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Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID‐19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF‐A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF‐A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP‐mRNAs between cells is functionally extended by cells’ own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF‐A mRNA delivery to cells via LNPs, a fraction of internalized VEGF‐A mRNA is secreted via EVs. The overexpressed VEGF‐A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA‐Seq analysis reveals that as cells’ response to LNP‐VEGF‐A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF‐A mRNA in vitro and in vivo. Upon equal amount of VEGF‐A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF‐A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF‐A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF‐A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this mRNA differently than LNPs. The study shows that a fraction of LNP‐mRNA that is cell‐endocytosed can be sent to other cells via the secretion of extracellular vesicles (EVs). LNPs transform these EVs  as functional extensions to distribute therapeutic mRNA between cells.Importantly, EVs can be isolated such as from cardiac progenitor cells (CPC‐EVs), and thus utilized for mRNA delivery in vivo. Upon mRNA delivery to cardiac tissue, CPC‐EVs cause less expression of inflammatory cytokines, compared to other vehicles used.

Anti-HER2 therapy is integral to the treatment of HER2-positive breast cancer, but drug resistance hampers its effectiveness. Although antibody-drug conjugates (ADCs) are increasingly used in clinical practice, their application is often hindered by adverse reactions and drug resistance. Therefore, it is crucial to enhance the bioavailability of ADCs and reduce their dosages to mitigate both adverse effects and resistance. Pyrotinib’s effect on HER2-positive breast cancer cell lines (SK-BR-3 and JIMT-1) was investigated via western blot, focusing on HER2 and downstream pathways. Pyrotinib’s influence on HER2 ubiquitination and internalization was assessed through RT-qPCR, western blot, and immunofluorescence. The ability of pyrotinib to augment trastuzumab emtansine (T-DM1) endocytosis and antiproliferative effects was studied via CCK-8 and immunofluorescence. In vivo experiments in nude mice were conducted to explore the therapeutic efficacy of T-DM1 combined with pyrotinib. The single-drug study showed that pyrotinib downregulated HER2 protein levels and HER2 downstream signaling pathways. The mechanism of downregulating HER2 protein levels involved the promotion of HER2 internalization and degradation through the ubiquitin-proteasome pathway. The two-drug combination study showed that pyrotinib promoted the endocytosis of T-DM1, which improved its bioavailability. Increased cellular uptake further enhanced the antitumor effects of T-DM1 in both in vitro and in vivo experiments. Our results reveal the molecular mechanism by which pyrotinib regulates HER2 levels by promoting HER2 internalization, thereby facilitating the endocytosis of T-DM1. These findings suggest a potential combination treatment strategy for the targeted therapy of HER2-positive breast cancer.

Background HER2-targeted therapies have improved the outcomes of HER2-positive gastric cancer (GC), yet resistance remains a challenge. We sought to explore the effects of reversible and irreversible HER2 tyrosine kinase inhibitors (TKIs) alone or in combination with the HER2-targeting antibody drug conjugate trastuzumab deruxtecan (T-Dxd). Methods The effects of HER2-TKIs on HER2 and downstream signaling were evaluated via Western blotting. Proteasomal inhibitors and co-immunoprecipitation assays were performed to explore the role of proteasomal degradation in HER2 expression modulation, and immunofluorescence assays were employed to explore mechanisms of HER2 internalization. The synergistic potential of the irreversible HER2-TKI pyrotinib in combination with T-Dxd was validated using growth and viability assays in anti-HER2-positive GC cell cultures and tumor growth and immunohistochemical staining assays in a mouse xenograft model. Results Our study revealed that reversible HER2-TKIs elevated HER2 protein levels, whereas irreversible HER2-TKIs decreased them. Pyrotinib triggered HER2 degradation within the proteasome by promoting ubiquitination and dissociation from HSP90. Furthermore, pyrotinib substantially induced HER2 internalization, which led to improved cellular uptake of T-Dxd. The increased T-Dxd uptake was accompanied by greater efficacy in suppressing the growth of GC cells and enhanced anti-tumor effects in an animal model. Conclusion In summary, our research reveals the molecular mechanisms of irreversible HER2-TKIs in regulating HER2 protein expression by promoting HER2 internalization. These findings advance our comprehension of targeted therapy for GC and provide a promising therapeutic combination strategy with enhanced efficacy against HER2-positive GC.

Endosomal sorting complex required for transport (ESCRT) complexes are involved in endosomal trafficking to the lysosome, cytokinesis, and viral budding. Extensive genetic, biochemical, and structural studies on the ESCRT system have been carried out in yeast and mammalian systems. However, the question of how the ESCRT system functions at the whole organism level has not been fully explored. In C. elegans, we performed RNAi experiments to knock-down gene expression of components of the ESCRT system and profiled their effects on protein degradation and endocytosis of YP170, a yolk protein. Targeted RNAi knock-down of ESCRT-Ⅰ (tsg-101 and vps-28) and ESCRT-Ⅲ (vps-24, and vps-32.2) components interfered with protein degradation while knock-down of ESCRT-Ⅱ (vps-25 and vps-36) and ESCRT-Ⅲ (vps-20 and vps-24) components hampered endocytosis. In contrast, the knockdown of vps-37, another ESCRT-Ⅰ component, showed no defect in either YP170 uptake or degradation. Depletion of at least one component from each complex - ESCRT-0 (hgrs-1), ESCRT-Ⅰ (tsg-101, vps-28, and vps-37), ESCRT-Ⅱ (vps-36), ESCRT-Ⅲ (vps-24), and Vps4 (vps-4) - resulted in abnormal distribution of embryos in the uterus of worms, possibly due to abnormal ovulation, fertilization, and egg-laying. These results suggest differential physiological roles of ESCRT-0, -Ⅰ, -Ⅱ, and -Ⅲ complexes in the context of the whole organism, C. elegans.

