Mesenchymal stem/stromal cells (MSCs) continue to drive innovation in regenerative medicine, yet the field is undergoing a critical evolution from the early concept of cellular engraftment to complex paracrine-mediated tissue repair. This review systematically evaluates the core scientific questions defining this transition, highlighting the latest advancements in MSC biology and the rapidly expanding therapeutic frontier of MSC-derived extracellular vesicles (MSC-EVs). We critically appraise recent progress in decoding MSC therapeutic mechanisms, specifically their profound capacity for immune modulation and microenvironmental remodeling. By synthesizing current findings across diverse and challenging clinical applications-encompassing oral tissue regeneration, osteoarthritis, and complex immune disorders-we provide expert insights into the discrepancies between promising preclinical data and variable clinical outcomes. Crucially, we address persistent translational bottlenecks, including inherent donor heterogeneity, manufacturing scalability, and the lack of standardized potency assays. It is our perspective that overcoming these limitations demands a strategic pivot towards precise cell-free MSC-EV therapies and advanced bioengineering strategies. Ultimately, this review highlights key directions for future research. By connecting basic mechanistic insights with clinical demands, we aim to accelerate the translation of effective MSC-based therapies.

Extracellular vesicles are promising biomarkers for non-invasive diagnostics, but their heterogeneity in size and composition challenges conventional analysis. Raman spectroscopy with optical tweezers can characterize individual vesicles, yet the full capabilities of this approach remain underexplored. Here, we investigate Raman spectroscopy integrated with optical tweezers for real-time characterization of submicron particles, focusing on vesicle trapping kinetics. Using 100-nm dipalmitoylphosphatidylcholine (DPPC) vesicles, we examine the effects of objective type (water immersion, oil immersion, dry), laser power, concentration, and polarization on trapping dynamics and Raman signals. Vesicle trapping kinetics were satisfactorily described using a kinetic balance model with a stretched exponential that separates the contributions of particle influx, thermal escape, and finite trap capacity. Our measurements reveal distinct regimes with different objectives: with water immersion the signal rapidly saturates because the deep, stiff trap suppresses escape; with oil immersion spherical aberrations reduce trap stiffness and depth, leading to a continuous increase in equilibrium signal with power. The characteristic trapping time universally depends on total laser power, independent of objective. At elevated concentrations, the stretching exponent parameter β drops from 1 to ∼0.6 and large-amplitude steps appear, indicating vesicle aggregation as a source of kinetic dispersion. Numerical simulations qualitatively support the interpretation that trap stiffness and depth govern the observed kinetic regimes. These findings provide practical guidelines for single-vesicle Raman experiments and show that kinetic analysis offers a sensitive label-free indicator of sample polydispersity and aggregation that can be applied to real biological samples.

Loncastuximab tesirine (LT) is an antibody-drug conjugate (ADC), a novel class of therapeutics that combines a target-specific monoclonal antibody with a cytotoxic agent via a chemical linker. LT has been approved in the United States as therapy for relapsed or refractory diffuse large B-cell lymphoma (DLBCL). Given the novelty of this drug, cutaneous adverse effects remain poorly characterized. In this case report, we present a 35-year-old male with relapsing and refractory DLBCL who developed a bullous eruption after his third cycle of LT. Physical exam revealed scattered erosions and erythema of the neck and extremities, along with multiple bullae and erosions on the hands and dorsal feet, and vesicles on the shin. Histopathological exam revealed basovacuolar alteration with epidermal dysmaturation and a re-epithelializing blister, features suggestive of toxic erythema of chemotherapy (TEC). Direct immunofluorescence was negative for immune complex deposition. To date, there are only three cases of LT-associated bullous eruptions in the literature, all demonstrating subepidermal blistering with granular IgA deposition along the basement membrane. This case lacks immunoglobulin deposition and instead exhibits TEC-like cytotoxic interface dermatitis. This case expands the clinicopathologic spectrum of LT-associated cutaneous toxicity and highlights a distinct non-immune mediated, TEC-like vesiculobullous presentation.

Traumatic brain injury triggers inflammatory processes that can persist long after the mechanical insult. This commentary examines the award-winning study by Kodali and colleagues, in which a single intranasal dose of human mesenchymal stromal cell-derived extracellular vesicles 90 min after controlled cortical impact suppressed NLRP3-p38/MAPK signalling and prevented chronic cognitive and affective dysfunction in mice. The study combines a clinically attractive administration route with durable functional effects, helping to explain its rapid influence. Translation will nevertheless depend on establishing human intranasal delivery, defining active cargo and mechanisms, and developing reproducible potency and release assays for a heterogeneous biological product. The work is important because it suggests that early modulation of post-traumatic inflammation may alter the long-term course of brain injury.

