Publications

Fibrosis is a serious unintended side effect of radiation therapy. In this study, we aimed to investigate whether amphiregulin (AREG) plays a critical role in fibrosis development after total-body irradiation (TBI). We found that the expression of AREG and fibrotic markers, such as α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1α1), was elevated in the kidneys of 6 Gy TBI mice. Expression of AREG and α-SMA was mainly elevated in the proximal and distal tubules of the kidney in response to TBI, which was confirmed by immunofluorescence staining. Knockdown of Areg mRNA using self-assembled-micelle inhibitory RNA (SAMiRNA) significantly reduced the expression of fibrotic markers, including α-SMA and COL1α1, and inflammatory regulators. Finally, intravenous injections of SAMiRNA targeting mouse Areg mRNA (SAMiRNA-mAREG) diminished radiation-induced collagen accumulation in the renal cortex and medulla. Taken together, the results of the present study suggest that blocking of AREG signaling via SAMiRNA-mAREG treatment could be a promising therapeutic approach to alleviate radiation-induced kidney fibrosis.

Resistive pulse sensing (RPS) is an analytical method that can be used to individually count particles from a small sample. RPS simply monitors the physical characteristics of particles, such as size, shape, and charge density, and the integration of RPS with biosensing is an attractive theme to detect biological particles such as virus and bacteria. In this report, a methodology of biosensing on RPS was investigated. Polydopamine (PD), an adhesive component of mussels, was used as the base material to create a sensing surface. PD adheres to most materials, such as noble metals, metal oxides, semiconductors, and polymers; as a result, PD is a versatile intermediate layer for the fabrication of a biosensing surface. As an example of a biological particle, human influenza A virus (H1N1 subtype) was used to monitor translocation of particles through the pore membrane. When virus-specific ligands (6′-sialyllactose) were immobilized on the pore surface, the translocation time of the virus particles was considerably extended. The detailed translocation data suggest that the viral particles were trapped on the sensing surface by specific interactions. In addition, virus translocation processes on different pore surfaces were distinguished using machine learning. The result shows that the simple and versatile PD-based biosensor surface design was effective. This advanced RPS measurement system could be a promising analytical technique.

Biological nanoparticles, such as exosomes, offer an approach to drug delivery because of their innate ability to transport biomolecules. Exosomes are derived from cells and an integral component of cellular communication. However, the cellular cargo of human exosomes could negatively impact their use as a safe drug carrier. Additionally, exosomes have the intrinsic yet enigmatic, targeting characteristics of complex cellular communication. Hence, harnessing the natural transport abilities of exosomes for drug delivery requires predictably targeting these biological nanoparticles. This manuscript describes the use of two chemical modifications, incorporating a neuropilin receptor agonist peptide (iRGD) and a hypoxia-responsive lipid for targeting and release of an encapsulated drug from bovine milk exosomes to triple-negative breast cancer cells. Triple-negative breast cancer is a very aggressive and deadly form of malignancy with limited treatment options. Incorporation of both the iRGD peptide and hypoxia-responsive lipid into the lipid bilayer of bovine milk exosomes and encapsulation of the anticancer drug, doxorubicin, created the peptide targeted, hypoxia-responsive bovine milk exosomes, iDHRX. Initial studies confirmed the presence of iRGD peptide and the exosomes’ ability to target the αvβ3 integrin, overexpressed on triple-negative breast cancer cells’ surface. These modified exosomes were stable under normoxic conditions but fragmented in the reducing microenvironment created by 10 mM glutathione. In vitro cellular internalization studies in monolayer and three-dimensional (3D) spheroids of triple-negative breast cancer cells confirmed the cell-killing ability of iDHRX. Cell viability of 50% was reached at 10 μM iDHRX in the 3D spheroid models using four different triple-negative breast cancer cell lines. Overall, the tumor penetrating, hypoxia-responsive exosomes encapsulating doxorubicin would be effective in reducing triple-negative breast cancer cells’ survival.

