Publications

Colorectal cancer (CRC) is the 2nd most fatal cancer in the United States, but when detected early it is highly curable. Stool-derived extracellular vesicles (EVs) are a novel biomarker source that could augment the sensitivity for detection of CRC precursors. However, standardization of isolation methods for stool-derived EVs remains underexplored. We previously reported that size-exclusion chromatography (SEC) followed by ultrafiltration (UF-100kDa) was suitable for human stool supernatant EV isolation. In this study, we first assess alternative EV concentration methods (ultrafiltration [UF]; 10 kDa, 30 kDa, 50 kDa, 100 kDa and speed vacuum [SV]). Second, we investigate the host/bacterial EV proteomes by mass spectrometry. We report no difference in recovery, RNA and soluble protein contamination among concentration methods. Proteomic analysis reveals a diverse bacterial proteome, while human-derived proteins are more abundant. Specifically, pancreatic enzymes are among the most abundant proteins, further exploration revealed that zymogen granules are likely co-isolated in stool EV preparations. To enable discovery of EV-based molecular signatures of CRC precursors with high sensitivity, immunocapture strategies will likely be needed. Notably, we identified 10 surface proteins that may serve as candidates for the purification of colon-derived EVs. This work serves as framework for the future discovery and validation of EV-based biomarkers for CRC.

Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. The cytokine transforming growth factor‐β (TGF‐β) facilitates cancer progression via EVs secreted by cancer cells, which act on recipient cells in the tumour microenvironment. However, the mechanisms of how TGF‐β affects cancer cell EV release and composition are incompletely understood. Here, we systematically investigate the effects of TGF‐β on the release and protein composition of EVs from breast cancer cells. TGF‐β suppresses the transcription of RAB27B mediated by SMAD3 and thereby hampers EV release. Using click chemistry and quantitative proteomics, we found that TGF‐β increases the quantity of protein cargo and changes the composition of EVs by downregulating RAB27B expression. The recomposed EVs, induced by TGF‐β or RAB27B depletion, inhibit CD8+ T cell‐mediated breast cancer killing. Our findings reveal the critical roles of TGF‐β and RAB27B in cancer development by regulating EV release and composition and thus provide potential targets to improve cancer immunotherapy.

The management of diabetic wounds presents a considerable challenge within the realm of clinical practice. Cellular-derived nanoparticles, or extracellular vesicles (EV), generated by human adipose-derived stem cells (hASCs) have been investigated as promising candidates for the treatment of diabetic wounds. Nevertheless, limitations on the yield, as well as the qualitative angiogenic properties of the EV produced, have been a persistent issue. In this study, a novel approach involving the use of various cell culture morphologies, such as cell spheroids, on hASC was used to promote both EV yield and qualitative angiogenic properties for clinical use, with an emphasis on the in vivo angiogenic properties exhibited by the EV. Moreover, an increase in the secretion of the EV was confirmed after cell spheroid culture. Furthermore, microRNA(miRNA) analysis of the produced EVs indicated an increase in the presence of wound healing-associated miRNAs on the cell spheroid EV. Analysis of the effectiveness of the treated EVs in vitro indicated a significant promotion of the biological function of fibroblast and endothelial cells, cell migration, and cell proliferation post-cell spheroid EV application. Meanwhile, in vivo experiments on diabetic rats indicated a significant increase in collagen production, re-epithelization, and angiogenesis of the diabetic wound after EV administration. In this investigation, we posit that the use of cell spheroids for the culture of hASC represents a novel approach to enhance the substantial secretion of extracellular vesicles while increasing the angiogenic wound healing properties. This innovation holds promise for augmenting the therapeutic potential of EVs in diabetic wound healing, aligning with the exigencies of clinical applications for these nanoparticles.

