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Background With the accumulating omics data, an efficient and time-saving transient assay to express target genes is desired. Mesophyll protoplasts, maintaining most stress-physiological responses and cellular activities as intact plants, offer an alternative transient assay to study target genes’ effects on heat and oxidative stress responses. Results In this study, a perennial ryegrass ( Lolium perenne L.) mesophyll protoplast-based assay was established to effectively over- or down-regulate target genes. The relative expression levels of the target genes could be quantified using RT-qPCR, and the effects of heat and H 2 O 2 -induced oxidative stress on protoplasts’ viability could be quantitatively measured. The practicality of the assay was demonstrated by identifying the potential thermos-sensor genes LpTT3.1 / LpTT3.2 in ryegrass that over-expressing these genes significantly altered protoplasts’ viability rates after heat stress. Conclusion This protoplast-based rapid stress regulatory gene identification assay was briefed as ‘PRIDA’ that will complement the stable genetic transformation studies to rapidly identify candidate stress-regulatory genes in perennial ryegrass and other grass species.

MicroRNAs (miRNAs) are emerging drivers in tumor progression, while the role of miR-503-3p in breast cancer (BC) remains largely unknown. We aimed to explore the impact of macrophage-derived exosomal miR-503-3p in the development of BC by regulating disheveled-associated binding antagonist of beta-catenin 2 (DACT2). miR-503-3p and DACT2 expression in BC tissues and cells was assessed, and the expression of Wnt/β-catenin signaling pathway-related proteins in BC cells was also evaluated. Macrophages were induced and exosomes were extracted. The screened BC cell lines were, respectively, treated with exosomes, miR-503-3p inhibitor/mimic or upregulated/inhibited DACT2, and then the phenotypes, glucose intake, oxygen consumption rate, and adenosine-triphosphate (ATP) level of BC cells were determined. Cell growth in vivo was also observed. MiR-503-3p was elevated, DACT2 was reduced, and Wnt/β-catenin signaling pathway was activated in BC cells. Macrophage-derived exosomes, upregulated miR-503-3p or inhibited DACT2 promoted malignant behaviors of BC cells, glucose intake, and activity of the Wnt/β-catenin signaling pathway, while repressed oxygen consumption rate and ATP level in BC cells. Reversely, reduced miR-503-3p or upregulated DACT2 exerted opposite effects. This study revealed that reduction of macrophage-derived exosomal miR-503-3p repressed glycolysis and promoted mitochondrial oxidative phosphorylation in BC by elevating DACT2 and inactivating Wnt/β-catenin signaling pathway. Our research may provide novel targets for BC treatment.

Background Hyperlipidemia is an established risk factor for cardiovascular disease. Limonin, a natural tetracyclic triterpenoid compound found in the traditional Chinese herb Tangerine peel and citrus fruits, has been shown to ameliorate hyperlipidemia, although the underlying mechanisms of action are unknown. The present study employed a comprehensive approach integrating to assess the efficacy of Limonin in the treatment of hyperlipidemia and to explore its molecular mechanisms. Methods Hyperlipidemia model was induced by high-fat diet (HFD). The effects of Limonin on hyperlipidemia were evaluated through serum, liver lipid, and Hematoxylin & eosin (H&E). Then, the mechanism of Limonin alleviates hyperlipidemia was explored by network pharmacology. Targeted metabolism was used to measure bile acids (BAs)’ changes in serum and fecal, and 16 S rDNA sequencing of gut microbiota. Finally, the expression of genes and proteins about FXR/FGF15, ASBT, and FGF15/FGFR4 signaling pathways in the distal ileum or liver was detected by qPCR, immunohistochemistry, and Western blotting. Results The results confirmed the significant anti-hyperlipidemia effect of Limonin. Network pharmacology analysis revealed that Limonine alleviates hyperlipidemia is possibly closely pertaining to BAs metabolism. In a mouse model of hyperlipidemia, Limonin altered the colonic BAs profile, especially in terms of elevated levels of conjugated BAs. Limonin reshaped the structure of the gut microbiome by decreasing bile salt hydrolase (BSH)-producing genera, including Lactobacillus , Bacteroides , Clostridium , Streptococcus , and Adlercreutzia . Decreased BSH activity increased levels of conjugated BAs, which inhibited activation of ileum FXR, facilitated BAs synthesis and fecal BAs excretion. The decreased FXR activity resulted in lower expression of FGF15 and ASBT in the distal ileum, lower expression of FGF15 and its receptor in the liver, and increased expression of CYP7A1 in the liver. Conclusion Limonin remodels the gut microbiota to reduce BSH activity and to activate BAs synthesis pathways, thereby ameliorating dyslipidemia. These results provide a theoretical basis for clinical investigations into the use of Limonin in anti-hyperlipidemia therapies.