Vesicle trafficking is critical for proper distribution of proteins and lipids within eukaryotic cells. This process relies on the selective incorporation of cargo into transport carriers by coat systems, including adaptor-clathrin complexes. While the molecular basis of cargo recognition is well established, the extent to which cargo contributes directly to coat assembly remains incompletely understood. Here, we present cryo-electron microscopy (cryo-EM) structures of the human AP-1 complex bound to a cargo containing tandem sorting motifs at resolutions of 3.5 and 4.0 Å. The structures reveal that a single cargo molecule bearing tandem motifs can bridge two neighboring AP-1 complexes through a previously unrecognized μ1-σ1 inter-complex interface, thereby promoting adaptor oligomerization. Critically, this interface contributes to efficient AP-1-clathrin cage assembly, cargo trafficking, and neuronal development in zebrafish. Together, these findings support a model in which cargo engagement can facilitate higher-order adaptor assembly under physiological conditions, complementing established coat-driven mechanisms.

This study characterized the pharmacokinetic interaction between Compound Danshen Tablets (CDT) and atorvastatin (ATV) and investigated absorption- and transporter-related mechanisms underlying this interaction. ATV pharmacokinetics were evaluated after oral and intravenous administration in normal and hyperlipidemic/diabetic rats, with or without repeated CDT treatment. Mechanistic studies included single-pass intestinal perfusion, Caco-2 bidirectional transport assays, human multidrug resistance protein 1 inside-out vesicle transport assays, ileal P-glycoprotein (P-gp) immunoblotting, an in vivo digoxin probe-substrate experiment, and molecular docking. Repeated CDT administration increased oral ATV exposure in both normal and model rats, with the area under the plasma concentration-time curve from 0 to 24 hours and maximum plasma concentration increasing by up to 85.83% and 306.53%, respectively, whereas intravenous ATV pharmacokinetics were not significantly altered. In Caco-2 monolayers, ATV exhibited marked directional efflux, with an efflux ratio of 39.32, which was reduced to 10.99 by verapamil. CDT increased the apical-to-basolateral apparent permeability coefficient of ATV from 0.41 to 1.31 × 10-6 cm/s and reduced the efflux ratio to 9.82, whereas the basolateral-to-apical apparent permeability coefficient decreased from 16.27 to 12.90 × 10-6 cm/s. In human multidrug resistance protein 1 inside-out vesicles, CDT concentration dependently reduced ATP-dependent N-methylquinidine transport, with inhibition rates ranging from 24.97% to 80.45% at 50-800 μg/mL. Together with the ileal P-gp protein data and the digoxin probe-substrate experiment, these findings indicate that CDT enhances intestinal absorption of ATV primarily by attenuating intestinal P-gp-mediated efflux through reducing both P-gp transport activity and protein abundance. These results provide mechanistic evidence that modulation of intestinal efflux transport contributes to the increased systemic exposure of ATV after repeated CDT administration. SIGNIFICANT STATEMENT: This study identifies a clinically relevant herb-drug interaction in which repeated administration of compound Danshen tablets increases oral atorvastatin exposure in normal and hyperlipidemic/diabetic rats without materially altering intravenous atorvastatin pharmacokinetics. Integrated pharmacokinetic, single-pass intestinal perfusion, Caco-2 transport, human multidrug resistance protein 1 vesicle, ileal P-glycoprotein, and digoxin probe-substrate data support attenuation of intestinal P-glycoprotein-mediated efflux, through reduced transporter activity and protein abundance, as a mechanism enhancing atorvastatin absorption.

Nitrogen mustard (NM) induces severe, refractory skin injuries lacking specific available therapeutics. Considering that extracellular vesicles (EVs) show promising effects in wound healing, we developed a split-type microneedle (MN-EVs@miR) loaded with miR-125b-enriched engineered EVs (EVs@miR) to treat NM-induced skin injury. EVs were isolated from human umbilical cord mesenchymal stem cells (hUMSCs), loaded with miR-125b via transfection, and then conjugated to hyaluronic acid methacryloyl (HAMA) hydrogel using acrylic acid-polyethylene glycol-N-hydroxysuccinimide (AC-PEG-NHS) to prepare microneedle tips, with polyvinyl alcohol (PVA) and sodium thiosulfate constituting the substrate. The prepared MN-EVs@miR exhibited sufficient mechanical strength (0.34 N/needle), sustained EVs@miR release over 21 days, and long-term in vivo retention (>21 days) with excellent biocompatibility. In vitro, EVs@miR enhanced cell viability, inhibited apoptosis, regulated inflammation (downregulating TNF-α, IL-1β, and IL-6; upregulating IL-10), and promoted angiogenesis. In vivo, MN-EVs@miR significantly mitigated NM-induced skin blistering and necrosis, with superior wound healing efficacy compared to that of EVs@miR injection and blank MNs. In conclusion, the split-type MN-EVs@miR provides a promising preventive and therapeutic strategy for NM-induced skin injuries with potential battlefield and clinical applications.

