Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases.

Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Extracellular Microvesicles vs. Mitochondria: Competing for the Top Spot in Cardiovascular Regenerative Medicine.

Regenerative medicine aims to restore, replace, and regenerate human cells, tissues, and organs. Despite significant advancements, many cell therapy trials for cardiovascular diseases face challenges like cell survival and immune compatibility, with benefits largely stemming from paracrine effects. Two promising therapeutic tools have been recently emerged in cardiovascular diseases: extracellular vesicles (EVs) and mitochondrial transfer. Concerning EVs, the first pivotal study with EV-enriched secretome derived from cardiovascular progenitor cells has been done treating heart failure. This first in man demonstrated the safety and feasibility of repeated intravenous infusions and highlighted significant clinical improvements, including enhanced cardiac function and reduced symptoms in heart failure patients. The second study uncovered a novel mechanism of endothelial regeneration through mitochondrial transfer via tunneling nanotubes (TNTs). This research showed that mesenchymal stromal cells (MSCs) transfer mitochondria to endothelial cells, significantly enhancing their bioenergetics and vessel-forming capabilities. This mitochondrial transfer was crucial for endothelial cell engraftment and function, offering a new strategy for vascular regeneration without the need for additional cell types. Combining EV and mitochondrial strategies presents new clinical opportunities. These approaches could revolutionize regenerative medicine, offering new hope for treating cardiovascular and other degenerative diseases. Continued research and clinical trials will be crucial in optimizing these therapies, potentially leading to personalized medicine approaches that enhance patient outcomes.

Extracellular Vesicles from Adipose Tissue-Derived Stromal Cells Stimulate Angiogenesis in a Scaffold-Dependent Fashion.

BACKGROUND: The extracellular vesicles (EVs) secreted by adipose tissue-derived stromal cells (ASC) are microenvironment modulators in tissue regeneration by releasing their molecular cargo, including miRNAs. However, the influence of ASC-derived extracellular vesicles (ASC-EVs) on endothelial cells (ECs) and vascularisation is poorly understood. The present study aimed to determine the pro-angiogenic effects of ASC-EVs and explore their miRNA profile. METHODS: EVs were isolated from normoxic and hypoxic cultured ASC conditioned culture medium. The miRNA expression profile was determined by miRseq, and EV markers were determined by Western blot and immunofluorescence staining. The uptake dynamics of fluorescently labelled EVs were monitored for 24 h. ASC-EVs' pro-angiogenic effect was assessed by sprouting ex vivo rat aorta rings in left ventricular-decellularized extracellular matrix (LV dECM) hydrogel or basement membrane hydrogel (Geltrex®). RESULTS: ASC-EVs augmented vascular network formation by aorta rings. The vascular network topology and stability were influenced in a hydrogel scaffold-dependent fashion. The ASC-EVs were enriched for several miRNA families/clusters, including Let-7 and miR-23/27/24. The miRNA-1290 was the highest enriched non-clustered miRNA, accounting for almost 20% of all reads in hypoxia EVs. CONCLUSION: Our study revealed that ASC-EVs augment in vitro and ex vivo vascularisation, likely due to the enriched pro-angiogenic miRNAs in EVs, particularly miR-1290. Our results show promise for regenerative and revascularisation therapies based on ASC-EV-loaded ECM hydrogels.

Hypoxic extracellular vesicles from hiPSCs protect cardiomyocytes from oxidative damage by transferring antioxidant proteins and enhancing Akt/Erk/NRF2 signaling.

BACKGROUND: Stem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection. METHODS: EVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385. RESULTS: We demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs. CONCLUSIONS: In this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.

Exosomal miRNA-21-5p and miRNA-21-3p as key biomarkers of myocardial infarction.

Objective: Coronary artery disease (CAD) is a debilitating condition that can lead to myocardial infarction (MI). Exosomal miRNAs (exo-miRNA) can be diagnostic biomarkers for detecting MI. Here, we conduct a study to evaluate the efficacy of exo-miRNA-21-5p/3p for early detection of MI. Methods: A total of 135 CAD patients and 150 healthy subjects participated in this study. Additionally, we randomly divided 26 male Wistar rats (12 weeks old) into two groups: control and induced MI. Angiographic images were used to identify patients and healthy individuals of all genders. In the following, serum exosomes were obtained, and exo-miRNA-21-5p/3p was measured by reverse-transcriptase polymerase chain reaction. Results: We observed an upregulation of exo-miRNA-21-5p/3p in CAD patient and MI-induced animal groups compared to controls. Analysis of the ROC curves defined 82% and 88% of the participants' exo-miRNA-21-5p and exo-miRNA-21-3p diagnostic power, respectively, which in the animal model was 92 and 82. Conclusion: This study revealed that the mean expression levels of exo-miRNA-21-5p/3p were significantly increased in CAD patients and animal models of induced MI. Also, these results are associated with the atherogenic lipid profile of CAD patients, which may play an important role in the progression of the disease. Therefore, they can be considered as novel biomarkers.

