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.

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.

With this "In the Limelight: Tumor-Stroma Interactions" special issue, FEBS Open Bio aims to highlight the relevance of the tumor stroma cells, and their interactions with the tumor cells, in the progression of cancer.

Lactoferrin (Lf) is a multifunctional glycoprotein of the transferrin family which has shown to efficiently block cell migration and/or invasion in a wide range of cancer cell models. The objective of this study was to further understand how Lf targets cancer cells by examining the effect of acetylcholine (ACh) levels on Lf signaling using A549 (p53 wild‐type) and H1299 (p53‐null) nonsmall cell lung cancer (NSCLC) cell lines. Treatment with Lf reduced cell viability more effectively in A549 cells than in H1299 cells. The half maximal inhibitory concentration (IC50) of Lf for A549 and H1299 was 8.97 ± 1.4 and 35.03 ± 4.2 mg·mL−1, respectively. To uncover the potential molecular mechanism involved in the decreased cell viability observed in A549 cell following Lf treatment, the activity of tumor suppressor (p53), acetylcholinesterase (AChE), and ACh levels were measured. Treatment of A549 cells with Lf led to ~ 1.50‐fold activation of p53, ~ 1.60‐fold activation of AChE, and ~ 1.80‐fold decrease in ACh levels. Vascular endothelial growth factor (VEGF) levels also decreased in cell culture supernatants upon treatment with Lf in both cell lines, and in A549 cells, the decrease occurred in a manner dependent on p53 and AChE. Given previous reports on the role of Lf in apoptosis induction, we examined AKT activity following Lf treatment and showed that AKT activity decreased ~ 1.95‐fold in A549 cells and ~ 1.50‐fold in H1299 cells. Furthermore, Lf‐induced activation of caspase‐3 was diminished by A549 cell cotreatment with siRNA targeted against p53 and/or AChE and increased by inhibiting the function of VEGF and/or AKT in both cell lines. In conclusion, this study identifies a mechanism wherein ACh concentrations in the cell culture supernatant attenuate the impact of Lf on NSCLC cell viability. These findings provide preliminary insight into the complex actions of Lf and suggest that the Lf‐AChE‐ACh pathway may warrant further study as a potential target in NSCLC.

The postsynaptic density (PSD) is a complex, multilayered protein network largely situated on the internal surface of the postsynaptic membrane. It is the first processing unit for incoming synaptic signals, and changes in its internal structure are associated with synaptic strength and plasticity. These structural changes are largely governed by multivalent interactions between its components. The in vitro characterization of such complexes requires unbiased methods that can be used to estimate the size of the emerging assemblies for systems with multiple possible stoichiometries. Here, we present an experimental method for detecting specific PSD proteins as well as their complexes based on their diffusion in a microfluidic environment. The method requires a fluorescent labeling technique that does not disrupt the function of labeled proteins, a microfluidic device that can maintain laminar flow for protein solutions, a microscope that can record the fluorescent signal emitted by these solutions, and an analytic software package that can process the collected experimental data and convert them into approximate particle sizes. We demonstrate the applicability of our method on protein constructs of various postsynaptic proteins, including the multivalent assembly between GKAP and LC8.

The 2nd MObility for Vesicle research in Europe (MOVE) Symposium, held in Belgrade—Serbia, from October 8 to 11, 2024, showcased the dynamic and interdisciplinary nature of extracellular vesicles (EVs) research in Europe. Organized by eight National EV Societies under the MOVE initiative, the event gathered over 280 attendees from 28 countries, promoting collaboration and scientific exchange. The symposium featured eight keynote lectures, 48 oral and 126 poster presentations, and sessions dedicated to EV‐related tools and industry innovations. The scientific program was structured around seven core themes: EV biogenesis and signal transmission, roles of EVs in health and disease, EV‐based biomarkers, interspecies communication, novel EV preparation and analysis techniques, therapeutic and regenerative applications, and the manufacturing of native and engineered EV products. Supported by 18 sponsors and the Ministry of science, technological development and innovation of the Republic of Serbia, the symposium also highlighted the MOVE Fellowship Program and offered rich networking opportunities. This landmark event reinforced MOVE's promising mission to promote excellence, mobility, and resource sharing in EV research across Europe.

Bcl‐2‐associated athanogene 3 (BAG3) is the only member of the BAG cochaperone family that is induced by stressful stimuli such as heat shock and heavy metals. In the present study, bag3 knockout (KO) HeLa cells were generated via the CRISPR‐Cas9 system, and the role of BAG3 in relation to p53 under heat stress conditions was investigated. Normally, the levels of p53 were low in both wild‐type (WT) and KO cells, while heat shock increased the levels of nuclear p53 in both cell lines. However, the increased level of p53 was much greater in WT cells than in KO cells, which suggested that BAG3 played a role in controlling the level of p53 under heat stress conditions. The mRNA level of p53 did not increase in either WT or KO cells during the heat stress period, which suggested that the differences in the levels of p53 were not due to transcriptional regulation. Both treatment with the proteasome inhibitor MG132 and heat shock drastically increased p53 levels to a similar extent in WT cells. Interestingly, both BAG3 and Hsp70 rapidly translocated to the nucleus and formed a complex with p53 upon heat stress. During a 1‐h recovery period from heat stress, the transcriptional activity of p53 increased up to 4‐fold in WT cells, but only 1.69‐fold in KO cells. These results demonstrate that Hsp70 and BAG3 are involved in the quality control of p53 under heat stress conditions and suggest a role for BAG3 as a cochaperone protein.

Long non‐coding RNAs (lncRNAs) are transcripts with a length more than 200 nt, which do not encode proteins and act just as RNA molecules. In general, lncRNAs have much more distinct tissue specificity than proteins, as they usually realize more peculiar regulatory functions. Their expression levels are often altered in a response to stress conditions, metabolic changes, development of different diseases, and carcinogenesis. Cancer‐associated lncRNAs are widely considered as perspective and useful biomarkers. Thus, development of clinical tests, which include tissue‐specific and cancer‐specific lncRNAs, might significantly contribute to cancer diagnostics and/or prognosis of the disease. A number of lncRNAs is known to be dysregulated in liver tumors and considered as probable biomarkers. However, most of them are rather universally well‐known lncRNAs associated with various cancers. In the present review, we aimed to shed light on other lncRNAs with preferential expression in liver and/or liver tumors, for example, LINC01554, LINC01093, LINC01348, LINC02428, FAM99B, etc. We summarized recent discoveries unveiling their dysregulation in liver malignancies and related cellular mechanisms in which they are involved and considered their significance as probable liver cancer biomarkers.