Membrane Vesicles Research Articles

Abstract C003: Outer membrane vesicles from Bacteroides fragilis contain coding and non-coding small RNA species that modulate inflammatory signaling in intestinal epithelial cells

Abstract Alterations to the community structure and function of the microbiome are associated with changes to host physiology, including immune responses. However, the contribution of microbial-derived RNAs carried by outer membrane vesicles (OMVs) to host immune responses remains unclear. This paper investigates the role of OMVs and OMV-associated small RNA (sRNA) species from pathogenic and commensal Bacteroides fragilis (ETBF and NTBF respectively) in eliciting different immune responses in intestinal epithelial cells. To distinguish the differences in the sRNA profiles of both strains and their OMVs, small RNA-seq was conducted, and identified enrichment of discrete sRNA species in OMVs that were also differentially expressed between the two strains. To understand the effects of OMVs on pattern recognition receptors, Toll-like receptor (TLR) reporter cells were treated with OMVs, and activation of TLRs 2, 3, 4, and 7 were observed. Treatment of Caco-2 and HT29-MTX cells with OMVs demonstrated increased expression of IL-8. Surprisingly, we discovered that degradation of RNase-accessible RNAs within ETBF OMVs, but not NTBF OMVs, resulted in vesicles with enhanced capacity to stimulate IL-8 expression, indicating that these extravesicular RNAs exert an immunosuppressive effect. This suggests a dual role for OMV-associated RNAs in modulating host immune responses, with implications for both bacterial pathogenesis and therapeutic applications. Citation Format: Aadil Sheikh, Colin Scano, Julia Xu, Tolulope Ojo, Jessica M. Conforti, Kayla L. Haberman, Bryan King, James Lotter, Alysia Martinez, Harry Ojeas, Michelle Pujol, Juli Watkins, Emily Lin, Bernd Zechmann, Amanda Sevcik, Christie Sayes, Elyssia S Gallagher, Joshua Mell, Garth Ehrlich, Joseph H Taube, K. Leigh Greathouse. Outer membrane vesicles from Bacteroides fragilis contain coding and non-coding small RNA species that modulate inflammatory signaling in intestinal epithelial cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C003.

Tackling immunosuppression by Neisseria gonorrhoeae to facilitate vaccine design.

Gonorrhoea, caused by Neisseria gonorrhoeae, is a common sexually transmitted infection. Increasing multi-drug resistance and the impact of asymptomatic infections on sexual and reproductive health underline the need for an effective gonococcal vaccine. Outer membrane vesicles (OMVs) from Neisseria meningitidis induce modest cross-protection against gonococcal infection. However, the presence of proteins in OMVs derived from N. gonorrhoeae that manipulate immune responses could hamper their success as a vaccine. Here we modified two key immunomodulatory proteins of the gonococcus; RmpM, which can elicit 'blocking antibodies', and PorB, an outer membrane porin which contributes to immunosuppression. As meningococcal PorB has adjuvant properties, we replaced gonococcal PorB with a meningococcal PorB. Immunisation with OMVs from N. gonorrhoeae lacking rmpM and expressing meningococcal porB elicited higher antibody titres against model antigens in mice compared to OMVs with native PorB. Further, a gonococcal protein microarray revealed stronger IgG antibody responses to a more diverse range of antigens in the Nm PorB OMV immunised group. Finally, meningococcal PorB OMVs resulted in a Th1-skewed response, exemplified by increased serum IgG2a antibody responses and increased IFNɣ production by splenocytes from immunised mice. In summary, we demonstrate that the replacement of PorB in gonococcal OMVs enhances immune responses and offers a strategy for gonococcal vaccine development.

Inserting Omp22 into the flagellin protein, replacing its hypervariable region, results in stronger protection against lethal Acinetobacter baumannii infection.