Biomimetic nanomedicine cocktail enhances selective targeting for ovarian Cancer chemo- and immunotherapy.

Ovarian cancer is one of the deadliest cancers, and combined chemo- and immunotherapies are potential strategies to combat it. However, the anti-cancer efficacy of the combined therapies may be limited by the non-selective co-delivery of chemotherapy and immunotherapy. Herein, a combined chemo- and immunotherapy is designed to selectively target ovarian tumor (ID8) cells and dendritic cells (DCs) using ID8 cell membrane (IM) and bacterial outer membrane vesicles (OMVs), respectively. Doxorubicin (DOX) and Ovalbumin (OVA) peptide (OVA257-264) are chosen as model chemotherapy and immunotherapy agents, respectively. A DNA nanocube capable of easily loading DOX or OVA257-264 is chosen as the carrier. Firstly, the DNA nanocube is used to load DOX or OVA257-264 to prepare cube-DOX or cube-OVA. This nanocube was then encapsulated with IM to form IM@Cube-DOX and with OMV to form OMV@Cube-OVA. IM@Cube-DOX can be selectively taken up by ID8 cells, leading to effective cell killing, while OMV@Cube-OVA targets and activates DC2.4 cells in vitro. Both IM@Cube-DOX and OMV@Cube-OVA show increased accumulation at ID8 tumors in C57BL/6 mice. Combined IM@Cube-DOX + OMV@Cube-OVA therapy demonstrates better anti-tumor efficacy than non-selective delivery methods such as OMV@(Cube-DOX + Cube-OVA) or IM@(Cube-DOX + Cube-OVA) in ID8-OVA tumor-bearing mice. In conclusion, this study demonstrates a biomimetic delivery strategy that enables selective drug delivery to tumor cells and DCs, thereby enhancing the anti-tumor efficacy of combined chemo- and immunotherapy through the selective delivery strategy.

Exosomal mRNA in plasma serves as a predictive marker for microvascular invasion in hepatocellular carcinoma.

BACKGROUND AND AIM: There is a pressing need for non-invasive preoperative prediction of microvascular invasion (MVI) in hepatocellular carcinoma (HCC). This study investigates the potential of exosome-derived mRNA in plasma as a biomarker for diagnosing MVI. METHODS: Patients with suspected HCC undergoing hepatectomy were prospectively recruited for preoperative peripheral blood collection. Exosomal RNA profiling was conducted using RNA sequencing in the discovery cohort, followed by differential expression analysis to identify candidate targets. We employed multiplexed droplet digital PCR technology to efficiently validate them in a larger sample size cohort. RESULTS: A total of 131 HCC patients were ultimately enrolled, with 37 in the discovery cohort and 94 in the validation cohort. In the validation cohort, the expression levels of RSAD2, PRPSAP1, and HOXA2 were slightly elevated while CHMP4A showed a slight decrease in patients with MVI compared with those without MVI. These trends were consistent with the findings in the discovery cohort, although they did not reach statistical significance (P > 0.05). Notably, the expression level of exosomal PRPSAP1 in plasma was significantly higher in patients with more than 5 MVI than in those without MVI (0.147 vs 0.070, P = 0.035). CONCLUSION: This study unveils the potential of exosome-derived PRPSAP1 in plasma as a promising indicator for predicting MVI status preoperatively.

Preparation and anti-tumor activity of paclitaxel silk protein nanoparticles encapsulated by biofilm.

