RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl-/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.

Safety and effectiveness are the cornerstone objectives of nanomedicine in developing nanotherapies. It is crucial to understand the biological interactions between nanoparticles and immune cells. This study focuses on the manufacture by the microfluidic technique of N-trimethyl chitosan/protein nanocarriers and their interaction with J774 cells to elucidate the cellular processes involved in absorption and their impact on the immune system, mainly through endocytosis, activation of lysosomes and intracellular degradation. TEM of the manufactured nanoparticles showed spherical morphology with an average diameter ranging from 36 ± 16 nm to 179 ± 92 nm, depending on the concentration of the cargo protein (0, 12, 55 μg/mL). FTIR showed the crosslinking between N-trimethyl chitosan and the sodium tripolyphosphate and the α-helix binding loss of BSA. TGA revealed an increase in the thermal stability of N-trimethyl chitosan/protein nanoparticles compared with the powder. The encapsulation of the cargo protein used was demonstrated using XPS. Their potential to improve cell permeability and use as nanocarriers in future vaccine formulations was demonstrated. The toxicity of the nanoparticles in HaCaT and J774 cells was studied, as well as the importance of evaluating the differentiation status of J774 cells. Thus, possible endocytosis pathways and their impact on the immune response were discussed. This allowed us to conclude that N-trimethyl chitosan nanoparticles show potential as carriers for the immune system. Still, more studies are required to understand their effectiveness and possible use in therapies.

Mitochondria are crucial for cellular ATP production. They are highly dynamic organelles, whose morphology and function are controlled through mitochondrial fusion and fission. The specific roles of mitochondria in podocytes, the highly specialized cells of the kidney glomerulus, remain less understood. Given the significant structural, functional, and molecular similarities between mammalian podocytes and Drosophila nephrocytes, we employed fly nephrocytes to explore the roles of mitochondria in cellular function. Our study revealed that alterations in the Pink1-Park (mammalian PINK1-PRKN) pathway can disrupt mitochondrial dynamics in Drosophila nephrocytes. This disruption led to either fragmented or enlarged mitochondria, both of which impaired mitochondrial function. The mitochondrial dysfunction subsequently triggered defective intracellular endocytosis, protein aggregation, and cellular damage. These findings underscore the critical roles of mitochondria in nephrocyte functionality.

Schisandra chinensis, a traditional Chinese medicine known for its antitussive and sedative effects, has shown promise in preventing various viral infections. Bovine herpesvirus-1 (BoHV-1) is an enveloped DNA virus that causes respiratory disease in cattle, leading to significant economic losses in the industry. Because the lack of previous reports on Schisandra chinensis resisting BoHV-1 infection, this study aimed to investigate the specific mechanisms involved. Results from TCID50, qPCR, IFA, and western blot analyses demonstrated that Schisandra chinensis could inhibit BoHV-1 entry into MDBK cells, primarily through its extract Methylgomisin O (Meth O). The specific mechanism involved Meth O blocking BoHV-1 entry into cells via clathrin- and caveolin-mediated endocytosis by suppressing the activation of PI3K-Akt signaling pathway. Additionally, findings from TCID50, qPCR, co-immunoprecipitation and western blot assays revealed that Schisandra chinensis blocked BoHV-1 gD transcription through enhancing m6A methylation of gD after virus entry, thereby hindering gD protein expression and preventing progeny virus entry into cells and ultimately inhibiting BoHV-1 replication. Overall, these results suggest that Schisandra chinensis can resist BoHV-1 infection by targeting the PI3K-Akt signaling pathway and inhibiting gD transcription.

The incorporation of different molecules by eukaryotic cells occurs through endocytosis, which is critical to the cell\'s survival and ability to reproduce. Although this process has been studied in greater detail in mammalian and yeast cells, several groups working with pathogenic protists have made relevant contributions. This review analysed the most relevant data on the endocytic process in anaerobic protists (Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, and Tritrichomonas foetus). Many protozoa can exert endocytic activity across their entire surface and do so with great intensity, as with E. histolytica. The available data on the endocytic pathway and the participation of PI-3 kinase, Rab, and Rho molecular complexes is reviewed from a historical perspective.

Acute respiratory infections contribute to morbidity and mortality worldwide. The common cause of this deadly disease is a virus, and one of the most commonly found is the influenza virus. Influenza viruses have several capabilities in infection, including utilizing the host\'s machinery to survive within cells and replicate safely. This review aims to examine the literature on how influenza viruses use host machinery, including endocytosis and autophagy, for their internalization and replication within cells. This review method involves a literature search by examining articles published in the PubMed and Scopus databases. The keywords used were \"Endocytosis\" OR \"Autophagy\" AND \"Influenza Virus\". Eighteen articles were included due to inclusion and exclusion criteria. GTPases switch, and V-ATPase plays a key role in the endocytic machinery hijacked by influenza viruses to enter host cells. On the other hand, LC3 and Atg5 facilitate influenza-induced apoptosis via the autophagic pathway. In conclusion, influenza viruses primarily use clathrin-mediated endocytosis to enter cells and avoid degradation during endosomal maturation by exiting endosomes for transfer to the nucleus for replication. It also uses autophagy to induce apoptosis to continue replication. The capability of the influenza viruses to hijack endocytosis and autophagy mechanisms could be critical points for further research. Therefore, we discuss how the influenza virus utilizes both endocytosis and autophagy and the approach for a new strategic therapy targeting those mechanisms.

