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Novel therapeutic approaches for wound healing have included biomaterials from the Atlantic cod (Gadus morhua L.), with promising results in wound management. The use of extracellular vesicles (EVs), which can be isolated from cod biofluids, remains to be studied. EVs play key roles in cellular communication, and their use both as biomarkers and as therapeutic agents is widely reported in human pathologies, particularly with respect to mesenchymal stem cells. This pilot study characterized the total proteomic cargo content of EVs from cod serum and mucus and assessed the EVs’ potential for regenerative activity in wound-healing processes, using human and mouse fibroblast and keratinocyte in vitro scratch injury models. The pro-regenerative potential of both cod serum EVs and mucus EVs was identified, with differing capacities for accelerating wound closure in fibroblast and keratinocyte cells. This was further supported by varying effects of the cod serum EVs and mucus EVs on cellular vimentin and FGF-2 levels. The serum EV and mucus EV protein cargoes differed with respect to abundance of protein hits and associated enriched functional GO and KEGG pathways, but both were associated with immune, stress and wound-healing processes. Cod EVs may present as innovative therapeutic options for regenerative medicine applications, and our reported findings provide valuable insights for future in-depth studies.

Pancreatic ductal adenocarcinoma remains an aggressive cancer with a low 5-year survival rate. Although gemcitabine has been a standard treatment for advanced pancreatic cancer, patients often develop resistance to this therapeutic. We have previously shown that treating pancreatic cancer cells in vitro with a combination of gemcitabine and the cytokine TRAIL significantly reduced both cell viability and survival. The data presented here demonstrate that this response to treatment is inhibited when cells are incubated with a conditioned medium derived from untreated cells. We show that this inhibition is specifically mediated by extracellular vesicles present in the conditioned medium, as seen by a significant decrease in apoptosis. Additionally, we further demonstrate that this effect can be reversed in the presence of GW4869, an inhibitor of exosome biogenesis and release. These results show that pancreatic cancer cell-derived extracellular vesicles can confer resistance to treatment with gemcitabine and TRAIL. The implications of these findings suggest that removal of EVs during treatment can improve the response of cells to gemcitabine and TRAIL treatment in vitro.

Electrically non-excitable astroglia take up neurotransmitters, buffer extracellular K + and generate Ca 2+ signals that release molecular regulators of neural circuitry. The underlying machinery remains enigmatic, mainly because the sponge-like astrocyte morphology has been difficult to access experimentally or explore theoretically. Here, we systematically incorporate multi-scale, tri-dimensional astroglial architecture into a realistic multi-compartmental cell model, which we constrain by empirical tests and integrate into the NEURON computational biophysical environment. This approach is implemented as a flexible astrocyte-model builder ASTRO. As a proof-of-concept, we explore an in silico astrocyte to evaluate basic cell physiology features inaccessible experimentally. Our simulations suggest that currents generated by glutamate transporters or K + channels have negligible distant effects on membrane voltage and that individual astrocytes can successfully handle extracellular K + hotspots. We show how intracellular Ca 2+ buffers affect Ca 2+ waves and why the classical Ca 2+ sparks-and-puffs mechanism is theoretically compatible with common readouts of astroglial Ca 2+ imaging. Astrocytes have gained increasing attention for their roles in regulating neural circuits via neurotransmitter uptake, K + buffering, and ability to signal via Ca 2 +  transients. Here, the authors develop a computational modelling environment for astrocytes, akin to the NEURON environment, called ASTRO.

An in vitro model of the human blood-brain barrier was developed, based on a collagen hydrogel containing astrocytes, overlaid with a monolayer of endothelium, differentiated from human induced pluripotent stem cells (hiPSCs). The model was set up in transwell filters allowing sampling from apical and basal compartments. The endothelial monolayer had transendothelial electrical resistance (TEER) values >700Ω.cm 2 and expressed tight-junction markers, including claudin-5. After differentiation of hiPSCs the endothelial-like cells expressed VE-cadherin ( CDH5 ) and von-Willebrand factor ( VWF ) as determined by immunofluorescence. However, electron microscopy indicated that at set-up (day 8 of differentiation), the endothelial-like cells still retained some features of the stem cells, and appeared immature, in comparison with primary brain endothelium or brain endothelium in vivo . Monitoring showed that the TEER declined gradually over 10 days, and transport studies were best carried out in a time window 24-72hrs after establishment of the model. Transport studies indicated low permeability to paracellular tracers and functional activity of P-glycoprotein ( ABCB1 ) and active transcytosis of polypeptides via the transferrin receptor ( TFR1 ).