Transfusion of stored erythrocytes is associated with the increased risk of morbidity and mortality in critical infections, but the mechanism is incompletely understood. Previous studies have suggested that RBC-derived extracellular vesicles (EVs) may be potential risk factors for the occurrence of transfusion-related immunomodulation. The purpose of our study was to evaluate the effects of RBC-derived EVs under inflammatory conditions and explore the underlying mechanisms. In vivo, the activity of EVs was evaluated in cecal ligation and puncture (CLP)-induced sepsis. Our results showed that EVs significantly aggravated the inflammatory response to sepsis in serum and lung tissue by promoting the production of the proinflammatory factors tumor necrosis factor-α (TNF-α）-interleukin-6(IL-6), and interleukin-1β (IL-1β) and reduced the survival rate of septic mice in vivo. Importantly, adoptive transfer of EVs-pretreated bone marrow-derived macrophages (BMDMs) obviously aggravated systemic proinflammatory factors in mice after CLP surgery. In vitro, the proinflammatory properties of EVs were shown to elevate TNF-α, IL-6, and IL-1β levels in lipopolysaccharide (LPS)-stimulated BMDMs. Moreover, EVs promoted LPS-induced macrophage polarization into a proinflammatory phenotype. The underlying mechanism might involve EV-mediated up-regulation of TLR4–MyD88–NF-κB–MAPK activity to favor macrophage cytokine production.

Exosomes, nanovesicles derived from cells, contain a variety of biomolecules that can be considered biomarkers for disease diagnosis, including microRNAs (miRNAs). Given knowledge and demand, inexpensive, robust, and easy-to-use tools that are compatible with downstream nucleic acid detection should be developed to replace traditional methodologies for point-of-care testing (POCT) applications. This study deploys a paper-based extraction kit for exosome and exosomal miRNA analytical system with some quantifying methods to serve as an easy sample preparation for a possible POCT process. Exosomes concentrated from HCT116 cell cultures were arrested on paper-based immunoaffinity devices, which were produced by immobilizing anti-CD63 antibodies on Whatman filter paper, before being subjected to paper-based silica devices for nucleic acids to be trapped by silica nanoparticles adsorbed onto Whatman filter paper. Concentrations of captured exosomes were quantified by enzyme-linked immunosorbent assay (ELISA), demonstrating that paper-based immunoaffinity devices succeeded in capturing and determining exosome levels from cells cultured in both neutral and acidic microenvironments, whereas microRNA 21 (miR-21), a biomarker for various types of cancers and among the nucleic acids absorbed onto the silica devices, was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) to prove that paper-based silica devices were capable of trapping exosomal nucleic acids. The developed paper-based kit and the devised procedure was successfully exploited to isolate exosomes and exosomal nucleic acids from different biological samples (platelet-poor plasma and lesion fluid) as clinical applications.

Abstract Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of extracellular vesicle inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed and high-throughput assays. We captured 4 subpopulations of EVs from colorectal cancer cell lines HT29 and SW403 based on EpCAM, CD9, CD63 and CD81 expression, and quantified the co-expression of 8 outer (integrins and tetraspanins) and 4 inner (heat shock, endosomal and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

Due to differences in geographic surveillance systems, chemical sanitization practices, and antibiotic stewardship (AS) implementation employed during the COVID-19 pandemic, many experts have expressed concerns regarding a future surge in global antimicrobial resistance (AMR). A potential beneficiary of these differences is the Gram-positive bacteria MRSA. MRSA is a bacterial pathogen with a high potential for mutational resistance, allowing it to engage various AMR mechanisms circumventing conventional antibiotic therapies and the host's immune response. Coupled with a lack of novel FDA-approved antibiotics reaching the clinic, the onus is on researchers to develop alternative treatment tools to mitigate against an increase in pathogenic resistance. Mitigation strategies can take the form of synthetic or biomimetic nanomaterials/vesicles employed in vaccines, rapid diagnostics, antibiotic delivery, and nanotherapeutics. This review seeks to discuss the current potential of the aforementioned nanomaterials in detecting and treating MRSA.

Extracellular vesicles (EVs) play important roles in diabetes mellitus (DM) via connecting the immune cell response to tissue injury, besides stimulation to muscle insulin resistance, while DM is associated with increased risks for major cardiovascular complications. Under DM, chronic hyperglycemia, and subsequent increase in the production of reactive oxygen species (ROS) further lead to cardiac growth remodeling and dysfunction. The purinergic drug ticagrelor is a P2Y12 receptor antagonist. Although it is widely used in cardioprotection, the underlying molecular mechanism of its inhibitory effect on diabetic cardiomyopathy is poorly elucidated. Here, we aimed to understand how ticagrelor exerts its cardio-regulatory effects. For this purpose, we investigated the anti-oxidative and cardioprotective effect of EVs derived from ticagrelor-pretreated cardiomyocytes under DM conditions. To mimic DM in cardiomyocytes, we used high glucose incubated H9c2-cells (HG). HG cells were treated with EVs, which were derived from either ticagrelor-pretreated or untreated H9c2-cells. Our results demonstrated that ticagrelor-pretreated H9c2-derived EVs significantly decreased the hyperglycemia-induced aberrant ROS production, prevented the development of apoptosis and ER stress, and alleviated oxidative stress associated miRNA-expression profile. Importantly, EVs derived from ticagrelor-pretreated H9c2-cells enhanced endothelial cell migration and tube formation, suggesting a modulation of the EV profile in cardiomyocytes. Our data, for the first time, indicate that ticagrelor can exert an important regulatory effect on diabetic cardiomyopathy through extracellular vesicular modulation behind its receptor-inhibition-related effects.

