Optimizing the dietary nitrogen-to-sulfur (N:S) ratio is crucial for enhancing ruminal microbial fermentation and nutrient digestibility. This study investigated the effects of different dietary N:S ratios on ruminal antioxidant indices, digestive enzyme activities, immune parameters, volatile fatty acid (VFA) profiles, microbial communities, and metabolomic profiles in plateau-type Tibetan sheep. Ninety 2-month-old male Tibetan sheep (initial body weight, 15.55 ± 0.20 kg) were randomly assigned to three dietary treatments formulated with N:S ratios of 9.5:1 (HP-H), 8.5:1 (HP-M), and 7.5:1 (HP-L). Sheep fed the HP-H diet exhibited improved ruminal antioxidant indices compared with the other treatments, with CAT and T-AOC activities being higher than those of both the HP-M and HP-L groups, while SOD and GSH-Px activities were significantly higher only compared with those in the HP-L (p < 0.05). Furthermore, ruminal cellulase activity, as well as propionate and butyrate levels, were significantly higher in the HP-H group (p < 0.05). Microbiome analysis revealed that the HP-H diet enriched the relative abundances of Candidatus Saccharimonas, the Rikenellaceae RC9 gut group, and uncultured rumen bacteria. Correspondingly, untargeted metabolomics identified higher signal intensities of 3-hydroxyphenylacetic acid, nervonic acid, arachidonic acid (peroxide-free), and calciferol in the ruminal fluid of the HP-H group relative to the other treatments. In conclusion, when the nitrogen-to-sulfur ratio was 9.5:1, ruminal antioxidant enzyme activities were increased, and the concentrations of propionic acid and butyric acid were increased. The differential microorganisms and metabolites were mainly associated with lipid metabolism, as well as cofactor and vitamin metabolism.

Breast cancer remains a major global health burden. Dactylospongia species have been explored for their cytotoxic potential. This study aims to evaluate the cytotoxic potential of compounds derived from the marine sponge Dactylospongia elegans. Dactylospongia elegans were collected from the Lembeh Strait, macerated using methanol, then partitioned to an ethyl acetate fraction. The cytotoxic activity of these fractions was assessed using MDA-MB-231 cells while toxicity testing was done using the BSLT. TLC was carried out to determine the groups of compounds, while LC-MS/MS was used to predict active compounds contained in the ethyl acetate fractions. In silico studies were conducted as preliminary studies to determine the antitumor mechanism. The ethyl acetate fraction and F4 subfraction of Dactylospongia elegans exhibited cytotoxicity toward MDA-MB-231 cells with IC50 values of 15.72 and 41.76 µg/mL, respectively. The BSLT indicated the strongest toxicity belongs to the F6 subfraction (LC50 = 32.831 µg/mL). TLC analysis confirmed the presence of major secondary metabolites as terpenoids, steroids, and alkaloids, then confirmed with LC-MS/MS including 5-epi-illimaquinone and calciferol. Molecular docking revealed that calciferol exhibited the strongest binding affinity toward tyrosine kinase and p53-MDM2 receptors, with binding energies of -10.13 and -10.28 kcal/mol, respectively. These findings suggest that Dactylospongia elegans contains bioactive constituents with potential anticancer activity, particularly against TNBC.

Vitamin D (VD) deficiency is prevalent globally. To improve dietary recommendations for food fortification, we used male rats fed with diets depleted of VD or supplemented/rehabilitated with either vitamin D2 or vitamin D3 or combination, and compared their impact on calcium homeostasis, skeletal muscle and heart. Body composition was assessed by DEXA, muscle strength by grip test. Serum calcium, parathyroid hormone and 25-hydroxyvitamin D [25(OH)D] were measured using standard methods. Skeletal muscle and left ventricle were used for histopathology; RNA and protein were extracted for analysis of gene expression and enzymatic activity, respectively. 25-hydroxylated form of D3 was significantly higher than that of D2 in serum. D2 was less efficacious than D3 at improving skeletal muscle fiber size, strength, expression of contractility genes [Myh2, Tnnc1] and metabolic enzymes [Cs, β-Had]. Under maintenance cardiac contractility gene expression [Myh7 & Serca2a] was comparable between the D2 and D3 groups, though 400 IU D2 notably suppressed Serca2 expression. Post-rehabilitation, the 2000 IU D3 group demonstrated significant improvement in these genes. Overall, our study demonstrates similar efficacy of D2 and D3 at regulating calcium homeostasis, but a better efficacy of D3 than D2 on structure, function and energy metabolism of skeletal muscle and heart.

Accurate analysis of vitamin D in human milk is essential for infant nutrition but remains analytically challenging due to low levels and a complex matrix. This study evaluates a practical multi-method strategy using LC-MS/MS, ELISA, and UPLC-PDA for vitamin D analysis in human milk. LC-MS/MS precisely quantified 25(OH)D2 and 25(OH)D3 with assay LOQ of 0.045 ng/mL and 0.100 ng/mL, respectively. ELISA showed a 11.58 ng/mL positive bias of vitamin D which limits use for accurate quantification. UPLC-PDA profiled vitamins A and E effectively but lacked sensitivity for vitamin D. Longitudinally, LC-MS/MS results show 25(OH)D3 and total 25(OH)D concentrations remained relatively stable, with medians ranging from 0.141 to 0.281 ng/mL and 0.263 to 0.315 ng/mL, respectively, across the study period, while 25(OH)D2 peaked at 8 weeks before declining. This study establishes LC-MS/MS as the preferred analytical strategy for quantifying 25(OH)D2 and 25(OH)D3 in human milk, providing critical data to assess infant nutritional intake and guide clinical interventions.

