Applied Biochemistry and Biotechnology最新文献

Gastric cancer poses a significant global health challenge, with current treatment options like chemotherapy often resulting in toxicity to healthy cells. In this context, nanomedicine emerges as a promising strategy to enhance drug delivery and minimize adverse effects. This study focuses on developing Zinc oxide nanoparticles (ZnO-NPs) that are functionalized with ascorbic acid and conjugated with chitosan (ZnO@AsA/CS) to improve their efficacy and cellular uptake for targeting gastric adenocarcinoma cells (AGS). The ZnO-NPs were synthesized and modified to form ZnO@AsA/CS. Various characterization techniques, including X-ray diffraction (XRD), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), polydispersity index (PDI), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), were utilized to assess their properties. The cytotoxic effects of the nanoparticles for AGS and HGF-1 normal cells were evaluated through MTT assays. For AGS cells, lactate dehydrogenase (LDH) assays were conducted to examine cell membrane integrity. Additionally, the activity of Caspase-3, Caspase-8, and Caspase-9 was analyzed to explore apoptotic pathways. The generation of reactive oxygen species was measured using DCFH-DA staining, and the expression of apoptotic markers was analyzed via quantitative reverse transcription polymerase chain reaction (qRT-PCR). The characterization process revealed that the ZnO@AsA/CS nanoparticles had a uniform size distribution, averaging between 100 to 200 nm. MTT assay results indicated a dose-dependent reduction in AGS cell viability, with an IC₅₀ of 78.4µg/ml. LDH assays confirmed the cytotoxicity profile consistent with the MTT results. Following treatment with ZnO@AsA/CS, significant changes in the expression of apoptotic genes were noted, including downregulation of Bcl-2 and upregulation of Caspase-3 and Caspase-8 and Caspase-9. Overall, the study suggests that ZnO@AsA/CS could serve as an effective therapeutic strategy for treating gastric adenocarcinoma.

Lignin-derived aromatic aldehydes are key intermediates in lignocellulosic biomass degradation but can exert cytotoxic effects on microorganisms. Ceriporiopsis subvermispora exhibited enhanced glucose consumption and mycelial growth when cultured in the presence of vanillin, suggesting its ability to metabolize lignin-derived aldehydes. We identified 16 putative aldehyde dehydrogenase genes (Cs-aldhA-P) from C. subvermispora, based on sequence homology to known aromatic aldehyde dehydrogenases (Pc-ALDH1 and Pc-ALDH2) from Phanerochaete chrysosporium. These Cs-Aldhs shared 65.7-89.7% amino acid identity with Pc-ALDHs and contained conserved functional motifs. They were grouped into six clades based on phylogenesis and AlphaFold2-based structural analyses, with pronounced divergence in the architecture of their substrate-binding pockets. Fourteen of these genes were successfully expressed in Rhodococcus erythropolis, and their enzymatic activities were evaluated using fluorescence-based NADH assays. Cs-AldhJ showed broad specificity and high activity (1.86 ± 0.34 to 5.22 ± 0.05 U/mg), whereas Cs-AldhA and Cs-AldhK exhibited lower activities, ranging from 0.02 ± 0.00 to 0.08 ± 0.01 U/mg and 0.05 ± 0.01 to 0.38 ± 0.01 U/mg, respectively. The corresponding oxidation products were confirmed by GC-MS, and kinetic analyses revealed mechanistic diversity among the enzymes. These findings highlight the functional diversification of the Cs-Aldh family and provide molecular insights into the cellular responses of C. subvermispora to lignin-derived aromatic compounds.

Background: Microglial autophagy is closely related to the development of Parkinson's disease (PD). The objective of this research was to investigate the effect of glycyrrhizin (Gly) combined with electroacupuncture (EA) on PD mice and its potential regulation of microglial autophagy.

Methods: A PD mouse model was constructed using the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), followed by the administration of Gly to PD mice and EA. The activation of BV2 cells was triggered by lipopolysaccharide (LPS). Western blotting, immunofluorescence, flow cytometry, Nissl staining, and immunohistochemistry were used to investigate the molecular mechanism that governs how microglial autophagy levels influence PD progression.

Results: Elevated levels of HMGB1 were observed in both patients with PD and PD mice. Gly inhibited HMGB1 expression and extracellular secretion by increasing SIRT1 activity and subsequently inhibit BV2 autophagy. EA inhibited the autophagy of microglia in PD mice by inhibiting the activation of TRPV1 and reducing the expression of HMGB1 in vivo. Gly combined with EA downregulated HMGB1 expression, inhibited microglial autophagy and improved the PD process, resulting in better effects than Gly or EA alone.