Background Plasma-derived Extracellular Vesicles (EVs) have been suggested as novel biomarkers in melanoma, due to their ability to reflect the cell of origin and ease of collection. This study aimed to identify novel EV biomarkers that can discriminate between disease stages. This was achieved by characterising the plasma-derived EVs of patients with melanoma, and comparing their proteomic and metabolomic profile to those from healthy controls. Methods EVs were isolated from the plasma of 36 patients with melanoma and 13 healthy controls using Size Exclusion Chromatography. Proteomic and Metabolomic Analyses were performed, and machine learning algorithms were used to identify potential proteins and metabolites to differentiate the plasma-derived EVs from melanoma patients of different disease stages. Results The concentration and size of the EV population isolated was similar between groups. Proteins (APOC4, PRG4, PLG, TNC, VWF and SERPIND1) and metabolites (lyso PC a C18:2, PC ae C44:3) previously associated with melanoma pathogenesis were identified as relevant in differentiating between disease stages. Conclusion The results further support the continued investigation of circulating plasma-derived EVs as biomarkers in melanoma. Furthermore, the potential of combined proteo-metabolomic signatures for differentiation between disease stages may provide valuable insights into early detection, prognosis, and personalised treatment strategies.

Background MicroRNAs (miRNAs) participate in diverse cellular changes following acute ischemic stroke (AIS). Circulating miRNAs, stabilized and delivered to target cells via extracellular vesicles (EVs), are potential biomarkers to facilitate diagnosis, prognosis, and therapeutic modulation. We aimed to identify distinctive expression patterns of circulating EV-miRNAs in AIS patients. Methods miRNA profiles from EVs, isolated from plasma samples collected within 24 h following AIS diagnosis, were examined between a dataset of 10 age-, gender- and existing comorbidities-matched subjects (5 AIS and 5 healthy controls, HC). We measured 2578 miRNAs and identified differentially expressed miRNAs between AIS and HC. An enrichment analysis was conducted to delineate the networks and biological pathways implicated by differentially expressed microRNAs. An enrichment analysis was conducted to delineate the networks and biological pathways implicated by differentially expressed microRNAs. Results Five miRNAs were differentially expressed between stroke (AIS) versus control (HC). hsa-let-7b-5p, hsa-miR-16-5p, and hsa-miR-320c were upregulated, whereas hsa-miR-548a-3p and hsa-miR-6808-3p, with no previously reported changes in stroke were downregulated. The target genes of these miRNAs affect various cellular pathways including, RNA transport, autophagy, cell cycle progression, cellular senescence, and signaling pathways like mTOR, PI3K-Akt, and p53. Key hub genes within these networks include TP53, BCL2, Akt, CCND1, and NF-κB. These pathways are crucial for cellular function and stress response, and their dysregulation can have significant implications for the disease processes. Conclusion Our findings reveal distinct circulating EV-miRNA expression patterns in AIS patients from Qatar, highlighting potential biomarkers that could aid in stroke diagnosis and therapeutic strategies. The identified miRNAs are involved in critical cellular pathways, offering novel insights into the molecular mechanisms underlying stroke pathology. Circulating EV-miRNAs differentially expressed in AIS may have a pathophysiological role and may guide further research to elucidate their precise mechanisms.

This work describes protocols for preparing specific forms of human platelet lysates from pooled platelet concentrates (PCs) and the isolation of platelet-derived extracellular vesicles (p-EVs). Clinical-grade PCs can be sourced from blood establishments immediately following expiration for transfusion use. Here, we describe methods to process PCs into specific lysates from which p-EVs can be isolated. Each lysate type is prepared using platelet activation and processing methods which produce distinct products that may be useful in different applications. For example, serum-converted platelet lysate (SCPL)-EVs were recently shown to have powerful therapeutic properties following myocardial infarction in mice. EVs can be isolated from all products using size exclusion chromatography, producing pure and consistent p-EVs from multiple batches. Together, these methods allow isolation of p-EVs with excellent potential for clinical and preclinical applications. • Platelet concentrates (PCs) obtained from local blood establishments are reliable and sustainable sources to generate biomaterials. • We outline five distinct methods of platelet lysate generation and one method for extracellular vesicle isolation. • Each platelet lysate form has different biological properties which may be suitable for certain applications.