Vitamin D receptor (VDR) agonists promote quiescence of cancer-associated fibroblasts and improve efficacy of chemotherapy in preclinical models of pancreatic cancer. We conducted a run-in phase trial with primary endpoint of safety when the VDR agonist paricalcitol is given with first-line gemcitabine and albumin-bound paclitaxel (GA) in patients with metastatic pancreatic cancer. Secondary endpoints included pharmacodynamic analyses. Thirty-six patients were randomized to GA plus placebo, GA plus intravenous paricalcitol or GA plus oral paricalcitol with pretreatment and on-treatment tumor biopsies. Paricalcitol was safely administered with GA, although five patients (42%) receiving oral paricalcitol had grade 2-4 hypercalcemia and required dose reduction. Nuclear VDR protein expression was heterogeneous across patients, and VDR was expressed in tumor, immune and stromal cells. Compared to pretreatment specimens, on-treatment biopsies had decreased proportion of αSMA+ fibroblasts, altered fibroblast VDR activation signature and greater density and spatial colocalization of CD8+ T cells with tumor cells in the GA-plus-paricalcitol arms. VDR expression was predictive of tumor response in the GA-plus-paricalcitol arms. Paricalcitol can be safely administered with chemotherapy to patients with metastatic pancreatic cancer, and on-treatment biopsies indicated favorable modulation of the tumor microenvironment by paricalcitol as predicted by preclinical models. ClinicalTrials.gov identifier: NCT03520790 .

The conversion of ergosterol to vitamin D₂ (VD2) in Agaricus bisporus is still limited by low efficiency. This study integrates a particle size-regulated powder photoconversion pathway and natural deep eutectic solvent (NADES) integrated extraction-photoconversion pathway to achieve a high VD₂ yield. In the powder pathway, combining UV-B irradiation with a median particle size of 130-140 μm maximized VD₂ yield, reaching 2.59% by balancing optical penetration depth, light shielding, and the resulting porous cellular structure. In the extract pathway, the menthol-based NADES system was developed to achieve simultaneous high-efficiency ergosterol extraction (85.41% extraction efficiency) and stabilization, as well as ergosterol photoconversion to VD₂ (4.08% yield), attributed to enhanced cell permeability and hydrogen bonding interactions. The NADES system also enhanced the storage stability of ergosterol and VD₂ by 24.79% and 20.53%, respectively, compared to the ethanol during storage. This work provides green and tunable dual-pathway strategies for developing high-value VD₂-enriched products.

This study aimed to explore the application and pedagogical innovation of liquid chromatography-tandem mass spectrometry （LC-MS/MS） in undergraduate thesis training for medical laboratory science students. Using the development of 25-hydroxyvitamin D2 （25（OH）D2） and 25-hydroxyvitamin D3 （25（OH）D3） external quality assessment （EQA） samples as a teaching platform， an experimental teaching model driven by research tasks and clinical practice was established. The teaching design encompassed the entire workflow， including sample preparation， instrument operation， data processing， and result evaluation， enabling students to acquire hands-on experience with core LC-MS/MS techniques and quality control procedures. Through the implementation of the experimental project， students participated in EQA sample preparation， reference method-based quantification， homogeneity and stability assessments， and EQA result analysis， thereby gaining comprehensive exposure to the process from experimental design to result interpretation. The results demonstrated that the prepared EQA samples exhibited satisfactory homogeneity and stability， meeting the quality standards established by the China National Accreditation Service for Conformity Assessment （CNAS）. In the "2024 Beijing Serum Vitamin D EQA" activity， a total of 16 medical laboratory institutions in Beijing participated. Teaching practice revealed that this project significantly enhanced students' comprehensive abilities in experimental design， instrument operation， data analysis， and quality assessment， while also fostering scientific thinking and teamwork. By introducing advanced analytical techniques into the undergraduate curriculum under the educational concept of "research-driven teaching and integration of industry and education"， this approach not only optimized course content and experimental procedures but also broadened students' practical perspectives in clinical laboratory and research applications. This study provides a replicable teaching model for undergraduate laboratory education and offers valuable insights for promoting the integration of advanced analytical technologies into medical laboratory science training.

This study establishes ultraviolet (UV) irradiation as a promising postharvest strategy for enhancing the nutritional quality of edible mushrooms. However, its efficacy depends strongly on wavelength, dose, tissue geometry, species physiology, and storage conditions. Evidence indicates that UV-driven changes follow distinct mechanistic routes: photochemical conversion of ergosterol to vitamin D2, oxidative signaling in phenolics and antioxidants, stress-mediated retention of water-soluble vitamins, and matrix-level alterations in polysaccharides and cell-wall structures. These responses were interpreted through unified pathways linking UV dose to molecular targets and physiological outcomes. A major limitation in previous studies is inconsistency in light parameters and incomplete reporting of UV conditions. To address this, quantitative modeling using response surface methodology (RSM) was applied to integrate heterogeneous datasets. A weighted meta-analysis revealed species- and geometry-specific UV-B optima. For sliced Agaricus bisporus, an optimum was identified at 16,664 J/m[2] and 3.2 W/m[2], yielding 16.27 μg/g vitamin D2 (95% CI: 13.69-18.85). These values fall within observed experimental ranges, confirming model validity. Dose-response patterns consistently showed that moderate UV exposure enhances vitamin D2, whereas excessive exposure leads to nutrient degradation or plateau effects. Human and animal trials demonstrate that UV-treated mushrooms significantly increase serum 25(OH)D2 levels without adverse effects, establishing them as a bioavailable and safe dietary source of vitamin D2. Regulatory bodies have confirmed the safety of UV-treated A. bisporus powder, with no elevation in agaritine levels. These findings support the inclusion of UV-treated mushrooms in evidence-based nutritional interventions to address global vitamin D deficiency.