Chronic unresolved inflammation is a core driver of diabetic kidney disease (DKD) progression, with gut microbiota dysbiosis and metabolite imbalance (via gut-kidney axis) as key pathogenic triggers. This review systematically elucidates the pathological link between gut microbiota-metabolite-axis dysfunction and DKD-related inflammation (centered on NF-κB/NLRP3 pathways) and summarizes multi-target intervention strategies-including traditional Chinese medicine (TCM), SGLT2 inhibitors, probiotics/prebiotics-targeting this axis. Literature search was conducted on PubMed using keywords [\"Gut microbiota\" or \"Gut microflora\" or \"Gut microbiota metabolites\"], [\"Diabetic kidney disease\" or \"Diabetic nephropathy\" or \"DKD\"], [\"immune regulation\"], [\"intestinal barrier\"], [inflammation\"], [\"Traditional Chinese Medicine\" or \"TCM\"], without date restrictions. Articles that do not meet the requirements are excluded. Gut microbiota dysbiosis in DKD is characterized by reduced SCFA-producing bacteria (Ruminococcaceae, Lachnospiraceae) and enriched pathogenic Proteobacteria, leading to metabolite imbalance: insufficient beneficial metabolites (SCFAs, IPA) and accumulation of harmful metabolites (TMAO, phenyl sulfate, BCAAs). This imbalance impairs intestinal barrier (ZO-1/Occludin downregulation), promotes endotoxin (LPS) translocation, and activates NF-κB (p65 phosphorylation) and NLRP3 inflammasome (NLRP3/ASC/caspase-1 complex), exacerbating renal inflammation via pro-inflammatory cytokines (IL-1β, TNF-α, IL-6). Intervention strategies (including TCM) suppress this cascade: TCM (eg, Astragalus membranaceus, Xiaoyaosan) reshapes microbiota, strengthens intestinal barrier, and inhibits NF-κB/NLRP3; SGLT2 inhibitors and probiotics/prebiotics complement via SCFA elevation and TMAO reduction. Clinically, these interventions lower UACR, improve eGFR, and correlate with reduced serum IL-1β/TNF-α. Gut microbiota-metabolite-intestinal barrier axis dysfunction is a pivotal pathological mechanism of DKD inflammation, mediated by NF-κB/NLRP3 pathways. Multi-pronged interventions targeting this axis effectively resolve inflammation, providing promising therapeutic approaches for DKD.

An eco-friendly composite coating was developed for blueberry preservation through the incorporation of thymol-loaded β-cyclodextrin microcapsules (THY@β-CD) into a potato starch (PO) matrix. Microencapsulation at an optimal wall-to-core ratio of 13:1 achieved a THY encapsulation efficiency of 73.24%. Structural analyses confirmed the successful formation of an inclusion complex, which enhanced thermal stability and provided a controlled release profile governed by Fickian diffusion mechanisms. When applied to blueberries, the coating significantly reduced weight loss by 22%, delayed softening, and more effectively preserved anthocyanin content compared to uncoated fruit during 10-day storage. Furthermore, it well-maintained the sensory quality and visual appeal of the fruit. These results demonstrate that the THY@β-CD/PO coating synergistically integrates sustained antimicrobial delivery with matrix compatibility, offering a promising natural alternative to synthetic preservatives for extending the shelf life of blueberries.

CCCH is a subfamily of zinc finger proteins involved in plant growth, development, and stresses response. The function of CCCH in regulating leaf senescence, especially its roles in abscisic acid (ABA)-mediated leaf senescence is largely unknown. The objective of this study was to determine functions and mechanisms of CCCH gene in regulating leaf senescence in switchgrass (Panicum virgatum). A CCCH gene, PvCCCH69 (PvC3H69), was cloned from switchgrass. Overexpressing PvC3H69 in rice suppressed both natural senescence with leaf aging and dark-induced leaf senescence. Endogenous ABA content, ABA biosynthesis genes (NCED3, NCED5, and AAO3), and ABA signaling-related genes (SnRKs, ABI5, and ABF2/3/4) exhibited significantly lower levels in senescencing leaves of PvC3H69-OE plants than those in WT plants. PvC3H69-suppression of leaf senescence was associated with transcriptional upregulation of genes mainly involved in the light-dependent process of photosynthesis, including light-harvesting complex proteins, PSI proteins, and PSII proteins and downregulation of ABA biosynthesis and signaling genes and senescence-associated genes. PvC3H69 could act as a repressor for leaf senescence via upregulating photosynthetic proteins and repressing ABA synthesis and ABA signaling pathways.

Wendan pomelo (Citrus maxima), valued for its unique quality and high nutritional value, is susceptible to postharvest decay caused by mechanical damage and fungal infection. This study developed a bio-based preservation strategy by incorporating Bacillus subtilis (B. subtilis) into a sodium alginate (SA)-based coating. An antagonistic B. subtilis strain, isolated from the pomelo growth environment, demonstrated effective inhibition against the pathogenic fungi P. digitatum and P. italicum. The B. subtilis/SA (2.0%) coating maintained high bacterial viability without adversely affecting the viscosity, gas barrier properties, or mechanical strength of the film. The application of the B. subtilis/SA coating significantly delayed fruit appearance deterioration, pulp softening, and decay in pomelo. Furthermore, the treatment enhanced flavonoid accumulation and increased the activity of antioxidant enzymes, thereby maintaining quality and extending storage life to 90 d. This study provides an effective bio-preservation strategy for the postharvest management of pomelo.