Urinary extracellular vesicles (U-EVs) are gaining increasing interest as non-invasive liquid biopsy tools for clinical use. Prostate cancer (PCa) is amongst the highest cancer-related cause of death in men, and therefore, the identification of non-invasive robust biomarkers is of high importance. This study assessed U-EV profiles from individuals affected by PCa at Gleason scores 6–9, compared with healthy controls. U-EVs were characterised and assessed for proteomic cargo content by LC-MS/MS analysis. The U-EV proteomes were compared for enrichment of gene ontology (GO), KEGG, and Reactome pathways, as well as disease–gene associations. U-EVs ranged in size from 50 to 350 nm, with the majority falling within the 100–200 nm size range for all groups. U-EV protein cargoes from the PCa groups differed significantly from healthy controls, with 16 protein hits unique to the GS 6–7 and 88 hits to the GS 8–9 U-EVs. Pathway analysis showed increased enrichment in the PCa U-EVs of biological process GO (5 and 37 unique to GS 6–7 and GS 8–9, respectively), molecular function GO (3 and 6 unique to GS 6–7 and GS 8–9, respectively), and cellular component GO (10 and 22 unique to GS 6–7 and GS 8–9, respectively) pathways. A similar increase was seen for KEGG pathways (11 unique to GS 8–9) and Reactome pathways (102 unique to GS 8–9). Enrichment of disease–gene associations was also increased in the PCa U-EVs, with highest differences for the GS 8–9 U-EVs (26 unique terms). The pathway enrichment in the PCa U-EVs was related to several key inflammatory, cell differentiation, cell adhesion, oestrogen signalling, and infection pathways. Unique GO and KEGG pathways enriched for the GS 8–9 U-EVs were associated with cell–cell communication, immune and stress responses, apoptosis, peptidase activity, antioxidant activity, platelet aggregation, mitosis, proteasome, mRNA stability oxytocin signalling, cardiomyopathy, and several neurodegenerative diseases. Our findings highlight U-EVs as biomarkers to inform disease pathways in prostate cancer patients and offer a non-invasive biomarker tool for clinical use.

Tenrecs are heterothermic burrowing mammals, which are capable of withstanding extreme environmental stressors, including during hibernation. Their phylogenetic position as reminiscent of an ancestral placental mammal makes tenrecs a unique model for evolutionarily conserved traits, with potential translatability to human physiology and pathobiology, including adaptations to extreme environments. In this study, we compared tenrec plasma for post-translational protein citrullination profiles (citrullinomes) and extracellular vesicle (EV) characteristics, including selected microRNA cargoes (miR-21, miR-155, miR-206, miR-210), between baseline active and hibernating states at low (12 °C) and high (28 °C) ambient temperatures. Our findings show considerable changes in citrullinome plasma profiles and associated Gene Ontology and KEGG pathway analysis linked to physiological and inflammatory processes, comparing hibernating and active states, also differing between the two ambient temperature groups. We furthermore identified modified EV profiles with respect to stress-related (miR-21, miR-155), hypoxia (miR-210) and metabolic/muscle related (miR-206) microRNA cargoes, which showed significant differences between active and hibernating animals, also comparing the two ambient temperature groups. Our findings show novel roles for post-translational protein citrullination in regulating immune and metabolic associated pathways in the tenrec, and highlight EV profiles, based on microRNA cargoes, as indicators for stress and metabolic responses in active versus hibernating states, including at different temperatures. Collectively our data highlights the tenrec as an evolutionary model for regulating pathobiological responses in extreme environments and may have translatable potential for human physiology and pathologies.

This paper examines the relationship between the morphological modality of 189 dendritic spines and the surrounding astroglia using full three-dimensional reconstructions of neuropil fragments. An integrative measure of three-dimensional glial coverage confirms that thin spine postsynaptic densities are more tightly surrounded by glia. This distinction suggests that diffusion-dependent synapse–glia communication near ‘learning’ synapses (associated with thin spines) could be stronger than that near ‘memory’ synapses (associated with larger spines).

