Applied Biochemistry and Biotechnology最新文献

Background As a commonly used anesthetic in clinical practice, sevoflurane (Sevo) has been found to have a certain protective effect on myocardial ischemia/reperfusion (MI/R) injury. However, the underlying molecular mechanisms deserve further elucidation. Methods Human cardiomyocytes were induced by hypoxia/reoxygenation (H/R), and MI/R rat model was established by ligation of left coronary artery. Cell viability and apoptosis were tested using CCK8 assay and flow cytometry. Inflammatory factors and ferroptosis-related markers were tested by corresponding kit. The levels of ALOX5, ferroptosis-related markers, and tripartite motif 65 (TRIM65) were determined by qRT-PCR or western blot. The interaction between TRIM65 and ALOX5 was evaluated by Co-IP assay. Results Sevo repressed H/R-induced cardiomyocyte apoptosis, inflammation, and ferroptosis. Sevo reduced the ALOX5 protein level, and ALOX5 overexpression reversed the inhibitory effects of Sevo on H/R-induced cardiomyocyte injury. E3 ubiquitin ligase TRIM65 could decrease ALOX5 protein stability by promoting its ubiquitination level. TRIM65 inhibited H/R-induced cardiomyocyte apoptosis, inflammation, and ferroptosis by downregulating ALOX5. Furthermore, TRIM65 knockdown reversed the protective effects of Sevo on H/R-induced cardiomyocyte injury. Additionally, Sevo alleviated MI/R injury in rat models by activating TRIM65-mediated ubiquitination of ALOX5. Conclusion Sevo restrained H/R-induced cardiomyocyte apoptosis, inflammation, and ferroptosis to alleviate MI/R injury, which might be associated with the TRIM65/ALOX5 axis.

Polycyclic aromatic hydrocarbons (PAHs) are relatively common in saline and hypersaline environments and pose a great threat to both the environment and human health. Biological treatment is an economically effective method for treating persistent organic pollutant pollution; however, traditional microbe species have limited ability to degrade PAHs in saline environments. In this study, strain Y3 was screened from the PAH-degrading bacterial consortium 5 H enriched in mixed saline soils in Shanxi and identified as belonging to the genus Altererythrobacter using 16 S rRNA sequencing. Using phenanthrene as the only carbon source and by changing the environmental factors, it was found that strain Y3 was highly tolerant to moderate to high salinity and highly efficient under neutral to weakly alkaline conditions. PAH-degrading genes, such as genes encoding ring hydroxylating dioxygenase (RHD) and gentisate 1,2-dioxygenase (G12O), were also annotated in the genome of strain Y3 and presented high novelty compared with other reported alleles. Moreover, the gene clusters of the PAH degradation pathway of strain Y3 were identified and annotated. Combined with the detected PAH-degrading intermediates, the degradation pathway of phenanthrene by strain Y3 was proposed. Phenanthrene was first activated by RHD and then further degraded through the G12O pathway. To our knowledge, this is the first report of phenanthrene degradation by Altererythrobacter sp. Y3 under high salinity. The PAH-degrading genes and pathways associated with Altererythrobacter are also reported for the first time. Strain Y3 is identified in this study to be able to degrade phenanthrene in saline and hypersaline environments, promoting the understanding of PAH degradation and providing a useful microbial resource for the remediation of PAHs in saline environments.

Kawasaki disease (KD) is classified as an acute febrile illness characterized primarily by systemic vasculitis. While the involvement of N6-methyladenosine (m⁶A) modification in various cardiovascular conditions has been established, its specific role in KD remains unexplored. This study aims to investigate the potential influence of m⁶A modification in KD and to elucidate the mechanisms involved. Clinically, 18 KD patients and 15 healthy volunteers were recruited. Reverse transcription-quantitative polymerase chain reactio (RT-qPCR) was performed to assess the expression of m⁶A-related enzymes. Lactate dehydrogenase (LDH) release, interleukin (IL)-1β and IL-18 concentrations were analyzed by commercial kits. Hoechst 33,342/PI staining was performed for cell death detection. Pyroptosis-related protein levels were detected by Western blot. RNA immunoprecipitation (RIP) and dual-luciferase reporter assays were performed to assess the interaction between methyltransferase-like protein 3 (METTL3) and Nod-like receptor (NLR) family CARD domain-containing protein 4 (NLRC4). Finally, a KD mouse model was induced by Candida albicans cell wall extracts. Results showed that serum from KD patients exhibited higher METTL3 expression. Additionally, KD serum-treated THP1 cells induced pyroptosis of human umbilical vein endothelial cells (HUVECs). Mechanistically, METTL3 promoted the stability of NLRC4 mRNA via m⁶A methylation. Furthermore, NLRC4 overexpression promoted pyroptosis in KD-treated HUVECs. Finally, METTL3 inhibition attenuated coronary arteritis and pyroptosis in a KD mouse model. In summary, this study concluded that METTL3-mediated m⁶A methylation modification promoted the endothelial cell pyroptosis in KD, which might provide a reference for the pathogenesis of KD.