Exosomes are now considered as important mediators of intercellular communication. The exosome cargo contains proteins; mRNA, microRNA (miRNA) and DNA that delivers the information between one cell to other cells can play an important role in identifying the pathophysiological conditions of any cell. Increased understanding of mechanism about exosome release and its communication could provide a novel strategy for the development of biomarkers in various health conditions. Recent shreds of evidence revealed that radiation induces the secretion and alters the composition of exosomes released from radiated cells. The exosomes released from radiated cells alters the signaling pathways in recipient cells. Unraveling the mechanisms related to radiation and exosomes would shed light on the unknown factors that are involved in radiation-induced non-targeted effects. This can provide pavement for the development of biomarkers in radiation emergency situations.

Background Extracellular vesicles (EVs) contain thousands of proteins and nucleic acids, playing an important role in cell–cell communications. Sertoli cells have been essential in the testis as a “nurse cell”. However, EVs derived from human Sertoli cells (HSerCs) have not been well investigated. Methods EVs were isolated from HSerCs via ultracentrifugation and characterized by transmission electron microscopy, tunable resistive pulse sensing, and Western blotting. The cargo carried by HSerCs-EVs was measured via liquid chromatography-mass spectrometry and GeneChip miRNA Arrays. Bioinformatic analysis was performed to reveal potential functions of HSerCs-EVs. Results A total of 860 proteins with no less than 2 unique peptides and 88 microRNAs with high signal values were identified in HSerCs-EVs. Biological processes related to molecular binding, enzyme activity, and regulation of cell cycle were significantly enriched. Specifically, many proteins in HSerCs-EVs were associated with spermatogenesis and regulation of immune system, including Septins, Large proline-rich protein BAG6, Clusterin, and Galectin-1. Moreover, abundant microRNAs within HSerCs-EVs (miR-638, miR-149-3p, miR-1246, etc.) had a possible impact on male reproductive disorders such as asthenozoospermia and oligozoospermia. Conclusions Our study has shown that HSerCs-EVs contain diverse components such as proteins and microRNAs. Further research is required to evaluate HSerCs-EVs in spermatogenesis, which are underutilized but highly potent resources with particular promise for male infertility.

Growth plate cartilage has limited self-repair ability, leading to poor bone bridge formation post-injury and ultimately limb growth defects in children. The current corrective surgeries are highly invasive, and outcomes can be unpredictable. Following growth plate injury, the direct loss of extracellular matrix (ECM) coupled with further ECM depletion due to the inhibitory effects of inflammation on the cartilage matrix protein greatly hinder chondrocyte regeneration. We designed an exosome (Exo) derived from bone marrow mesenchymal stem cells (BMSCs) loaded ECM-mimic hydrogel to promote cartilage repair by directly supplementing ECM and anti-inflammatory properties. Aldehyde-functionalized chondroitin sulfate (OCS) was introduced into gelatin methacryloyl (GM) to form GMOCS hydrogel. Our results uncovered that GMOCS hydrogel could significantly promote the synthesis of ECM due to the doping of OCS. In addition, the GMOCS-Exos hydrogel could further promote the anabolism of chondrocytes by inhibiting inflammation and ultimately promote growth plate injury repair through ECM remodeling.

Marine phages have been applied to trace ground- and surface water flows. Yet, information on their transport in soil and related particle intactness is limited. Here we compared the breakthrough of two lytic marine tracer phages (Pseudoalteromonas phages PSA-HM1 and PSA-HS2) with the commonly used Escherichia virus T4 in soil- and sand-filled laboratory percolation columns. All three phages showed high mass recoveries in the effluents and a higher transport velocity than non-reactive tracer Br−. Comparison of effluent gene copy numbers (CN) to physically-determined phage particle counts or infectious phage counts showed that PSA-HM1 and PSA-HS2 retained high phage particle intactness (Ip > 81%), in contrast to T4 (Ip < 36%). Our data suggest that marine phages may be applied in soil to mimic the transport of (bio-) colloids or anthropogenic nanoparticles of similar traits. Quantitative PCR (qPCR) thereby allows for highly sensitive quantification and thus for the detection of even highly diluted marine tracer phages in environmental samples.