Extracellular vesicles (EVs) are implicated in inter-organ communication, which becomes particularly relevant during aging and exercise. DNA methylation-based aging clocks reflect lifestyle and environmental factors, while regular exercise is known to induce adaptive responses, including epigenetic adaptations. Twenty individuals with High-fitness (aged 57.7 ± 9.8 years) and twenty Medium–Low-fitness (aged 57.5 ± 9.7 years) subjects provided blood samples. EVs were isolated from the samples using a size exclusion chromatography (SEC)-based method, and their protein content was analyzed by mass spectrometry (MS). Acceleration of the biological age estimator DNAmFitAge (AgeAccelFit) was associated with the protein cargo of EVs, whereas PhenoAge and GrimAge acceleration did not show a significant relationship. This finding suggests that the epigenetic aging-modulating role of exercise may involve inter-organ communication via EVs. Set Enrichment Analysis was performed to identify enriched Gene Ontology (GO) terms for sets of proteins that were either correlated with AgeAccelFit or detected exclusively in individuals with high levels of aerobic fitness. The protein cargo of EVs further suggests that inter-organ communication influences inflammation, the immune system, cellular repair, adhesion, metabolism and coagulation. Our findings help to understand the preventive role of exercise, which could be mediated in part by EVs.

Extracellular vesicles (EVs) are widely investigated for their implications in cell-cell signaling, immune modulation, disease pathogenesis, cancer, regenerative medicine, and as a potential drug delivery vector. However, maintaining integrity and bioactivity of EVs between Good Manufacturing Practice separation/filtration and end-user application remains a consistent bottleneck towards commercialization. Milk-derived extracellular vesicles (mEVs), separated from bovine milk, could provide a relatively low-cost, scalable platform for large-scale mEV production; however, the reliance on cold supply chain for storage remains a logistical and financial burden for biologics that are unstable at room temperature. Herein, we aim to characterize and engineer a freeze-dried, mEV formulation that can be stored at room temperature without sacrificing structure/bioactivity and can be reconstituted before delivery. In addition to undertaking established mEV assays of structure and function on our preparations, we introduce a novel, efficient, high throughput assay of mEV bioactivity based on Electric Cell Substrate Impedance Sensing (ECIS) in Human dermal fibroblast monolayers. By adding appropriate excipients, such as trehalose and tryptophan, we describe a protective formulation that preserves mEV bioactivity during long-term, room temperature storage. Our identification of the efficacy of tryptophan as a novel additive to mEV lyophilization solutions could represent a significant advancement in stabilizing small extracellular vesicles outside of cold storage conditions.

Background Previous studies found that cigarette smoke (CS) exposure could induce NSCLC malignancy and miRNA dysregulation. Yet, the association of CS-induced miRNA dysregulation and NSCLC malignancy has not been clearly understood. This study aimed to evaluate the effect of CS exposure in smokers on the expression of miR-10b-5p and miR-320b in extracellular vesicles (EVs) from NSCLC patients. Material and methods Bioinformatic analysis was conducted to validate miRNA candidates. Blood and tissue samples were collected from NSCLC patients (n = 21) with smoking and non-smoking history. EVs were isolated from plasma and miRNAs were extracted from the isolated EVs. The miRNAs relative expression was analyzed and compared. Results In silico analysis identified miR-320b and miR-10b-5p as potential biomarkers for diagnosing NSCLC in smokers. Experimental analysis revealed differential expression of EVs-associated miRNAs in NSCLC patients with smoking and non-smoking histories. EVs-associated miR-10b-5p was significantly overexpressed in smoker NSCLC patients (p = 0.000), while miR-320b expression was significantly lower in this group (p = 0.018). Additionally, smoking intensity influenced miRNA expression, with higher smoking intensity correlating with increased miR-10b-5p expression and decreased miR-320b expression. ROC analysis demonstrated that EVs were a superior source of miRNAs compared to plasma for NSCLC diagnostics. miR-10b-5p and miR-320b in EVs showed higher diagnostic performance (AUC 0.878; 0.739) compared to plasma (AUC 0.628; 0.559). Conclusion CS exposure induces different expression of miR-10b-5p and miR-320b in EVs of NSCLC patients with smoking history. EV-related miR-10b-5p and miR-320b showed potential to be utilized as prognostic biomarker for smokers NSCLC patients.