Cutinase (EC 3.1.1.74), a serine esterase that degrades a variety of ester substrates into fatty acids, has significant potential for various applications. Bacillus subtilis is a food-safe strain known for its exceptional protein secretion capabilities. This study aims to enhance the expression of cutinase from Humicola insolens (HIC) in B. subtilis. Specifically, B. subtilis WS9 was selected as the expression host, with pHY300PLK as the expression vector, achieving an initial enzymatic activity of 35.00 U·mL^-1 in shake flask fermentation. Subsequently, the effect of different promoters on HIC expression was investigated, and the optimal promoter, P_amyQ', was screened, achieving an enzyme activity of 75.85 U·mL^-1. Then, by co-expressing the chaperone protein PrsA and the intracellular chaperone proteins DnaK and GroE, the enzyme activity increased to 121.43 U·mL^-1. Based on that, we optimized the fermentation medium and conditions for cutinase production, and the highest activity achieved was 232.31 U·mL^-1, which was 6.63 times higher than the original level. The recombinant strain was further subjected to high-density fermentation in a 3-L fermenter. After 96 h, the enzymatic activity reached 1548.53 U·mL^-1, which was 6.67 times higher than that achieved in the shake flask. Notably, the cutinase expressed by B. subtilis exhibited comparable efficacy to that expressed by Pichia pastoris in the application of tomato preserves. This study establishes a robust framework for industrial-scale HIC production, advancing its application in food technology.

Inhibiting c-Abl kinase pharmacologically is necessary because of its role in oxidative stress and neurodegeneration. When activated, it causes the accumulation of α-synuclein and dopaminergic neuron damage, leading to Parkinson's disease (PD). Reports of the effectiveness of c-Abl inhibitors repurposed for PD were accompanied by both hope and numerous concerns. Therefore, there is an urgent need for alternative c-Abl inhibitors. We employed a machine-learning-based QSAR model to identify potential actives against c-Abl kinase, screening selected FDA-approved and phase 1 drugs; optimizing the compounds' structures through bioisostere replacement; conducting molecular docking algorithms (HTVS, SP, XP, and Prime's Molecular Mechanics with Generalized Born and Surface Area (Prime-MMPBSA)); in silico pharmacokinetic profiling; and structural stability and dynamics studies for 200 ns. From 3605 drugs and 1456 bioisosteres, two bioisosteres of indobufen (indobufen 25 and 22) showed promising potential against c-Abl kinase. As the two bioisosteres returned the closest docking scores (14.880 and - 14.265 kcal mol^-1, respectively) to the control drug (nilotinib, - 15.312 kcal mol^-1), the Prime-MMGBSA calculations returned - 81.92 and - 84.07 kcal mol^-1, respectively; MMPBSA calculations after a 200-ns MD simulation run returned - 48.20 ± 3.69 kcal mol^-1 and - 49.94 ± 3.05 kcal mol^-1, respectively. This indicates their stability compared to other test compounds, as supported by the RMSD, RMSF, PCA, and DCCM results. Finally, both bioisosteres interacted with MET 318, ASP 381, TYR 253, ALA 269, and PHE 317 in the c-Abl active site. We present these bioisosteres as potential candidates for the treatment or management of PD targeting c-Abl kinase. However, in vitro and in vivo experiments to validate the findings are urgently required.