Ischemic conditioning and exercise have been suggested for protecting against brain ischemia-reperfusion injury. However, the endogenous protective mechanisms stimulated by these interventions remain unclear. Here, in a comprehensive translational study, we investigated the protective role of extracellular vesicles (EVs) released after remote ischemic conditioning (RIC), blood flow restricted resistance exercise (BFRRE), or high-load resistance exercise (HLRE). Blood samples were collected from human participants before and at serial time points after intervention. RIC and BFRRE plasma EVs released early after stimulation improved viability of endothelial cells subjected to oxygen-glucose deprivation. Furthermore, post-RIC EVs accumulated in the ischemic area of a stroke mouse model, and a mean decrease in infarct volume was observed for post-RIC EVs, although not reaching statistical significance. Thus, circulating EVs induced by RIC and BFRRE can mediate protection, but the in vivo and translational effects of conditioned EVs require further experimental verification.

Ovarian cancer is highly prevalent and has high mortality rates due to metastasis and relapse. The cross communication between cancer-associated fibroblasts (CAFs) and cancer-associated macrophages (CAMs) in the ovarian tumor microenvironment leads to cancer cell invasion and metastasis. However, the role of overproduction of IL-33/ST2 in the CAFs of ovarian cancer is still unclear. The expression of IL-33, ST2, apoptosis-related proteins and epithelial-mesenchymal transition (EMT) markers was measured by western blotting. Primary normal fibroblasts and CAFs from ovarian cancerous tissue were isolated and cultured in vitro, and the medium was used to stimulate blood-derived monocytes. Flow cytometry analysis was used to detect the frequency of M2-like macrophages in blood-derived monocytes from patients with ovarian cancer. Cell invasion were evaluated using Transwell assays. A xenograft model was used to study tumor growth in ST2-knockout and wild-type NOD-SCID mice. The results demonstrated higher expression of IL-33 and ST2 in carcinoma tissues compared with in para-carcinoma tissues, and there was a survival improvement associated with elevated IL-33. IL-33 and culture supernatants from CAFs, rather than normal ovarian fibroblasts, led to a higher expression of M2 macrophage marker genes in human blood-derived monocytes. Invasion and migration were aggravated in COC1 cells co-cultured with CAF-induced CAMs, and the EMT marker genes were upregulated. It was reported that EMT marker genes were downregulated and tumor volumes were significantly reduced in ST2-deficient mice. Overall, the IL-33/ST2 axis in ovarian cancer might integrate IL-33-expressing CAFs with M2 type-like CAMs, which aggravated invasion and metastasis by promoting EMT.

Extracellular vesicles (EVs) including exosomes act as intercellular communicators by transferring protein and microRNA cargoes, yet the role of EV lipids remains unclear. Here, we show that the pro-tumorigenic action of lymphoma-derived EVs is augmented via secreted phospholipase A2 (sPLA2)-driven lipid metabolism. Hydrolysis of EV phospholipids by group X sPLA2, which was induced in macrophages of Epstein-Barr virus (EBV) lymphoma, increased the production of fatty acids, lysophospholipids, and their metabolites. sPLA2-treated EVs were smaller and self-aggregated, showed better uptake, and increased cytokine expression and lipid mediator signaling in tumor-associated macrophages. Pharmacological inhibition of endogenous sPLA2 suppressed lymphoma growth in EBV-infected humanized mice, while treatment with sPLA2-modified EVs reversed this phenotype. Furthermore, sPLA2 expression in human large B cell lymphomas inversely correlated with patient survival. Overall, the sPLA2-mediated EV modification promotes tumor development, highlighting a non-canonical mechanistic action of EVs as an extracellular hydrolytic platform of sPLA2.

