EV Uptake Research Articles

Genetically engineered human induced pluripotent stem cells for the production of brain-targeting extracellular vesicles

BackgroundExtracellular vesicles (EVs) are cell-secreted membrane vesicles that have become a promising, natural nanoparticle system for delivering either naturally carried or exogenously loaded therapeutic molecules. Among reported cell sources for EV manufacture, human induced pluripotent stem cells (hiPSCs) offer numerous advantages. However, hiPSC-EVs only have a moderate ability for brain delivery. Herein, we sought to develop a stable hiPSC line for producing EVs with substantially enhanced brain targeting by genetic engineering to overexpress rabies viral glycoprotein (RVG) peptide fused to the N terminus of lysosomal associated membrane protein 2B (RVG-Lamp2B) which has been shown capable of boosting the brain delivery of EVs via the nicotinic acetylcholine receptor.MethodsAn RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. Western blot was used to detect the expression of RVG-Lamp2B-HA in RVG-edited hiPSCs as well as EVs derived from RVG-edited hiPSCs. Uptake of EVs by SH-SY5Y cells in the presence of various endocytic inhibitors was analyzed using flow cytometry. Biodistribution and brain delivery of intravenously injected control and RVG-modified EVs in wild-type mice were examined using ex vivo fluorescent imaging.ResultsHere we report that an RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. The RVG-edited iPSCs have normal karyotype, express pluripotency markers, and have differentiation potential. Expression of RVG-Lamp2B-HA was detected in total cell extracts as well as EVs derived from RVG-edited (vs. control) hiPSCs. The RVG-modified EVs enter neuronal cells via distinct endocytic pathways, compared with control EVs. The biodistribution study confirmed that EVs derived from RVG-edited hiPSCs possess higher brain delivery efficiency.ConclusionTaken together, we have established stable, genetically engineered hiPSCs for producing EVs with RVG expression, offering the improved ability for brain-targeted drug delivery.

Supramolecular hydrogels for sustained extracellular vesicle delivery

AbstractExtracellular vesicles (EVs) have been explored as promising drug delivery platforms and cell-free therapies for a range of diseases. Despite their therapeutic potential, challenges persist in achieving sustained EV delivery. Here, we integrate EVs into a supramolecular and injectable hydrogel-based drug delivery system based on dodecyl- or octadecyl-modified hydroxypropyl methylcellulose (HPMC-C12 or -C18) that form non-covalent crosslinks with liposomes. Hydrogel mechanics and EV-release kinetics were tunable by varying liposome concentrations. Using mesenchymal stem cell-derived EVs (MSC-EVs), we confirm effective, hydrogel-mediated sustained EV delivery and uptake and a ~ 20% greater anti-inflammatory response in pathogenic vascular smooth muscle cells than bolus EV-only treatment. Graphical abstract

Transition to green mobility: a twin investigation behind the purchase reasons of electric vehicles in the Indian market