Acinetobacter baumannii, a common nosocomial pathogen, is known for its rapid acquisition of antimicrobial resistance, underscoring the urgent need to develop an effective vaccine against this pathogen. Outer membrane protein 22 (Omp22) regulates the biogenesis of outer membrane vesicles to transport virulence-promoting factors into the host cells and facilitates the progression of A. baumannii infection. In this study, we used a mouse model to assess a vaccine's immunogenicity and protective efficacy using recombinant Omp22 protein within the hypervariable region of flagellin (FliC-Omp22). FliC-Omp22 demonstrated superior protection following challenge with a lethal dose of multidrug-resistant (MDR) A. baumannii strain 58ST compared to Omp22 alone. In addition, it elicited increased IgG1/IgG2a and IL-4/IFN-γ ratios, indicating a predominant Th2 immune response. Furthermore, the FliC-Omp22 vaccination elicited strong specific antibodies that inhibited the adhesion and invasion of A. baumannii 58ST and enhanced the opsonic killing activity against the pathogen. FliC-Omp22 immunization significantly reduced bacterial loads in infected mice's spleen, lungs, and liver, thereby improving their survival against the lethal infection caused by MDR A. baumannii 58ST. This study suggests that integrating Omp22 into the hypervariable domain of flagellin holds promise for developing an effective vaccine against A. baumannii infections.

Probiotic-derived extracellular vesicles alleviate AFB1-induced intestinal injury by modulating the gut microbiota and AHR activation.

Aflatoxin B1 (AFB1) is a mycotoxin that widely found in the environment and mouldy foods. AFB1 initially targets the intestine, and AFB1-induced intestinal injury cannot be ignored. Lactobacillus amylovorus (LA), a predominant species of Lactobacillus, plays a role in carbohydrate metabolism. Extracellular vesicles (EVs), small lipid membrane vesicles, are widely involved in diverse cellular processes. However, the mechanism by which Lactobacillus amylovorus-QC1H-derived EVs (LA.EVs) protect against AFB1-induced intestinal injury remains unclear. In our study, a new strain named Lactobacillus amylovorus-QC1H (LA-QC1H) was isolated from pig faeces. Then, EVs derived from LA-QC1H were extracted via ultracentrifugation. Our results showed that LA.EVs significantly alleviated AFB1-induced intestinal injury by inhibiting the production of proinflammatory cytokines, decreasing intestinal permeability and increasing the expression of tight junction proteins. Moreover, 16S rRNA analysis revealed that LA.EVs modulated AFB1-induced gut dysbiosis in mice. However, LA.EVs did not exert beneficial effects in antibiotic-treated mice. LA.EVs treatment increased intestinal levels of indole-3-acetic acid (IAA) and activated intestinal aryl hydrocarbon receptor (AHR)/interleukin-22 (IL-22) signalling in AFB1-exposed mice. Inhibition of intestinal AHR signalling markedly weakened the protective effect of LA.EVs in AFB1-exposed mice. LA.EVs alleviated AFB1-induced intestinal injury by modulating the gut microbiota, activating the intestinal AHR/IL-22 signalling, reducing the inflammatory response and promoting intestinal barrier repair in mice.

Plant-Derived Exosome-like Nanoparticles: A Comprehensive Overview of Their Composition, Biogenesis, Isolation, and Biological Applications

Plant-derived exosome-like nanoparticles (PELNs) are a type of membranous vesicle isolated from plant tissues. They contain proteins, lipids, nucleic acids, and other components. PELNs are involved in the defensive response to pathogen attacks by exerting anti-inflammatory, antiviral, antifibrotic, and antitumor effects through the substances they contain. Most PELNs are edible and can be used as carriers for delivering specific drugs without toxicity and side effects, making them a hot topic of research. Sources of PELNs are abundantly, and they can be produced in high yields, with a low risk of developing immunogenicity in vivo. This paper summarizes the formation, isolation, and purification methods; physical properties; and composition of PELNs through a comprehensive literature search. It also analyzes the biomedical applications of PELNs, as well as future research directions. This paper provides new ideas and methods for future research on PELNs.

Nano Plasma Membrane Vesicle-Lipid Nanoparticle Hybrids for Enhanced Gene Delivery and Expression.

Lipid nanoparticles (LNPs) have emerged as the leading nonviral nucleic acid (NA) delivery system, gaining widespread attention for their use in COVID-19 vaccines. They are recognized for their efficient NA encapsulation, modifiability, and scalable production. However, LNPs face efficacy and potency limitations due to suboptimal intracellular processing, with endosomal escape efficiencies (ESE)below 2.5%. Additionally, up to 70% of NPs undergo recycling and exocytosis after cellular uptake. In contrast, cell-derived vesicles offer biocompatibility and high-delivery efficacy but are challenging to load with exogenous NAs and to manufacture at large-scale. To leverage the strengths of both systems, a hybrid system is designed by combining cell-derived vesicles, such as nano plasma membrane vesicles (nPMVs), with LNPs through microfluidic mixing and subsequent dialysis. These hybrids demonstrate up to tenfold increase in ESE and an 18-fold rise in reporter gene expression in vitro and in vivo in zebrafish larvae (ZFL)and mice, compared to traditional LNPs. These improvements are linked to their unique physico-chemical properties, composition, and morphology. By incorporating cell-derived vesicles, this strategy streamlines the development process, significantly enhancing the efficacy and potency of gene delivery systems without the need for extensive screening.