Recent studies indicate that endocytosis of Eph-ephrin complexes may be one of the mechanisms by which a high affinity cell-cell adhesion is converted to a repulsive interaction. In this study, we show that EphA8 undergoes clathrin-mediated endocytosis upon treatment with ephrin-A5, and that EphA8 is associated tightly with Tiam-1, a Rac-specific guanine nucleotide exchange factor. Analysis of EphA8 deletion mutants revealed that a juxtamembrane region in EphA8 is critically involved in endocytosis of EphA8-ephrinA5 complexes. An EphA8 mutant lacking this juxtamembrane portion was defective for endocytosis with ephrinA5, and also displayed a weak association with Tiam-1. Expression of an endocytosis-defective version of EphA8 resulted in a low level of Rac activity in response to ephrin-A5 stimulation. More importantly, down-regulation of Tiam-1 resulted in inefficient endocytosis of EphA8-ephrinA5 complexes. These results suggest that Tiam-1 plays a role in clathrin-dependent endocytosis of EphA8-ephrinA5 complexes.

To initiate defense responses against invasion of pathogenic organisms, animals and plants must recognize microbe-associated molecular patterns (MAMPs). In this study, the elicitor activity of bacterial DNA on the model plant Arabidopsis thaliana was examined. EcoRI-digested plasmid DNA induced defense responses such as generation of reactive oxygen species and deposition of callose, whereas SmaI- and HapII-digested plasmid DNA and EcoRI-digested herring DNA did not remarkably induce these responses. Further, methylation of the CpG sequence of plasmid DNA and Escherichia coli DNA reduced the level of the defense responses. The endocytosis inhibitors wortmannin and amantadine significantly inhibited DNA-induced defense responses. These results suggest that nonmethylated CpG DNA, as a MAMP, induced defense responses in Arabidopsis and that non-methylated DNA seems to be translocated into the cytoplasm by endocytosis.

C. elegans coelomocytes are macrophage-like scavenger cells that provide an excellent in vivo system for the study of clathrin-mediated endocytosis. Using this in vivo system, several genes involved in coelomocyte endocytosis have been identified previously. However, the detailed mechanism of endocytic pathway is still unknown. Here, we report a new function of calcineurin, an evolutionarily conserved Ca²+/calmodulin-dependent Ser/Thr protein phosphatase, in coelomocyte endocytosis. We found that calcineurin mutants show defective coelomocyte endocytosis. Genetic analysis suggests that calcineurin and a GTPase, dynamin (DYN-1), may function upstream of an orphan receptor, CUP-4, to regulate endocytosis. Therefore, we propose a model in which calcineurin may regulate coelomocyte endocytosis via DYN-1 and CUP-4 in C. elegans.

The aim of this work was to characterize the placental uptake of folic acid from the maternal circulation. Using 2 human trophoblast cell lines (BeWo and JAR), we verified that uptake of 3 H-folic acid was pH-dependent, increasing significantly with decreasing extracellular pH. In BeWo cells, uptake of 3 H-folic acid at pH 5.5 was (i) Na + -independent;; (ii) inhibited by folic acid, 5-methyltetrahydrofolate (5-MTHF), and methotrexate (MTX);; (iii) inhibited by the anion transport inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) and 4-acetamido-4′-isothiocyano-2,2′-disulfonic acid stilbene (SITS);; (iv) inhibited by the proton ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP);; (v) not inhibited by blockers of receptor-mediated endocytosis (cytochalasin D and monensin);; (vi) trans-inhibited by MTX and folic acid;; and (vii) not affected by an anti-reduced folate transporter-1 (RFC) antibody. At pH 7.5, uptake of 3 H-folic acid was (i) Na + -independent;; (ii) inhibited by folic acid and MTX, but not by 5-MTHF;; (iii) inhibited by SITS, but not by DIDS;; (iv) not affected by FCCP;; (v) inhibited by monensin (but not by cytochalasin D);; (vi) trans-inhibited by folic acid (but not by MTX);; and (vii) inhibited by an anti-RFC antibody. In conclusion, in BeWo cells, both RFC and receptor-mediated endocytosis seem to be involved in 3 H-folic acid uptake at pH 7.5, whereas at pH 5.5, RFC and (or) a low pH-operating transporter distinct from RFC are involved.

Large amounts of lipofuscin-like pigments were demonstrated by means of the Schmorl's technique in endocardial cells enveloping the heart muscle trabeculae in adult platyfish (Xiphophorus maculatus). Such pigment granules occurred in a low number in the corresponding cell layers in the heart of 6-mm long prenatal platies. The endocardial cells contained numerous lysosome-like bodies, bristle-coated vesicles, and tubules of agranular endoplasmic reticulum, particularly in adult specimens. The lipofuscin-rich endocardial cell layers displayed deep blue precipitations in hearts from the ferritin-injected adult platies 6 h or more post-injection, when treated with acid ferrocyanide instead of Schmorl's solution, i.e., this cell type is able to take up foreign particles. The occurrence of much lipofuscin-like pigments in endocardial cell layers in adult platies provides evidence that these cells have performed important scavenger functions at this stage of development.