Applied Biochemistry and Biotechnology最新文献

This work describes the development of a novel biosensor obtained by immobilizing laccase from Pleurotus ostreatus Florida onto a glassy carbon electrode platform modified with zinc oxide quantum dots. For enzyme immobilization, the exopolysaccharide botryosphaeran from Botryosphaeria rhodina MAMB-05 was used. Although both biomaterials are from different fungal sources, laccase immobilization was guaranteed, which was demonstrated by the excellent stability of the fabricated biosensor device for the voltammetric determination of 2,6-dimethoxyphenol (2,6-DMP). Under the optimal experimental conditions, the cathodic current from the square-wave voltammograms presented a linear dependence on the 2,6-DMP concentration within the range of 10-400 nmol L^-1, with a limit of detection of 9 nmol L^-1. This bioanalytical device exhibited excellent repeatability and long-term storage stability.

The incidence of laryngeal squamous cell carcinoma (LSCC) has been rising recently. LSCC is one of the most prevalent malignant tumors of the head and neck. In this study, we aimed to investigate whether tripartite motif containing 71 (TRIM71) could serve as a molecular target for the treatment of LSCC. The mRNA and protein levels were examined by using real-time qPCR and Western blot, respectively. Cell proliferation was determined by cell-counting kit 8 assay. To further confirm the function of TRIM71 in LSCC, an in vivo cell line-derived xenograft study was conducted. The half-life of eukaryotic translation initiation factor 5A2 (eIF5A2) protein was measured by cycloheximide chase assay. Our results showed that TRIM71 was significantly downregulated in LSCC tumor tissues. TRIM71 overexpression significantly inhibited LSCC cell growth and suppressed tumor volume and weight in the xenograft models. The interaction between TRIM71 and eIF5A2 was verified by co-immunoprecipitation assay. Moreover, overexpression of TRIM71 in LSCC cells significantly inhibited the protein expression of eIF5A2 by down-regulating its stability, while it did not affect its mRNA level. In contrast, overexpression of eIF5A2 abolished the anti-tumor effects of TRIM71. In summary, TRIM71 may exert its anti-tumor effects through regulating eIF5A2, highlighting the potential of TRIM71 as an effective therapeutic target for the treatment of LSCC.

Syringaldehyde derived from lignin is one of the essential intermediates for the production of basic chemicals. However, it was poorly understood for the direct microbial conversion of syringaldehyde. Here, this study tried to use cold plasma technique to enhance syringaldehyde conversion for the bacterium Pseudomonas putida. It illustrated that cell growth and syringaldehyde conversion were separately increased by 1.49 times at 3 h and 1.60 times at 6 h for 35 s, 1.16 and 3.44 times for 140 W, and 1.63 and 4.02 times for 105 Pa for P. putida through single factor assays of cold plasma treatment. To be sure, cell growth and syringaldehyde conversion were enhanced by 1.14 and 5.54 times at 3 h under the optimum parameters (35 s, 140 W, and 105 Pa) for P. putida. Furthermore, genome re-sequencing further discovered single-nucleotide polymorphisms of P. putida, such as PP_2589 (A428V), PP_5651 (V82F), and PP_0545 (W335R), and thus indicated that the potential genetic changes derived from cold plasma treatment would be responsible for the acceleration of syringaldehyde conversion. This work would provide a robust strain catalyst and the potential candidate mutation sites for genetic manipulation for microbial bioconversion of the value-added and lignin-based biochemicals.