GelMA microneedle-loaded bio-derived nanovaccine shows therapeutic potential for gliomas

ABSTRACT Glioma is the most common primary malignant tumor of the central nervous system in adults. Although immunotherapy, especially tumor vaccines, has made some progress in the treatment of gliomas compared with surgery and radiotherapy. However, the lack of specific or relevant tumor antigens severely limits the further development of tumor vaccines. Here, we report a bio-derived vaccine (TMV@CpG) derived from glioma cell membrane vesicles and carrying TLR9 agonist CpG as adjuvant, which was loaded onto the GelMA microneedle to obtain the microneedle vaccine (MN-TMV@CpG). Microneedle vaccine fully utilize the innate immune cells rich in the skin, inducing stronger cellular immune responses. In subcutaneous tumor models, MN-TMV@CpG reversed the immune-suppressing microenvironment of tumor, and effectively inhibited tumor progression. In an intracranial tumor model, MN-TMV@CpG significantly prolonged the survival duration and induced stronger immune memory responses in tumor bearing mice when combined with anti-PD1 mAb. These results suggest that bio-derived nanovaccines can be used as a potential antitumor immunotherapy strategy.

Development of High-Production Bacterial Biomimetic Vesicles for Inducing Mucosal Immunity Against Avian Pathogenic Escherichia coli

To evaluate the immunoprotective effect of bacterial biomimetic vesicles (BBVs) against avian pathogenic Escherichia coli (APEC), a ΔtolA J11 mutant strain was generated by deleting the tolA gene in the low pathogenic O78 serotype J11 strain. The total protein content of outer membrane vesicles (OMVs) derived from the ΔtolA J11 strain exhibited a sevenfold increase compared to the wild-type strain. Additionally, high-pressure homogenization technology was employed to produce BBVs, resulting in a sixfold increase in total protein content compared to spontaneously secreted OMVs from ΔtolA J11. The immunogenicity of both OMVs and BBVs was assessed through intranasal or intramuscular immunization in specific pathogen-free (SPF) chickens. Results demonstrated that intranasal immunization with OMVs or BBVs in chickens elicited specific IgY antibodies against APEC outer membrane proteins and specific sIgA antibodies in the nasal cavity and trachea, as well as a significant increase in the proliferation response of chicken peripheral blood lymphocytes. The bacterial load in the blood and various organs of the challenged chickens were significantly reduced, resulting in a 66.67% and 58.30% survival rate against a high pathogenic serotype O78 strain challenge, while the control group exhibited only a 16.67% survival rate. The intramuscular immunization with OMVs or BBVs in chickens only induced specific IgY antibodies, with a survival rate of only 33.33% for challenged chickens during the same period. Therefore, intranasal vaccination of the highly productive BBVs is capable of eliciting an immune response similar to that of OMVs and providing protection against APEC infection, thus offering innovative insights for the advancement of APEC vaccines.

Helicobacter pylori outer membrane vesicles directly promote Aβ aggregation and enhance Aβ toxicity in APP/PS1 mice

Helicobacter pylori (H. pylori) infection has been found associated with Alzheimer’s disease (AD) with unclear mechanisms. Outer Membrane Vesicles (OMVs) are spherical particles secreted by Gram-negative bacteria. Here we explore the effect of H. pylori OMVs on Aβ aggregation and toxicity. We show intraperitoneally-injected H. pylori OMVs enter the brain and co-localize with Aβ plaques in APP/PS1 mice, accompanied by aggravated Aβ pathology, exacerbated cognitive deficits and synaptic impairment, indicating that H. pylori OMVs promote β-amyloidosis and AD development. The in vitro results further identify that H. pylori OMVs significantly accelerate Aβ aggregation and increase Aβ-induced neurotoxicity. Through lipidomic analysis, we reveal that lipid components, particularly LPC 18:0 in H. pylori OMVs accelerate Aβ aggregation and enhance Aβ neurotoxicity. Moreover, H. pylori OMVs-enhanced Aβ neurotoxicity is mediated by Ca2+. These findings reveal a mechanism of H. pylori OMVs in accelerating AD development in which the bacterial OMVs-originated lipid components play a key role in promoting Aβ aggregation and neurotoxicity.