Purpose The study aims to fulfil two objectives, namely, to explore what dimensions comes into play in the current consumer EV purchase behaviour based on relevant literature identified in the past and to find out whether there are other reasons that are peculiar to the Indian automobile market which influenced consumers to adopt this eco-friendly technology. Design/methodology/approach Mixed method approach for collecting the primary data was used. The study was conducted in five cities across India, through a questionnaire administered to 266 existing EV owners using judgemental sampling, that included dimensions as to what made them purchase EVs (any variant whether hybrid or battery) and encouraged their green behaviour. Moreover, the survey also took care of gathering other motivational constructs that may have played a significant role in the Indian EV market. To take care of this, use of single open-ended question was done. Statistical techniques like exploratory factor analysis (EFA) and Partial Least Square-Structural Equation Modelling (PLS-SEM) version 4.0 were performed to gain meaningful insights from this investigation. Findings Based on our analysis, EV purchase behaviour in the Indian market could be explained by six configurational paths identified in conceptual framework of this study. The data analysed revealed long-term operational benefits or relative advantages, trialability or prior driving experience and positive attitude towards EVs playing an important role towards EV purchase decision followed by environmental concern, social norms and personal values of Indian consumers. Besides, several other factors were identified in open comments like favourable government policies, brand consciousness, compatibility and functional benefits in terms of comfortable and noiseless driving which tend to drive the decision of Indian consumers towards buying EVs. The identification of these motivations can help policymakers in targeting the customer market accordingly. Research limitations/implications Although the study is restricted by its scope of sampling criteria, yet is distinct in its methodological approach and thereby adds value to existing literature on marketing and diffusion of EVs. The study explores the untapped gap in the current literature by helping identify reasons on the basis of which consumers made their choices of different EVs. Instead of analysing the potential antecedents of EV uptake, the study brings to light the actual reasons of EV purchase behaviour in the context of an emerging economy like India. That is to say, the horizon of EV motivations spans across technological, individual and regulatory attributes in the Indian automobile market. Practical implications This study is relevant and fulfils the underlying gap in the existing literature related to actual motivations that consumers look for before buying a high involvement product such as EVs. The results can be beneficial for marketers to design effective marketing strategies from the perspective of consumers. Promotional strategies addressing ease of using EVs and how battery technology is better than before needs to be clearly communicated by the marketers. Consumer education is necessary, in this regard, to make people aware of the rising air pollution levels. They have to be made understood the real advantages of owning EVs and how these non-conventional vehicles are far better when compared to traditional cars. In a nutshell, the study underscored potential determinants or reasons that made Indian consumers to go for EVs, based on statistically significant evidences which can be crucial for stakeholders operating in the Indian EV industry. Social implications The study has important implications for members of the community. By sacrificing their egoistic tendencies or individual preferences for energy intensive products such as ICE based vehicles, people can imbibe altruistic orientation and can contribute to the collective benefit of all by shifting towards energy rich vehicles powered by electricity. This is necessary to achieve the broader goals of sustainable development and to curtail the rising climate change phenomenon. Originality/value The study applied both quantitative (deductive) and qualitative (inductive) approach to study into the reasons for consumers’ purchase motivations towards electric cars. Taking a distinct methodological approach, the study enhances the knowledge on EVs by asking consumers to comment in their own words as to why they purchased EVs. This was necessary so as to better explore the needs of the Indian automobile market and which factors govern the purchase of EVs in domestic market. Past literature majorly focused on purchase intention towards EVs but this study in unexampled and analysed the purchase behaviour towards EVs. It should be noted that our investigation reveals the complexity of EV purchase reasons than those identified in past literature and it adds to the current pool of literature on EVs by explaining factors with unrivalled illustrative power.

Unraveling new players in helminth pathology: extracellular vesicles from Fasciola hepatica and Dicrocoelium dendriticum exert different effects on hepatic stellate cells and hepatocytes

Fasciola hepatica and Dicrocoelium dendriticum are parasitic trematodes residing in the bile ducts of mammalian hosts, causing, in some cases, impairment of liver function and hepatic fibrosis. Previous studies have shown that extracellular vesicles released by F. hepatica (FhEVs) and D. dendriticum (DdEVs) induce a distinct phenotype in human macrophages, but there is limited information on the effect of parasitic EVs on liver cells, which interact directly with the worms in natural infections. In this study, we isolated FhEVs and DdEVs by size exclusion chromatography and labeled them with a lipophilic fluorescent dye to analyze their uptake by human hepatic stellate cells (HSC) and hepatocytes, important cell types in liver pathology, using synthetic liposomes as internal labeling and uptake control. We analyzed EV uptake and the proteome profiles after the treatment with EVs for both cell types. Our results reveal that EVs establish unique and specific interactions with stellate cells and hepatocytes, suggesting a different role of EVs derived from each parasite, depending on the migration route to reach their final niche. FhEVs have a cytostatic effect on HSCs, but induce the extracellular matrix secretion and elicit anti-inflammatory responses in hepatocytes. DdEVs have a more potent anti-proliferative effect than FhEVs and trigger a global inflammatory response, increasing the levels of NF-κB and other inflammatory mediators in both cell types. These interactions may have a major influence on the progression of the disease, serving to generate conditions that may favor the establishment of the helminths in the host.

Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid.

Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes or ostomies. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super-resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β-casein and lactalbumin. HMEV and some dEV fractions contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin-mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.

Molecular Determinants Involved in the Docking and Uptake of Tumor-Derived Extracellular Vesicles: Implications in Cancer.

Extracellular vesicles produced by tumor cells (TEVs) influence all stages of cancer development and spread, including tumorigenesis, cancer progression, and metastasis. TEVs can trigger profound phenotypic and functional changes in target cells through three main general mechanisms: (i) docking of TEVs on target cells and triggering of intra-cellular signaling; (ii) fusion of TEVs and target cell membranes with release of TEVs molecular cargo in the cytoplasm of recipient cell; and (iii) uptake of TEVs by recipient cells. Though the overall tumor-promoting effects of TEVs as well as the general mechanisms involved in TEVs interactions with, and uptake by, recipient cells are relatively well established, current knowledge about the molecular determinants that mediate the docking and uptake of tumor-derived EVs by specific target cells is still rather deficient. These molecular determinants dictate the cell and organ tropism of TEVs and ultimately control the specificity of TEVs-promoted metastases. Here, we will review current knowledge on selected specific molecules that mediate the tropism of TEVs towards specific target cells and organs, including the integrins, ICAM-1 Inter-Cellular Adhesion Molecule), ALCAM (Activated Leukocyte Cell Adhesion Molecule), CD44, the metalloproteinases ADAM17 (A Disintegrin And Metalloproteinase member 17) and ADAM10 (A Disintegrin And Metalloproteinase member 10), and the tetraspanin CD9.

Lung Fibroblasts Take up Breast Cancer Cell-derived Extracellular Vesicles Partially Through MEK2-dependent Macropinocytosis.

Through a phenotypic screen, we found that MEK inhibitor Trametinib suppressed EV uptake and macropinocytosis in lung fibroblasts, and that EV uptake is mediated by MEK2 in these cells. Our results suggest that MEK2 inhibition could serve as a strategy to block cancer EV uptake by lung fibroblasts.

Blockade of CD93 in pleural mesothelial cells fuels anti-lung tumor immune responses.

Background: CD93 reportedly facilitates tumor angiogenesis. However, whether CD93 regulates antitumor immunity remains undeciphered. Methods: Lung tumor tissues, malignant pleural effusions (MPEs) were obtained from lung cancer patients. Blood was obtained from healthy volunteers and lung cancer patients with anti-PD-1 therapy. Furthermore, p53fl/flLSL-KrasG12D, Ccr7-/-, Cd93-/- mice and CD11c-DTR mice were generated. Specifically, EM, NTA and western blotting were utilized to identify Tumor extracellular vesicles (TEVs). EV labeling, detection of EV uptake in vitro and in vivo, degradation of EV proteins and RNAs were performed to detect the role of TEVs in tumor progression. Pleural mesothelial cells (pMCs) were isolated to investigate related signaling pathways. Recombinant proteins and antibodies were generated to test which antibody was the most effective one to increase CCL21a in p-pMCs. RNA-Seq, MiRNA array, luciferase reporter assay, endothelial tube formation assay, protein labeling and detection, transfection of siRNAs and the miRNA mimic and inhibitor, chemotaxis assay, immunohistochemical staining, flow cytometry, Real-time PCR, and ELISA experiments were performed. Results: We show that CD93 of pMCs reduced lung tumor migration of dendritic cells by preventing pMCs from secreting CCL21, thereby suppressing systemic anti-lung tumor T-cell responses. TEV-derived miR-5110 promotes CCL21 secretion by downregulating pMC CD93, whereas C1q, increasing in tumor individuals, suppresses CD93-mediated CCL21 secretion. CD93-blocking antibodies (anti-CD93) inhibit lung tumor growth better than VEGF receptor-blocking antibodies because anti-CD93 inhibit tumor angiogenesis and promote CCL21 secretion from pMCs. Anti-CD93 also overcome lung tumor resistance to anti-PD-1 therapy. Furthermore, lung cancer patients with higher serum EV-derived miR-5193 (human miR-5110 homolog) are more sensitive to anti-PD-1 therapy, while patients with higher serum C1q are less sensitive, consistent with their regulatory functions on CD93. Conclusions: Our study identifies a crucial role of CD93 in controlling anti-lung tumor immunity and suggests a promising approach for lung tumor therapy.

