FEBS Open Bio最新文献

Porphyromonas gingivalis (Pg) is a periodontal pathogen that has been implicated in the development and progression of head and neck squamous cell carcinoma (HNSCC). However, studying its interaction with HNSCC in vitro is challenging because of the obligatory anaerobic nature of Pg. To overcome this limitation, we developed a coculture system that enables the viability of both HNSCC cells and Pg using spheroid culture combined with Hippo pathway inhibition via treatment with a LATS1/2 inhibitor. In spheroid cultures, many HNSCC cell lines failed to grow in a normal medium. However, stable growth of these cells was achieved through Hippo pathway inhibition, which maintained the cells in an undifferentiated state. Furthermore, the addition of Pg to HNSCC spheroids maintained Pg viability in three out of four HNSCC cell lines, even after 3 days. Hippo pathway inhibition further enhanced Pg survival within the spheroids, likely by suppressing the differentiation-induced expression of antimicrobial genes S100A8 and S100A9. Coculturing HNSCC cells with Pg did not promote spheroid growth but induced p38 activation, leading to increased expression of the proinflammatory cytokines IL-1α and IL-8. Database analysis using The Cancer Microbiome Atlas corroborated these findings, showing upregulation of p38 phosphorylation, IL-1α, and IL-8 in Porphyromonas-positive HNSCC samples. These findings suggest that the established coculture system is a representative model of the clinical properties of Pg-positive HNSCC and can serve as a valuable tool for investigating the long-term interactions between HNSCC and viable Pg.

Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome associated with pathogenic variants affecting the GTPase-activating protein neurofibromin. Genetic variants affect neurofibromin through targeted protein degradation, failed aggregation of the monomers or failure of specific domains depending on the functional state. In addition to the occurrence in NF1, there is evidence of pathogenic variants occurring in various solid tumors. We collected data from 63 patients from our molecular tumor board for NF1 gene sequencing and detected 72 NF1 variants, thereby 32% of those being pathogenic. They occurred most often in lung cancer, glioma, melanoma, sarcoma, and gynecological cancer and affected women more often. Pathogenic NF1 variants appeared at low frequency except in malignant melanoma and glioma (10%). We present common pathogenic variants, their types, and association with tumor entities, their frequency, and domain localization and focus on common recurrent variants and their probable result and predictive quality in somatic mutation screening. We detected variants in different tumor entities without NF disease, covering more frequent truncating mutations than reported for germline. We question whether all NF1 variants reported in tumors without the presence of NF1 are somatic. To conclude, recognition of NF1 mosaicism requires multitissue sampling, precise sequencing technologies, and inclusion of genetic counseling.

Recombinant protein production in Escherichia coli is a fundamental aspect of biotechnology. Fusion tags are commonly used to enhance solubility and facilitate purification. However, these tags can lead to challenges such as low yields, complicated purification processes, and the necessity for tag removal, especially when dealing with peptides. This study introduces a novel fusion tag called SmallTalk, a truncated version of the small metal-binding protein SmbP. Weighing in at 5 kDa, SmallTalk includes two of the four α-helices found in SmbP. It retains the ability to bind Ni(II) ions, which enables purification through IMAC. In this work, we assessed the efficiency of SmallTalk in expressing and purifying both a model protein, the green fluorescent protein, and the antimicrobial peptide Bin1b. Both proteins were effectively expressed and purified using IMAC, demonstrating SmallTalk's value as an affinity tag, yielding 7.2 mg·L^-1 of cell culture for the green fluorescent protein and up to 9.8 mg·L^-1 for Bin1b. Antimicrobial assays conducted with SmallTalk-tagged Bin1b showed activity against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, with minimum inhibitory concentrations ranging from 7.5 to 22.5 μm. Importantly, SmallTalk enabled the full retention of Bin1b's antimicrobial activity without the need for its removal, significantly simplifying the production process. These findings indicate that SmallTalk provides a promising strategy for the recombinant production of peptides. This tag has the potential to enhance the expression, purification, and functional analysis of antimicrobial peptides, which are increasingly being pursued as alternatives to antibiotics in the fight against antimicrobial resistance.

Autophagy-mediated secretion contributes to the maintenance of intracellular homeostasis by releasing cytoplasmic components into the extracellular space. However, several aspects of the process remain unclear. In this study, we developed an ultrasensitive detection system using HiBiT tag/NanoBiT technology to analyze the conditions that trigger the secretion of LC3, an autophagosome marker. In HiBiT-tagged knock-in cells, a detectable amount of HiBiT-dependent NanoLuc luciferase activity (HiBiT activity) from HiBiT-fused LC3 was observed in the culture supernatants. However, the levels were lower than those of CD63. HiBiT activity was detected only in the presence of detergent, indicating that LC3 was released from the lipid membranes. Treatment with bafilomycin A1 significantly increased the extracellular HiBiT activity, which was diminished in ATG5 or FIP200 knockout cells, suggesting that this release depends on autophagosome formation. However, some HiBiT-LC3 was detected in these knockout cells, indicating that LC3 may be released via an autophagy-independent mechanism. The introduction of a C-terminal truncation (ΔG) or the K51A/L53A mutation also reduced LC3 release, but did not completely inhibit it, suggesting that multiple pathways exist for LC3 release. This system is expected to elucidate the mechanisms underlying autophagy-mediated secretion.