In order to overcome the poor bioavailability of paclitaxel (PTX), in this study, self-assembled paclitaxel silk fibronectin nanoparticles (PTX-SF-NPs) were encapsulated with outer membrane vesicles of Escherichia coli (E. coil), and biofilm-encapsulated paclitaxel silk fibronectin nanoparticles (OMV-PTX-SF-NPs) were prepared by high-pressure co-extrusion, the size and zeta potential of the OMV-PTX-SF-NPs were measured. The antitumor effects of OMV-PTX-SF-NPs were evaluated by cellular and pharmacodynamic assays, and pharmacokinetic experiments were performed. The results showed that hydrophobic forces and hydrogen bonding played a major role in the interaction between paclitaxel and filipin proteins, and the size of OMV-PTX-SF-NPs was 199.8 ± 2.8 nm, zeta potential was -17.8 ± 1.3 mv. The cellular and in vivo pharmacokinetic assays demonstrated that the OMV-PTX-SF-NPs possessed a promising antitumor effect. Pharmacokinetic experiments showed that the AUC0-∞ of OMV-PTX-SF-NPs was 5.314 ± 0.77, which was much larger than that of free PTX, which was 0.744 ± 0.14. Overall, we have successfully constructed a stable oral formulation of paclitaxel with a sustained-release effect, which is able to effectively increase the bioavailability of paclitaxel, improve the antitumor activity, and reduce the adverse effects.

Extracellular vesicle transfer of lncRNA H19 splice variants to cardiac cells.

The delivery of therapeutic long non-coding RNAs (lncRNA) to the heart by extracellular vesicles (EVs) is promising for heart repair. H19, a lncRNA acting as a major regulator of gene expression within the cardiovascular system, is alternatively spliced, but the loading of its different splice variants into EVs and their subsequent uptake by recipient cardiac cells remain elusive. Here, we dissected the cellular expression of H19 splice variants and their loading into EVs secreted by Wharton-Jelly mesenchymal stromal/stem cells (WJ-MSCs). We demonstrated that overexpression of the mouse H19 gene in WJ-MSCs induces the expression of H19 splice variants at different levels. Interestingly, EVs isolated from the H19-transfected WJ-MSCs (EV-H19) showed similar expression levels for all tested splice variant sets. In vitro, we further demonstrated that EV-H19 was taken up by cardiomyocytes, fibroblasts, and endothelial cells (ECs). Finally, analysis of EV tropism in living rat myocardial slices indicated that EVs were internalized mostly by cardiomyocytes and ECs. Collectively, our results indicated that EVs can be loaded with different lncRNA splice variants and successfully internalized by cardiac cells.

tRF-His-GTG-1 enhances NETs formation and interferon-α production in lupus by extracellular vesicle.

BACKGROUND: Hyperactive neutrophil extracellular traps (NETs) formation plays a crucial role in active severe systemic lupus erythematosus (SLE). However, what triggers the imbalance in dysregulated NETs formation in SLE is elusive. Transfer RNA-derived small RNAs (tsRNAs) are novel non-coding RNAs, which participate in various cellular processes. We explore the role of tsRNAs on NETs formation in SLE. METHODS: We analyzed the levels of NETs DNA and platelet-derived extracellular vesicles (pEVs) from 50 SLE patients and 20 healthy control subjects. The effects of pEVs on NETs formation were evaluated by using immunofluorescence assay and myeloperoxidase-DNA PicoGreen assay. The regulatory mechanism of pEVs on NETs formation and inflammatory cytokines production were investigated using an in vitro cell-based assay. RESULTS: Increased circulating NETs DNA and pEVs were shown in SLE patients and were associated with disease activity (P < 0.005). We demonstrated that SLE patient-derived immune complexes (ICs) induced platelet activation, followed by pEVs release. ICs-triggered NETs formation was significantly enhanced in the presence of pEVs through Toll-like receptor (TLR) 8 activation. Increased levels of tRF-His-GTG-1 in pEVs and neutrophils of SLE patients were associated with disease activity. tRF-His-GTG-1 interacted with TLR8 to prime p47phox phosphorylation in neutrophils, resulting in reactive oxygen species production and NETs formation. Additionally, tRF-His-GTG-1 modulated NF-κB and IRF7 activation in neutrophils upon TLR8 engagement, resulting IL-1ß, IL-8, and interferon-α upregulation, respectively. CONCLUSIONS: The level of tRF-His-GTG-1 was positively correlated with NETs formation in SLE patients; tRF-His-GTG-1 inhibitor could efficiently suppress ICs-triggered NETs formation/hyperactivation, which may become a potential therapeutic target.