Conclusion: Gly combined with EA can reduce the level of autophagy in microglia by downregulating HMGB1, thereby improving the PD process and providing a theoretical basis for the clinical treatment of PD.

Crop straw, a significant agricultural waste, is produced in large quantities but has limited digestibility due to its lignocellulosic structure, limiting its use as animal feed. Cellulase degrades cellulose by breaking glycosidic bonds, while microorganisms secrete hydrolase enzymes to enhance straw degradation. However, the synergistic effects of microorganisms and enzymes on straw degradation have received limited attention. In this study, we investigated the combined effects of microflora and enzymes on oat grass straw degradation through a 9-day constant-temperature fermentation experiment. Our results showed that the combined addition of microflora and enzymes improved straw degradation by disrupting its structural integrity, altering lignocellulose-related groups, and breaking chemical bonds. Microflora contributed to a significant reduction in Acid Detergent Lignin (ADL), hemicellulose, and Crude Ash (CA), while enzymes reduced Crude Fiber (CF), Neutral Detergent Fiber (NDF), and Acid Detergent Fiber (ADF). The synergistic combination of both microflora and enzymes resulted in substantial reductions in CF, NDF, ADF, ADL, hemicellulose, and CA, with degradation rates increasing by 16.13%, 27.83%, 18.86%, 1.82%, 8.96%, and 1.58%, respectively, compared to the control. In conclusion, the synergistic effects of microflora and enzymes significantly enhance straw cellulose degradation, providing valuable insights into improving straw utilization as animal feed and developing more effective straw fermentation agents.

Flavin-dependent halogenases (FDHs) are a class of enzymes renowned for their regioselective ability to precisely insert halogen atoms into small aromatic compounds. Halogen incorporation can enhance the physicochemical and biological properties of molecules, making them valuable for agrochemical and pharmaceutical applications. Through bioinformatic mining of bacterial genomes, we discovered and functionally characterized SnFDHal, an efficient tryptophan 5-halogenase from Streptomyces noursei NRRL B-1714. This halogenase operates across a broad pH range and exhibits a melting temperature of 46.7 °C at both pH 6 and pH 8, which is comparable to thermophilic halogenases such as Th-Hal and BorH, and notably higher than those of mesophilic counterparts. Steady-state kinetic analysis revealed that SnFDHal displays superior catalytic efficiency for the chlorination of L-tryptophan compared to other FDHs reported to date. Structural modeling of its active site suggests that a conserved bulky phenylalanine residue (F49) promotes halogenation at the C-5 position of L-tryptophan, consistent with experimental findings. The combination of high catalytic efficiency and thermostability positions SnFDHal as a promising biocatalyst for applications in agrochemical and pharmaceutical industries.

The purine analogue, 2-fluoroadenine-9-β-D-arabinofuranoside, better known as fludarabine, is a widely used drug for cancer treatment. Nowadays, it is mainly synthesized by chemical methods, but it has been proved that biocatalysis has several advantages over chemical synthesis. In this work, Cellulosimicrobium cellulans was studied due to its fludarabine biosynthesis ability, inferring the activity of a NDT type II enzyme capable of using arabinose as sugar donor. Reaction parameters were optimized, achieving bioconversion rates of 75% at two hours, significantly higher than previously reported. Additionally, various mixed entrapment techniques for whole-cell immobilization were analyzed as a strategy to stabilize the biocatalyst for potential industrial applications. Natural matrices such as agar, agarose, and alginate combined with different nanocomposites were assayed. Finally, we developed an innovative mixed nanobiocatalyst using an agarose matrix to which a mixture of nanocomposites bentonite and montmorillonite was added. We characterized the biocatalyst to demonstrate that the nanocomposite and Cellulosimicrobium cellulans cells were successfully incorporated into the matrix. The developed mixed biocatalyst was able to synthesize more than 59% of fludarabine in six hours of reaction and its reusability under operational conditions was improved more than tenfold. This technology offers an efficient, cheap and sustainable method for producing fludarabine, with the potential to meet regional demand and reduce reliance on pharmaceutical monopolies.

