Process Of Endocytosis Research Articles

Coupling of nanostraws with diverse physicochemical perforation strategies for intracellular DNA delivery

Effective intracellular DNA transfection is imperative for cell-based therapy and gene therapy. Conventional gene transfection methods, including biochemical carriers, physical electroporation and microinjection, face challenges such as cell type dependency, low efficiency, safety concerns, and technical complexity. Nanoneedle arrays have emerged as a promising avenue for improving cellular nucleic acid delivery through direct penetration of the cell membrane, bypassing endocytosis and endosome escape processes. Nanostraws (NS), characterized by their hollow tubular structure, offer the advantage of flexible solution delivery compared to solid nanoneedles. However, NS struggle to stably self-penetrate the cell membrane, resulting in limited delivery efficiency. Coupling with extra physiochemical perforation strategies is a viable approach to improve their performance. This study systematically compared the efficiency of NS coupled with polyethylenimine (PEI) chemical modification, mechanical force, photothermal effect, and electric field on cell membrane perforation and DNA transfection. The results indicate that coupling NS with PEI modification, mechanical force, photothermal effects provide limited enhancement effects. In contrast, NS-electric field coupling significantly improves intracellular DNA transfection efficiency. This work demonstrates that NS serve as a versatile platform capable of integrating various physicochemical strategies, while electric field coupling stands out as a form worthy of primary consideration for efficient DNA transfection.

Abstract 6533: Pivotal role of specific LYVE1+ tissue resident macrophages in PDAC progression & immunotherapy resistance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense fibrotic stroma and abundant myeloid cell infiltration, which render it resistant to any therapeutic interventions, including immunocheckpoint blockade (ICB) therapy. Understanding the composition and function of myeloid cells within the PDAC microenvironment may help identify promising targets for PDAC immunotherapy. Tissue resident macrophages (TRMs) contribute to organogenesis and tissue regeneration and metabolism. In this study, we discovered a distinct population of pancreas TRMs that highly express lymphatic vessel hyaluronan receptor 1 (LYVE-1). Single cell RNAseq data reveal that LYVE-1+ TRM cluster in the pancreas expresses high levels of complement activation genes and transcription factors such as c-Maf and Stab1. RNAseq data also confirm that LYVE-1+ TRMs express enriched genes related to mannose binding, endocytosis, and glutathione metabolic process, while LYVE-1 negative macrophages express enriched genes related to antigen processing and presentation, T cell activation, and phagocytosis. Multiparameter flow cytometric analysis demonstrates that LYVE-1+ TRMs express M2-like markers, including increased CD163, CD206 and decreased MHC Class II molecule. During PDAC development and progression, LYVE-1+ TRMs expand and localize around the tumor border, promoting fibrotic stroma formation. Imaging mass cytometry (IMC) also reveals abundant infiltration of LYVE-1+CD14+CD68+ macrophages in human PDAC tumors and colocalized with collagen deposition. This subset of macrophages also highly express c-Maf. Through the use of Lys-Cre-c-Maf f/f mice, we demonstrate that depletion of c-Maf from myeloid cells significantly reduces LYVE-1+ TRMs and polarizes them to resemble an M1-like phenotype. In conclusion, our findings suggest that LYVE-1+ macrophages are a unique subset of myeloid cells within the pancreas and likely play a critical role in PDAC progression and immunotherapeutic resistance. Future work will focus on targeting LYVE1+ TRMs using various approaches, such as conditional knockout mice and small molecule inhibitors, to determine their therapeutic potential for PDAC treatment. Presented by Dr. Hong Li Citation Format: Hong Li, Jun Yan. Pivotal role of specific LYVE1+ tissue resident macrophages in PDAC progression & immunotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6533.

Abstract 43: Targeting trogocytosis through cathepsin B inhibition enhances CAR T cell persistence