Neutrophils and platelets are key members in the immunopathogenesis of SLE. EVs play a key role in intercellular communication. Abnormal NETs formation promotes vascular complications and organ damage in SLE patients. tsRNA is a novel regulatory small non-coding RNA and participates in diverse pathological processes. Herein, we showed that SLE patient-derived ICs activates platelets directly, followed by intracellular tRF-His-GTG-1 upregulation, which is loaded into pEVs. The pEV-carried tRF-His-GTG-1 could interact with TLR8 in neutrophils, followed by activation of the downstream signaling pathway, including p47phox-NOX2-ROS, which causes NETs enhancement, while IRF7 promotes the expression of IFN-α. The tRF-His-GTG-1 inhibitor could suppress efficiently SLE ICs-induced NETs formation and pEVs primed NETs enhancement. This study offers new molecular machinery to explain the association between the platelets-derived tsRNAs, pEVs, and hyperactive NETs formation in lupus. tRF-His-GTG-1 may serve as a potential therapeutic target and help to advance our understanding of tsRNAs in SLE pathogenesis.

Chitosan/PLGA-based tissue engineered nerve grafts with SKP-SC-EVs enhance sciatic nerve regeneration in dogs through miR-30b-5p-mediated regulation of axon growth.

Extracellular vesicles from skin-derived precursor Schwann cells (SKP-SC-EVs) promote neurite outgrowth in culture and enhance peripheral nerve regeneration in rats. This study aimed at expanding the application of SKP-SC-EVs in nerve grafting by creating a chitosan/PLGA-based, SKP-SC-EVs-containing tissue engineered nerve graft (TENG) to bridge a 40-mm long sciatic nerve defect in dogs. SKP-SC-EVs contained in TENGs significantly accelerated the recovery of hind limb motor and electrophysiological functions, supported the outgrowth and myelination of regenerated axons, and alleviated the denervation-induced atrophy of target muscles in dogs. To clarify the underlying molecular mechanism, we observed that SKP-SC-EVs were rich in a variety of miRNAs linked to the axon growth of neurons, and miR-30b-5p was the most important among others. We further noted that miR-30b-5p contained within SKP-SC-EVs exerted nerve regeneration-promoting effects by targeting the Sin3a/HDAC complex and activating the phosphorylation of ERK, STAT3 or CREB. Our findings suggested that SKP-SC-EVs-incorporating TENGs represent a novel type of bioactive material with potential application for peripheral nerve repair in the clinic.

Optimizing mesenchymal stem cell extracellular vesicles for chronic wound healing: Bioengineering, standardization, and safety.

Chronic wounds represent a significant global burden, afflicting millions with debilitating complications. Despite standard care, impaired healing persists due to factors like persistent inflammation and impaired tissue regeneration. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) offer an innovative regenerative medicine approach, delivering stem cell-derived therapeutic cargo in engineered nanoscale delivery systems. This review examines pioneering bioengineering strategies to engineer MSC-EVs into precision nanotherapeutics for chronic wounds. Emerging technologies like CRISPR gene editing, microfluidic manufacturing, and biomimetic delivery systems are highlighted for their potential to enhance MSC-EV targeting, optimize therapeutic cargo enrichment, and ensure consistent clinical-grade production. However, key hurdles remain, including batch variability, rigorous safety assessment for potential tumorigenicity, immunogenicity, and biodistribution profiling. Crucially, collaborative frameworks harmonizing regulatory science with bioengineering and patient advocacy hold the key to expediting global clinical translation. By overcoming these challenges, engineered MSC-EVs could catalyze a new era of off-the-shelf regenerative therapies, restoring hope and healing for millions afflicted by non-healing wounds.

Extremely high spatiotemporal resolution microscopy for live cell imaging by single photon counting, noise elimination, and a novel restoration algorithm based on probability calculation.

Optical microscopy is essential for direct observation of dynamic phenomena in living cells. According to the classic optical theories, the images obtained through light microscopes are blurred for about half the wavelength of light, and therefore small structures below this "diffraction limit" were thought unresolvable by conventional optical microscopy. In reality, accurately obtained optical images contain complete information about the observed objects. Temporal resolution is also important for the observation of dynamic phenomena. A challenge exists here to overcome the trade-off between the time required for measurement and the accuracy of the measurement. The present paper describes a concrete methodology for reconstructing the structure of an observed object, based on the information contained in the image obtained by optical microscopy. It is realized by accurate single photon counting, complete noise elimination, and a novel restoration algorithm based on probability calculation. This method has been implemented in the Super-resolution Confocal Live Imaging Microscopy (SCLIM) we developed. The new system named SCLIM2M achieves unprecedented high spatiotemporal resolution. We have succeeded in capturing sub-diffraction-limit structures with millisecond-level dynamics of organelles and vesicles in living cells, which were never observed by conventional optical microscopy. Actual examples of the high-speed and high-resolution 4D observation of living cells are presented.

Stem Cell-Derived Extracellular Vesicle-Bearing Injectable Hydrogel for Collagen Generation in Dermis.

