presentation and had an excellent outcome at 6 years of follow-up. The third patient, undiagnosed for 10 years, suffered from neurological sequalae. The molecular profile was remarkable in two patients for exon 2 deletion in the FBP1 gene, a founder mutation reported in Turkish and Armenian patients, and a rare frameshift mutation in the third case. Conclusions: The use of next-generation sequencing as as a first-tier test for FBP deficiency is a non-invasive and rapid method for early diagnosis and management of this rare yet treatable disorder. It can detect both disease-causing variants and large deletions, founder mutations as well, delineating the molecular profile in populations where this disorder is highly prevalent.

Background/objectives: Volatile organic compounds (VOCs) are emerging as non-invasive biomarkers of metabolic and disease-related processes, yet their reliable detection from complex biological matrices such as urine remains analytically challenging. This study aimed to establish a robust, non-targeted headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME GC-MS) workflow optimized for very small-volume urinary samples.

Methods: We systematically evaluated the effects of pH adjustment and NaCl addition on VOC extraction efficiency using a 75 µm CAR/PDMS fiber and a sample volume of only 0.75 mL. Method performance was further assessed using concentration-dependent experiments with representative VOC standards and by application to real human urine samples analyzed in technical triplicates.

Results: Acidification to pH 3 markedly improved extraction performance, increasing both total signal intensity and the number of detectable VOCs, whereas alkaline conditions and additional NaCl produced only minor effects. Representative VOC standards showed compound-specific linear dynamic ranges with minimal carry-over within the relevant analytical range. Application to real urine samples confirmed high analytical reproducibility, with triplicates clustering tightly in principal component analysis and most metabolites exhibiting relative standard deviations below 25%.

Conclusions: The optimized HS-SPME GC-MS method enables comprehensive, non-targeted urinary VOC profiling from limited sample volumes. This workflow provides a robust analytical foundation for exploratory volatilomics studies under sample-limited conditions and supports subsequent targeted method refinement once specific compounds or chemical classes have been prioritized.

Background/Objectives: Breast cancer is a prevalent and heterogeneous disease with multiple subtypes, which are defined by characteristics such as molecular biomarkers and metastatic status. This study aimed to profile the metabolic activity of various breast cancer subtypes, both with and without chemotherapy (doxorubicin) application. Methods: Six human breast cell lines were evaluated, two non-tumorigenic controls and four cancerous lines. The cancer lines were clustered as primary-derived, metastasis-derived, triple-negative (TNBC), and strong hormone receptor-positive (ER+/PR+) and analyzed using the Biolog phenotype mammalian microarrays (PM-M1 to PM-M8) to assess metabolic activity via NADH production under a wide array of substrate parameters. Results: Unique metabolic profiles emerged across the subtypes and clusters; the TNBC and metastatic cells demonstrated enhanced utilization of glycolytic and anaerobic substrates consistent with the Warburg effect. The ER+/PR+ cells showed heightened glucose utilization and unique sensitivity to metabolic effectors and doxorubicin. Additionally, significant metabolic differences were observed in nucleoside and amino acid utilization between cancer and control cells, particularly in metastatic and TNBC lines. Conclusions: Our findings reveal the profound metabolic diversity among breast cancer subtypes and highlight distinct substrate dependencies for proliferation. The results additionally provide a framework for developing metabolic biomarkers and targeted therapies for chemotherapy resistance in breast cancer subtypes.