Alzheimer's Disease-Derived Outer Membrane Vesicles Exacerbate Cognitive Dysfunction, Modulate the Gut Microbiome, and Increase Neuroinflammation and Amyloid-β Production.

Although our understanding of the molecular biology of Alzheimer's disease (AD) continues to improve, the etiology of the disease, particularly the involvement of gut microbiota disturbances, remains a challenge. Outer membrane vesicles (OMVs) play a key role in central nervous system diseases, but the impact of OMVs on AD progression remains unclear. In this study, we hypothesized that AD-derived OMVs (OMVsAD) were a risk factor in AD pathology. To test our hypothesis, young APP/PS1 mice (AD mice) were given OMVsAD by gavage. Young AD mice were euthanized 120days after gavage to assess the intestinal barrier, gut microbiota diversity, mediators of neuroinflammation, glial markers, amyloid burden, and short-chain fatty acid (SCFA) levels. Our results showed that OMVsAD accelerated cognitive dysfunction after 120days of intragastric administration. Morris water maze experiment and new object recognition test showed that OMVsAD caused significantly poorer spatial ability learning and memory of the AD mice. We observed the OMVsAD-treated APP/PS1 mice display OMVs disrupting the intestinal barrier compared with controls of normal human-derived OMVs. Compared with the OMVsHC group, claudin-5 and ZO-1 related to the intestinal barrier were significantly downregulated in the OMVsAD group. The OMVsAD activate microglia in the cerebral cortex and hippocampus of AD mice, and the levels of IL-1β, IL-6, TNF-α, and NF-Κb were upregulated. We also found that OMVsAD increased Aβ production. 16S rRNA sequencing showed that OMVsAD negatively regulated the α- and β-diversity index of intestinal flora and reduced the levels of SCFA. OMVsAD may change the intestinal flora of young AD, damage the intestinal mucosa and blood-brain barrier, and accelerate AD neuropathological damage.

AKK-derived outer membrane vesicles alleviate indomethacin-induced mucin secretion reduction in LS174T cells by inhibiting endoplasmic reticulum stress

IntroductionAKK-derived outer membrane vesicles (AKK-OMVs) have shown potential in modulating intestinal mucosal immunity by increasing the number of intestinal goblet cells. However, it remains unclear whether AKK-OMVs can directly regulate MUC2 secretion in goblet cells exposed to indomethacin in vitro and the underlying mechanisms involved.MethodsThe abnormal mucin secretion model in LS174T cells was established using indomethacin, with treatment including Akkermansia muciniphila (AKK) supernatant, AKK-OMVs, and extracellular vesicle removal supernatant. The effects of these treatment on MUC2 expression were observed. Transcriptomic sequencing analysis was used to explore the underlying regulatory mechanisms, which were further validated through qRT-PCR and western blotting.ResultsThe treatment with AKK supernatant and AKK-OMVs alleviated the indomethacin-induced reduction in MUC2 secretion in goblet cells. Mechanistically, transcriptomic analysis showed that the gene expression associated with endoplasmic reticulum (ER) stress were upregulated after indomethacin treatment in LS174T cells. This suggests that AKK-OMVs, as the active component in the supernatant, improved MUC2 expression by inhibiting ER stress.ConclusionAKK-OMVs can directly stimulate goblet cells to promote MUC2 secretion, providing potential for further in vivo studies to confirm their protective effects against indomethacin-induced intestinal injury.