Abstract Chimeric Antigen Receptor (CAR) T cell therapy is an effective treatment for cancer patients. While CAR T cells have shown remarkable efficacy, most patients receiving CAR T cell therapy eventually relapse. CAR-mediated trogocytosis (CMT) is a potential tumor escape mechanism involving the transfer of cell surface proteins from tumor cells to CAR T cells. CMT results in antigen-negative tumor cells and correlates with reduced CAR T cell persistence, possibly due to increased CAR T cell fratricide or exhaustion. To date it has not been conclusively demonstrated that antigens transferred by CMT are the cause of fratricide or exhaustion. To answer this question, we developed a system to rapidly degrade trogocytosed protein in CAR T cells. Using this system, we show that the transfer of CD19 to CAR T cells directly causes CAR T cell fratricide and exhaustion. We hypothesize that reducing CMT will increase CAR T cell persistence. To identify molecular mechanisms driving CMT as potential therapeutic targets, we inhibited various proteins essential for cell adhesion, endocytosis, actin polymerization, or antigen processing using small molecule inhibitors. We found that inhibition of the cysteine protease Cathepsin B (CTSB), with a membrane-permeable (Ca-074) or membrane-impermeable (Ca-074-Me) inhibitor, significantly reduced CMT without affecting CAR T cell cytotoxicity. We hypothesize that CTSB inhibition could be a potential therapeutic approach to limit CMT.CTSB activity is regulated by the protein Cystatin A (CSTA). Both Ca-074-Me and CSTA sterically block access to the active site cysteine of CTSB. We found that overexpression of CSTA in CAR T cells significantly reduced CTSB activity and reduced antigen transfer. CSTA overexpression minimized antigen loss on tumor cells, indicating that CTSB inhibition blocks CMT early during the trogocytic process. CSTA overexpression reduced CAR T cell exhaustion but did not alter their expansion, tumor cell killing, or phenotype, indicating that this approach selectively inhibits CMT. Next, we assessed the effect of CSTA overexpression on CAR T cell persistence in vivo. NALM6 tumor-bearing NSG mice were injected with conventional or CSTA-overexpressing CD19 CAR T cells. Four weeks after CAR T cell injection, we observed substantially increased CAR T cell numbers in the blood (10.6x, p=0.0317), bone marrow (7.3x, p=0.0159), and spleens (5.6x, p=0.0556) of mice treated with CSTA-overexpressing CAR T cells. Taken together, we provide the first experimental evidence that CMT directly causes fratricide and exhaustion, reducing CAR T cell persistence. We demonstrate that CMT can be targeted efficiently by overexpressing the CTSB inhibitor CSTA in CAR T cells, resulting in substantially increased CAR T cell persistence. This represents a promising approach to improve CAR T cell efficacy and limit the occurrence of relapse in patients receiving CAR T cell therapy. Citation Format: Kenneth A. Dietze, Michael L. Olson, Etse Gebru, Djordje Atanackovic, Aaron P. Rapoport, Tim Luetkens. Targeting trogocytosis through cathepsin B inhibition enhances CAR T cell persistence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 43.

Abstract 2045: Novel TFRC humanized mouse model for drug delivery to the brain

Abstract Gliomas are the most common neoplasm in the central nervous system (CNS). Although immunotherapy has shown efficacy in several tumor types, no immunotherapies are approved by the US Food and Drug Administration for treatment of gliomas. One of the biggest challenges in using immunotherapies in the CNS is the existence of the blood-brain barrier (BBB), which limits drug delivery and the subsequent therapeutic drug efficacy. In addition to biodistribution, assessment of PK in preclinical models do not reflect the clinical findings. Conjugation of therapeutic agents with molecules binding to TRFC expressed along the BBB are being used as one of the alternative approaches to deliver drugs into the CNS. Herein, we describe a novel TFRC humanized mouse model (hTFRC), developed to enable assessment of compounds targeting human TFRC and aiming to deliver drug through the BBB. hTFRC model was developed by knock-in at the mouse endogenous locus to enable a physiological expression of TFRC. Splenocytes from hTFRC mice stimulated with labelled pHrodo Red human transferrin show internalization of human transferrin, suggesting that humanization of the transferrin receptor does not change the endocytosis process. In vivo, treatment of hTFRC mice with anti-BACE1/anti-TFRC antibodies enables the transfer of anti-BACE1 to the brain, while anti-BACE1 antibody is not. As a consequence of the drug delivery, mice treated with anti-BACE1/anti-TFRC show a marked reduction in Amyloid-β 1-40 levels in the brain. Altogether, the data suggest that hTFRC is expressed in the BBB and enables the efficient shuttling of therapeutic antibodies to the brain. While assessment of TFRC-targeting antibodies in a glioma model remains to be performed, the hTFRC model is a promising tool to assess delivery and efficacy of immunotherapies in gliomas. Moreover, the hTFRC model is also being intercrossed with the well-established hSA/FcRn humanized mouse model (Viuff et al., 2016), to enable a more translatable PK and biodistribution of compounds targeting TFRC. Citation Format: Fabiane Sonego, Angela Pappalardo, Gaëlle Martin, Burak Ozgür, Peter Ravn, Yacine Cherifi, Florence Sotty, Patricia Isnard-Petit, Kader Thiam. Novel TFRC humanized mouse model for drug delivery to the brain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2045.

Macropinocytic cups function as signal platforms for the mTORC2-AKT pathway to modulate LPS-induced cytokine expression in macrophages.

