P-glycoprotein and breast cancer resistance protein are key efflux transporters relevant to drug-drug interactions (DDIs) for orally administered drugs. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use M12 guidance recommends using unbound in vitro inhibition potency (IC50,u) for clinical risk assessment, yet the influence of nonspecific binding to membrane vesicles remains uncharacterized. Herein, vesicle binding for a panel of efflux transporter inhibitors was systematically quantified using equilibrium dialysis and compared with conventional microsomal binding. Nonspecific binding to vesicles reduced free inhibitor concentrations, resulting in right-shifted IC50 values, particularly for high-binding compounds (itraconazole and dronedarone) compared with low-binding compounds (telmisartan) in breast cancer resistance protein assays with Sf9 control vesicles. Similar right shifts were observed for P-glycoprotein inhibition assays with human embryonic kidney 293 control vesicles, although less pronounced. Correction of IC50 values across different vesicle concentrations generally resulted in aligned IC50,u values. Microsomal binding correlated strongly (Pearson correlation coefficient = 0.965) with vesicle binding, supporting its use as a surrogate for binding correction. Incorporating binding-corrected IC50 values into static DDI risk calculations increased false positives but did not affect false negatives. These findings demonstrate that accounting for fraction unbound in vesicle-based assays can substantially influence the estimation of in vitro inhibitory potency and subsequent static DDI risk assessment. SIGNIFICANT STATEMENT: This study systematically characterizes nonspecific binding to membrane vesicles in P-glycoprotein and breast cancer resistance protein inhibition assays and demonstrates its substantial impact on IC50 determination. The strong correlation between vesicles and microsomal binding provides a practical surrogate for implementing International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use M12 unbound potency recommendations for vesicle-based inhibition assays.

Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have substantially improved the treatment of lung cancer, yet durable responses remain limited by primary and acquired resistance. Increasing evidence identifies the PI3K/AKT/mTOR pathway as a central link between oncogenic signaling, metabolic adaptation, and tumor immune suppression. Genetic and molecular alterations, including PIK3CA activation, PTEN loss, and aberrant EGFR or ALK signaling, can activate this pathway and regulate PD-L1 expression at transcriptional, translational, and post-translational levels. Conversely, tumor-intrinsic PD-L1 signaling can reinforce PI3K/AKT/mTOR activity, promoting proliferation, survival, glycolysis, epithelial-mesenchymal transition, and resistance to therapy. This reciprocal interaction establishes a feedforward circuit that supports lung cancer progression and immune escape. The pathway also reshapes the tumor microenvironment by promoting regulatory T-cell expansion, M2-like macrophage polarization, myeloid-cell recruitment, CD8[+] T-cell dysfunction, and resistance to immune checkpoint blockade. This review critically examines the bidirectional crosstalk between PI3K/AKT/mTOR and PD-1/PD-L1 signaling in lung cancer and summarizes the molecular regulators that connect these pathways. We further discuss therapeutic strategies designed to disrupt this network, including PI3K, AKT, and mTOR inhibitors; targeted therapies; chemotherapy and radiotherapy combinations; antiangiogenic approaches; nanomedicine; extracellular-vesicle-based delivery; cell-based interventions; and natural compounds. Although preclinical evidence supports combining pathway inhibition with immune checkpoint blockade, clinical translation is challenged by pathway complexity, context-dependent regulation of PD-L1, systemic toxicity, and the absence of validated predictive biomarkers. Biomarker-guided patient selection, optimized dosing and sequencing, and selective targeting of tumor- or immune-cell-specific pathway components will therefore be essential for converting this mechanistic rationale into durable clinical benefit.