This study introduces a two-stage correlated analysis model to overcome clinical information oversights in raw data manipulation for canonical correlation analysis extensions. The novel algorithm integrates whole slide images, gene expression data, and pathway scoring data to unveil high-order correlations linked to sarcoma recurrence. This study employs data from 259 sarcoma samples (TCGA and UCSC databases). A two-stage analysis was introduced, using a deep self-reconstruction model for multi-modal data integration, followed by a hypergraph-based adaptive sparse multi-view canonical correlation analysis to explore higher-order correlations among modal features. This study validates DSR-AdaSMCCA's effectiveness with error variance and correlation coefficient analyses, demonstrating faster convergence and higher coefficients, confirming the success of the deep subspace reconstruction strategy. Bioinformatics analysis confirms the algorithm's ability to discover the genes enriched in the sarcoma recurrence-related diseases and uncovered potential biological mechanisms in predicting sarcoma recurrence through the association between WSI features and genetic characteristics. The proposed model in this study successfully integrates imaging genetics data, accurately identifies key features associated with local recurrence of sarcoma, and reveals pathways closely linked to immune response and inflammation through enrichment analysis. The study deepens the understanding of sarcoma recurrence, providing valuable insights for personalized treatment strategies and unraveling the intricate networks influencing tumor relapse. The complete source code is openly available at https://github.com/babykai12345/HB-AdaSMCCA .

Given the critical involvement of intestinal macrophages in ulcerative colitis (UC) pathogenesis and Shikonin's (SHK) established anti-inflammatory properties, this study investigated whether SHK inhibits macrophage proinflammatory (M1) polarization in UC and elucidated its downstream mechanisms. Dextran sulfate sodium (DSS)-induced UC model in mice was treated with SHK by gavage, and the therapeutic effect of SHK was evaluated by observing the changes in body weight, colon length, and Disease Activity Index (DAI) in mice. The pathological changes of colon tissue were observed by HE staining, and tight junction (TJ) proteins and inflammatory cytokines in colon tissue were detected. In vitro experiments were conducted to observe the inhibitory effect of SHK intervention on M1 macrophage polarization using LPS/IFN-γ-induced RAW264.7 cell model. Immunofluorescence, RT-qPCR, and Western blot were used to detect changes in NOD2 and CARD9 levels. SHK treatment significantly ameliorated murine colitis, evidenced by reduced DAI, attenuated DSS-induced colon histopathology, and preserved TJ integrity. SHK downregulated colonic expression of macrophage activation markers (F4/80), M1 polarization markers (iNOS, MCP-1), and pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), while elevating anti-inflammatory mediators (IL-10, Arg1). In LPS/IFN-γ-stimulated RAW264.7 macrophages, SHK consistently suppressed M1 polarization markers/pro-inflammatory mediators (TNF-α, IL-6, MCP-1, iNOS) while enhancing M2 markers (IL-10, Arg1, CD206, Ym1). Mechanistically, SHK inhibited the NOD2/CARD9 pathway in both in vivo and in vitro models. Crucially, NOD2/CARD9 overexpression attenuated SHK's therapeutic effects in murine colitis and blocked its suppression of M1 polarization in vivo and in vitro. This study demonstrates that SHK alleviates UC by inhibiting NOD2/CARD9-mediated macrophage M1 polarization, revealing a novel therapeutic mechanism for UC management.

Biofilm structure enables microorganisms to survive in adverse environmental conditions, and biofilm-related infections are an increasing global public health concern. Information on the structure and interactions of polymicrobial biofilms, which generally contain more than one species in nature and in vivo, is also quite limited. Candida albicans and Staphylococcus aureus, important opportunistic pathogens, are frequently co-isolated together in infections and exhibit increased resistance to treatment. Silver nanoparticles (AgNPs) have been proposed as effective agents against antimicrobial-resistant strains, but synthesis by traditional methods has negative effects on the environment and health. In this study, the effects of cyanobacterium Chroococcus-mediated AgNPs on planktonic and biofilm-formed single and dual S. aureus ATCC 25923 and C. albicans ATCC 14053 strains were investigated for the first time to our knowledge. The MIC test results obtained showed that S. aureus ATCC 25923 was much more sensitive to AgNPs, whereas resistance to amphotericin B and ampicillin increased in dual culture. Scanning and transmission electron microscopic findings, biofilm reduction test, hydrophobicity determination, and confocal microscopic data revealed that AgNPs acted with multi-targets on cell wall and membrane structures, cytoplasm, and organelles in single and dual cultures and had antibiofilm effects. XTT test results demonstrated the biofilm-reducing effect of AgNPs, but this effect was diminished in dual species biofilms compared to single biofilms. In addition, application of AgNPs before biofilm formation is much more effective than application after biofilm formation. Interestingly, the effect of ampicillin on dual biofilm structure was greater than on single biofilm, suggesting that bacterial lysis induced by ampicillin may disrupt fungal biofilm structure or that environmental changes following lysis suppress biofilm development. Our data support that Chroococcus-mediated AgNPs have promising potential in biofilm management, although elucidating the mechanisms underlying the observed limited resistance in dual biofilms remains important.

