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Tunneling nanotubes (TNT) are dynamic, membranous cellular structures that mediate intercellular communication. However, conventional coculture methods present significant limitations for TNT observation and analysis. In this study, a Transwell membrane (TM)-based three-dimensional (3D) coculture system was designed to support TNT formation and minimize extracellular vesicle-mediated influence. Using this system, 3T3-L1 preadipocytes were seeded on the upper side of a 1.0 μm pore-size polyethylene terephthalate (PET) or polycarbonate (PC) TM and differentiated into mature adipocytes, followed by seeding of MDA-MB-231 breast cancer cells on the opposite side. High-resolution laser scanning confocal microscopy combined with 3D reconstruction in Imaris were applied to produce precise visualization and spatial analysis of TNT. Immunofluorescence staining revealed that TNT readily formed between mature adipocytes and MDA-MB-231 cells across the 1.0 μm TM pores, with α-tubulin identified as one of the main TNT structural component. Quantitative morphometric analysis showed that TNT diameters primarily ranged from 50 to 600 nm, and lengths extended up to 5.5 μm. Directional analysis indicated that TNT significantly extended from the cells above the TM membrane to the cells below, suggesting that the direction of gravity might affect its growth direction. Notably, classical F-actin probes such as rhodamine-phalloidin exhibited strong nonspecific binding to TM, whereas immunofluorescence staining showed high signal specificity. The optimized 3D coculture platform, leveraging TM as a scaffold and 1.0 μm pores as TNT-permissive channels, can effectively support the formation and transmembrane extension of TNT, and can achieve high-resolution three-dimensional imaging and spatial quantitative analysis of TNT. This approach overcomes key limitations of traditional 2D coculture models and establishes a reliable and reproducible framework for studying TNT-mediated intercellular communication.
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An electrochemical biosensor was constructed for detection of protein kinase A (PKA) activity based on bio-barcode and terminal extension reaction dual signal amplification strategy. Gold nanoparticles (AuNPs) were modified on the surface of glassy carbon electrode (GCE) by electrodeposition method. Cysteine-labeled substrate peptide was self-assembled on GCE surface by Au—S bonds, which was phosphorylated in the presence of PKA and ATP. Bio-barcode probe of S1-AuNPs-S2 was linked with phosphorylated substrate peptide through phosphate-Zr4+-phosphate chemistry. In the presence of terminal deoxynucleotidyl transferase (TdTase) and dATP, TdTase-mediated terminal extension reaction of DNA S2 was initiated. The TdTase extension product hybridized with biotin-labeled DNA S3 (biotin-S3), which was further linked with horseradish peroxidase labeled streptavidin (SA-HRP) by biotin and streptavidin interaction. HRP catalyzed hydrogen peroxide (H2O2) and hydroquinone (HQ) to generate an amplified electrochemical response signal, achieving quantitative analysis of PKA activity. The response current of electrochemical biosensor is proportional to PKA concentrations in the range of 0.01~100 U/mL, with a detection limit of 0.005 U/mL. The proposed electrochemical biosensor exhibits good selectivity and reproducibility, and can be applied for PKA activity assay in cell lysates samples, inhibitor screening and discovery of protein kinase related drugs.
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Photosensitizers, as a core component of photodynamic therapy (PDT), exhibit physicochemical properties that significantly influence therapeutic outcome of PDT. In this study, fluorescence spectroscopy, UV-Vis absorption spectroscopy, and synchronous fluorescence spectroscopy were employed to compare the interactions between PCN and PCN-AuPt with bovine serum albumin (BSA). It was found that both PCN and PCN-AuPt spontaneously formed 1∶1 complex with BSA, reducing BSA fluorescence through a static quenching mechanism and enhancing the hydrophobicity of BSA microenvironment. PCN-AuPt exhibited higher binding constant (Ka) and binding site number with BSA than those of PCN, which were driven primarily by electrostatic and hydrophobic interactions, whereas PCN-BSA binding depended predominantly on electrostatic interaction.In addition, pharmacokinetic results showed that PCN-AuPt was metabolized in the blood of mice for a longer period of time compared to PCN, correlating with its stronger protein binding property. The findings reveal that in situ reductive doping of AuPt nano-enzyme could significantly strengthen the binding affinity of PCN to BSA, providing a theoretical basis for the surface modification and performance optimization of novel photosensitizers.