Oral delivery of water-repelling drugs for acute liver failure (ALF) often runs into problems like poor solubility, instability, and low absorption. In this work, we found that extracellular vesicles derived from honey (HEVs) can serve as a natural nanocarrier that works orally and targets the liver on its own. After isolating and characterizing these vesicles, we saw they are membrane-bound nanoparticles roughly 160 nm in size with a surface charge around -32 mV, and they contain proteins and RNAs. Oral HEVs mainly ended up in the liver and showed some anti-inflammatory activity without any drug loaded. To get better therapeutic effects, we loaded curcumin (Cur), a hydrophobic anti-inflammatory compound, into HEVs to make Cur@HEVs. These loaded vesicles greatly improved how well curcumin is absorbed and released in a sustained manner. In acetaminophen (APAP) caused ALF mice, oral Cur@HEVs improved liver function and survival much more than plain curcumin or empty HEVs did. Specifically, Cur@HEVs simultaneously put the brakes on pyroptosis (via the NLRP3/GSDMD/caspase-1 route) and necroptosis (through the RIPK1/RIPK3/MLKL pathway), which in turn cut down the release of inflammatory cytokines and kept liver cells from dying. Overall, HEVs come across as a safe, food-based nanocarrier that can be taken orally, and Cur@HEVs offer a bioactive platform for dually targeting inflammatory cell death in ALF. Our findings suggest that dietary extracellular vesicles warrant further investigation as potential therapeutic biomaterials, though additional translational studies are needed.

The Enterococcus genus comprises an unusual subset of the human microbiota with the dual role of beneficial commensals and opportunistic pathogens. The biological activity of Enterococcus species is not limited to colonization and is mainly driven by numerous bioactive compounds. Metabolites, extracellular vesicles (EVs), exopolysaccharides, and cell wall structures serve as mediators of complex host-microbe interplay. Through the transfer of these bioactive substances, Enterococcus-derived EVs shape host signaling pathways and help bacteria to adapt to environmental conditions. Additionally, structural molecules such as peptidoglycan, lipoteichoic acid, and adhesins bind to pattern recognition receptors, triggering signaling cascades responsible for both innate and adaptive immunity. Moreover, biological roles of metabolites, exopolysaccharides, and bacteriocins include the modulation of oxidative stress, antimicrobial activity, regulation of immunometabolic pathways, maintenance of intestinal barrier integrity, and others. This review presents a summary of biological activities and molecular mechanisms of Enterococcus-derived bioactive compounds with a focus on their role in health and the pathophysiology of diseases.

Sugarcane bagasse is an abundant agricultural by-product containing bioactive phenolic compounds such as p-coumaric acid (CA), although their practical utilization is often limited by poor bioavailability. In this study, food-grade liposomes encapsulating purified CA (LCA) and bagasse-derived phenolic extract (LBC) were prepared to investigate whether liposomal delivery enhances intracellular antioxidant function. The physicochemical properties of the liposomes, including particle size, zeta potential, membrane morphology, and encapsulation efficiency, were characterized using dynamic light scattering, confocal laser scanning microscopy, Fourier transform infrared spectroscopy, and HPLC/MS/MS analysis. Both LCA and LBC formed stable nanosized vesicles (approximately 70-125 nm) with high encapsulation efficiencies exceeding 70 %. Optimization of the ultrasonication process substantially reduced particle size and improved particle uniformity compared with our previous formulation, producing liposomes suitable for cellular evaluation. Cellular antioxidant activity (CAA) assays using HepG2 cells demonstrated that both LCA and LBC exhibited greater intracellular antioxidant activity than their corresponding non-encapsulated samples under both AAPH- and H2O2-induced oxidative stress conditions. Liposomal encapsulation also modified cellular responses at higher concentrations, consistent with improved intracellular delivery of encapsulated phenolic compounds. These findings indicate that food-grade liposomal encapsulation effectively enhances the intracellular functionality of both purified CA and complex bagasse-derived phenolic extracts by improving cellular delivery. This study provides new evidence that physicochemically optimized food-grade liposomes can enhance the intracellular functionality of phenolic compounds and supports their potential as practical delivery systems for sustainable functional food ingredients derived from agricultural by-products.