As an interspecies-signaling molecule, indole that is also regulating the microbial community quorum sensing (QS) can be an indispensable factor in influencing the performance of an anaerobic digestion process. Mainly released by Gram-negative bacteria, the impact of indole regulation on methane production in such a system is hardly exposed. This research intends to analyze the methane production affected by the microbial community fluctuations in the waste sewage sludge (WSS) in response to QS repression by indole and its analogs: 4-fluoroindole (4-FI), 5-fluoroindole (5-FI), 6-fluoroindole (6-FI), 3-indoleacetic acid (3-IAA), and 3-indoleacrylic acid (3-IARA). Illumina MiSeq platform was utilized to delve into the active microbes, with ribonucleic acid (RNA) used as the template to generate the 16S metagenomic library. As results, all of them inhibit methane generation even with substrates (acetic acid) availability, and this phenomenon can be led by the slight imbalance of Gram-positive/negative bacterial composition and the inactivation of the viable core fermenters: Firmicutes, Proteobacteria, and Chloroflexi. Plus, the following compounds, indole, 3-IAA, and 6-FI directly disrupted methane production by both acetoclastic and hydrogenotrophic methanogenic archaea. Methanosarcina acetivorans C2A, as a prominent methane synthesizer, was also characterized in terms of its viability and methane synthesis activity against them. Notably, Methanosarcina acetivorans C2A was non-viable in the presence of indole and 6-FI, thus lowering methane production. From here, the affirmation of the direct and indirect inhibitions of methanogenic archaea by indole and its derivatives will pave the way for a valuable future exploration of QS mechanism mapping in archaea during methanogenesis.

Emerging evidence underscores the pivotal yet context-dependent role of butyrate metabolism in oncogenic progression; however, the mechanistic landscape of its associated genetic determinants in prostate carcinogenesis remains poorly characterized. This gap presents a critical opportunity to delineate novel metabolic checkpoints that may offer therapeutic vulnerabilities for advanced prostate malignancies. Analyzing prostate cancer (PC) patients from TCGA and GEO databases, we identified 320 BMGs and stratified tumors into two butyrate metabolism-associated clusters. Machine learning and single-cell transcriptome analysis are used for further study. Luciferase reporter assay, qRT-PCR, FISH, and functional assays are applied to investigate the role of FOS. BMC1 correlated with aggressive phenotypes and stromal-rich tumor microenvironments, while BMC2 was linked to cell cycle regulation and DNA repair. A machine learning-derived RSF + GBM prognostic model demonstrated robust predictive accuracy for biochemical recurrence (training C-index: 0.85). BM scores are further associated with tumor mutation burden and differential drug sensitivities. FOS is overexpressed in PC, promotes proliferation and migration via transcriptional suppression of tumor-suppressive miR-27b. Clinical cohorts confirmed FOS's correlation with advanced T stages and recurrence risk. These findings establish BM-based stratification as a prognostic tool and implicate the FOS-miR-27b axis as a therapeutic target, bridging metabolic heterogeneity with molecular mechanisms in PC progression. Our research offers a critical opportunity to delineate novel metabolic checkpoints that may offer therapeutic vulnerabilities for advanced prostate malignancies.

Propagation in in vitro conditions offers significant advantages such as faster, mass production and disease, virus free plant production. In in vitro culture, the addition of auxin and cytokinin plant growth regulators to the nutrient medium is very efficient for cell differentiation, shoot multiplication, organ formation and tillering. In this context, it was aimed to determine the effects of different plant growth regulators (BAP and NAA) combinations added to MS nutrient medium on shoot regeneration and development of shoots in in vitro culture of Aronia melanocarpa (Michx) Elliott. Rootless shoot explants were cultured in prepared MS nutrient media containing different concentrations of BAP (0.6, 0.7, 1.0, 2.0, 3.0 and 4.0 mg.L^- 1) and NAA (0.05, 0.01, 0.5, 0.1 and 1.0 mg.L^- 1) for 8 weeks. At the end of culture, the regeneration situation in the cultured explants and the morphological characteristics of the developing shoots were examined. Within the scope of morphological characteristics; the highest length of shoot (cm), average shoot length (cm), number of shoots per explant (tillering) (number), longest root length (cm), number of leaves per shoot (unit), chlorophyll density, rooted explant rate (%) and callus forming explant rate (%) parameters were taken into account in the measurements. As a result of the research; the highest values were determined for the average shoot length in MS medium containing 3 mg.L^- 1 BAP + 0.5 mg.L^- 1 NAA (M3) as 2.52 cm; the number of shoots per explant in MS medium containing 2 mg.L^- 1 BAP (M10) and in MS medium containing 3 mg.L^- 1 BAP + 0.1 mg.L^- 1 NAA (M7). And also M8 medium containing 0.7 mg.L^- 1 BAP + 0.05 mg.L^- 1 NAA gave the best result in terms of longest root length. However, the rate of explants forming callus, chlorophyll density, number of leaves per shoot and the highest shoot length were not found to be different with the doses of BAP and NAA added to the nutrient media. As a result; It was determined that the dose of BAP added to the MS nutrient medium contributed to shoot and organ formation and development, being higher than the dose of NAA.