Despite the remarkable advances of dermal fillers that reduce wrinkles caused by dermis thickness reduction, they still lack effective hydrogel systems that stimulate collagen generation along with injection convenience. Here, we develop a stem cell-derived extracellular vesicle (EV)-bearing thermosensitive hydrogel (EVTS-Gel) for effective in vivo collagen generation. The TS-Gel undergoes sol-gel transition at 32.6 °C, as demonstrated by the storage and loss moduli crossover. Moreover, the TS-Gel and the EVTS-Gel have comparable rheological properties. Both hydrogels are injected in a sol state; hence, they require lower injection forces than conventional hydrogel-based dermal fillers. When locally administered to mouse skin, the TS-Gel extends the retention time of EVs by 2.23 times. Based on the nature of the controlled EV release, the EVTS-Gel significantly inhibits the dermis thickness reduction caused by aging compared to the bare EV treatment for 24 weeks. After a single treatment, the collagen layer thickness of the EVTS-Gel-treated dermis becomes 2.64-fold thicker than that of the bare EV-treated dermis. Notably, the collagen generation efficacy of the bare EV is poorer than that of the EVTS-Gel of a 10× lesser dose. Overall, the EVTS-Gel shows potential as an antiaging dermal filler for in vivo collagen generation.

Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening.

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 µm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

Comprehensive proteomic analysis of HCoV-OC43 virions and virus-modulated extracellular vesicles.

Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles. IMPORTANCE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.

Advances in preparation and engineering of plant-derived extracellular vesicles for nutrition intervention.

Plant-derived extracellular vesicles (PLEVs), as a type of naturally occurring lipid bilayer membrane structure, represent an emerging delivery vehicle with immense potential due to their ability to encapsulate hydrophobic and hydrophilic compounds, shield them from external environmental stresses, control release, exhibit biocompatibility, and demonstrate biodegradability. This comprehensive review analyzes engineering preparation strategies for natural vesicles, focusing on PLEVs and their purification and surface engineering. Furthermore, it encompasses the latest advancements in utilizing PLEVs to transport active components, serving as a nanotherapeutic system. The prospects and potential development of PLEVs are also discussed. It is anticipated that this work will not only address existing knowledge gaps concerning PLEVs but also provide valuable guidance for researchers in the fields of food science and biomedical studies, stimulating novel breakthroughs in plant-based therapeutic options.

Neurodegenerative diseases associated with the disruption of proteostasis and their therapeutic strategies using chemical chaperones.

Aberrant proteostasis is thought to be involved in the pathogenesis of neurodegenerative diseases. Some proteostasis abnormalities are ameliorated by chaperones. Chaperones are divided into three groups: molecular, pharmacological, and chemical. Chemical chaperones intended to alleviate stress in organelles, such as the endoplasmic reticulum (ER), are now being administered clinically. Of the chemical chaperones, 4-phenylbutyrate (4-PBA) has been used as a research reagent, and its mechanism of action includes chaperone effects and the inhibition of histone deacetylase. Moreover, it also binds to the B-site of SEC24 and regulates COPII-mediated transport from the ER. Although its therapeutic effect may not be strong, elucidating the mechanism of action of 4-PBA may contribute to the identification of novel therapeutic targets for neurodegenerative diseases.

Multilayered collagen-lipid hybrid nanovesicles for retinol stabilization and efficient skin delivery.

Lipid-based nanocarriers have been extensively utilized for the solubilization and cutaneous delivery of water-insoluble active ingredients in skincare formulations. However, their practical application is often limited by structural instability, leading to premature release and degradation of actives. Here we present highly robust multilamellar nanovesicles, prepared by the polyionic self-assembly of unilamellar vesicles with hydrolyzed collagen peptides, to stabilize all-trans-retinol and enhance its cutaneous delivery. Our results reveal that the reinforced multilayer structure substantially enhances dispersion stability under extremely harsh conditions, like freeze-thaw cycles, and stabilizes the encapsulated retinol. Interestingly, these multilamellar vesicles exhibit significantly lower cytotoxicity to human dermal fibroblasts than their unilamellar counterparts, likely due to their smaller particle number per weight, minimizing potential disruptions to cellular membranes. In artificial skin models, retinol-loaded multilamellar vesicles effectively upregulate collagen-related gene expression while suppressing the synthesis of metalloproteinases. These findings suggest that the robust multilamellar vesicles can serve as effective nanocarriers for the efficient delivery and stabilization of bioactive compounds in cutaneous applications.