Applied Biochemistry and Biotechnology最新文献

Cordyceps polysaccharides, as bioactive macromolecules derived from Cordyceps sinensis, exhibit pharmacological activities such as immune modulation and anti-tumor properties. However, the understanding of their biosynthetic mechanisms remains limited. To elucidate the molecular changes associated with differentially expressed proteins (DEPs) during Cordyceps polysaccharide biosynthesis, a total of 1,242 DEPs enriched during this process were identified and annotated using the TMT-based quantitative proteomics. Subsequently, comparative analyses showed that Cordyceps polysaccharide biosynthesis was most active during the logarithmic growth phase, with high activity in sugar metabolism pathways, including galactose metabolism. Notably, DEPs with glycosyltransferase activity were identified and annotated, as they play a pivotal role in polysaccharide biosynthesis. Furthermore, within KEGG pathways closely associated with polysaccharide biosynthesis, several significantly up-regulated DEPs were identified, including NOS, ID-RCD, Arp2, NAD-GDH, phaA, GGT, CYP53A1, LAD, lacZ, and AAO. Notably, several key DEPs closely associated with polysaccharide biosynthesis showed fold-changes exceeding tenfold in the logarithmic phase relative to the early stage. Additionally, qPCR analysis showed that transcriptional changes were largely consistent with protein-level differences. Interestingly, the lacZ gene may play a critical role in Cordyceps polysaccharide biosynthesis through its glycosyltransferase activity. Collectively, these findings provide a scientific basis for the metabolic regulation of Cordyceps polysaccharides.

Plant-based therapeutics are of immense importance in modern drug discovery owing to their multi-targeted mechanisms and favourable safety profiles. Among these, Petroselinum crispum has long been utilised in traditional medicine, yet remains underexplored for its potential against liver fibrosis, a progressive condition lacking effective antifibrotic therapies. In this study, a comprehensive computational strategy was employed to systematically investigate the hepatoprotective potential of P. crispum metabolites. A total of 177 phytochemicals from P. crispum were screened based on drug-likeness properties, while 10,014 liver fibrosis-associated genes were retrieved from established databases. Integration of compound-target predictions with disease-related gene sets identified key overlapping inflammatory mediators. Network pharmacology analysis, followed by molecular docking and dynamics simulations, revealed CXCL8, IL1B, and CSF2 as central targets potentially modulated by P. crispum flavonoids, apigenin and luteolin. The findings demonstrated stable binding interactions, supported by favourable binding energies. Although apigenin's interaction with IL1B has been reported in inflammatory conditions, its specific modulatory role on CXCL8 and CSF2 within the context of liver fibrosis remains unexplored. Notably, to the best of our knowledge, this is the first study to demonstrate that luteolin stably interacts with all three targets in the context of hepatic fibrosis. These findings highlight the polypharmacological nature of these flavonoids in modulating multiple cytokine-driven signaling pathways for the therapeutic repositioning of P. crispum constituents in chronic liver disease.

ε-Poly-L-lysine (ε-PL) is a L-lysine homopolymer with wide applications in the fields of food, cosmetics, and pharmaceuticals due to its excellent antimicrobial properties and biodegradability. It is primarily produced through microbial fermentation by Streptomyces species, with its biosynthesis catalyzed by the membrane-bound enzyme ε-PL synthetase (Pls). However, Pls purification from industrial strains has not been achieved, and its enzymatic properties, particularly the effects of metal ions on its activity, remain insufficiently characterized. To address this gap, we successfully expressed, purified, and characterized full-length Pls from the industrial strain Streptomyces albulus GS114 for the first time, achieving milligram-scale yields of high-purity protein. A systematic evaluation of His-tag length and position revealed their impact on recombinant membrane protein expression and purification. To improve purification efficiency, we developed a novel strategy integrating DEAE anion exchange chromatography with Ni-affinity chromatography, enabling efficient Pls isolation. Preliminary enzymatic assays showed that Pls exhibits optimal activity at 20 °C and pH 8.5. Both Mn²⁺ and Mg²⁺ effectively support Pls catalytic activity, marking the first report on the impact of metal ions on Pls activity. These findings lay the foundation for further research on enzyme engineering, biosynthetic mechanisms, and potential improvements in ε-PL biomanufacturing.

Cisplatin is mainly used for treating terminal hepatocellular carcinoma (HCC), but the development of drug resistance limits its clinical efficacy in treating this disease. The helicase activity of DDX11 can prevent hypersensitivity to chemotherapy drugs and DNA damage. The aim of this study was to explore the potential molecular mechanism through which DDX11 mediates cisplatin resistance in HCC. HepG2 and Huh7 cisplatin-resistant strains were established and named HepG2-DDP and Huh7-DDP, respectively. Cell proliferation and DNA damage were measured by CCK-8, colony formation and comet assays, and the expression of related proteins and genes was detected by Western blotting, immunohistochemistry, immunofluorescence and RT‒qPCR. The HepG2-DDP and Huh7-DDP cell lines were xenografted subcutaneously into nude mice to investigate the role of DDX11 in vivo. DDX11 expression was upregulated in HCC clinical samples and cell lines. DDX11 knockdown inhibited HCC cell proliferation and promoted sensitivity to cisplatin, DNA damage and endoplasmic reticulum (ER) stress, and SKP2 knockdown weakened the effects of DDX11 overexpression and promoted cisplatin-induced ER stress. Mechanistically, DDX11 inhibited cisplatin-induced DNA damage and ER stress by upregulating SKP2 expression, ultimately reducing the sensitivity of HCC cells to cisplatin. This study revealed that the DDX11/SKP2 axis may be a promising therapeutic target for improving resistance to cisplatin in patients with HCC.