Surfaceome analysis of extracellular vesicles from senescent cells uncovers uptake repressor DPP4.

Senescent cells are beneficial for repairing acute tissue damage, but they are harmful when they accumulate in tissues, as occurs with advancing age. Senescence-associated extracellular vesicles (S-EVs) can mediate cell-to-cell communication and export intracellular content to the microenvironment of aging tissues. Here, we studied the uptake of EVs from senescent cells (S-EVs) and proliferating cells (P-EVs) and found that P-EVs were readily taken up by proliferating cells (fibroblasts and cervical cancer cells) while S-EVs were not. We thus investigated the surface proteome (surfaceome) of P-EVs relative to S-EVs derived from cells that had reached senescence via replicative exhaustion, exposure to ionizing radiation, or treatment with etoposide. We found that relative to P-EVs, S-EVs from all senescence models were enriched in proteins DPP4, ANXA1, ANXA6, S10AB, AT1A1, and EPHB2. Among them, DPP4 was found to selectively prevent uptake by proliferating cells, as ectopic overexpression of DPP4 in HeLa cells rendered DPP4-expressing EVs that were no longer taken up by other proliferating cells. We propose that DPP4 on the surface of S-EVs makes these EVs refractory to internalization by proliferating cells, advancing our knowledge of the impact of senescent cells in aging-associated processes.

Extracellular Vesicles from Neural Stem Cells Carry microRNA-16-5p to Reduce Corticosterone-induced Neuronal Injury in Depression Rats

ObjectiveDepression is a common mental illness. Neural stem cell-derived extracellular vesicles (NSC-EVs) are involved in repairing neuronal injury. We estimated the mechanism of miR-16-5p in depression rats. MethodsEVs were extracted from NSCs. The depression rat model was established by corticosterone (CORT) induction and treated with NSC-EVs. The depression behavioral/pathological changes in rats were assessed using forced swimming test, open field test, sucrose consumption test and western blotting. The neuronal apoptosis in hippocampal tissue were detected. CORT-induced PC12 cell model was established. EV uptake by PC12 cells was measured and PC12 cell apoptosis was detected. The downstream targets of miR-16-5p were predicted and verified. The expressions of miR-16-5p and MYB in rats, PC12 cells, and EVs were measured. Functional rescue experiments were conducted to verify the role of miR-16-5p and MYB in PC12 cell apoptosis. ResultsCORT induction increased neuronal apoptosis in hippocampal tissue and induced depression-like behaviors in rats, while NSC-EV treatment improved depression-like behaviors and apoptosis in rats. In PC12 cells, NSC-EVs decreased CORT-induced PC12 cell apoptosis. NSC-EVs carried miR-16-5p into PC12 cells. miR-16-5p knockdown in EVs partially reversed the inhibitory effects of NSC-EVs on CORT-induced PC12 cell apoptosis. miR-16-5p targeted to inhibit MYB to repress CORT-induced PC12 cell apoptosis. In vivo experiments further verified that NSC-EVs reduced neuronal injury in CORT-induced depression rats via the miR-16-5p/MYB axis. ConclusionNSC-EVs-mediated alleviation on neuronal injury by carrying miR-16-5p to target MYB was highly likely one of the mechanisms by which NSC-EVs mediated miR-16-5p in neuroprotection of depression rats.

Biodistribution of mesenchymal stromal cell-derived extracellular vesicles administered during acute lung injury

BackgroundMesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are a promising cell-free therapy for acute lung injury (ALI). To date, no studies have investigated their biodistribution in ALI or discerned the timing of administration for maximal lung targeting, which are crucial considerations for clinical translation. Our study aimed to characterize a mouse model of ALI and establish the distribution kinetics and optimal timing of MSC-EV delivery during lung injury.MethodsMSC-EVs were isolated by ultracentrifugation alone (U/C) or tangential flow filtration with ultracentrifugation (TFF-U/C) and characterized by nanoparticle tracking analysis and western blot. A lipopolysaccharide (LPS)-induced mouse model of ALI was established to study the inflammatory response over 72 h. ALI was assessed by histological lung injury score, bronchoalveolar lavage fluid cell count and inflammatory cytokines. For biodistribution studies, ALI mice were intravenously administered fluorescently labeled MSC-EVs to determine the optimal timing of administration and organ-specific biodistribution. Live in vivo and ex vivo fluorescence imaging was conducted at various timepoints post-EV injection.ResultsEVs isolated by either ultracentrifugation alone or TFF-U/C displayed comparable size distribution (~ 50–350 nm) and EV marker expression (CD63/81). TFF-U/C generated a 5.4-fold higher particle concentration and 3.9-fold higher total protein when compared to ultracentrifugation alone. From the inflammatory time-course study, cell count and IL-1β peaked in bronchoalveolar lavage fluid at 24 h after ALI induction. MSC-EVs delivered at 24 h (as opposed to 0.5 h, 5 h or 10 h) after disease induction resulted in a 2.7–4.4-fold higher lung uptake of EVs. Biodistribution studies comparing organ-specific MSC-EV uptake showed progressive lung accumulation up to 48 h post-delivery (threefold higher than the spleen/liver), with a decline at 72 h. Importantly, lung EV fluorescence at 48 h in ALI mice was significantly elevated as compared to control mice. The lung tropism of MSC-EVs was further validated as therapeutically inert EVs derived from HEK293T cells accumulated mainly to the spleen and liver with a 5.5-fold lower distribution to the lungs as compared to MSC-EVs.ConclusionMSC-EVs exhibit maximal lung accumulation when administered during heightened inflammation at 24 h after ALI induction. This lung tropism suggests that MSC-EVs may serve as a practical rescue treatment for acute inflammatory respiratory conditions.

Characterisation of extracellular vesicles isolated from hydatid cyst fluid and evaluation of immunomodulatory effects on human monocytes.

Hydatidosis is a disease caused by the larval stage of Echinococcus granulosus, which involves several organs of intermediate hosts. Evidence suggests a communication between hydatid cyst (HC) and hosts via extracellular vesicles. However, a little is known about the communication between EVs derived from HC fluid (HCF) and host cells. In the current study, EVs were isolated using differential centrifugation from sheep HCF and characterized by western blot, electron microscope and size distribution analysis. The uptake of EVs by human monocyte cell line (THP-1) was evaluated. The effects of EVs on the expression levels of pro- and anti-inflammatory cytokines were investigated using quantitative real-time PCR (RT-PCR), 3 and 24 h after incubation. Moreover, the cytokine level of IL-10 was evaluated in supernatant of THP-1 cell line at 3 and 24 h. EVs were successfully isolated and showed spherical shape with size distribution at 130.6 nm. After 3 h, the expression levels of pro-inflammatory cytokine genes (IL1Β, IL15 and IL8) were upregulated, while after 24 h, the expression levels of pro-inflammatory cytokines were decreased and IL13 gene expression showed upregulation. A statistically significant increase was seen in the levels of IL-10 after 24 h. The main mechanism of the communication between EVs derived from HCF and their host remains unclear; however, time-dependent anti-inflammatory effects in our study suggest that HC may modulate the immune responses via EVs.