Understanding the motion of colloidal particles flowing in small spaces is a general issue in various fields such as thermal engineering and micro/nanofluidics. In the present study, we investigated the motion of fluorescent submicrometer particles in a 3- μm microchannel by defocusing nanoparticle image velocimetry. An optical measurement system with controlled spherical aberration and an algorithm for processing defocused particle images with multiple diffraction rings were developed. By detecting the centroid position and the diameter of the outermost diffraction ring, which is proportional to the distance between the focal plane and the particle, the position of particles was determined with the spatial resolutions of 154–204 nm in the streamwise direction and 76–311 nm in the depthwise direction, which are comparable to or smaller than the optical diffraction limit. A reusable microfluidic device containing a size-regulated microchannel made of glass was developed, which is suitable for optical measurements and precise flow control. By controlling the strength of low-temperature glass bonding, detachment of the bonded glass substrates, washing, and reuse were achieved. Based on this method and technology, the velocity of particles with diameters of 199, 457, and 1114 nm was successfully measured in pressure-driven laminar flow. Results suggested that for larger particles comparable to the channel size, the particle velocity is slowed from the flow velocity by particle–wall hydrodynamic interactions. Therefore, the motion of colloidal particles in 10 0 - μm spaces is considered to be affected by particle–wall hydrodynamic interactions, as well as 10 2 - μm spaces reported previously.

Tumour cells are characterized by having lost their differentiation state. They constitutively secrete small extracellular vesicles (sEV) called exosomes when they come from late endosomes. Dendrogenin A (DDA) is an endogenous tumour suppressor cholesterol-derived metabolite. It is a new class of ligand of the nuclear Liver X receptors (LXR) which regulate cholesterol homeostasis and immunity. We hypothesized that DDA, which induces tumour cell differentiation, inhibition of tumour growth and immune cell infiltration into tumours, could functionally modify sEV secreted by tumour cells. Here, we have shown that DDA differentiates tumour cells by acting on the LXRβ. This results in an increased production of sEV (DDA-sEV) which includes exosomes. The DDA-sEV secreted from DDA-treated cells were characterized for their content and activity in comparison to sEV secreted from control cells (C-sEV). DDA-sEV were enriched, relatively to C-sEV, in several proteins and lipids such as differentiation antigens, "eat-me" signals, lipidated LC3 and the endosomal phospholipid bis(monoacylglycero)phosphate, which stimulates dendritic cell maturation and a Th1 T lymphocyte polarization. Moreover, DDA-sEV inhibited the growth of tumours implanted into immunocompetent mice compared to control conditions. This study reveals a pharmacological control through a nuclear receptor of exosome-enriched tumour sEV secretion, composition and immune function. Targeting the LXR may be a novel way to reprogram tumour cells and sEV to stimulate immunity against cancer.

Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. They have important roles in numerous physiological and pathological processes, and show considerable promise as novel biomarkers of disease, as therapeutic agents and as drug delivery vehicles. Intriguingly, however, understanding of the cellular and molecular mechanisms that govern the many observed functions of EVs remains far from comprehensive, at least partly due to technical challenges in working with these small messengers. Here, we highlight areas of consensus as well as contentious issues in our understanding of the intracellular and intercellular journey of EVs: from biogenesis, release and dynamics in the extracellular space, to interaction with and uptake by recipient cells. We define knowledge gaps, identify key questions and challenges, and make recommendations on how to address these.

Background Subclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively. Results We found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals. Conclusions Our results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.

BackgroundSubclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively.ResultsWe found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals.ConclusionsOur results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.

Synaptic dysfunction is an early mechanism in Alzheimer's disease that involves progressively larger areas of the brain over time. However, how it starts and propagates is unknown. Here we show that amyloid-β released by microglia in association with large extracellular vesicles (Aβ-EVs) alters dendritic spine morphology in vitro, at the site of neuron interaction, and impairs synaptic plasticity both in vitro and in vivo in the entorhinal cortex-dentate gyrus circuitry. One hour after Aβ-EV injection into the mouse entorhinal cortex, long-term potentiation was impaired in the entorhinal cortex but not in the dentate gyrus, its main target region, while 24 h later it was also impaired in the dentate gyrus, revealing a spreading of long-term potentiation deficit between the two regions. Similar results were obtained upon injection of extracellular vesicles carrying Aβ naturally secreted by CHO7PA2 cells, while neither Aβ42 alone nor inflammatory extracellular vesicles devoid of Aβ were able to propagate long-term potentiation impairment. Using optical tweezers combined to time-lapse imaging to study Aβ-EV-neuron interaction, we show that Aβ-EVs move anterogradely at the axon surface and that their motion can be blocked through annexin-V coating. Importantly, when Aβ-EV motility was inhibited, no propagation of long-term potentiation deficit occurred along the entorhinal-hippocampal circuit, implicating large extracellular vesicle motion at the neuron surface in the spreading of long-term potentiation impairment. Our data indicate the involvement of large microglial extracellular vesicles in the rise and propagation of early synaptic dysfunction in Alzheimer's disease and suggest a new mechanism controlling the diffusion of large extracellular vesicles and their pathogenic signals in the brain parenchyma, paving the way for novel therapeutic strategies to delay the disease.