Background/Objectives: Equol, a gut microbial metabolite of the soy isoflavone, daidzein, is associated with estrogenic activity and potential benefits for women's health. While equol production depends on individual gut microbial composition, its dietary and clinical correlates in young women remain incompletely characterized. This study explored the relationship between urinary equol production, dietary habits, and premenstrual symptom severity in healthy university-aged women. Methods: We conducted a cross-sectional study of 41 Japanese women, aged 19-20 years. Urinary equol was measured using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, following enzymatic hydrolysis. Participants were classified as either equol producers or non-producers, based on urinary concentration thresholds. Dietary intake was evaluated using a dietary questionnaire focused on soy products and dietary fiber sources. Premenstrual symptoms were assessed using a standardized Japanese questionnaire for premenstrual syndrome and premenstrual dysphoric disorder. Results: Twelve percent of participants were classified as equol producers. Compared with non-producers, equol producers reported higher consumption of pumpkin, soybean sprouts, and green tea. Among non-producers, higher consumption of certain vegetables and fiber-rich foods, including broccoli, pickled radish, konjac, and konjac jelly, was associated with greater premenstrual symptom severity, whereas such associations were not observed among equol producers. The analytical method demonstrated high sensitivity and reproducibility for urinary equol measurement. Conclusions: These findings suggest that equol production status may be associated with distinct dietary patterns and with differences in the relationship between food intake and premenstrual symptom severity in young women. Although the cross-sectional design and limited sample size preclude causal inference, these findings suggest that urinary equol is a promising candidate biomarker for future research on diet-related modulation of premenstrual symptoms.

Background/Objectives: Elevated circulating non-esterified fatty acids (NEFAs) are closely associated with hepatic inflammatory injury in dairy cattle, simultaneously with the entry of lipopolysaccharide (LPS) into the liver. This study aimed to investigate the synergistic effects of NEFAs and LPS on pyroptosis in bovine hepatocytes. Methods: Primary bovine hepatocytes were allocated into control, NEFA, NEFA + LPS, NEFA + LPS + Caspase-1 inhibitor, and NEFA + LPS + NLRP3 inhibitor groups. Levels and activation of pyroptosis-related markers (NLRP3, ASC, Caspase-1, GSDMD, IL-18 and IL-1β) were measured. Results: NEFAs alone upregulated these markers in a dose-dependent manner. Compared to NEFAs alone, NEFA + LPS co-treatment significantly enhanced levels of the markers, increased IL-1β secretion, and promoted NLRP3/Caspase-1 co-localization and Caspase-1activity. Notably, these effects of NEFA + LPS were attenuated by the NLRP3 or Caspase-1 inhibitors. Similar results were obtained when repeating the experiments in carcinoma HepG2 cells. Also, a random liver section from the subclinical ketotic cows displayed a higher fluorescence intensity of NLRP3 and Caspase-1 and stronger co-localization than that from a healthy cow. Conclusions: NEFAs and LPS synergistically contribute to pyroptosis in bovine hepatocytes by enhancing NLRP3 inflammasome assembly and subsequent Caspase-1 activation, providing a potential target for mitigating hepatic injury.

Background:Beauveria bassiana infection of silkworm forms Bombyx Batryticatus (BB). It is a medicinal material with significant pharmacological potential. While artificial diet feeding improves the production efficiency of BB, it might alter host metabolism, consequently affecting its bioactive components and efficacy. To address this, we conducted a metabolomics analysis of BB reared under different feeding conditions; Methods: UPLC-MS/MS was employed to conduct metabolomic analysis of BB under three rearing conditions: all instars mulberry leaf feeding (MF), all instars artificial diet feeding (AF), and mixed feeding (AMF). The sample collection time was selected as the time when silkworms died after infection (D0), and the fifth day after death (D5), which is the time when fungus produces biologically active secondary metabolites to reach a stable state; Results: Compared to MF, AF did not significantly alter the levels of the index component induced by B. bassiana infection-beauvericin. Moreover, the overall metabolic profile differences between the two groups decreased at the later stage (D5). Specifically, the average Pearson correlation between these groups was 0.659 ± 0.102, and the first two principal components of PCA explained 49.6% of the total variance. This suggests a reduction in the differences in their pharmacological active components. KEGG enrichment analysis revealed that AF promoted the accumulation of certain flavonoids (e.g., apigenin, luteolin), but, overall, the biosynthesis of flavone and flavonol is suppressed. Additionally, several metabolites, including N,N'-diferuloylputrescine, N-methyl-4-aminobutyric acid, and 3,4-dimethoxyphenylacetic acid, were identified to be significantly positively correlated with artificial diet supplementation; Conclusions: This study reveals metabolic differences in BB under different rearing methods at the metabolomic level, providing a scientific basis for evaluating the quality of this medicinal material.