Inflammatory Bowel Disease (IBD) refers to the inflammatory disorders of the colon and small intestine. The effect of the essential oil from Tamarix aphylla (TAEO) on inflammation and apoptosis has been investigated using a rat TNBS-induced colitis model. The study utilized macroscopic and histopathological evaluations, cytokine profiling by ELISA, and protein expression assays to assess the effects of TAEO on ulceration, cytokine levels, apoptotic proteins, and anti-apoptotic proteins. Sound experimental groups received several dosages of TAEO (10, 30, and hundred mg/kg) and dexamethasone serving as a comparative control. TAEO significantly reduced mucosal damage in a dose-dependent manner, with dosages of 30 and 100 mg/kg effectively decreasing the ulcer index compared to controls (p < 0.001). It also modulated cytokine profiles, notably reducing TNF-α (p < 0.05 for 30 mg/kg; p < 0.001 for 100 mg/kg), IL-1β, and TGF-β (p < 0.05 for 30 mg/kg; p < 0.001 for 100 mg/kg), while increasing IL-10 at higher doses (p < 0.01 for 30 mg/kg; p < 0.001 for 100 mg/kg). Furthermore, TAEO reduced the expression of pro-apoptotic proteins Bax and caspase-3 (p < 0.001 for both at 100 mg/kg), and enhanced the anti-apoptotic protein Bcl-2. Reductions in NF-κB and p38 MAPK activation were also significant (p < 0.01 and p < 0.001, respectively, for 100 mg/kg). Notably, high-dose TAEO (100 mg/kg) significantly activated the Nrf2 pathway more than dexamethasone (p < 0.001), promoting antioxidant defenses. Histopathological assessments confirmed these findings, showing substantial improvements in tissue architecture and reductions in inflammatory markers. TAEO possesses strong anti-inflammatory, anti-apoptotic, and antioxidant actions in TNBS-induced colitis with high therapeutic potential against IBD. The effects are specific towards higher doses, suggesting a dose-dependent mechanism of action, and justifying further testing.

The protein encoded by the Disco-interacting protein 2 homolog B (DIP2B) gene contains DNA methyltransferase and adenosine monophosphate (AMP) binding sites. Although DIP2B has been implicated in tumorigenesis, its diagnostic and prognostic significance across various cancers remains unclear. A comprehensive data analysis was performed using multiple bioinformatics platforms and tools, including the TCGA and GTEx databases, the R programming language, STRING, Cytoscape, TISIDB, cBioPortal, GSCALite, HPA, NetworkAnalyst, and CancerSEA. DIP2B expression was elevated in multiple cancer types, with variations observed across different immune and molecular subtypes. DIP2B was implicated in numerous cancer-related signaling pathways and showed enhanced diagnostic and prognostic significance across malignancies. Transcription factor analysis identified specificity protein 1 (SP1), specificity protein 3 (SP3), and histone deacetylase 1 (HDAC1) as potential regulators of DIP2B expression.In addition, a positive correlation was found between DIP2B and central memory CD8 T cells in cancers, suggesting a potential role in immune modulation. DIP2B shows promise as a prognostic biomarker and may represent anovel target for immunotherapy.

Multicopper oxidases such as laccases have emerged as valuable biocatalysts in environmental and industrial processes due to their ability to oxidize a variety of phenolic and aromatic compounds using molecular oxygen as the sole electron acceptor. In the present study, laccase from different Achromobacter species, having close 16 S rRNA identity related to Achromobacter sp. strain 206,011 (accession no. MK949376.1), was screened. Maximum microbial growth and enzyme production were both observed at 24 h under optimized conditions. Optimum enzyme activity was observed at 40 °C and pH 5.0. The laccase maintained over 85% activity with metal ions up to 10 mM concentration, demonstrating strong metal tolerance and suitability for biotechnological applications in metal-rich environments. Partial purification of the crude enzyme revealed two prominent bands on SDS-PAGE, approximately at 50 kDa and 200 kDa, suggesting the possible presence of laccase isozymes. Native-PAGE further confirmed enzymatic activity through guaiacol oxidation. The laccase was subsequently evaluated for its bioremediation potential through dye decolorization assays, demonstrating promising activity across different dye types. These findings support the potential of this laccase as a robust biocatalyst for environmental applications, particularly in the treatment of dye-laden effluents.