Bone marrow mesenchymal stem cells (BMSCs) can boost osteosarcoma (OS) cell proliferation and invasion, yet the function of extracellular vesicles (EVs) derived from BMSCs on OS is scarcely known. This study is aimed at examining the role of BMSC-EVs in OS cells. BMSCs and BMSC-EVs were isolated and identified. The effect of EVs and EVs-si-NORAD on OS cell proliferation, invasion, migration, and angiogenesis was determined. Expressions of NORAD, miR-877-3p, and CREBBP were detected. The binding relationship among NORAD, miR-877-3p, and CREBBP was verified. The miR-877-3p inhibitor or pc-CREBBP was delivered into OS cells treated with EVs-si-NORAD for in vitro analysis. The nude mouse model of the subcutaneous tumor xenograft was established for in vivo analysis. BMSC-EVs promoted OS cell proliferation, invasion, migration, and angiogenesis. BMSC-EVs carried NORAD into OS cells and upregulated CREBBP by sponging miR-877-3p. miR-877-3p downregulation or CREBBP overexpression partly inverted the inhibitory effect of EVs by silencing NORAD on OS cell proliferation, invasion, migration, and angiogenesis. In vivo experiments validated that BMSC-EV-derived NORAD facilitated tumor growth by upregulating CREBBP via miR-877-3p. To conclude, BMSC-EV-derived NORAD facilitated OS cell proliferation, invasion, migration, and angiogenesis by modulating CREBBP via miR-877-3p, which may offer new insights into OS treatment.

Development of nanotechnology and corresponding industries during the last decade resulted in a new challenge for analytical science. This includes an ultrasensitive detection and characterization of nanoparticles of different origin and other nanomaterials in various media, including so complex ones as food, biological or environmental samples. The goal of this review is a systematic analysis of possible approaches and description of physical principles behind these methods. The main attention is paid to optical methods which are considered by authors to be mostly effective for the formulated task. Different approaches for detection and analysis of nanoparticles in a volume as well as of those adsorbed on a surface are discussed. While the technologies based on direct analysis of nanoparticle suspensions belong to the established approaches whose development potential has been in large extent exhausted, the novel technologies based on the surface sensing of adsorbed nanoparticles demonstrate intensive development. Therefore, the final part of the review is focused on the wide-field surface plasmon resonance microscopy. It allows one an ultrasensitive detection and characterization of individual nanoparticles of different origin in complex media and provides numerous possibilities for subsequent chemical identification of the detected particles using a hyphenation with other analytical technologies.

Lymphoma is a common malignant tumor globally. Tumor-derived extracellular vesicles (Evs) participate in genetic information exchange between tumor cells. We investigated the role and mechanism of human Burkitt lymphoma cells Raji-derived Evs (Raji-Evs) in lymphoma cells. Effects of Evs on lymphoma cell proliferation, invasion, autophagy, and apoptosis were assessed using Cell Counting Kit-8 method, Transwell assay, laser confocal microscopy, Western blotting, and flow cytometry. microRNA (miR)-106a expression in lymphoma cells was determined using reverse transcription-quantitative polymerase chain reaction and then downregulated in Raji cells and then Evs were isolated (Evs-in-miR-106a) to evaluate its role in lymphoma cell growth. The binding relationship between miR-106a and Beclin1 was verified using RNA pull-down and dual-luciferase assays. Beclin1 was overexpressed in SU-DHL-4 and Farage cells and SU-DHL-4 cell autophagy and apoptosis were detected. The levels of miR-106a and Beclin1 in SU-DHL-4 cells were detected after adding autophagy inhibitors. The tumorigenicity assay in nude mice was performed to validate the effects of Raji-Evs in vivo. Raji-Evs promoted lymphoma cell proliferation and invasion and increased miR-106a. miR-106a knockdown reversed Evs-promoted lymphoma cell proliferation and invasion. miR-106a carried by Raji-Evs targeted Beclin1 expression. Beclin1 overexpression or miR-106a inhibitor reversed the effects of Evs on lymphoma cell autophagy and apoptosis. Autophagy inhibitors elevated miR-106a expression and lowered Beclin1 expression. Raji-Evs-carried miR-106a inhibited Beclin1-dependent autophagy and apoptosis in lymphoma cells, which were further verified in vivo, together with promoted tumor growth. We proved that Raji-Evs inhibited lymphoma cell autophagy and apoptosis and promoted cell growth via the miR-106a/Beclin1 axis.