Long-lasting synaptic changes enable memory storage and regulate recall in the brain. Our previous work established that generation of multi-innervated dendritic spines (MISs), spines with typically two excitatory presynaptic inputs, underlies hippocampal memory formation in aged, but not young mice. The identification of MIS generation was done by ultrastructural analysis in hippocampal CA1 stratum radiatum 24 h after contextual fear conditioning (CFC). However, our analysis did not consider multi-spine boutons (MSBs), which were recently shown to increase in complexity (complex MSBs are pre-synaptic boutons connecting with more than two post-synapses) at a later time point after CFC in young age. Therefore, we re-analyzed our three-dimensional electron microscopy images and show that, unexpectedly, MSB complexity, decreases in CA1 stratum radiatum 24 h after CFC. The decrease in MSB complexity occurred both in young and aged mice, indicating that aging has no impact on this synaptic change. Considering that complex MSBs link the activity of multiple postsynaptic neurons, we suggest that after CFC a decrease in MSB complexity may be required for specific memory recall.

Oysters and clams are important for food security and of commercial value worldwide. They are affected by anthropogenic changes and opportunistic pathogens and can be indicators of changes in ocean environments. Therefore, studies into biomarker discovery are of considerable value. This study aimed at assessing extracellular vesicle (EV) signatures and post-translational protein deimination profiles of hemolymph from four commercially valuable Mollusca species, the blue mussel (Mytilus edulis), soft shell clam (Mya arenaria), Eastern oyster (Crassostrea virginica), and Atlantic jacknife clam (Ensis leei). EVs form part of cellular communication by transporting protein and genetic cargo and play roles in immunity and host–pathogen interactions. Protein deimination is a post-translational modification caused by peptidylarginine deiminases (PADs), and can facilitate protein moonlighting in health and disease. The current study identified hemolymph-EV profiles in the four Mollusca species, revealing some species differences. Deiminated protein candidates differed in hemolymph between the species, with some common targets between all four species (e.g., histone H3 and H4, actin, and GAPDH), while other hits were species-specific; in blue mussel these included heavy metal binding protein, heat shock proteins 60 and 90, 2-phospho-D-glycerate hydrolyase, GTP cyclohydrolase feedback regulatory protein, sodium/potassium-transporting ATPase, and fibrinogen domain containing protein. In soft shell clam specific deimination hits included dynein, MCM3-associated protein, and SCRN. In Eastern oyster specific deimination hits included muscle LIM protein, beta-1,3-glucan-binding protein, myosin heavy chain, thaumatin-like protein, vWFA domain-containing protein, BTB domain-containing protein, amylase, and beta-catenin. Deiminated proteins specific to Atlantic jackknife clam included nacre c1q domain-containing protein and PDZ domain-containing protein In addition, some proteins were common as deiminated targets between two or three of the Bivalvia species under study (e.g., EP protein, C1q domain containing protein, histone H2B, tubulin, elongation factor 1-alpha, dominin, extracellular superoxide dismutase). Protein interaction network analysis for the deiminated protein hits revealed major pathways relevant for immunity and metabolism, providing novel insights into post-translational regulation via deimination. The study contributes to EV characterization in diverse taxa and understanding of roles for PAD-mediated regulation of immune and metabolic pathways throughout phylogeny.

Prostate cancer (PCa) progression is shaped by the tumour microenvironment, where hypoxia promotes aggressiveness and contributes to therapy resistance. Extracellular vesicles (EVs), secreted under hypoxia, can deliver modified bioactive cargo that reprograms recipient cells. This study examined whether EVs from hypoxia-conditioned metastatic PCa cells enhance malignant traits in cancerous and non-tumorigenic prostate cell lines via Wnt signalling and epithelial–mesenchymal transition (EMT). EVs from PC3 cells cultured under hypoxia (1% O2) or normoxia (21% O2) as control were applied to LNCaP (low metastatic potential) and PNT2 (non-tumorigenic) cells. PC3 hypoxia-derived EVs increased HIF-1α, upregulated mesenchymal markers (Vimentin, N-cadherin) and Wnt-related genes (Wnt3A, Wnt5A, Fzd7), and suppressed the epithelial marker E-cadherin. Functional assessment showed that LNCaP cells treated with PC3 hypoxia EVs showed greater motility and invasiveness, and PNT2 cells displayed transcriptomic reprogramming. These findings show that hypoxia-driven EVs can propagate pro-metastatic signalling in less aggressive and normal prostate cells. The findings highlight EVs as a potential therapeutic target in PCa progression.