Thermal stress is a prevalent challenge in the aquaculture of aquatic species. To characterize the hepatopancreatic proteomic differences in Procambarus clarkii under stress conditions, we applied an iTRAQ-based quantitative proteomic assay to identify differentially abundant proteins (DAPs). This analysis was conducted on the hepatopancreas of crayfish exhibiting strong stress resistance (SSR), representing a sustained thermal stress-tolerance trajectory, and weak stress resistance (WSR), representing an early stress-intolerance trajectory. In total, 1198 proteins were quantified; 184 proteins were identified as having at least a 1.2-fold change in abundance and adjusted P-value (FDR) <0.05, with 102 upregulated and 82 downregulated in SSR relative to WSR. Upregulated proteins were predominantly enriched in translation and aromatic compound catabolic processes, as well as pathways such as ribosome, retinol metabolism, linoleic acid metabolism, hormone biosynthesis, and tryptophan metabolism. Conversely, downregulated proteins were mainly involved in fatty acid biosynthesis, vacuolar transport, signal transduction, ATP hydrolysis coupled proton transport processes, and with significant enrichment in pathways, including oxidative phosphorylation, NF-kappa B signaling pathway, and synaptic vesicle cycle pathways. A pathway-level comparison between the DAPs and previously identified differential metabolites revealed that pathways containing both altered proteins and metabolites were predominantly involved in proteostasis/stress response, energy metabolism, and glutathione metabolism. Transcript-level assessment of selected proteomic candidates showed consistent upregulation patterns for genes involved in energy metabolism (such as MDH), antioxidant factors (including sod), and consistent downregulation patterns of ER stress markers (such as GRP78). These results suggest that differences in protein abundance between WSR and SSR are associated with alterations in energy metabolism, antioxidant-related processes, and cellular homeostasis. Our findings may provide candidate molecular features and pathways for further investigation of stress-associated phenotypes in crayfish.

Liposomes are vesicular structures consisting of one or more lipid bilayers that enclose an aqueous inner core, allowing the encapsulation of both hydrophilic compounds in the inner compartment and lipophilic and amphiphilic molecules within the lipid bilayer. In recent years, microfluidic-based manufacturing techniques have emerged as a highly attractive approach for liposome production, as they allow precise control over fluid dynamics at the micrometer scale, thereby enabling reproducible and scalable fabrication of nanocarriers with narrow size distributions. Since even minor variations in liposomal formulations can markedly influence their critical features, a well-defined manufacturing process and stringent process control are essential to ensure that the final product consistently meets the required quality standards. The selection of the organic solvent plays a critical role in governing lipid solubilization, solvent-antisolvent mixing, and lipid self-assembly kinetics, ultimately affecting vesicle nucleation, growth, and bilayer organization. Likewise, microfluidic operating parameters critically influence the physicochemical characteristics of the resulting liposomes, including particle size, polydispersity index, surface charge, lamellarity, and colloidal stability. Within a rational formulation-development approach, a comprehensive understanding of the relationships between critical material attributes (e.g., lipid composition and solvent properties) and critical process parameters (e.g., Reynolds number; flow rate ratio, FRR; total flow rate, TFR; and mixing efficiency) is essential for rational process optimization. Specifically, this work highlights how lipid composition, organic solvent selection, microfluidic operating conditions, and purification methods collectively influence the structural and functional behavior of liposomes, thereby supporting the development of robust and well-controlled microfluidic manufacturing processes.

Extracellular vesicles (EV) secreted by living cells are important players in cell-to-cell communication, particularly in the diffuse immune system, where they contribute to local and systemic coordination. Indeed, EV are in the ideal size range for diffusion in interstitial and lymphatic fluids, which is the main route of tissue EV trafficking towards secondary lymphoid organs that orchestrate immune responses. In the lymph nodes, EV interact with multiple immune and stromal cell populations sensitive to antigens and co-delivered tissue messages via degradative and non-degradative pathways. Natural conveyers of bioactive compounds inherited from their mother cells and their surroundings, EV modulate innate and adaptive immune responses in health and disease. The aim of this scoping review is to aggregate information from the literature on cellular effectors and molecular pathways of EV-mediated immune responses in the lymph node environment and to gain insight into practices using EV for immune intervention .