First report on the physicochemical and proteomic characterization of Proteus mirabilis outer membrane vesicles under urine-mimicking growth conditions: comparative analysis with Escherichia coli

IntroductionUropathogenic bacteria employ multiple strategies to colonize the urinary tract, including biofilm formation, invasion of urothelial cells, and the production of adhesins, toxins, and siderophores. Among the most prevalent pathogens causing urinary tract infections (UTIs) are Uropathogenic Escherichia coli and Proteus mirabilis. A notable feature of Gram-negative bacteria is their ability to produce outer membrane vesicles (OMVs), which play critical roles in bacterial survival, virulence, and host-pathogen interactions, including UTIs.MethodsIn this study, OMVs were isolated and characterized from two clinical strains, E. coli U144 and P. mirabilis 2,921, cultured in both Luria-Bertani broth and artificial urine.Result and discussionThe OMVs ranged in size from 85 to 260 nm, with the largest vesicles observed in artificial urine. Proteomic analysis allowed the identification of 282 proteins in OMVs from E. coli and 353 proteins from P. mirabilis when cultured LB medium, while 215 were identified from E. coli and 103 from P. mirabilis when cultured in artificial urine. The majority of these proteins originated from the bacterial envelope, while others were linked to motility and adhesion. Notably, the protein composition of OMVs varied depending on the growth medium, and proteins associated with zinc and iron uptake being more prominent in artificial urine, suggesting their importance in the urinary environment. Crucially, this is the first report to characterize P. mirabilis OMVs under different culture conditions, offering novel insights into the role of OMVs in UTI pathogenesis. These findings provide a deeper understanding of the molecular mechanisms by which OMVs contribute to bacterial virulence, establishing the foundation for potential therapeutic interventions targeting OMV-mediated processes in UTIs.

The Coordinated Changes in Platelet Glycan Patterns with Blood Serotonin and Exosomes

The structures of glycans, specifically their terminal positions, play an important role as ligands for receptors in regulating the adhesion ability of platelets. Recent advances in our understanding of free/unbound serotonin (5-HT) in blood plasma at supraphysiological levels implicate it as one of the most profound influencers in remodeling the platelet’s surface N-glycans. Proteomic analysis of the membrane vesicles identified enzymes, specifically glycosyltransferases, only on the surface of the platelets isolated from the supraphysiological level of 5-HT-containing blood plasma. However, these enzymes can only be effective on the cell surface under certain biological conditions, such as the level of their substrates, temperature, and pH of the environment. We hypothesize that exosomes released from various cells coordinate the required criteria for the enzymatic reaction on the platelet surface. The elevated plasma 5-HT level also accelerates the release of exosomes from various cells, as reported. This review summarizes the findings from a wide range of literature and proposes mechanisms to coordinate the exosomes and plasma 5-HT in remodeling the structures of N-glycans to make platelets more prone to aggregation.

Development and Biological Characterization of Cancer Biomimetic Membrane Nanovesicles for Enhancing Therapy Efficacy in Human Glioblastoma Cells

As nanocarriers of a new generation, biomimetic nanovesicles are an emerging class of therapeutic tools whose surface is integrated or fabricated with biomaterials capable of mimicking the biological features and functions of native cells. Thanks to this, biomimetic nanovesicles, in particular, those made by plasma membrane moieties, possess greatly improved biocompatibility, high target specificity, a long retention time, and minimal undesired immune responses. For these reasons, a multitude of progenitor cells including cancer ones were employed as templates to generate biomimetic or membrane-camouflaged nanovesicles hosting different therapeutic compounds. In this contribution, different membrane-derived biomimetic vesicles (M-NVs) were generated by osmotic lysis or plasma membrane isolation approaches from normal and cancer cell lines and assayed against in vitro models of human glioblastoma. M-NVs were compared in their cellular internalization degrees of DNA and proteins, morphologically and molecularly characterized, expressing an extracellular membrane-associated marker. Then, Rose Bengal (RB), a photoactivable drug characterized by a relatively low cellular uptake, was incorporated into nascent glioblastoma-derived M-NVs and finally administered to homotypic receiving cells, showing an increased degree of internalization as well as induced cytotoxic effects, even in the absence of photodynamic direct stimulation. Similar results were also obtained assaying lyophilized M-NVs loaded with RB. In conclusion, M-NVs generated by cell membranes effectively deliver several cargoes, including therapeutic molecules, maintain functionality after lyophilization, and show significant internalization effects, making them a promising strategy for therapeutic applications against human glioblastoma cells.

Targeted intervention in nerve-cancer crosstalk enhances pancreatic cancer chemotherapy.