11:10-11:20 Uptake of preeclampsia-derived EVs activates microglia in vitro

11:10-11:20 Uptake of preeclampsia-derived EVs activates microglia in vitro

Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression

BackgroundExtracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression.MethodsEVs were isolated by differential ultracentrifugation from cell culture supernatant as well as plasma samples from healthy individuals (n = 27) and breast cancer patients (n = 41). EVs were thoroughly characterized by electron microscopy, nanoparticle tracking analysis, immunoblot, and flow cytometry. ROR transfer to target cells was observed using microscopy-based assays and biodistribution experiments were conducted in syngeneic mice. EV impact on cancer cell migration and invasion was tested in functional assays.ResultsWe observed that the supernatant of ROR-overexpressing cells was sufficient for transferring the receptors to ROR-negative cells. Analyzing the secretome of the ROR-overexpressing cells, we detected a high enrichment of ROR1/2 on large and small EVs, but not on large oncosomes. Interestingly, the majority of ROR-positive EVs remained attached to the target cell surface after 24 h of stimulation and was quickly removed by treatment with trypsin. Nonetheless, ROR-positive EVs increased migration and invasion of breast cancer cells, even after chemically inhibiting EV uptake, in dependence of RhoA downstream signaling. In vivo, ROR-depleted EVs tended to distribute less into organs prone for the formation of breast cancer metastases. ROR-positive EVs were also significantly elevated in the plasma of breast cancer patients and allowed to separate them from healthy controls.ConclusionsThe oncogenic Wnt receptors ROR1/2 are transferred via EVs to the surface of ROR-negative cancer cells, in which they induce an aggressive phenotype supporting tumor progression.2c9NzHjw1WgkMJnzAAfwiwVideo

Targeting extracellular vesicle delivery to the lungs by microgel encapsulation

AbstractExtracellular vesicles (EVs) secreted by stem and progenitor cells have significant potential as cell‐free ‘cellular’ therapeutics. Yet, small EVs (<200 nm) are rapidly cleared after systemic administration, mainly by the liver, presenting challenges targeting EVs to a specific organ or tissue. Microencapsulation using natural nano‐porous hydrogels (microgels) has been shown to enhance engraftment and increase the survival of transplanted cells. We sought to encapsulate EVs within microgels to target their delivery to the lung by virtue of their size‐based retention within the pulmonary microcirculation. Mesenchymal stromal cell (MSC) derived EVs were labelled with the lipophilic dye (DiR) and encapsulated within agarose‐gelatin microgels. Endothelial cells and bone marrow derived macrophages were able to take up EVs encapsulated in microgels in vitro, but less efficiently than the uptake of free EVs. Following intrajugular administration, microgel encapsulated EVs were selectively retained within the lungs for 72h, while free EVs were rapidly cleared by the liver. Furthermore, microgel‐loaded EVs demonstrated greater uptake by lung cells, in particular CD45+ immune cells, as assessed by flow cytometry compared to free EVs. Microencapsulation of EVs may be a novel tool for enhancing the targeted delivery of EVs for future therapeutic applications.

Technology for the Tolling Boom

As EV uptake grows, fuel tax revenues are falling and so tolling is more important than ever, which is why the best tech is of increasing importance

Integrating Diffusion of Innovations and Theory of Planned Behavior to Predict Intention to Adopt Electric Vehicles in Rwanda

Purpose: Integration of the theory of planned behaviour and the Diffusion of innovations (DOI) theory to examine EVs low adoption intention in Rwanda.
 Methodology: The research model involved the integration of the Theory of planned behaviour and the Diffusion of innovation. To build the conceptual framework, over 50 articles were collected from Google scholar and summarised using meta-analysis. Two hundred and twenty participants were surveyed in person using a questionnaire, with each construct having at least three questions to run the Partial least squares (PLS) Structural Equation Modelling (SEM). Data were analysed using SmartPLS 4.0.
 Findings: The study found that two factors of the Theory of planned behaviour TPB- attitudes and perceived behavioural control- positively impact EVs adoption intention while subjective norm does not. Three factors of diffusion of innovation (DOI)- trialability, compatibility, and observability- positively impact attitude towards EV adoption.
 Unique Contribution to Theory, Practice and Policy: Findings from the research explain the low adoption of electric vehicles in Rwanda. They can serve as an objective basis for policymakers to upscale EV uptake in Rwanda and brings to perspective the context of Rwanda on factors of EV adoption.