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In order to investigate liver damage in mouse from high-salt and high-fat diet and the therapeutic effects of Chrysanthemum morifolium cv. Fubaiju aqueous extract, the model of inflammatory injury of mouse liver induced by high-salt and high-fat diet was established, and treated with aqueous extract of Chrysanthemum morifolium cv. Fubaiju. In the 3rd, 6th and 9th months of the experiment, the mice were sacrificed, and the potential mechanism of the therapeutic effect of Chrysanthemum morifolium cv. Fubaiju was explored through organ coefficients, serum biochemical indexes, pathological sections, and network pharmacology. Results showed that high-salt and high-fat diet can induce inflammatory injury in mouse liver. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (γ-GT) in the liver of high-salt and high-fat mouse group were significantly increased, and the level of reduced glutathione(GSH) was significantly decreased.The intervention of Chrysanthemum morifolium cv. Fubaiju could significantly reverse the disorder of liver serum indexes induced by high-salt and high-fat diet, suggesting that Chrysanthemum morifolium cv. Fubaiju could effectively alleviate the liver injury induced by high-salt and high-fat diet. The results of network pharmacology showed that Chrysanthemum morifolium cv. Fubaiju could act on EGFR, TNF, TP53 and other targets through active ingredients such as caffeic acid, 4,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, apigenin and luteolin, and regulate IL-17, Toll-like receptors and other signaling pathways to exert anti-hepatitis effects. In summary, high-salt and high-fat diet can cause inflammatory damage to the liver of mouse, leading to the occurrence of hepatitis. The aqueous extract of Chrysanthemum morifolium cv. Fubaiju has therapeutic effects on mouse liver inflammatory injury induced by high-salt and high-fat diet and can be considered as a potential preventive drug for liver inflammatory injury and homology of medicine and food.
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To elucidate the differences in metabolite composition and relative abundance of Aurantii Fructus (Zhiqiao) from two representative producing regions, Jiangxi and Chongqing, non-targeted metabolomics based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was employed. Multivariate statistical analysis, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were applied to identify significantly different metabolites. These metabolites were subsequently annotated and subjected to pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In total, 1 861 metabolites belonging to 17 chemical classes were detected, among which 857 metabolites were identified as significantly different between the two regions. Notably, samples from Jiangxi exhibited higher relative abundances in compounds such as vitamin K3, methyl chlorogenic acid, narirutin, and nobiletin, whereas samples from Chongqing were enriched in 1,4-naphthoquinone, pseudo-yohimbine, and luteolin. KEGG pathway analysis revealed that 103 differential metabolites were involved in 66 metabolic pathways, with “biosynthesis of flavonoids and flavonols” and “flavonoid biosynthesis” being the most significantly enriched pathways(p<0.05). The study provides a systematic metabolomic characterization of Aurantii Fructus from two different regions, offering a scientific basis for its quality evaluation, geographical origin traceability, and geoherbalism studies, as well as supporting the exploration and utilization of its bioactive constituents.
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Spatiotemporal distribution pattern of biodiversity at the county scale are crucial for formulating and implementing biodiversity conservation measures. Taking the arid river valley regions of Aba Prefecture, Sichuan Province (including Wenchuan, Li, and Mao Counties) as the study area, this research employed the InVEST model combined with remote sensing and GIS techniques,and selected six indicators, including Shannon’s diversity index, habitat quality index, net primary productivity, normalized difference vegetation index, surface water content index, and land surface temperature, to evaluate the spatiotemporal characteristics of biodiversity in the arid river valley regions of Aba Prefecture from 2010 to 2020. The results indicate that all six indicators exhibited an overall “higher in the east and lower in the west” spatial pattern with a trend toward stabilization amid fluctuations. The biodiversity index demonstrated a consistent distribution pattern, with high-value areas mainly located in forested zones in eastern Wenchuan County, southwestern and northern Li County, and southern and northern Mao County, maintaining a stable area proportion of approximately 10.50%.Low-value areas were concentrated in low-coverage grasslands and human-disturbed valleys in western Wenchuan County and Li County, occupying over 11.00% of the area since 2015 after an initial increase. From 2010 to 2020, biodiversity exhibited an overall upward trend. Significant improvements occurred in southeastern Wenchuan County and eastern Mao County, while noticeable degradation was observed in central Wenchuan County and southeastern Mao County. Li County remained relatively stable. These spatial patterns suggest that ecological engineering projects and human disturbances jointly shape the pronounced spatial heterogeneity of biodiversity at the county scale. Based on these findings, and considering spatial differentiation characteristics and management feasibility, targeted biodiversity conservation strategies are proposed.