Indocyanine green J-aggregate (IJA) is a promising photothermal (PTT) agent that has recently been utilised in preclinical studies for cancer diagnostics and treatment. The unique properties, such as the red-shift absorption band and longer wavelengths, are behind IJA's superior thermal stability compared to its monomeric ICG. Loading IJA into nanoparticles (NPs) has proven advantageous in enhancing its in vivo targeting of various cancer models. However, the loading of IJA into more complex lipids, such as lipid nanocapsules (LNCs) and solid lipid nanoparticles (SLNs), has not been reported. The present work focuses on investigations of the effect of formulation parameters on pre-formed IJA (p-IJA) stability and the formation of p-IJA-loaded LNCs and SLNs, thus enhancing their theranostic applications. We investigated the effect of the lipid shell of LNCs and the lipid core of SLN on p-IJA stability. Our findings demonstrated the significant role of lipophilic surfactants (Span 85) and a high-melting-point lipid core (sodium stearate) in enhancing the p-IJA ratio and heating capacity following loading into SLNs. More importantly, p-IJA-SLN enhanced the optical stability of p-IJA in a range of biological media, such as serum proteins, blood, and collagen. Furthermore, lyophilised p-IJA-SLNs were successfully obtained after long-term storage. Overall, p-IJA-loaded lipid NPs could provide a promising platform for various applications, including photoacoustic imaging, PTT, photodynamic therapy (PDT), and combination therapy with chemotherapeutics.

Small extracellular vesicles (sEVs) play crucial roles in intercellular communication. However, the internalization of individual sEVs by recipient cells has not been directly observed. Here, we examined these mechanisms using state-of-the-art imaging techniques. Single-molecule imaging shows that tumor-derived sEVs can be classified into several subtypes. Simultaneous single-sEV particle tracking and observation of super-resolution movies of membrane invaginations in living cells reveal that all sEV subtypes are internalized via clathrin-independent endocytosis mediated by galectin-3 and lysosome-associated membrane protein-2C, while some subtypes that recruited raft markers are internalized through caveolae. Integrin β1 and talin-1 accumulate in recipient cell plasma membranes beneath all sEV subtypes. Paracrine, but not autocrine, sEV binding triggers Ca2+ mobilization induced by the activation of Src family kinases and phospholipase Cγ. Subsequent Ca2+-induced activation of calcineurin–dynamin promotes sEV internalization, leading to the recycling pathway. Thus, we clarified the detailed mechanisms of sEV internalization driven by paracrine adhesion signaling.

Extracellular vesicles (EVs) have emerged as promising therapeutics with broad clinical applications as diagnostic biomarkers and therapeutic drug delivery systems. Yet, these biopharmaceuticals pose a challenge in terms of manufacturing due to their complexity and heterogeneity. Despite advancements in the field, current purification technologies lack scalability and/or selectivity. Affinity chromatography (AC) − coupling unmatched specificity and scalability − could be used to simplify purification processing and generate clinical-grade EVs with higher titers and purity. In the present work, we report the implementation of an immuno-AC resin to capture and purify EVs directly from clarified cellular feedstocks. Firstly, to guide and support marker selection, vesicle phenotype characterization was conducted using single particle interferometric reflectance image sensing (SP-IRIS) coupled with immunofluorescence. CD81 was the marker which shown to be more present and more likely to have the other markers (CD63 and CD9). Thus, anti-CD81 VHH ligand was generated and evaluated towards recombinant CD81 protein and CD81 bearing EV particles using surface plasmon resonance (SPR). Different chromatographic studies with Anti-CD81 ligand immobilized onto agarose beads resin were conducted to optimize the process parameters (residence time, dynamic binding capacity and impurity clearance). At residence time of 2 min, on average 40 % of pure triple tetraspanin-positive EV fraction was recovered. The enrichment in EV particles herein obtained, based on scale-up calculations, it would be possible to produce 1 × 1013 EVs from a 1L cell culture, while meeting impurity requirements in a single-step purification process (impurity removal over 2 log reduction value). A single-step purification process is possible, enabling the successful isolation of homogeneous EVs population, counting with a final HCP titer of 60 ng/mL and 9 ng/mL of dsDNA impurities. EV’s morphological integrity and internalization ability were also demonstrated, showcasing elution’s efficiency under mild conditions. Overall, this work contributes to the development of a novel, highly specific, AC technology using a camelid-derived affinity ligand which, bridging the scalability requirements demanded of large-scale production, could potentiate the advent of EV-based therapies.