Tumour-draining lymph nodes (LNs) undergo massive remodelling including expansion of the lymphatic sinuses, a process that has been linked to lymphatic metastasis by creation of a pre-metastatic niche. However, the signals leading to these changes have not been completely understood. Here, we found that extracellular vesicles (EVs) derived from melanoma cells are rapidly transported by lymphatic vessels to draining LNs, where they selectively interact with lymphatic endothelial cells (LECs) as well as medullary sinus macrophages. Interestingly, uptake of melanoma EVs by LN-resident LECs was partly dependent on lymphatic VCAM-1 expression, and induced transcriptional changes as well as proliferation of those cells. Furthermore, melanoma EVs shuttled tumour antigens to LN LECs for cross-presentation on MHC-I, resulting in apoptosis induction in antigen-specific CD8+ T cells. In conclusion, our data identify EV-mediated melanoma-LN LEC communication as a new pathway involved in tumour progression and tumour immune inhibition, suggesting that EV uptake or effector mechanisms in LECs might represent a new target for melanoma therapy.

The aim of this work was to co-nanoencapsulate Lactobacillus acidophilus (LCFE) and Bifidobacterium bifidum (BCFE) cell-free extract and zenyan (Carum copticum L.) seed water (ZWE) and ethanolic (ZEE) extract in electrospun cellulose acetate (CA) nanofibers and evaluate antimicrobial potential. The zeta potential, SEM image, antibacterial (MIC and MBC), and antifungal (MIC and MFC) activities were evaluated. TPC (total phenol content) of water and ethanol extract of zenyan seed were 14.05 and 136.44 mg GAE/g, respectively. A zeta potential of −40.25, −45.80, −43.71, 48.55, 35.50, 47.93, 31.50, 44.69, and −29.61 mV was found for nanofibers of pure CA (cellulose acetate), CA/LCFE, CA/BCFE, CA/ZWE, CA/ZEE, CA/LCFE/ZWE, CA/LCFE/ ZEE, CA/BCFE/ZWE, and CA/LCFE/ZEE, respectively. CA electrospun nanofiber loaded with different extracts showed nanosized diameter and uniform structure. Nanoencapsulated extracts showed considerably higher antibacterial and antifungal activity compared to free extracts. Antibacterial activity of lactobacilli cell-free extract was higher than bifidobacteria, which indicated the presence of the higher amount of antibacterial compounds in lactobacilli extract. Gram-positive bacteria (S. aureus and L. monocytogenes) had the lowest MIC and MBC of free and nanoencapsulated extracts while Gram-negatives (E. coli, S. dysenteriae, and S. enteritidis) had higher MIC and MBC. CA-coated zenyan extracts (water and ethanolic) inhibited the growth of the assayed fungi at the MIC ranging 0.25 to 0.95%. These concentrations were 1.5–2 times lower than those obtained for pure extracts. For nanoencapsulated cellfree extracts of both probiotics, the MIC values were about five times lower than the free extracts. The highest antimicrobial activity obtained for CA nanofibers contained zenyan ethanolic extract and cell-free extract of lactobacilli or bifidobacteria.

Virus-like particles (VLPs) are excellent platforms for the development of influenza vaccine candidates. Nonetheless, their characterization is challenging due to VLPs' unique biophysical and biochemical properties. To cope with such complexity, multiple analytical techniques have been developed to date (e.g., single-particle analysis, thermal stability, or quantification assays), most of which are rarely used or have been successfully demonstrated for being applicable for virus particle characterization. In this study, several biophysical and biochemical methods have been evaluated for thorough characterization of monovalent and pentavalent influenza VLPs from diverse groups (A and B) and subtypes (H1 and H3) produced in insect cells using the baculovirus expression vector system (IC-BEVS). Particle size distribution and purity profiles were monitored during the purification process using two complementary technologies - nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS). VLP surface charge at the selected process pH was also assessed by this last technique. The morphology of the VLP (size, shape, and presence of hemagglutinin spikes) was evaluated using transmission electron microscopy. Circular dichroism was used to assess VLPs' thermal stability. Total protein, DNA, and baculovirus content were also assessed. All VLPs analyzed exhibited similar size ranges (90-115 nm for NTA and 129-141 nm for TRPS), surface charges (average of -20.4 mV), and morphology (pleomorphic particles resembling influenza virus) exhibiting the presence of HA molecules (spikes) uniformly displayed on M1 protein scaffold. Our data shows that HA titers and purification efficiency in terms of impurity removal and thermal stability were observed to be particle dependent. This study shows robustness and generic applicability of the tools and methods evaluated, independent of VLP valency and group/subtype. Thus, they are most valuable to assist process development and enhance product characterization.