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Taking Hedysarum scoparium in the declining growth stage under different stubble treatments in Otog Banner, Ordos City, as the research subject, the research quantitatively analyzed root-system architecture indices of Hedysarum Scoparium, including spatial distribution, morphological parameters (root length, surface area, volume), topological indices, and diameter-class composition, using root scanner coupled with WinRHIZO software. Comprehensive evaluation of the stubble treatments was conducted via correlation analysis, principal component analysis, and membership-function analysis. Results showed that different stubble treatments (stubble heights of 0 cm, 10 cm and 20 cm) significantly influenced root architecture. When stubble was retained at 10 cm, the root system was dominated by oblique roots, and all morphological parameters peaked with total root length of 2 967.11 cm(79.82% above the length of uncut control), total surface area of 777.63 cm2(53.42% above), and total volume of 42.61 cm3(24.34% above). The topological index was the lowest at 10 cm stubble height (0.61), indicating denser branches and a highly efficient dichotomous structure. After stubble, the proportion of fine roots increased markedly, enhancing resource-use efficiency.Membership-functions of stubble treatments were ranked in descending order as follows:10 cm stubble (0.833),0 cm stubble(0.442),uncut stubble(0.259),20 cm stubble (0.109). Thus, retaining 10 cm stubble is the optimal cutting regime for improving quality, increasing productivity, and rejuvenating Hedysarum scoparium. These findings provide a scientific basis for the tending and management of artificial Hedysarum scoparium vegetation in western Ordos.
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Molasses yeast wastewater (MYW) contains a large amount of melanoidin, caramel pigment and other macromolecular pigments. The removal of these pigments is a challenge in the treatment of MYW. In this study, a native decolorizing bacterial consortium was developed through extended natural fermentation of MYW. The decolorization conditions were optimized by single-factor experiment and response surface method, and it was determined that the main factors influencing the decolorization rate were sucrose concentration, fermentation temperature and fermentation time. Under conditions of 10% wastewater concentration, static culture, 4% inoculum amount, and a pH of 10, the addition of 15 g/L sucrose followed by fermentation at 43 ℃ for 5 d achieved a decolorization rate of 45.03% for MYW, and simultaneously decreased 33.72% of the chemical oxygen demand (COD). In addition, the bacterial consortium removed 25.57% melanoidin and 9.70% caramel pigment. Metagenomic sequencing results showed that the addition of sucrose altered the composition and relative abundance of the decolorizing bacterial consortium. It significantly increased the abundance of Firmicutes and enriched the genus Lactiplantibacillus. Additionally, it significantly upregulated the expression levels of gallate decarboxylase encoding genes (lpdB, lpdC, lpdD) and glycoside hydrolase.Functional analysis revealed Lactiplantibacillus is the core contributor to carbohydrate-active enzymes (CAZymes). The inhibitory effect of decolorized MYW on the germination of maize and rice seeds was eliminated. In conclusion, the optimization of fermentation conditions altered the composition and functions of the decolorizing bacterial consortium, reduced wastewater toxicity, and provided a theoretical basis for the biological decolorization of MYW.
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To investigate the pollen biological characteristics of different red-flowered oil-tea Camellia, this study used Camellia chekiangoleosa, Camellia qrossideulata Chang Sect., Camellia pitardii, and Camellia pitardii var. compressa as experimental materials, and the numbers of stamens and pollen grains per flower were recorded. The effects of gibberellin A3 (GA3),1-naphthaleneacetic acid (NAA) and 2, 4-dichlorophenoxyacetic acid (2, 4-D) of different concentrations on pollen germination and pollen tube growth in these four red-flowered Camellia species were systematically analyzed. The results showed that among the four species, Camellia chekiangoleosa had the highest numbers of stamens and pollen grains per flower, followed by Camellia qrossideulata Chang Sect.. Camellia pitardii exhibited a higher number of stamens per flower than Camellia pitardii var. compressa, while the pollen grain number per flower was the opposite. The optimal hormone treatments and concentrations for promoting pollen germination of the four species were as follows: NAA at 0.5 mg/L for Camellia chekiangoleosa, 2, 4-D at 2 mg/L for Camellia qrossideulata Chang Sect., and NAA at 0.5 mg/L for both Camellia pitardii and Camellia pitardii var. compressa. The best hormone treatments and concentrations for inducing pollen tube growth were NAA at 0.5 mg/L for Camellia chekiangoleosa, 2, 4-D at 2.5 mg/L for Camellia qrossideulata Chang Sect., and 2, 4-D at 3 mg/L for both Camellia pitardii and Camellia pitardii var. compressa. These findings provide valuable data for understanding the pollen physiological characteristics in different red-flowered Camellia species and determining the optimal germination conditions and timing.