This study aims to investigate the cognition and application status of DeepSeek among surgical medical staff in Class III Grade A hospitals and analyse its influencing factors to optimise its clinical application. From February to March 2025, a questionnaire survey was conducted among 440 surgical medical staff from Class III Grade A hospitals in 18 provinces in China by a convenience sampling method. The questionnaire covered, among others, basic information, cognition and use status, attitudes and barriers and training needs, and also evaluated the degree of understanding, user experience (operation convenience, results accuracy, system stability) and degree of trust concerning DeepSeek. The SPSS 25.0 software was used for data analysis, which included frequency, percentage, Mann-Whitney -test, Kruskal-Wallis -test and multivariate ordinal logistic regression analysis. A total of 424 valid questionnaires were collected (96.4%). The results indicated that 67.0% of the medical staff understood the basic functions of DeepSeek, and 70.3% used DeepSeek occasionally. It was mainly used for teaching and research support (43.2%), other life services (35.6%) and patient services (29.2%). Multivariate analysis showed that medical staff working in operating rooms and neurosurgery departments, those who were occasional users, and medical staff who primarily used DeepSeek for other life services demonstrated significantly higher levels of knowledge about DeepSeek. Despite widespread awareness of DeepSeek's capabilities (67.0% understanding basic functions), significant implementation gaps persist, with limited clinical utilisation and predominant usage in low-risk applications. Key barriers include insufficient training (94.8% untrained), data privacy concerns (57.5%) and over-reliance fears (58.5%). These findings reveal a substantial untapped potential for AI integration in surgical practice, highlighting critical needs for targeted training interventions, enhanced data security frameworks and staged implementation protocols to bridge the awareness-utilisation gap and facilitate meaningful clinical adoption.

Background The incidence of parathyroid injury in patients with differentiated thyroid carcinoma (DTC) who underwent reoperation is significantly higher than that incurred from the initial surgery. The aim of this study was to investigate the clinical value of carbon nanoparticles (CN) as tracers for lymph nodes to guide cervical lymph node dissection and protect the parathyroid during reoperation for DTC. Methods Our study recruited 116 patients with DTC who previously underwent thyroidectomy and later received remedial surgical treatment at the Department of Breast and Thyroid Surgery, Hunan Provincial People’s Hospital, China, between February 2011 and February 2014. Those patients were randomly divided into the experimental group (the CN group) with 64 cases and the control group with 52 cases. Carbon nanoparticle suspension (CNS, 0.1–0.3 ml) was intraoperatively injected into the residual thyroid or enlarged lymph nodes in the CN group; in the control group, CNS was not applied intraoperatively. The differences in identification of the parathyroid glands, the number of lymph nodes resected intraoperatively, and the incidence of common complications after thyroidectomy in both groups were recorded and analyzed. Results The accuracy of identification of the parathyroid in the CN group and control group was 92.2 % and 28.8 %, respectively, and the identification rate of three glands or more in both groups was 75 % and 36.5 %, respectively; those differences were statistically significant between the two groups ( P  < 0.05). There was also a significant difference between the two groups in the number of lymph nodes removed in the central and lateral cervical compartments ( P  < 0.05). There was no increase in common complications after the second surgery compared with the previous surgery; in addition, there was a decline in the incidence of transient hypoparathyroidism (HPT) ( P  < 0.05). Conclusions By tracing the thyroid and cervical regional lymph nodes with carbon nanoparticles, the parathyroid glands can be easily identified and protected to reduce the complications of transient hypoparathyroidism resulting from reoperation for residual or missed DTC. Carbon nanoparticle tracers also facilitate radical resection of lymph nodes at the central and lateral compartments of the neck.

Brigatinib and brigatinib-analog are potent and selective ALK inhibitors with the similar structure. A simple and sensitive high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of brigatinib and brigatinib-analog in rat plasma and brain homogenate was developed and validated. Chromatographic separation was carried out on an ODS column with acetonitrile and 0.1% formic acid in water as the mobile phase with gradient elution at a flow rate of 0.5 mL/min. Detections were performed using a TSQ Quantum Ultra mass spectrometric detector with electrospray ionization (ESI) interface, which was operated in the positive ion mode. A simple protein precipitation preparation process was used. The lower limits of quantification (LLOQs) were 1.0 ng/mL and 0.5 ng/mL for analytes in rat plasma and brain homogenate, respectively. The intrabatch and interbatch precision and accuracy of brigatinib and brigatinib-analog were well within the acceptable limits of variation. The simple and sensitive LC-MS/MS method was successfully applied to the pharmacokinetic and brain distribution studies following a single oral administration of brigatinib and brigatinib-analog to rats. The above studies would lay a good foundation for the further applications of brigatinib and brigatinib-analog.