Nanotechnology leveraging renewable plant biomass to develop silver nanoparticles offers eco-friendly solutions with more sustainable applications. The present research explores the employment of an aqueous extract of Cynoglossum creticum leaves in the green synthesis of silver nanoparticles (Ccl-AgNPs) for potential biological and sensing purposes. The synthesis parameters were controlled spectrophotometrically, and the physicochemical characterizations of Ccl-AgNPs were evaluated. Phytochemicals in the extract served dual roles, as confirmed by infrared spectroscopy analysis. Thermogravimetric analysis (TGA) and energy-dispersive X-ray spectroscopy (EDS) further revealed the presence of a plant-derived organic matter on AgNPs. Morphological descriptions indicated the spherical form of Ccl-AgNPs, comprising a median size of 32 nm. Furthermore, the biological characteristics of green Ccl-AgNPs were investigated, as they showed promising antioxidant activities in DPPH, ABTS, reducing power, and phenanthroline assays. Ccl-AgNPs also exhibited a good inhibitory impact on the growth of four tested Gram-positive and Gram-negative bacteria. Pseudomonas aeruginosa was most susceptible to the inhibitory activity of Ccl-AgNPs, with a minimum inhibitory dose of 31.25 µg/mL. Colloidal AgNPs capped by Ccl-extract have established the potential for use as an SPR-based colorimetric sensing system for the selective detection of neomycin sulfate, with a limit of detection (LOD) of 2.88 µM. The colorimetric probe was successfully tested for neomycin sulfate detection in environmental and biological fluids and veterinary pharmaceutical preparations, with recovery rates ranging between 92.62 and 105.76%. This study underscores the potential of Cynoglossum creticum, a livestock-hazardous weed, as a sustainable resource for synthesizing silver nanoparticles for potential ecological and biomedical uses.

Exosomes derived from mesenchymal stem cells (MSC EXO) have emerged as promising therapeutic candidates for pneumonia. However, the molecular mechanisms underlying MSC EXO-mediated pneumonia protection remain incompletely elucidated. WI-38 fibroblasts were exposed to lipopolysaccharide (LPS) in vitro, while an animal model of pneumonia was generated through intratracheal LPS administration in mice. MSC EXO were isolated and used to treat the pneumonia models. The efficacy of MSC EXO was evaluated by detecting cell viability, proliferation, apoptosis, and pro-inflammatory cytokine secretion. Glutathione S-transferase (GST) pull-down, co-immunoprecipitation (Co-IP), and immunoprecipitation (IP) assays were performed to verify the RNF144A/thyroid-stimulating hormone receptor (TSHR) interaction. TSHR was upregulated in pneumonia serum samples and LPS-stimulated WI-38 fibroblasts. TSHR knockdown attenuated LPS-triggered apoptosis and inflammatory damage in WI-38 fibroblasts. Moreover, ring finger protein 144A (RNF144A) destabilized TSHR through ubiquitination in WI-38 cells. MSC EXO increased RNF144A expression in LPS-stimulated WI-38 fibroblasts. Downregulation of RNF144A diminished the protective effects of MSC EXO against LPS-triggered damage in WI-38 fibroblasts and LPS-induced pneumonia in mice. Additionally, re-expression of TSHR reversed the protective effects of MSC EXO against LPS-triggered injuries in WI-38 fibroblasts. Our findings suggest that MSC EXO protect against LPS-triggered injuries in WI-38 fibroblasts and LPS-evoked pneumonia in mice through RNF144A upregulation-mediated suppression of TSHR expression. This study provides a novel theoretical foundation for the application of MSC EXO in pneumonia treatment.