Applied Biochemistry and Biotechnology最新文献

Objective This study aimed to explore the possible mechanism of adiponectin on the insulin signaling in streptozotocin (STZ)-induced gestational diabetes mellitus (GDM) rats. Methods The GDM rats were induced by injection of 40 mg/kg STZ, and then orally treated with adiponectin (5, 10, 20 mg/kg) every day from gestation day (GD)7 to GD20. The body weight and fasting blood glucose (FBG) were observed every 3 days from GD9 to GD18. Meanwhile, the insulin tolerance test (ITT), homeostasis model assessment insulin resistance (HOMA-IR), and adiponectin expression were observed in rats. Moreover, the insulin signaling-related factors, sex hormone-binding globulin (SHBG), PPAR-α, phospho-AMPK alpha, glucose transporter-3 (GLUT3), and phospho-insulin receptor substrate-1 (p-IRS1) in placental tissues were measured in rats. In vitro, HTR-8-Svneo cells were induced by 30 mM glucose and then treated with adiponectin (50, 100, 200 ng/mL) to observe the changes in cell viability, SHBG expression, and the insulin signaling-related factors. Moreover, silencing or overexpression of SHBG was achieved via transfection with siRNA or pEX-4 SHBG, respectively. The effects of SHBG on the insulin signaling-related factors were observed in cells. Results Adiponectin treatment significantly improved the STZ-induced decrease in body weight, increase in the FBG, ITT, and HOMA-IR with increasing doses. Specifically, adiponectin treatment upregulated the SHBG expression and the insulin signaling-related factors (PPAR-α, p-AMPK, GLUT3, and p-IRS1) in the placental tissues of GDM rats with increasing doses. In line with the results of animal experiments, adiponectin treatment alleviated the high glucose-induced decrease in cell viability, SHBG expression, and the levels of GLUT3 and p-IRS1 in HTR-8-Svneo cells with increasing doses. Moreover, the levels of GLUT3 and p-IRS1 were regulated by the SHBG expression. SHBG silencing weakened the effect of adiponectin in improving the levels of GLUT3 and p-IRS in the high-glucose-induced cells. Conclusion The study showed that adiponectin upregulated the SHBG expression and improved the insulin signaling in STZ-induced GDM rats, as well as adiponectin upregulated the insulin signaling-related factors via SHBG in high-glucose-induced trophoblast cells. This study suggests that adiponectin may be a potential therapeutic target in GDM.

1,8-cineole, a bicyclic monoterpenoid compound with significant application value, is widely used in the fields of flavoring, pharmaceuticals, and biofuels. Although recent advances have enabled its biosynthesis in model microorganisms (Escherichia coli and Saccharomyces cerevisiae), the inherent cytotoxicity of terpenoids and the low catalytic efficiency of terpene synthases remain major bottlenecks limiting further improvement in biosynthetic efficiency. To address these limitations, we employed Serratia marcescens HBQA7-a non-model microorganism with high terpene tolerance, previously isolated in our lab-as a chassis for constructing an efficient 1,8-cineole biosynthetic system. Heterologous expression and functional screening of six 1,8-cineole synthase (CinS) genes from different sources were performed, among which SoCinS from Salvia officinalis exhibited the highest catalytic activity. Rational enzyme engineering through site-directed mutagenesis further improved SoCinS activity. In addition, fusion with high-expression tag proteins significantly improved enzyme expression levels, leading to increased 1,8-cineole titers. Under 72-hour shake flask fermentation conditions, the 1,8-cineole concentration reached 2.1 g/L. Finally, fed-batch fermentation in a 5-L bioreactor yielded a final 1,8-cineole titer of 10.2 g/L, demonstrating a higher productivity and greater industrial application potential than current model microbial systems. This work highlights the promising potential of non-model microorganisms in the efficient biosynthesis of terpenoid natural products.

Plant-mediated synthesis is a most recent approach toward green synthesis of copper-based nanoparticles. In this approach nanoparticles are capped with phytochemicals, suggesting that plants with unique biological properties might transfer these benefits to the nanoparticles. However, there is no experimental evidences to directly corelate bioactivities of nanoparticles to their capping phytochemicals. This study aims to assess the impacts of herbal antimicrobial compounds on the antimicrobial potency of resulted nanoparticles. Cu(OH)₂ nanoparticles (CuNPs) were synthesized by leaf extracts of eucalyptus and tobacco. Resulted particles were characterized to exhibit similar physicochemical properties which are determinative for antimicrobial activity. Antimicrobial effects of the leaf extracts and CuNPs were tested against Staphylococcus aureus and Escherichia coli. Eucalyptus leaf extract was found effective against S. aureus with growth inhibition zone of 13.1 ± 0.6 mm. CuNPs exhibited efficacy against both bacterial strains with inhibition zones exceeding 15 mm. Notably, no significant difference in antimicrobial effectiveness of CuNPs was observed, suggesting that the antimicrobial superiority of eucalyptus extract was not transferred to nanoparticles. These results could reshape our understanding about the impacts of herbal bioactive molecules on the bioactivity of plant-mediated synthesized nanoparticles.