What EV users say about policy efficacy: Evidence from Shanghai

Sustainable EV adoption is critical to urban low-carbon future. The in-depth analysis of urban incentives' role in encouraging EV purchases is less examined. The perspectives of EV adopters can provide unique insights into policy implementation and effectiveness, informing future policy design and learning. Taking Shanghai—a city with roughly 4% of global EV stock—as the case, we aim to fill the above gaps via an online survey among existing EV owners. Our analysis shows that (a) urban EV strategies played a critical role in Shanghai's early adoption success, encouraging many first-time buyers to choose EVs over petrol cars, avoiding future lock-in emissions; (b) locally tailored measures such as free license plates and access to highways during rush hours significantly encouraged EV purchases; and (c) users' feedback on policy implementation reveals challenges for next-step scale adoption, especially regarding an unsupportive installation environment for private chargers, and inadequate services, e.g., infrastructure maintenance and the building up of a used-car market. These findings suggest city-level incentives can increase EV uptake but are insufficient to support a complete EV transition. Research needs to look beyond incentives for early adopters and into policy and social aspects that can support upscaling and social embedding of EVs.

Health impacts from TRAPs and carbon emissions in the projected electric vehicle growth and energy generation mix scenarios in Malaysia

Road transport contributes over 70% of air pollution in urban areas and is the second largest contributor to the total carbon dioxide emissions in Malaysia at 21% in 2016. Transport-related air pollutants (TRAPs) such as NOx, SO2, CO and particulate matter (PM) pose significant threats to the urban population's health. Malaysia has targeted to deploy 885,000 EV cars on the road by 2030 in the Low Carbon Mobility Blueprint (LCMB). This study aims to quantify the health co-benefits of electric vehicle adoption from their impacts on air quality in Malaysia. Two EV uptake projections, i.e. LCMB and Revised EV Adoption (REVA) projections, and five electricity generation mix scenarios were modelled up to 2040. We used comparative health risk assessment to estimate the potential changes in mortality and burden of diseases (BoD) from the emissions in each scenario. Intake fractions and exposure-risk functions were used to calculate the burden from respiratory diseases (PM2.5, NOx, SO2, CO), cardiovascular diseases and lung cancer (PM2.5). Results showed that along with a net reduction of carbon emissions across all scenarios, there could be reduced respiratory mortality from NOx by 10,200 mortality (176,200 DALYs) and SO2 by 2600 mortality (45,400 DALYs) per year in 2040. However, there could also be additional 719 mortality (9900 DALYs) per year from PM2.5 and 329 mortality (5600 DALYs) from CO per year. The scale of reduction in mortality and BoD from NOx and SO2 are significantly larger than the scale of increase from PM2.5 and CO, indicating potential net positive health impacts from the EV adoption in the scenarios. The health cost savings from the reduced BoD of respiratory mortality could reach up to RM 7.5 billion per year in 2040. In conclusion, EV is a way forward in promoting a healthy and sustainable future transport in Malaysia.

AQP4-dependent glioma cell features affect the phenotype of surrounding cells via extracellular vesicles

BackgroundExtracellular vesicles (EVs) are membrane-enclosed particles released systemically by all cells, including tumours. Tumour EVs have been shown to manipulate their local environments as well as distal targets to sustain the tumour in a variety of tumours, including glioblastoma (GBM).We have previously demonstrated the dual role of the glial water channel aquaporin-4 (AQP4) protein in glioma progression or suppression depending on its aggregation state. However, its possible role in communication mechanisms in the microenvironment of malignant gliomas remains to be unveiled.ResultsHere we show that in GBM cells AQP4 is released via EVs that are able to affect the GBM microenvironment. To explore this role, EVs derived from invasive GBM cells expressing AQP4-tetramers or apoptotic GBM cells expressing orthogonal arrays of particles (AQP4-OAPs) were isolated, using a differential ultracentrifugation method, and were added to pre-seeded GBM cells. Confocal microscopy analysis was used to visualize the interaction and uptake of AQP4-containing EVs by recipient cells. Chemoinvasion and Caspase3/7 activation assay, performed on recipient cells after EVs uptake, revealed that EVs produced by AQP4-tetramers expressing cells were able to drive surrounding tumour cells toward the migratory phenotype, whereas EVs produced by AQP4-OAPs expressing cells drive them toward the apoptosis pathway.ConclusionThis study demonstrates that the different GBM cell phenotypes can be transferred by AQP4-containing EVs able to influence tumour cell fate toward invasiveness or apoptosis.This study opens a new perspective on the role of AQP4 in the brain tumour microenvironment associated with the EV-dependent communication mechanism.Graphical