Head and neck squamous cell carcinoma (HNSCC) is a common malignant tumor occurring in various sites such as the oral cavity, pharynx, larynx, and nasal cavity. This study aimed to explore the biological functions and prognostic value of E2F transcription factor 1 (E2F1) in HNSCC. Transcriptome and single-cell sequencing (scRNA-seq) data of HNSCC patients were analyzed using data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). All samples were divided into high and low expression groups based on the expression levels of E2F1. A risk model was constructed based on Lasso-Cox regression, and the differences between the two groups in terms of prognosis were explored. The scRNA-seq data of HNSCC samples were analyzed using the Seurat package to identify cell types. AUCell was used to score different types of cells, and subsequently, the interaction pathways between the high-scoring cell population and other cell populations were explored using the CellChat package. The expression level of E2F1 in tumor tissues was higher than that in normal tissues, which was confirmed by in vitro experiments. Analysis of transcriptome data from TCGA revealed significant differences in overall survival (OS) between the high and low expression groups. Prognostic genes were selected based on DEGs between the two groups, and a risk model was constructed. Subsequently, a nomogram model was constructed based on clinical factors and risk scores, which exhibited good predictive performance. The expression landscape of prognostic genes in different cell types was explored using scRNA-seq data of HNSCC samples. Dendritic cell populations were identified as high-scoring cell populations, and the pathways of interaction between this cell population and other cell populations were explored. We identified E2F1 as an independent prognostic factor closely associated with the prognosis and immune response of HNSCC.

Canine distemper, a viral disease with a global impact on various animals including dogs, foxes, wolves, lions, and leopards, requires early diagnosis for effective treatment and outbreak control. Common laboratory methods, such as enzyme-linked immunosorbent assay, polymerase chain reaction, and viral isolation, face challenges such as extended turnaround times, high costs, and the expertise required. This study has developed a field-based biosensor for detecting the canine distemper virus (CDV), utilising a screen-printed carbon electrode (SPCE) and a computer-assisted portable potentiostat. A 30-mer oligonucleotide capture probe, designed using Primer3 Plus software version 3.3.0, detected hybridisation with the CDV complementary strand through electrochemical analysis via differential pulsed voltammetry. The developed biosensor exhibited good linearity in quantifying the target analyte concentration (0.1 to 12.8 µM), with a detection limit of 0.05 µM, indicating high sensitivity. Specificity tests using complementary and non-complementary sequences confirmed the biosensor's accuracy. The electrode can be reused up to eight times with a residual capacity of 93.72 ± 5.45% after regeneration using a 50 mM NaOH solution. The developed biosensor was also used to detect CDV in biological samples after RNA extraction and amplification. Results from the biosensor aligned with those from reverse transcriptase polymerase chain reaction (RT-PCR) findings, showing 100% agreement. These findings support the potential development of a field-deployable portable device for effectively diagnosing canine distemper in biological samples.

Cultured plant cells often biosynthesize secondary metabolites to a lesser extent relative to the mother plants. This phenomenon is associated with epigenetic alterations of the biosynthetic gene(s). Here we investigated the effectiveness of epigenetic modifiers, such as inhibitors of histone deacetylase (HDAC) and DNA methyltransferase (DNMT), to activate cryptic secondary metabolite biosynthesis in tobacco (Nicotiana tabacum) BY-2 cells. The BY-2 suspension cells cultured with an HDAC inhibitor, suberoyl bis-hydroxamic acid, exhibited strong biosynthesis of four compounds that were originally present at trace concentrations. The induced compounds were identified as caffeoylputrescine (1), 4-O-β-D-glucopyranosylferulic acid (2), 5-O-caffeoylquinic acid (3), and feruloylputrescine (4). Biosynthetic activation of compounds 1-4 was reproduced by two other HDAC inhibitors. Treatment of the cells with a DNMT inhibitor (zebularine) also activated the biosynthesis of compounds 1-4, but had a limited effectiveness relative to the HDAC inhibitors, indicating that histone acetylation levels are involved more than DNA methylation levels in the epigenetic regulation of the biosynthesis of compounds 1-4 in the BY-2 cells. Following our previous demonstration using cultured cells of a monocotyledonous plant, this study demonstrates the utility of epigenetic modifiers to activate cryptic secondary metabolite biosynthesis in cultured cells of a dicotyledonous plant.