Ferroptosis is a critical contributor to ischemia-reperfusion (I/R) injury and subsequent organ failure. While ENPP2 has been implicated in regulating ferroptosis in cardiomyocytes, its specific role in myocardial I/R injury remains unclear. This study aims to elucidate the function of ENPP2-mediated ferroptosis in myocardial ischemia-reperfusion injury (MI/RI), to provide novel insights into potential therapeutic strategies. A mouse model of MI/RI was established and subjected to interventions with ENPP2 overexpression and/or SIRT1 knockdown. In vitro, cardiomyocytes were treated with palmitate and subjected to hypoxia/reoxygenation (H/R) to simulate I/R injury. These cells received treatments with ENPP2 overexpression (oe-ENPP2), SIRT1 overexpression (oe-SIRT1), PGC-1α silencing (si-PGC-1α), and/or SIRT1 knockdown (sh-SIRT1). Additionally, Erastin-induced ferroptosis in cardiomyocytes was used to assess the protective effects of oe-ENPP2. Ferroptosis was assessed through the lipid peroxidation (MDA, 4-HNE), iron and Fe²⁺ assays, GPx4 and SLC7A11 expression, and transmission electron microscope. Overexpression of ENPP2 significantly alleviated myocardial infarction in MI/RI mice, as indicated by the upregulation of GPx4 and SLC7A11 protein levels. In cardiomyocytes subjected to hypoxia/reoxygenation (H/R) or erastin-induced ferroptosis, oe-ENPP2 reduced apoptosis rates, preserved Fe²⁺ content, and restored GPx4 and SLC7A11 expression. Silencing PGC-1α blocked the protective effect of oe-ENPP2 against H/R-induced ferroptosis in HL-1 cells. Additionally, SIRT1 overexpression inhibited PGC-1α acetylation, whereas SIRT1 knockdown similarly reversed the anti-ferroptotic effects of oe-ENPP2 in H/R-treated HL-1 cells. SIRT1 silencing blocked the protective effects of oe-ENPP2 against myocardial infarction and fibrosis in MI/RI mice via the PGC-1α/NRF1 pathway. ENPP2 overexpression protects the mouse myocardium from I/R-induced ferroptosis injury via the SIRT1/PGC-1α/NRF1 pathway. These findings suggest a novel gene therapy strategy for mitigating myocardial I/R injury.

Hypertrophic cardiomyopathy (HCM) is a complex genetic disorder characterized by left ventricular hypertrophy and impaired cardiac function. Although progress has been made in understanding its genetic basis, the discovery of novel biomarker candidates and therapeutic targets remains crucial for improving diagnosis and treatment. Recent studies have demonstrated that copper metabolism plays an important role in various diseases, suggesting that copper-related genes (CRGs) may be of significant importance in HCM. In this study, we analyzed bulk RNA-seq and single-cell RNA-seq (scRNA-seq) data from healthy controls (HC) and HCM patients. Differentially expressed genes (DEGs) were identified through differential expression analysis, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to identify associated pathways. Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify gene modules related to the HCM phenotype, and a diagnostic model was constructed based on these modules. Additionally, cell type-specific expression patterns were explored through single-cell analysis, and Gene Set Enrichment Analysis (GSEA) and MR analyses were conducted to evaluate the causal relationships between genes and HCM risk. The study found that DEGs associated with HCM were significantly enriched in pathways related to immune responses. WGCNA identified gene modules highly correlated with HCM, among which the blue module exhibited the strongest correlation with HCM. The diagnostic model constructed based on DEGs and WGCNA module genes demonstrated good diagnostic performance, with FCN3, TIPARP, and PROM1 emerging as potential diagnostic biomarker candidates for HCM. Additionally, single-cell analysis revealed the expression characteristics of different cell types in HCM, and causal relationships between key genes and HCM risk were confirmed through GSEA and MR analyses. This study identified FCN3, TIPARP, and PROM1 as cuproptosis-associated biomarker candidates that showed reproducible expression patterns across cohorts and experimental validation. These findings are associative and hypothesis-generating; they suggest potential diagnostic utility that warrants prospective clinical evaluation and mechanistic studies (e.g., perturbation of copper homeostasis and gene manipulation) before any therapeutic inference can be made.