Virus-like particles (VLPs) are excellent platforms for the development of influenza vaccine candidates. Nonetheless, their characterization is challenging due to VLPs’ unique biophysical and biochemical properties. To cope with such complexity, multiple analytical techniques have been developed to date (e.g., single-particle analysis, thermal stability, or quantification assays), most of which are rarely used or have been successfully demonstrated for being applicable for virus particle characterization. In this study, several biophysical and biochemical methods have been evaluated for thorough characterization of monovalent and pentavalent influenza VLPs from diverse groups (A and B) and subtypes (H1 and H3) produced in insect cells using the baculovirus expression vector system (IC-BEVS). Particle size distribution and purity profiles were monitored during the purification process using two complementary technologies — nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS). VLP surface charge at the selected process pH was also assessed by this last technique. The morphology of the VLP (size, shape, and presence of hemagglutinin spikes) was evaluated using transmission electron microscopy. Circular dichroism was used to assess VLPs’ thermal stability. Total protein, DNA, and baculovirus content were also assessed. All VLPs analyzed exhibited similar size ranges (90–115 nm for NTA and 129–141 nm for TRPS), surface charges (average of −20.4 mV), and morphology (pleomorphic particles resembling influenza virus) exhibiting the presence of HA molecules (spikes) uniformly displayed on M1 protein scaffold. Our data shows that HA titers and purification efficiency in terms of impurity removal and thermal stability were observed to be particle dependent. This study shows robustness and generic applicability of the tools and methods evaluated, independent of VLP valency and group/subtype. Thus, they are most valuable to assist process development and enhance product characterization.

Neutrophils are classically characterized as merely reactive innate effector cells. However, the microbiome is known to shape the education and maturation process of neutrophils, improving their function and immune‐plasticity. Recent reports demonstrate that murine neutrophils possess the ability to exert adaptive responses after exposure to bacterial components such as LPS (Gram‐ negative bacteria) or LTA (Gram‐positive bacteria). We now ask whether small extracellular vesicles (EVs) from the gut may directly mediate adaptive responses in neutrophils in vitro. Murine bone marrow‐derived neutrophils were primed in vitro by small EVs of high purity collected from colon stool samples, followed by a second hit with LPS. We found that low‐dose priming with gut micro‐ biota‐derived small EVs enhanced pro‐inflammatory sensitivity as indicated by elevated levels of TNF‐α, IL‐6, ROS and MCP‐1 and increased migratory and phagocytic activity. In contrast, high‐ dose priming resulted in a tolerant phenotype, marked by increased IL‐10 and decreased transmi‐ gration and phagocytosis. Alterations in TLR2/MyD88 as well as TLR4/MyD88 signaling were cor‐ related with the induction of adaptive cues in neutrophils in vitro. Taken together, our study shows that small EVs from stools can drive adaptive responses in neutrophils in vitro and may represent a missing link in the gut–immune axis.

Virus-like particles resemble infectious virus particles in size, shape, and molecular composition; however, they fail to productively infect host cells. Historically, the presence of virus-like particles has been inferred from total particle counts by microscopy, and infectious particle counts or plaque-forming-units (PFUs) by plaque assay; the resulting ratio of particles-to-PFUs is often greater than one, easily 10 or 100, indicating that most particles are non-infectious. Despite their inability to hijack cells for their reproduction, virus-like particles and the defective genomes they carry can exhibit a broad range of behaviors: interference with normal virus growth during co-infections, cell killing, and activation or inhibition of innate immune signaling. In addition, some virus-like particles become productive as their multiplicities of infection increase, a sign of cooperation between particles. Here, we review established and emerging methods to count virus-like particles and characterize their biological functions. We take a critical look at evidence for defective interfering virus genomes in natural and clinical isolates, and we review their potential as antiviral therapeutics. In short, we highlight an urgent need to better understand how virus-like genomes and particles interact with intact functional viruses during co-infection of their hosts, and their impacts on the transmission, severity, and persistence of virus-associated diseases.