Introduction: Diffuse parenchymal lung diseases (DPLD) cover heterogeneous types of lung disorders. Among many pathological phenotypes, pulmonary fibrosis is the most devastating and represents a characteristic sign of idiopathic pulmonary fibrosis (IPF). Despite a poor prognosis brought by pulmonary fibrosis, there are no specific diagnostic biomarkers for the initial development of this fatal condition. The major hallmark of lung fibrosis is uncontrolled activation of lung fibroblasts to myofibroblasts associated with extracellular matrix deposition and the loss of both lung structure and function. Methods: Here, we used this peculiar feature in order to identify specific biomarkers of pulmonary fibrosis in bronchoalveolar lavage fluids (BALF). The primary MRC-5 human fibroblasts were activated with BALF collected from patients with clinically diagnosed lung fibrosis; the activated fibroblasts were then washed rigorously, and further incubated to allow secretion. Afterwards, the secretomes were analysed by mass spectrometry. Results: In this way, the CD44 protein was identified; consequently, BALF of all DPLD patients were positively tested for the presence of CD44 by ELISA. Finally, biochemical and biophysical characterizations revealed an exosomal origin of CD44. Receiver operating characteristics curve analysis confirmed CD44 in BALF as a specific and reliable biomarker of IPF and other types of DPLD accompanied with pulmonary fibrosis.

Background Drug delivery to the brain is challenging due to the restrict permeability of the blood brain barrier (BBB). Recent studies indicate that BBB permeability increases over time during physiological aging likely due to factors (including extracellular vesicles (EVs)) that exist in the bloodstream. Therefore, inspiration can be taken from aging to develop new strategies for the transient opening of the BBB for drug delivery to the brain. Results Here, we evaluated the impact of small EVs (sEVs) enriched with microRNAs (miRNAs) overexpressed during aging, with the capacity to interfere transiently with the BBB. Initially, we investigated whether the miRNAs were overexpressed in sEVs collected from plasma of aged individuals. Next, we evaluated the opening properties of the miRNA-enriched sEVs in a static or dynamic (under flow) human in vitro BBB model. Our results showed that miR-383-3p-enriched sEVs significantly increased BBB permeability in a reversible manner by decreasing the expression of claudin 5, an important tight junction protein of brain endothelial cells (BECs) of the BBB, mediated in part by the knockdown of activating transcription factor 4 (ATF4). Conclusions Our findings suggest that engineered sEVs have potential as a strategy for the temporary BBB opening, making it easier for drugs to reach the brain when injected into the bloodstream.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is adapting to continuous presence in humans. Transitions to endemic infection patterns are associated with changes in the spike (S) proteins that direct virus-cell entry. These changes generate antigenic drift and thereby allow virus maintenance in the face of prevalent human antiviral antibodies. These changes also fine tune virus-cell entry dynamics in ways that optimize transmission and infection into human cells. Focusing on the latter aspect, we evaluated the effects of several S protein substitutions on virus-cell membrane fusion, an essential final step in enveloped virus-cell entry. Membrane fusion is executed by integral-membrane “S2” domains, yet we found that substitutions in peripheral “S1” domains altered late-stage fusion dynamics, consistent with S1-S2 heterodimers cooperating throughout cell entry. A specific H655Y change in S1 stabilized a fusion-intermediate S protein conformation and thereby delayed membrane fusion. The H655Y change also sensitized viruses to neutralization by S2-targeting fusion-inhibitory peptides and stem-helix antibodies. The antibodies did not interfere with early fusion-activating steps; rather they targeted the latest stages of S2-directed membrane fusion in a novel neutralization mechanism. These findings demonstrate that single amino acid substitutions in the S proteins both reset viral entry—fusion kinetics and increase sensitivity to antibody neutralization. The results exemplify how selective forces driving SARS-CoV-2 fitness and antibody evasion operate together to shape SARS-CoV-2 evolution.