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MYB transcription factors represent one of the largest transcription factor families in plants, playing critical roles in plant growth and development, metabolic regulation, and responses to biotic and abiotic stresses. The R3-MYB subfamily remains systematically uncharacterized in kiwifruit. This study identified 167 R3-MYB subfamily genes in Actinidia chinensis ‘Donghong’ through bioinformatics methods, with comprehensive analysis of their chromosomal localization, protein physicochemical properties, collinearity, and promoter cis-acting elements. Results showed that members of this family are distributed across 29 chromosomes, with the highest enrichment observed on chromosome 23. The encoded proteins are predominantly hydrophilic and unstable, with molecular weights ranging from 7.9 to 122.8 kDa. Phylogenetic analysis classified them into four subgroups, and subgroup Ⅲ clustered in the same branch with anthocyanin repressors (Arabidopsis thaliana AtCPC and Malus domestica MdMYB17). Collinearity analysis revealed that the homology between kiwifruit and Arabidopsis thaliana is higher than that of Oryza sativa, and family expansion is primarily driven by segmental duplication. Gene structures exhibited high diversity, with the number of coding sequences (CDS) ranging from 1 to 30, and Motif 2 was identified as a conserved core motif. Promoter analysis uncovered enrichment of cis-acting elements responsive to hormones (abscisic acid, jasmonic acid methyl ester), stress (low temperature, drought), and light. Protein-protein interaction network analysis indicated that AcR3MYB24 acts as a central regulator. Transcriptome analysis combined with qRT-PCR demonstrated significantly elevated expression of AcR3MYB158 and AcR3MYB99 during the late stage of fruit development (110~140 d after anthesis).The expression patterns of genes such as AcR3MYB83 and AcR3MYB56 coincided with that of AcMYB10, a known transcriptional activator for anthocyanin accumulation, suggesting their potential role in promoting anthocyanin biosynthesis. This study represents the first comprehensive genome-wide identification of the R3-MYB family in kiwifruit, providing crucial genetic resources for deciphering the molecular mechanisms underlying fruit color formation and quality improvement of kiwifruit.
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To explore the unique quality of the common meat pieces of Jiami donkey, taking the front leg meat, hind leg meat, neck meat and donkey belly of Jiami donkey as materials, and the corresponding common meat pieces of Dezhou donkey as controls, the differences in nutritional quality (moisture, protein, fat, fatty acid, amino acid, minerals), edible quality (pH value, color, texture, flavor) and microstructure of common donkey meat pieces were compared and analyzed. In terms of nutritional quality, the hind leg meat of Jiami donkey had the highest water content (74.46%±0.05%). The front leg meat of Jiami donkey had the highest protein content (22.79±0.46 g/100 g). The donkey belly of Jiami donkey had the highest fat content (1.73%±0.43%), ash content (5.07±0.30 g/100 g) as well as the contents of potassium (235.16±3.11 mg/100 g), sodium (683.57±13.16 mg/100 g), magnesium (248.37±8.12 mg/kg) and selenium (0.71±0.01 mg/kg). The calcium content (174.00±0.01 mg/kg) in neck meat of Jiami donkey was the highest. The relative content of unsaturated fatty acids in the front leg meat of Jiami donkey was the highest, reaching 71.43%±3.14%. The essential amino acid contents of Jiami donkey meat ranged from 42.57% to 43.89%, among which the contents of isoleucine, leucine and lysine were relatively high. In terms of edible quality, the L* value of the donkey belly of Jiami donkey was significantly higher than those of other parts, and was significantly higher than that of the donkey belly of Dezhou donkey. The a* values of the hind leg meat and neck meat of Jiami donkey were significantly higher than those of other parts. The hardness of the donkey belly of Jiami donkey was significantly lower than those of other parts, and the elasticity of the front leg meat of Jiami donkey was greater than those of other parts. The types of volatile substances detected in Jiami donkey meat and Dezhou donkey meat were the same, mainly including alkanes, short-chain alkanes, aromatic compounds, aldehydes and sulfides. In terms of microstructure, the degree of muscle aggregation in the donkey belly of Jiami donkey was less than that of the donkey belly of Dezhou donkey. Therefore, Jiami donkey meat is rich in protein and unsaturated fatty acids, low in fat, and abundant in minerals. It also has good color, physical properties and flavor, and thus has high edible value.