Synaptic vesicle glycoprotein 2C (SV2C) is enriched in dopaminergic neurons and implicated in Parkinson's disease, but no selective small-molecule probes exist for it. Lacking a full-length SV2C structure, we built a homology model from SV2A cryo-EM templates and used molecular dynamics to characterize its conformational landscape. An AI-enhanced virtual screening pipeline, validated on a curated SV2A benchmark, was applied to 5.96 million commercial compounds, prioritizing 94 candidates for experimental testing. Of 71 compounds tested in an orthogonal biophysical assay cascade, 22 were active (31% hit rate), and five advanced to isoform-selectivity profiling. Compounds 36 and 56 emerged as leads, with SV2C K i values of 24.6 μM and 3.25 μM and greater than 10-fold selectivity over SV2A. An unpublished SV2A cryo-EM structure independently confirmed the predicted binding mode. This AI-driven pipeline delivered selective SV2C ligands from a general chemical library, providing tools to probe SV2C biology in Parkinson's disease.

Traumatic brain injury (TBI) is among the leading causes of mortality and long-term disability worldwide, particularly in younger populations. Despite extensive research, there are currently no clinically effective neuroprotective therapies capable of fully preventing secondary neurological damage after TBI. The pathological progression of TBI involves both primary and secondary injury mechanisms. Primary injury occurs immediately after mechanical trauma and results in irreversible structural damage, whereas secondary injury develops progressively over hours to days and significantly contributes to neuronal degeneration and neurological dysfunction. Secondary injury cascades include excitotoxicity, oxidative stress, mitochondrial dysfunction, blood-brain barrier (BBB) disruption, cerebral edema, neuroinflammation, calcium overload, and apoptotic cell death. These interconnected mechanisms aggravate tissue injury and impair cognitive, behavioural, and motor functions. Experimental animal models have substantially improved the understanding of TBI pathophysiology and facilitated the development of potential therapeutic approaches; however, successful translation from experimental studies to clinical application remains limited. Recent advances in molecular diagnostics have identified extracellular vesicles, microRNAs, and other circulating biomarkers as promising tools for early diagnosis and prognosis. In addition, growing interest has emerged in natural and herbal neuroprotective compounds because of their antioxidant and anti-inflammatory properties. This review comprehensively discusses the major pathophysiological mechanisms underlying TBI, translational limitations of current therapeutic strategies, and future directions for developing effective multimodal neuroprotective interventions.

Salivary extracellular vesicles (sEVs) are accessible candidates for non-invasive biomarker analysis, but their measurement commonly requires multistep, label-dependent workflows. Here a label-free gold extended-gate field-effect transistor (EG-FET) functionalized with an antibody that binds specifically to the Cluster of Differentiation 63 (anti-CD63), was developed for electrical detection of isolated salivary sEVs. Gold gates were modified with an 11-mercaptoundecanoic acid self-assembled monolayer and [1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide] EDC/NHS-mediated antibody immobilization. Binding-induced changes were quantified as the ratio of areas under transfer curves (AUC) acquired before and after incubation. Inter-chip coefficient of variation decreased from 10.5% for bare gates to 1.5% after antibody immobilization. The AUC ratio decreased with sEV concentration across 0.01X-1X (R[2] = 0.980); using the blank mean minus three standard deviations, the estimated detection threshold was approximately 0.1X (about 9.8 × 10[7] particles/mL for the representative stock). Anti-CD63 gates exposed to 0.1X sEVs produced a significant response, whereas Phosphate-Buffered Saline (PBS), Bovine Serum Albumin (BSA), and heterologous-antibody controls remained near baseline. Four independent sessions across a 16-day interval yielded coefficients of variation of 1.4% for PBS and 1.7% for sEV measurements. Response was retained when isolated sEVs were spiked into 0.2X clarified saliva. These proof-of-concept findings support reproducible label-free sEV detection while defining the need for direct whole-saliva interference testing, storage studies, and validation in clinically characterized cohorts.