The endosomal-lysosomal system represents a crucial degradation pathway for various extracellular substances, and its dysfunction is linked to cardiovascular and neurodegenerative diseases. This degradation process involves multiple steps: (1) the uptake of extracellular molecules, (2) transport of cargos to lysosomes, and (3) digestion by lysosomal enzymes. While cellular uptake and lysosomal function are reportedly regulated by the mTORC1-TFEB axis, the key regulatory signal for cargo transport remains unclear. Notably, our previous study discovered that isorhamnetin, a dietary flavonoid, enhances endosomal-lysosomal proteolysis in the J774.1 cell line independently of the mTORC1-TFEB axis. This finding suggests the involvement of another signal in the mechanism of isorhamnetin. This study analyzes the molecular mechanism of isorhamnetin using transcriptome analysis and reveals that the transcription factor GATA3 plays a critical role in enhanced endosomal-lysosomal degradation. Our data also demonstrate that mTORC2 regulates GATA3 nuclear translocation, and the mTORC2-GATA3 axis alters endosomal formation and maturation, facilitating the efficient transport of cargos to lysosomes. This study suggests that the mTORC2-GATA3 axis might be a novel target for the degradation of abnormal substances.

This study focused on developing an optimal formulation of liposomes loaded with bee venom (BV) and coated with PEG (BV-Lipo-PEG). The liposomes were characterized using dynamic light scattering, transmission electron microscopy, and Fourier transform infrared spectroscopy. Among the liposomal formulations, F3 exhibited the narrowest size distribution with a low PDI value of 193.72 ± 7.35, indicating minimal agglomeration-related issues and a more uniform size distribution. BV-Lipo-PEG demonstrated remarkable stability over 3 months when stored at 4 °C. Furthermore, the release of the drug from the liposomal formulations was found to be pH-dependent. Moreover, BV-Lipo-PEG exhibited favorable entrapment efficiencies, with values reaching 96.74 ± 1.49. The anticancer potential of the liposomal nanocarriers was evaluated through MTT assay, flow cytometry, cell cycle analysis, and real-time experiments. The functionalization of the liposomal system enhanced endocytosis. The IC50 value of BV-Lipo-PEG showed a notable decrease compared to both the free drug and BV-Lipo alone, signifying that BV-Lipo-PEG is more effective in inducing cell death in A549 cell lines. BV-Lipo-PEG exhibited a higher apoptotic rate in A549 cell lines compared to other samples. In A549 cell lines treated with BV-Lipo-PEG, the expression levels of MMP-2, MMP-9, and Cyclin E genes decreased, whereas the expression levels of Caspase3 and Caspase9 increased. These findings suggest that delivering BV via PEGylated liposomes holds significant promise for the treatment of lung cancer.

Signal transduction and homeostasis are regulated by complex protein interactions in the intracellular environment. Therefore, the transportation of impermeable macromolecules (nucleic acids, proteins, and drugs) that control protein interactions is essential for modulating cell functions and therapeutic applications. However, macromolecule transportation across the cell membrane is not easy because the cell membrane separates the intra/extracellular environments, and the types of molecular transportation are regulated by membrane proteins. Cell-penetrating peptides (CPPs) are expected to be carriers for molecular transport. CPPs can transport macromolecules into cells through endocytosis and direct translocation. The transport mechanism remains largely unclear owing to several possibilities. In this review, we describe the methods for investigating CPP conformation, translocation, and cargo transportation using artificial membranes. We also investigated biomolecular transport across living cell membranes via CPPs. Subsequently, we show not only the biochemical applications but also the synthetic biological applications of CPPs. Finally, recent progress in biomolecule and nanoparticle transportation via CPPs into specific tissues is described from the viewpoint of drug delivery. This review provides the opportunity to discuss the mechanism of biomolecule transportation through these two platforms.

A small molecule disulfide unit technology platform based on dynamic thiol exchange chemistry at the cell membrane has the potential for drug delivery. However, the alteration of the CSSC dihedral angle of the disulfide unit caused by diverse substituents directly affects the effectiveness of this technology platform as well as its own chemical stability. The highly stable open-loop relaxed type disulfide unit plays a limited role in drug delivery due to its low dihedral angle. Here, we have built a novel disulfide unit starship based on the 3,4,5-trihydroxyphenyl skeleton through trigonometric bundling. The intracellular delivery results showed that the trigonometric bundling of the disulfide unit starship effectively promoted cellular uptake without any toxicity, which is far more than 100 times more active than that of equipment with a single disulfide unit in particular. Then, the significant reduction in cell uptake capacity (73-93%) using thiol erasers proves that the trigonometric bundling of the disulfide starship is an endocytosis-independent internalization mechanism via a dynamic covalent disulfide exchange mediated by thiols on the cell surface. Furthermore, analysis of the molecular dynamics simulations demonstrated that trigonometric bundling of the disulfide starship can significantly change the membrane curvature while pushing lipid molecules in multiple directions, resulting in a significant distortion in the membrane structure and excellent membrane permeation performance. In conclusion, the starship system we built fully compensates for the inefficiency deficiencies induced by poor dihedral angles.