Plant-based nanoparticles have potential uses and advantages over traditional physico-chemical techniques. In this study, zinc oxide nanoparticles (ZnO-NPs) were biosynthesized from Tagetes erecta flower extract and Allium cepa peel extract, characterized and evaluated their antioxidant, and antidiabetic activities. UV-visible absorption spectroscopy (UV-Vis) revealed a characteristic absorption peak at 355 nm, while X-ray diffraction (XRD) confirmed the presence of crystalline ZnO-NPs having an average size of 35.14 nm. Fourier transform infrared (FTIR) spectroscopy provided evidence for presence of functional groups that stabilizes nanoparticles, elemental composition was confirmed by energy-dispersive X-ray spectroscopy (EDX). Scanning electron microscopy (SEM) provided evidence of the rod-like morphology of nanoparticles, and dynamic light scattering determined the particle size distribution and polydispersity index. The biosynthesized ZnO-NPs presented a range of biological activities that included key antioxidant properties and strong inhibition of α-glucosidase activity (IC₅₀ = 35.9 ± 2.49 µg/mL). The MTT assay was performed for cytotoxicity screening MIN6 pancreatic β-cells that confirmed high viability from 99.4% at 10 µg/mL to 85.6% at 100 µg/mL, indicating non-toxic behaviour. Furthermore, the biosynthesized ZnO-NPs also induce dose-dependent stimulation of insulin secretion, with fold-increases ranging from 2.29 to 7.64 (p ≤ 0.0001) compared to controls. The ZnO-NPs derived from peel of Allium cepa and the floral extracts of Tagetes erecta shown strong antioxidant and antidiabetic characteristics, suggesting that they have the potential to be effective therapeutic agents with minimal side effects for advanced biomedical applications.

Screening plant-based alkaloids is one of the alternate therapeutic approaches to control antibiotic-resistant micro-pathogens. Our research highlighted beta carboline alkaloids as one of the most promising small molecules to established anti-virulent and anti-biofilm efficacy to regulate resistant bacterial infection. In vitro, in vivo assay and molecular docking were employed. Result Among six different bacterial strains, harmine showed 160 ± 2.07 µg/ml as the minimum inhibitory concentrations (MIC), followed by harmalol (190 ± 2.46) and harmaline (270 ± 3.04) against Staphylococcus aureus 96 (SA 96). Methicillin-resistant Staphylococcus aureus MRSA strain also showed inhibition of growth (MIC) by harmine, harmalol and harmaline at 250 ± 3.10, 320 ± 3.39 and 390 ± 4.90 µg/ml, respectively. MRSA is a prominent source of nosocomial infections, forming biofilms. The growth of biofilm got decreased with exposure to the sub-MIC concentrations (60, 80 and 100 µg/mL) of harmine, suppressing protein, targeting EPS and inhibiting extracellular protease. Harmine promote biofilm cell detachment by targeting cell surface hydrophobicity. Harmine causes depolarization of bacteria's cell membrane. Bacterial cell viability was further studied by propidium iodide (PI), DNA leakage and Acridine Orange (A/O)-Ethidium Bromide (EtBr) assay. Harmine treatment leads to increased reactive oxygen species (ROS) levels in biofilm cells. The binding affinities by molecular docking and dynamics indicated highest affinity with AgrC (-6.17 kcal/mol). Harmine treatment (32.0 mg/ kg bw, IP for five days) further recovered MRSA infected lungs in BALB/c mice. The findings revealed that among the three beta carboline alkaloids, harmine might be employed as a potential antibiofilm and antimicrobial agent for successful control of clinical S. aureus infection.

This study explores the mechanism of DNMT3A in inflammatory response in allergic rhinitis (AR) mice. A mouse model of AR was established by ovalbumin induction, followed by injection of DNMT3A overexpression vector. The times of nose rubbing and sneezing within 15 min were recorded. The nasal mucosa tissues were observed by H&E staining. The serum histamine, IgE, IL-1β, IL-10, IFN-γ, and TNF-α were detected by ELISA. The proportion of Th17/Treg cells in lymphocytes was detected by flow cytometry. DNMT3A, IRF4, and CD44 expressions were tested by qRT-PCR or Western blot. ChIP evaluated the DNMT3A enrichment on IRF4 promoter and IRF4 enrichment on CD44 promoter. Methylation-specific PCR determined the methylation level of IRF4 promoter. The binding of IRF4 to CD44 was verified by dual-luciferase assay. DNMT3A was poorly expressed in AR mice. DNMT3A overexpression reduced the times of nose rubbing and sneezing in AR mice, alleviated nasal mucosal tissue injury, reduced the proportion of Th17/Treg cells, and diminished serum inflammatory factors. DNMT3A was enriched on IRF4 promoter and repressed IRF4 expression by enhancing IRF4 methylation level. IRF4 bound to CD44 promoter to elevate CD44 expression. In conclusion, DNMT3A-mediated methylation of IRF4 reduces AR inflammatory response by elevating CD44 expression.