Individuals with homozygous laminA/C p.R527C mutations manifest a severe form of Mandibuloacral dysplasia‐(MAD) and exhibit overlapping progeroid symptoms, for which the underlying molecular pathology remains unknown. Herein, it is shown that MAD patients achieved inflammaging with different pro‐inflammatory cytokines compared to progeria‐(HGPS) patient. Characterization of MAD iPSC‐derived Mesenchymal stem cells (MAD‐iMSC) uncovers deregulated mitochondrial Ca+2 as the primary cause of inflammaging, mediated through inflammasome formation rather than the cGAS‐STING pathway. Moreover, MAD‐iMSCs extracellular vesicles (EVs) can also upregulate mitochondrial Ca+2 in healthy cells. This deregulated Ca+2 homeostasis is indirectly mediated by mitochondrial calcium mediator, signal transducer, and activator of transcription‐3 (STAT3), situated on the mitochondrial associated membrane (MAM). Inflammaging is mitigated by various FDA‐approved MAM‐STAT3 upstream inhibitors, such as (Tocilizumab) or by correcting R527C mutation with CRISPR/CAS9. These results provide new insights into MAD disease and propose targeting defective mitochondrial Ca+2 homeostasis as a promising therapy for reversing immunosenescence.

Objectives: Small extracellular vesicles (sEVs) are nanosized vesicles with biological activities that exist in milk, playing functional roles in immunity, gut balance, and the nervous system. Currently, little is known about the impact of processing on milk sEVs. Methods: In this study, sEVs were collected from raw goat milk (g-sEV), pasteurized goat milk (pg-sEV), and goat milk powder (p-sEV) using a sucrose cushion centrifugation combined with qEV chromatography. Then, the sEVs were identified and compared using NTA, Western blot, and TEM. After extracting RNA and the total proteome from sEVs derived from different samples, the RNA was subjected to high-throughput sequencing, and peptide fragments were analyzed using mass spectrometry. Finally, GO and KEGG pathway analyses were performed on the results. Results: The characterization results revealed a decrease in diameter as the level of processing increased. High-throughput sequencing results showed that all three types of small extracellular vesicles were found to be rich in miRNA, and no significant differences were observed in the most abundant sEV species. Comparing with g-sEV, there were 3938 and 4645 differentially expressed miRNAs in pg-sEV and p-sEV, respectively, with the majority of them (3837 and 3635) being downregulated. These differentially expressed miRNAs were found to affect biological processes or signaling pathways such as neurodevelopment, embryonic development, and transcription. Proteomic analysis showed that there were 339 differentially expressed proteins between g-sEV and pg-sEV, with 209 proteins being downregulated. Additionally, there were 425 differentially expressed proteins between g-sEV and p-sEV, with 293 proteins being downregulated. However, no significant differences were observed in the most abundant protein species among the three types of sEVs. Enrichment analysis indicated that the differentially expressed proteins were associated with inflammation, immunity, and other related processes. Conclusions: These results indicate that extracellular vesicles have a protective effect on their cargo, while processing steps can have an impact on the size and quantity of the sEVs. Furthermore, processing can also lead to the loss of immune-related miRNA and proteins in sEVs.