Haemodynamic shear stress, sensed by vascular endothelial cells (ECs), is crucial for the functional integrity of ECs and the systemic benefits of exercise. Here, we observed that shear stress triggers the release of medium/large extracellular vesicles (m/lEVs), from ECs via activation of the mechanosensitive ion channel Piezo1. Endothelial Piezo1 activation induces Piezo1 translocation to the cell periphery, facilitating its trafficking from the vascular ECs to the smooth muscle cells in the splenic capsule. Within the splenic capsule, the translocated Piezo1 acts as a downstream effector of α1-adrenergic receptor, boosting exercise-induced spleen contraction and enhancing exercise endurance. Genetic or pharmacological inhibition of Piezo1 trafficking from ECs impaired spleen contraction during exercise and reduced exercise endurance in mice. Additionally, exercise-induced m/lEV release contributes to safeguard endothelial function through modulating intracellular Piezo1 homeostasis. Furthermore, positive correlations between exercise-induced increases in circulating Piezo1-enriched m/lEVs and the degree of spleen contraction were also observed in human volunteers. These findings unveil a vasculo-splenic axis during exercise, in which EC-derived m/lEVs shuttle Piezo1 to maintain endothelial function and exercise endurance, highlighting a novel role of Piezo1 in inter-organ communication.

Helicobacter pylori infection is characterized by chronic gastric inflammation driven by persistent activation of innate immune signalling pathways. Outer membrane vesicles (OMVs) released by H. pylori are important mediators of host-pathogen interactions. Although several mechanisms of OMV internalization have been described, how specific OMV-associated bacterial factors engage host innate immune receptors and couple vesicle association to inflammatory signalling remains poorly understood. Here, we investigated the contribution of OMV-associated virulence factors and Toll-like receptor 2 (TLR2) heterodimers to OMV-host cell interactions in gastric epithelial cells. Using engineered AGS and GES-1 reporter cells expressing TLR2/1 or TLR2/6, together with genetically defined OMVs, we found that H. pylori OMVs preferentially associated with TLR2-expressing cells and induced robust NF-κB activation. Comparative analysis of OMVs from wild-type and isogenic mutant strains identified Neutrophil-Activating Protein A (NapA) as a major determinant of TLR2-dependent OMV association and inflammatory signalling, whereas urease had a more limited contribution. Disruption of NapA or neutralization of TLR2 markedly reduced OMV association with gastric epithelial cells. Purified H. pylori lipopolysaccharide elicited a comparatively weak and TLR2/6-biased response, indicating that it does not account for the robust TLR2-dependent signalling induced by intact OMVs. Furthermore, OMV-associated NapA exhibited greater pro-inflammatory activity than soluble recombinant NapA, supporting a role for the vesicular context in enhancing TLR2-dependent signalling. Pharmacological inhibition with sulforaphane significantly attenuated OMV-induced TLR2/NF-κB activation. Collectively, these findings identify a previously unrecognized role for OMV-associated NapA in linking TLR2-dependent vesicle association with gastric epithelial cells to inflammatory signalling and support a model in which vesicular presentation.

The gut microbiota plays a critical role in regulating intestinal lipid metabolism, yet the mechanisms governing microbiota-host interactions during lipid uptake remain unclear. Here, we examined the relationship between fat digestibility and gut microbial composition, and found that piglets with higher apparent crude fat digestibility exhibited enhanced lipid absorption and oxidation, accompanied by reduced abundance of Clostridium butyricum in the ileal mucosa. Furthermore, oral administration of C. butyricum suppressed intestinal lipid absorption and oxidation by inhibiting PPARα signaling. Mechanistically, extracellular vesicles (EVs) derived from C. butyricum can enter intestinal epithelial cells and mediate the anti-lipid absorption effects by delivering miRNA-like RNAs (milRNAs), particularly milRNA-3020179, which primarily targeted host genes including PPARα and Rxra. Supplementation with C. butyricum or its EVs prevented lipid accumulation and attenuated the development of metabolic abnormalities in high-fat diet-fed mice, whereas deletion of milRNA-3020179 in C. butyricum abolished its effects on lipid deposition in intestinal organoids. Collectively, these findings identify C. butyricum-derived EV milRNAs as candidate microbial regulators of host lipid metabolism through modulation of PPARα signaling, providing insights into microbiota-driven control of intestinal lipid homeostasis.