Giant unilamellar vesicles (GUVs) are a powerful model system whose response to electric fields constitutes a strategy to directly assess membrane material properties. In the last few years, the field has advanced in both conceptual scope and experimental precision. Electric-field manipulation of GUVs remains a versatile platform for extracting membrane mechanical, electrical, and rheological properties with single-vesicle resolution, while increasingly extending from a diagnostic tool into an actuation mechanism. Here, we summarize classical electrodeformation and electroporation methodologies and review recent developments that push these approaches toward greater biological realism, including compound vesicles as nucleate-cell mimics, resting-membrane-potential- and ion-dependent poration, phase-transition electromechanics, and cytoskeleton-loaded, field-responsive membranes. We also discuss how electric fields are combined with other stimuli, such as light and biochemical cues, and repurposed to drive active, motile vesicle assemblies and to fabricate tailored lipid architectures at high throughput. We further highlight theoretical work on electromechanical coupling, membrane charging dynamics, and vesicle shape landscapes under electric stress. We close by outlining remaining challenges, from reproducibility across preparation protocols to bridging model membranes and living cells, and future opportunities for automated, high-throughput, and multimodal electromechanical studies of biomimetic membranes.

Carmofur, a lipidated urethane derivative and prodrug of 5-fluorouracil, has an established clinical application as an antineoplastic agent for the treatment of colorectal cancer, primarily through inhibition of acid ceramidase and subsequent intracellular drug release. Beyond this oncological application, recent drug repurposing efforts have identified covalent inhibition of the SARS-CoV-2 main protease as an additional antiviral mechanism. The pronounced amphiphilic character of carmofur, arising from a polar fluoropyrimidine headgroup appended to a hydrophobic aliphatic tail, raises the possibility of an additional membrane-directed mechanism orthogonal to canonical target engagement. Driven by observations that certain lipidated carmofur analogs induce potent disruption in giant unilamellar vesicles (GUVs), which serve as model-membrane systems, we evaluated nine analogs with differing alkyl tail lengths and saturation to characterize the capacity of such lipidated compounds to directly engage with biological membranes. To efficiently quantify the membrane-disrupting capabilities of these analogs, we benchmarked the detection accuracy of a transformer-based model and a CNN-based vision model, where YOLOv11 matched RT-DETRv3's detection accuracy while running at a fraction of its computational cost. Through both cell-free and in vitro experiments, we demonstrate that lipidated analogs of carmofur exhibit a chain-length-dependent membrane disruption mechanism, with maximal cell-free disruption peaking at the decyl and dodecyl tail lengths. By performing critical micelle concentration and dynamic light scattering analyses, we found that hydrophobic interactions drive self-assembly, with alkyl chain length influencing micelle formation and size distribution. Collectively, these properties may underlie their biological activity, revealing the significance of a previously underappreciated mode of action of lipidated anticancer agents.

Burn injuries cause deep, extensive skin damage and are associated with high morbidity and mortality. Effective treatment goes beyond superficial wound coverage; it requires regenerative repair that progresses from deeper tissue layers to complete skin restoration. Because of extreme trauma and severity, the fastest treatment strategies play a key role in the treatment of burn injuries. Recently, hydrogel dressings have gained attention as promising wound care options because they maintain a moist environment, provide cooling and pain relief, support autolysis, and promote tissue regeneration. However, the lack of biological function and, consequently, slower tissue regeneration limit the applications of hydrogels. In recent years, collaboration between doctors, materials scientists, and biologists has substantially improved the biological functionality of hydrogels. Integrating bioactive agents, such as proteins, peptides, antimicrobial compounds, drugs, metal nanoparticles, cell-based vesicles, and extracellular matrix, enables the development of multifunctional, active burn wound dressings. This review highlights advances in the functionalization of hydrogel dressings for burn care, including fabrication methods, biological efficacy, and therapeutic mechanisms. The literature provides a comprehensive overview of the area where further advancement could be pursued.