Nerve-cancer crosstalk has gained substantial attention owing to its impact on tumour growth, metastasis and therapy resistance. Effective therapeutic strategies targeting tumour-associated nerves within the intricate tumour microenvironment remain a major challenge in pancreatic cancer. Here we develop Escherichia coli Nissle 1917-derived outer membrane vesicles conjugated with nerve-binding peptide NP41, loaded with the tropomyosin receptor kinase (Trk) inhibitor larotrectinib (Lar@NP-OMVs) for tumour-associated nerve targeting. Lar@NP-OMVs achieve efficient nerve intervention to diminish neurite growth by disrupting the neurotrophin/Trk signalling pathway. Moreover, OMV-mediated repolarization of M2-like tumour-associated macrophages to an M1-like phenotype results in nerve injury, further accentuating Lar@NP-OMV-induced nerve intervention to inhibit nerve-triggered proliferation and migration of pancreatic cancer cells and angiogenesis. Leveraging this strategy, Lar@NP-OMVs significantly reduce nerve infiltration and neurite growth promoted by gemcitabine within the tumour microenvironment, leading to augmented chemotherapy efficacy in pancreatic cancer. This study sheds light on a potential avenue for nerve-targeted therapeutic intervention for enhancing pancreatic cancer therapy.

Identification and characterization of the lipoprotein N-acyltransferase in Bacteroides

Members of the Bacteroidota compose a large portion of the human gut microbiota, contributing to overall gut health via the degradation of various polysaccharides. This process is facilitated by lipoproteins, globular proteins anchored to the cell surface by a lipidated N-terminal cysteine. Despite their importance, lipoprotein synthesis by these bacteria is understudied. In Escherichia coli, the α-amino-linked lipid of lipoproteins is added by the lipoprotein N-acyltransferase Lnt. Herein, we have identified a protein distinct from Lnt responsible for the same process in Bacteroides, named lipoprotein N-acyltransferase in Bacteroides (Lnb). Deletion of Lnb yields cells that synthesize diacylated lipoproteins, with impacts on cell viability and morphology, growth on polysaccharides, and protein composition of membranes and outer membrane vesicles (OMVs). Our results not only challenge the accepted paradigms of lipoprotein biosynthesis in gram-negative bacteria but also suggest the existence of a new family of lipoprotein N-acyltransferases.

A novel multiplexed immunoassay for surface-exposed proteins in plasma extracellular vesicles.

Small membranous extracellular vesicles (EV) incorporate proteins and nucleic acids from the parent cell. Proteins exposed on EV surface are dictated by cellular origin and biogenesis pathway. To better understand the EV origin and function, it is important to develop methods that reveal surface protein composition of heterogeneous EV populations in culture supernatants and in biofluids. Tetraspanins CD9, CD63, and CD81 are common and abundant EV markers. However, their relative enrichment (profile) on EVs of specific cellular origins is not fully elucidated. We introduce LuminEV, a novel version of the Luminex assay for the multiplexed analysis of EV surface proteins. Optimized LuminEV reagents enable direct, specific, and sensitive measurements of EV markers in biofluids and in culture supernatants, bypassing EV isolation step. LuminEV assay for CD9, CD63, and CD81 was validated by comparing simplex and multiplex measurements, establishing linearity, spike-in recovery, inter- and intra-assay precision, and reproducibility between operators. LuminEV measurements of CD9, CD63, and CD81 in conditioned media from 15 cell lines revealed strong variations between cell types and showed high sensitivity, which enabled EV detection without prior concentration. Using tetraspanin levels as a readout, we noted suppression and induction of EV release from the cultured cells by GW6869 and monensin. Measurement of EV CD9, CD63, and CD81 in blood plasma from 70 disease-free donors showed respective abundance of 72, 16, and 12%. CD63 displayed weak, albeit significant, negative correlation with age and was slightly lower in female samples. The assay was then used to detect cell type-specific EV surface markers, including CD235a (erythrocytes), GAP43 (neurons), and CD68 (macrophages), and to detect differences in tetraspanin profiles between healthy and diseased donors. In summary, LuminEV offers robust and sensitive approach for multiplexed assessment of EV surface proteins, to facilitate the research into EV biology, biomarker, and therapeutic applications.

Isolation of Small Extracellular Vesicles (sEVs) from the Apoplastic Wash Fluid of Nicotiana benthamiana Leaves.