TXNIP is closely associated with diabetic peripheral neuropathy (DPN). Gynostemma pentaphyllum (Thunb.) Makino (GP), a perennial herb with five leaves, is considered to have medicinal values. However, it is unknown whether GP alleviates DPN by modulating TXNIP-mediated autophagy. The aim of this study was to evaluate the effect of GP on Schwann cell injury during DPN and to investigate the mechanism of GP in DPN for the first time. High-fat diet-fed GK rats and high-glucose-cultured RSC96 cells were used to establish DPN models. The effects of GP on DPN were investigated by blood glucose assay, neurological function assay, pathology assay, and immunohistochemistry. To investigate the effect of GP on autophagy and upstream PI3K/AKT/mTOR signaling pathway in Schwann cells, Western blot and immunofluorescence assay were performed on RSC96 cells to detect the expression of beclin-1 and LC3. Western blot method was used to detect the expression of PI3K, p-Akt/Akt, p-mTOR/mTOR, and RT-qPCR method and was used to detect the expression of PI3K. Apoptosis was detected by flow cytometry. The effects of TXNIP on the above indicators were also detected in RSC96 cells. Finally, the mechanism of GP regulation of autophagy and apoptosis in RSC96 cells was verified. GP reduced blood glucose level, attenuated peripheral nerve myelin damage, and improved nerve function in DPN rats. In addition, GP enhanced autophagy activity and reduced apoptosis in RSC96 cells. GP promoted autophagy by regulating TXNIP-mediated PI3K/AKT/mTOR signaling pathway, and GP reduced apoptosis in RSC96 cells by promoting cellular autophagy. GP attenuates DPN myelin damage in RSC96 cells by enhancing autophagy, and its mechanism may be related to the inhibition of PI3K/AKT/mTOR signaling pathway by up-regulating the expression of TXNIP.

A new lipopeptide-producing strain Cytobacillus sp. R3-1 was isolated from the production water of the Daqing oilfield in China and identified based on 16S rRNA gene sequence analyses. The strain R3-1 is capable of simultaneously producing both of the surfactin and fengycin, the two major families of the lipopeptide biosurfactant. The chemical structures of the surfactin and fengycin were confirmed by a combination of the ESI-MS, FT-IR, and amino acid analyses, and the impact of various temperatures, pH, and NaCl concentrations on the emulsifying activity (E₂₄) was investigated. The lipopeptide biosurfactant produced by the strain R3-1 exhibited strong emulsifying activity with E₂₄ value over 60% on crude oil and different hydrocarbons, including the cyclohexane, hexadecane, benzene, toluene, kerosene, diesel oil, and liquid paraffin. Meanwhile, it showed excellent emulsifying activity across a broad range of conditions of the temperature up to 60 °C, NaCl tolerance up to 100 g/L, and pH values between 5 and 9, which suggests that the strain R3-1 is a valuable microbial candidate for the simultaneous production of the surfactin and fengycin lipopeptide biosurfactant with strong emulsifying properties and stability under diverse environmental conditions and is a potential application in environmental bioremediation and enhanced oil recovery.

The incidence and mortality rates of colorectal cancer (CRC) are alarmingly high, and the scientific community is consistently engaged in developing newer therapeutic options for cancer cure or prevention. The fluoropyrimidine drug, 5-fluorouracil (5FU), remains the first line of treatment against CRC; nevertheless, relapses frequently occur since the cells gain resistance over time through various mechanisms. Studies have highlighted the significance of combinatorial treatment of a Wnt signaling inhibitor and 5FU as a better treatment strategy to overcome 5FU resistance. Small molecules that specifically target and disrupt β-catenin-TCF interaction, a crucial step of the Wnt signaling, are promising in CRC treatment. In this study, we investigated the synergistic cytotoxic activity of menadione with 5FU as the former has previously been shown to downregulate Wnt signaling in CRC cells. Docking and experimental results suggest that the drug combination interfered with key protein-protein interactions in the β-catenin-TCF complex, exerted synergistic anti-cancerous effects in CRC cells, and downregulated the expression of Wnt signaling proteins. Taken together, our data suggest that the simultaneous binding of 5FU and menadione to β-catenin can block Wnt signaling by disrupting β-catenin-TCF interaction and inhibit the proliferation of CRC cells.