Malaria parasites replicate inside red blood cells, degrading hemoglobin within a specialized digestive vacuole. Efficient hemoglobin processing is essential for parasite survival and influences antimalarial drug susceptibility. The vacuole constantly fuses with incoming hemoglobin-filled vesicles, yet the mechanisms that balance cargo influx with membrane homeostasis remain unclear. Here, using conditional reverse genetics, quantitative live-cell imaging, and 3D electron microscopy, we characterize the autophagy-related protein 18 of Plasmodium falciparum (PfATG18) as a key regulator of vacuolar membrane dynamics. Loss of PfATG18 caused vacuole fragmentation, accumulation of hemoglobin-filled vesicles, and parasite death. These defects were preceded by broad architectural destabilization of the parasite's V-ATPase, a proton pump controlling organelle acidification and the vacuole's fusion-fission equilibrium. Direct interference with its membrane sector phenocopied PfATG18 deficiency. We found that PfATG18 does not interact directly with the proton pump but instead associates with a putative V-ATPase assembly factor and with complexes regulating phosphoinositide balance and vesicle trafficking. The breadth of these interactions indicates a multifaceted role at the vacuolar membrane and a regulatory influence on V-ATPase mediated through associated protein machinery. Although a point mutation in PfATG18 has been linked to artemisinin resistance, its complete knockout did not decrease sensitivity. Instead, it rendered ring-stage parasites hypersensitive to dihydroartemisinin, while leaving their susceptibility to other antimalarial drugs unchanged. Together, these findings establish PfATG18 as a central regulator of endocytic membrane homeostasis, essential for V-ATPase function and asexual parasite proliferation in the human blood.

We report the rational design and synthesis of nitrogen-doped sialic acid-derived carbon dots (SiaMPDA CDs) with excitation-independent green emission and high quantum yield and demonstrate their application as efficient Förster resonance energy transfer (FRET) donors for the selective detection of influenza A virus (IAV) subtypes. The surface functionalization of the CDs with sialic acid moieties enables specific molecular recognition of the viral hemagglutinin (HA) protein via multivalent hydrogen-bonding interactions. In combination with Rhodamine B (RhB) as an acceptor, the SiaMPDA-RhB pair exhibits efficient FRET under native conditions. Upon interaction with IAV (H3N2 and H1N1), the FRET system displays anomalous spectral behavior characterized by a simultaneous increase in both donor and acceptor emission intensities without any spectral shift. Mechanistic investigations suggest the formation of a ternary IAV-SiaMPDA-RhB complex, wherein long-range charge transfer coupled with FRET governs the observed fluorescence response. Control experiments with proteins, membrane vesicles, and liposomes confirm that the signal specificity arises from targeted interactions between the sialic acid functionalities of the CDs and the viral surface proteins. The sensing platform enables selective and sensitive detection of IAV subtypes in the picomolar range, comparable to existing carbon dot-based detection systems. These findings establish N-doped sialic acid carbon dots as promising nanoscale probes for virus detection and provide insights into charge-transfer-coupled FRET mechanisms in complex bionano interfaces.

Frankia strain AiPa1T was isolated from root nodules of Alnus incana induced from soil under Picea abies in Finland. Strains AgKG'84/4T, AgB32T and AgB1.8T were isolated from Alnus glutinosa nodules from Germany. AiPa1T, AgB32T and AgKG'84/4T are infective on A. glutinosa and fix nitrogen in symbiotic nodules and pure culture, and strain AgB1.8T is infective on A. glutinosa but does not fix nitrogen and does not have the nif genes. Colonies of the four strains are white, and they form hyphae and sporangia. N2-fixing vesicles are formed by AiPa1T, AgB32T and AgKG'84/4T in NH4+-free medium but not by AgB1.8T. Comparative 16S rRNA gene sequence analyses showed AiPa1T, AgB32T and AgKG'84/4T were in cluster 1 (Fig. S1), while strain AgB1.8T was in cluster 4. A whole-genome phylogeny confirmed this positioning. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the four strains and all validly named Frankia species were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 64 and 71%, respectively. Strains AiPa1T, AgB32T and AgKG'84/4T have genome sizes of 7.00, 6.40 and 6.71 Mb, respectively, while the genome size of AgB1.8T is 9.82 Mb. Phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain AiPa1T (=DSM 114742T=LMG 32596T) to a novel species, for which the name Frankia abietis sp. nov. is proposed. Strain AgKG'84/4T (=DSM 114736T=LMG 32597T) is assigned to a novel species, for which the name Frankia limosa sp. nov. is proposed. Strain AgB32T (=DSM 114735T=LMG 32598T) is assigned to a novel species, with AgB32T as the type strain, for which the name Frankia palustris sp. nov. is proposed. Strain AgB1.8T (=DSM 114738T=LMG 33088T) is assigned to a novel species, for which the name Frankia luti sp. nov. is proposed.