Colorectal cancer is a common malignant tumor, whose growth and metastasis are influenced by numerous factors. MicroRNAs have garnered increasing attention in recent years due to their involvement in tumor development. Exosomes are involved in intercellular signaling and influence tumor development by promoting tumor cell proliferation and metastasis through activation of angiogenesis and other mechanisms. This study aimed to investigate how the exosomes containing miR-320d from colorectal cancer (CRC) cells promote colorectal cancer metastasis by regulating angiogenesis. CRC-derived exosomes containing miR-320d can be transferred to vascular endothelial cells, facilitating their proliferation, invasion, migration, and angiogenesis. By targeting GNAI1, miR-320d in these exosomes reduces GNAI1 levels in endothelial cells, causing more JAK2/STAT3 activation and VEGFA production. This ultimately enhances the migration and angiogenic capacity of vascular endothelial cells. Moreover, CRC patients with high levels of miR-320d in their blood respond better to treatment with bevacizumab. In vivo experiments further proved the role of miR-320d from CRC exosomes in increasing tumor size, blood vessel formation, and the spread of cancer to the liver. In this study, we have demonstrated that exosomal miR-320d promotes cancer cell metastasis and enhances angiogenesis by downregulating GNAI1 expression and enhancing JAK2/STAT3.

Acute myeloid leukemia (AML) is an aggressive disease with a high relapse rate. In this study, we map the metabolic profile of CD34+(CD38low/-) AML cells and the extracellular vesicle signatures in circulation from AML patients at diagnosis. CD34+ AML cells display high antioxidant glutathione levels and enhanced mitochondrial functionality, both associated with poor clinical outcomes. Although CD34+ AML cells are highly dependent on glucose oxidation and glycolysis for energy, those from intermediate- and adverse-risk patients reveal increased mitochondrial dependence. Extracellular vesicles from AML are mainly enriched in stem cell markers and express antioxidant GPX3, with their profiles showing potential prognostic value. Extracellular vesicles enhance mitochondrial functionality and dependence on CD34+ AML cells via the glutathione/GPX4 axis. Notably, extracellular vesicles from adverse-risk patients enhance leukemia cell engraftment in vivo. Here, we show a potential noninvasive approach based on liquid ‘cell-extracellular vesicle’ biopsy toward a redefined metabolic stratification in AML.

The increasing occurrence of infections caused by multidrug‐resistant (MDR) bacteria drives the need for new antibacterial drugs. Due to the current lack of antibiotic discovery and development, new strategies to fight MDR bacteria are urgently needed. Efforts to develop new antibiotic adjuvants to increase the effectiveness of existing antibiotics and design delivery systems are essential to address this issue. Here, a bioinspired delivery system equipped with combination therapy and paracellular transport is shown to enhance the efficacy against bacterial infections by improving oral delivery. A screening platform is established using an in vitro‐induced high polymyxin‐resistant strain to acquire plumbagin, which enhances the efficacy of polymyxin. Functionalized milk extracellular vesicles (FMEVs) coloaded with polymyxin and plumbagin cleared 99% of the bacteria within 4 h. Mechanistic studies revealed that the drug combination damaged the membrane, disrupted energy metabolism, and accelerated bacterial death. Finally, FMEVs are efficiently transported transcellularly through the citric acid‐mediated reversible opening of the tight junctions and showed high efficacy against an MDR Escherichia coli‐associated peritonitis–sepsis model in mice. These findings provide a potential therapeutic strategy to improve the efficacy of combination therapy by enhancing oral delivery using a biomimetic delivery platform.