Background/objectives: Chimeric antigen receptor (CAR) T cells are a powerful cancer therapy, but their function depends heavily on internal signaling domains and metabolic adaptability. Most studies evaluate CAR behavior upon antigen exposure, yet intrinsic signaling properties may pre-program CAR T cell states even in the absence of stimulation. This study investigates how CAR design and metabolic support shape baseline transcriptional programs, focusing on tonic signaling and NF-κB-related pathways.

Methods: We engineered CAR T cells targeting HER2 or GPC3 antigens, incorporating either 4-1BB or CD28 co-stimulatory domains, respectively. A subset of cells was further modified with adenosine deaminase 1 (ADA1) and CD26 to degrade extracellular adenosine and supply inosine, a metabolic strategy termed metabolic refueling (MR). Bulk RNA-seq was performed on resting T cells without antigen stimulation. We analyzed differential gene expression, gene set enrichment (GO, KEGG, Hallmarks), and transcription factor activity (DoRothEA) to assess the impact of CAR design and MR on T cell programming.

Results: All CAR T cells exhibited activation of NF-κB-centered inflammatory programs at baseline, indicating tonic signaling. GPC3 CAR T cells showed stronger baseline activation than HER2 CAR T cells. Metabolic refueling amplified these programs without altering their directionality, enhancing inflammatory, survival, and effector modules. Transcription factor activity scores mirrored these trends, highlighting RELA, FOS, and STATs as key regulatory nodes.

Conclusions: CAR-intrinsic features, notably co-stimulatory domain choice, define the tonic NF-κB activation tone in resting CAR T cells. Metabolic refueling boosts these baseline states without overstimulation, suggesting it may be especially valuable for weaker CAR constructs. These findings provide a framework for tuning CAR T cell function through combinatorial design strategies targeting signaling and metabolism.

Background: Psoriasis is a chronic, immune-mediated skin disorder characterized by accelerated epidermal turnover. Galectins are a family of carbohydrate-binding proteins that play crucial roles in various biological processes. Methods: This study aimed to assess the plasma concentrations of galectin 7 and 8 (gal-7 and 8) in 60 psoriatic patients compared to the control group of 30 individuals without dermatoses. Results: The median gal-7 plasma concentration in patients was 188.8 (11.43-1406) pg/mL, and it was significantly higher than in controls (p < 0.001). There was a positive correlation between gal-7 concentration and psoriasis area and severity index (PASI; R = 0.3, p = 0.0199), and a negative with RBC (R = -0.41, p < 0.001), hemoglobin concentration (R = -0.34, p < 0.01), total cholesterol (R = -0.38, p < 0.01) and LDL concentration (R = -0.36, p < 0.05). In contrast, gal-7 was not correlated with psoriasis duration or patients' age or sex (p > 0.05). The median gal-8 plasma concentration in patients was 0.07 (0.02-0.5) ng/mL, and was significantly higher in patients than controls (p < 0.05). There was a positive correlation between gal-8 concentration and glucose concentration (R = 0.26, p < 0.05). Gal-8 concentration was not correlated with PASI, BMI, age or sex of patients (p > 0.05). We also analyzed the receiver operating characteristic (ROC) curve to evaluate the predictive power of gal-7 and 8 for psoriasis. Gal-7 achieved statistical significance in predicting psoriasis and had an area under the curve (AUC) value of 0.842 (p < 0.001), a sensitivity of 80%, and a specificity of 86.7%, whereas gal-8 had an AUC value of 0.644 (p = 0.025), a sensitivity of 81%, and a specificity of 47%. Conclusions: Gal-7 and gal-8 could potentially serve as psoriasis biomarkers, whereby gal-7 could also serve as a marker of its severity. Future studies are needed to clarify their actual role or potential as therapeutic targets in psoriasis. Understanding their precise functions may open new perspectives for personalized treatment strategies in psoriatic patients.