Extracellular vesicles (EVs) are small membranous vesicles secreted by cells into their surrounding extracellular environment. Similar to mammalian EVs, plant EVs have emerged as essential mediators of intercellular communication in plants that facilitate the transfer of biological material between cells. They also play essential roles in diverse physiological processes including stress responses, developmental regulation, and defense mechanisms against pathogens. In addition, plant EVs have demonstrated promising health benefits as well as potential therapeutic effects in mammalian health. Despite the plethora of potential applications using plant EVs, their isolation and characterization remains challenging. In contrast to mammalian EVs, which benefit from more standardized isolation protocols, methods for isolating plant EVs can vary depending on the starting material used, resulting in diverse levels of purity and composition. Additionally, the field suffers from the lack of plant EV markers. Nevertheless, three main EV subclasses have been described from leaf apoplasts: tetraspanin 8 positive (TET8), penetration-1-positive (PEN1), and EXPO vesicles derived from exocyst-positive organelles (EXPO). Here, we present an optimized protocol for the isolation and enrichment of small EVs (sEVs; <200 nm) from the apoplastic fluid from Nicotiana benthamiana leaves by ultracentrifugation. We analyze the preparation through transmitted electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. We believe this method will establish a basic protocol for the isolation of EVs from N. benthamiana leaves, and we discuss technical considerations to be evaluated by each researcher working towards improving their plant sEV preparations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation and enrichment of small extracellular vesicles (sEVs) from the apoplastic fluid of Nicotiana benthamiana leaves.

Ticagrelor modestly raises plasma riboflavin concentration in humans and inhibits riboflavin transport by BCRP and MRP4.

Riboflavin (vitamin B2) has been proposed as a biomarker for breast cancer resistance protein (BCRP) activity. In recent studies in mice, cynomolgus monkeys, and humans, BCRP-inhibiting drugs increased the plasma concentration of riboflavin. We showed recently that ticagrelor inhibits BCRP and raises the plasma concentrations of the BCRP substrate rosuvastatin in healthy volunteers. In the same drug-drug interaction study, we now investigated whether ticagrelor affects the plasma concentrations of riboflavin. Intake of 90 mg ticagrelor increased the ratio between the peak plasma riboflavin concentration and the fasting riboflavin concentration before ticagrelor administration by 1.20-fold (90% confidence interval, 1.10-1.32; P = 0.006) compared to placebo. In vitro, riboflavin was transported by BCRP and multidrug-resistance-associated protein 4 (MRP4) but no clear transport was observed by MRP2, MRP3, or the P-glycoprotein. Moreover, ticagrelor inhibited the transport of riboflavin in BCRP- and MRP4-expressing membrane vesicles with unbound 50% inhibitory concentrations of 0.020 and 1.1μM, respectively. Based on vesicle and tissue protein expression data, the small intestinal MRP4-mediated efflux clearance of riboflavin (1.2-1.4 nL/min/mg) was estimated to be similar to that mediated by BCRP (0.23-1.3 nL/min/mg). As MRP4 is expressed in the basolateral membrane of enterocytes, it may facilitate the absorption of riboflavin and impair the utility of riboflavin as a biomarker of intestinal BCRP. To conclude, ticagrelor modestly raises the plasma concentration of riboflavin probably by inhibiting intestinal BCRP. Inhibition of intestinal MRP4 may have reduced the absorption of riboflavin and limited the effect of ticagrelor on riboflavin levels.

Unraveling the Emerging Niche Role of Extracellular Vesicles (EVs) in Traumatic Brain Injury (TBI).

Extracellular vesicles or exosomes, often known as EVs, have acquired significant attention in the investigations of traumatic brain injury (TBI) and have a distinct advantage in actively researching the fundamental mechanisms underlying various clinical symptoms and diagnosing the wide range of traumatic brain injury cases. The mesenchymal stem cells (MSCs) can produce and release exosomes, which offer therapeutic benefits. Exosomes are tiny membranous vesicles produced by various cellular entities originating from endosomes. Several studies have reported that administering MSC-derived exosomes through intravenous infusions improves neurological recovery and promotes neuroplasticity in rats with traumatic brain damage. The therapeutic advantages of exosomes can be attributed to the microRNAs (miRNAs), which are small non-coding regulatory RNAs that significantly impact the regulation of posttranscriptional genes. Exosome-based therapies, which do not involve cells, have lately gained interest as a potential breakthrough in enhancing neuroplasticity and accelerating neurological recovery for various brain injuries and neurodegenerative diseases. This article explores the benefits and drawbacks of exosome treatment for traumatic brain injury while emphasizing the latest advancements in this field with clinical significance.