Inflammation significantly influences cellular communication in the oral environment, impacting tissue repair and regeneration. This study explores the role of small extracellular vesicles (sEVs) derived from lipopolysaccharide (LPS)-treated stem cells from the apical papilla (SCAP) in modulating macrophage polarization and osteoblast differentiation. SCAPs were treated with LPS for 24 h, and sEVs from untreated (SCAP-sEVs) and LPS-treated SCAP (LPS-SCAP-sEVs) were isolated via ultracentrifugation and characterized using transmission electron microscopy, Western blot, and Tunable Resistive Pulse Sensing. LPS-SCAP-sEVs exhibited characteristic exosome morphology (~100 nm diameter) and expressed vesicular markers (CD9, CD63, CD81, and HSP70). Functional analysis revealed that LPS-SCAP-sEVs promoted M1 macrophage polarization, as evidenced by the increased pro-inflammatory cytokines (IL-6 and IL-1β) and the reduced anti-inflammatory markers (IL-10 and CD206), while impairing the M2 phenotype. Additionally, LPS-SCAP-sEVs had a minimal impact on SCAP metabolic activity or osteogenic gene expression but significantly reduced mineralization capacity in osteogenic conditions. These findings suggest that sEVs mediate the inflammatory interplay between SCAP and macrophages, skewing macrophage polarization toward a pro-inflammatory state and hindering osteoblast differentiation. Understanding this sEV-driven communication axis provides novel insights into the cellular mechanisms underlying inflammation in oral tissues and highlights potential therapeutic targets for modulating extracellular vesicle activity during acute inflammatory episodes.

Multiple myeloma is a plasma cell malignancy characterized by an abnormal increase in monoclonal immunoglobulins. Despite significant advances in treatment, some patients progress to more aggressive forms of multiple myeloma, including extramedullary disease or plasma cell leukemia. Although the exact molecular mechanisms are not known, several studies have confirmed the involvement of small extracellular vesicle‐enriched microRNAs in multiple myeloma progression. Therefore, we performed expression profiling of these molecules in bone marrow plasma of multiple myeloma, extramedullary disease, and plasma cell leukemia patients using small RNA sequencing to identify novel molecules involved in disease pathogenesis. In total, 42 microRNAs were significantly dysregulated among analyzed subgroups. Independent validation by RT‐qPCR confirmed elevated levels of miR‐140‐3p, miR‐584‐5p, miR‐191‐5p, and miR‐143‐3p in multiple myeloma patients compared to extramedullary disease and plasma cell leukemia patients. Subsequent statistical analysis revealed significant correlations between patient clinical characteristics or flow cytometry parameters and microRNA expression. These results indicate that dysregulation of microRNAs could contribute to multiple myeloma progression.

Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Extracellular vesicles are lipid-bilayer encapsulated nanovesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived extracellular vesicles (KDEVs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEVs in a well-established high-fat diet mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEVs through size-exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of high-fat diet KDEVs compared to KDEVs obtained from control mice on a regular diet, including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEVs are retrogradely trafficked into the dorsal root ganglion (DRG) neuron cell bodies. This study presents the first comprehensive isolation and molecular characterization of the KDEV protein and microRNA cargo in RD and HFD mice. Our findings suggest a potential novel communication pathway between keratinocytes and DRG neurons in the skin, which could have implications for PDN.

Parasitic helminths secrete extracellular vesicles (EVs) into their host tissues to modulate immune responses, but the underlying mechanisms are poorly understood. We demonstrate that Ascaris EVs are efficiently internalised by monocytes in human peripheral blood mononuclear cells and increase the percentage of classical monocytes. Furthermore, EV treatment of monocytes induced a novel anti‐inflammatory phenotype characterised by CD14+, CD16−, CC chemokine receptor 2 (CCR2−) and programmed death‐ligand 1 (PD‐L1)+ cells. In addition, Ascaris EVs induced T cell anergy in a monocyte‐dependent mechanism. Targeting professional phagocytes to induce both direct and indirect pathways of immune modulation presents a highly novel and efficient mechanism of EV‐mediated host‐parasite communication. Intra‐peritoneal administration of EVs induced protection against gut inflammation in the dextran sodium sulphate model of colitis in mice. Ascaris EVs were shown to affect circulating immune cells and protect against gut inflammation; this highlights their potential as a subject for further investigation in inflammatory conditions driven by dysregulated immune responses. However, their clinical translation would require further studies and careful consideration of ethical implications.