Circadian rhythms orchestrate physiological processes, including immune function, across a 24-h cycle. This study investigates the temporal distribution of immune cell populations in healthy mice entrained to a 12:12-h light-dark cycle. Inguinal lymph node (iLN) samples were collected at zeitgeber times (ZT) 0 (lights on) and ZT12 (lights off) to assess immune cell composition. A significantly higher proportion of natural killer T (NKT) cells and neutrophils was observed at ZT0 compared to ZT12, while dendritic cells, macrophages, and natural killer (NK) cells showed no significant temporal variation. Additionally, adaptive immune cells, particularly Programmed cell Death protein 1⁺ (PD1⁺) Cluster of differentiation 4⁺ (CD4⁺) and PD1⁺ Cluster of differentiation 8⁺ (CD8⁺) T cells, were more abundant during the light phase. These findings suggest a diurnal pattern in immune readiness, with implications for optimizing immunotherapeutic interventions based on circadian timing.

Endothelial-to-mesenchymal transition (EndoMT), a widely recognized biological process leading to abnormal endothelial function, has been implicated in various cardiovascular pathologies. DEAD-box proteins represent the largest family of RNA helicases associated with multiple physiological and pathophysiological processes; however, their role in the homeostasis of endothelial cells (ECs) remains largely unexplored. Here, we show that the levels of DEAD-box protein 3 X-linked (DDX3X), a DEAD-box RNA helicase protein, were significantly increased during EC transition in vivo and in vitro. DDX3X overexpression promoted EndoMT as well as endothelial dysfunction and inflammation, whereas its downregulation effectively inhibited this transition in ECs. Mechanistically, elevated DDX3X resulted in downregulation of bone morphogenetic protein receptor type 2 (BMPR2), a protein that is pivotal for maintaining endothelial homeostasis and function. Furthermore, our co-immunoprecipitation assays demonstrated a molecular interplay between DDX3X and BMPR2. Importantly, DDX3X was shown to promote the lysosomal degradation of BMPR2, thereby interrupting its downstream signal transduction. These findings identify DDX3X as a novel regulator of EndoMT by modulating BMPR2 signaling.

Intracellular Ca²⁺ regulates insulin secretion from pancreatic β-cells and is influenced by cannabinoid signaling. However, the hydrophobicity and complex pharmacology of cannabinoid ligands prevent precision receptor targeting, limiting our understanding of their roles in modulating insulin release. Here, we use fluorescent Ca²⁺ imaging to examine how the light-activatable CB2 receptor agonist azo-HU308 modulates Ca²⁺ dynamics in INS-1 β-cells. UV-A photoactivation of azo-HU308 triggered robust, repeatable Ca²⁺ transients, and pharmacological profiling revealed this effect was independent of CB2 receptor activation but was instead mediated by extracellular Ca²⁺ influx through TRPC channels. These findings position azo-HU308 as a novel optical tool for controlling β-cell Ca²⁺ levels and highlight a non-GPCR pathway by which synthetic cannabinoids can modulate Ca²⁺ dynamics in excitable cells.

The ability of Acinetobacter baumannii to form biofilm is correlated with its antimicrobial resistance. The identification of antimicrobial drugs acting on biofilm is crucial to develop effective therapies. Previously, we determined that a chionodracine-derived peptide, KHS-Cnd, was able to impair A. baumannii biofilm formation. Here, to investigate the physiological changes underlying this activity, extracellular metabolite profiles of four A. baumannii strains were analyzed by NMR during biofilm formation in the presence of KHS-Cnd. Metabolites involved in biofilm energy metabolism were found extracellularly after KHS-Cnd treatment. Significantly altered pathways were associated with glyoxylate/dicarboxylate and branched-chain/aromatic amino acid metabolism. Overall, differences in extracellular metabolites reflect modifications of biofilm metabolism due to peptide treatment, thus highlighting its therapeutic potential against A. baumannii biofilm-sustained infections.

The aim of this study was to address the lack of protocols for nuclear magnetic resonance (NMR)-based metabolomics in the agri-food sector by providing a reproducible workflow for the preparation and analysis of water-soluble metabolite fractions. These fractions, rich in primary metabolites such as sugars, amino acids, and organic acids, are key to assessing agri-food products' composition, quality, as well as to monitor their manufacturing and development processes. The protocol differentiates solid and liquid matrices, optimizing extraction procedures accordingly. Representative agri-food products-strawberry leaves (solid) and wine (liquid)-were analyzed to demonstrate the method's versatility and applicability. Key steps include tailored sample preparation, optimization of NMR acquisition, and spectral quality control, ensuring high data quality and reproducibility. The proposed workflow enhances reproducibility across agri-food metabolomics studies and facilitates integration into broader food quality, traceability, and safety frameworks.

The rapid shift to online and blended learning in higher education has led to the development and use of digital tools that support student engagement and learning outcomes. This systematic review examines the effectiveness of these digital tools across various disciplines in higher education, focussing on factors that promote or hinder student engagement. A criteria-based comprehensive systematic search of three databases (Scopus, Web of Science and ProQuest, last date of enquiry 20 August 2024) identified 25 studies, inclusion criteria focussing on primary studies describing and evaluating interactive digital tools designed to enhance learning and/or assessment in higher education. Papers were analysed for bias using JBI checklists, and the papers' findings were analysed using a thematic analysis approach. Analysis of the papers uncovered four key design features that foster engagement with effective digital tools: interactivity, ease of use, immediate feedback and personalised learning experiences. Based on these findings, this review proposes a cyclic model for designing digital tools, emphasising an initial needs analysis, integration with course content, active engagement of students and educators, and ongoing refinement based on feedback. This model offers actionable guidelines for educators and institutions aiming to optimise digital tool development in higher education. The papers identified were typically short-term studies, on specific cohorts of students, and more long-term studies of the impact of digital resources are needed to determine long-term learning gain. The systematic review underscores practical strategies for leveraging digital tools to promote active, self-directed learning by focussing on evidence-based principles.