Macropinocytosis is a large-scale endocytosis process primarily observed in phagocytes as part of their cellular function to ingest antigens. Once phagocytes encounter gram-negative bacteria, the receptor proteins identify lipopolysaccharides (LPSs), which trigger radical membrane ruffles that gradually change to cup-like structures. The open area of the cups closes to generate vesicles called macropinosomes. The target bacteria are isolated by the cups and engulfed by the cells as the cups close. In addition to its ingestion function, macropinocytosis also regulates the AKT pathway in macrophages. In the current study, we report that macropinocytic cups are critical for LPS-induced AKT phosphorylation (pAKT) and cytokine expression in macrophages. High-resolution scanning electron microscope observations detailed the macropinocytic cup structures induced by LPS stimulation. Confocal microscopy revealed that AKT and the kinase molecule mTORC2 were localized in the cups. The biochemical analysis showed that macropinocytosis inhibition blocked LPS-induced pAKT. RNA sequencing, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay analyses revealed that the inhibition of macropinocytosis or the AKT pathway causes a decrease in the expression of proinflammatory cytokines interlukin-6 and interlukin-1α. Moreover, activation of the transcription factor nuclear factor κB, which regulates the cytokine expression downstream of the AKT/IκB pathway, was hindered when macropinocytosis or AKT was inhibited. These results indicate that LPS-induced macropinocytic cups function as signal platforms for the AKT pathway to regulate the cytokine expression by modulating nuclear factor κB activity in LPS-stimulated macrophages. Based on these findings, we propose that macropinocytosis may be a good therapeutic target for controlling cytokine expression.

Dynein functions in galectin-3 mediated processes of clathrin-independent endocytosis

Dynein functions in galectin-3 mediated processes of clathrin-independent endocytosis

Materiobiomodulated ROS Therapy for De Novo Hair Growth.

Hair loss is characterized by the inability of hair follicles (HFs) to enter the telogen-anagen transition (TAT) and lack of de novo HFs. Current pharmaceutical therapies and surgical modalities have been largely limited to regulating hair regrowth efficiently without side effects and lacking treatment compliance. Here, this work proposes a materiobiomodulation therapy (MBMT), wherein polydopamine (PDA) nanoparticles with redox activity can be modulated to have a stoichiometric ROS (H2O2) donating ability. These nanoparticles can intracellularly deliver ROS with high-efficiency via the clathrin-dependent endocytosis process. Utilizing homozygote transgenic HyPerion (a genetically-encoded H2O2 biosensor) mice, this work also achieves in vivo dynamic monitoring of intracellular H2O2 elevation induced by ROS donators. Subcutaneous administration with ROS donators results in rapid onset of TAT and subsequent hair regrowth with a specific ROS "hormesis effect." Mechanistically, ROS activate β-catenin-dependent Wnt signaling, upregulating hair follicle stem cell expression. This work further develops a microneedles patch for transdermal ROS delivery, demonstrating long-term, low-dose ROS release. Unlike photobiomodulation therapy (PBMT), MBMT requires no external stimuli, providing a convenient and efficient approach for clinical hair loss treatment. This material-HF communication implicates new avenues in HF-related diseases, achieving targeted ROS delivery with minimal side effects.

Integrated physio-biochemical and transcriptomic analyses reveal the mechanism underlying ABA-mediated alleviation of salt stress in Limonium bicolor seedlings

Abscisic acid (ABA) plays an important role in regulating plant stress responses. However, there are currently no reports on ABA in regulating the salt tolerance of Limonium bicolor. Here, we analyzed the effects of exogenous ABA on growth, photosynthesis, salt secretion, and physiological and transcriptome changes of L. bicolor seedlings under 300 mM NaCl. Exogenous 10 µM ABA treatment in L. bicolor seedlings treated with salt upregulated the expression of genes related to stomatal development, increased the density and number of stomata, and improved plant gas exchange capacity, thereby increasing the net photosynthesis rate. Exogenous ABA treatment in L. bicolor seedlings under salt stress also upregulated the expression of genes related to salt gland development, promoted salt glands development, increased salt glands density, increased the salt secretion capacity of leaves, and reduced the accumulation of Na+ in the plant. ABA application enhanced salt tolerance of L. bicolor seedlings by increasing content of soluble solutes, enhancing antioxidant enzymes activities, decreasing accumulation of reactive oxygen species, and increasing ABA and indole acetic acid contents. Transcriptome sequencing revealed that genes differentially expressed in L. bicolor seedlings during ABA-induced salt tolerance are involved in many metabolic pathways, including plant hormone signal transduction, proline metabolism, endocytosis and protein processing in the endoplasmic reticulum. Furthermore, some transcription factors, MYB, bHLH, and WRKY, might contribute to the improved salt stress tolerance of L. bicolor conferred by ABA. These results provide a basis for further clarifying the salt tolerance mechanism mediated by exogenous ABA in L. bicolor seedlings.

Mannose Ligands for Mannose Receptor Targeting.

The mannose receptor (MR, CD 206) is an endocytic receptor primarily expressed by macrophages and dendritic cells, which plays a critical role in both endocytosis and antigen processing and presentation. MR carbohydrate recognition domains (CRDs) exhibit a high binding affinity for branched and linear oligosaccharides. Furthermore, multivalent mannose presentation on the various templates like peptides, proteins, polymers, micelles, and dendrimers was proven to be a valuable approach for the selective and efficient delivery of various therapeutically active agents to MR. This review provides a detailed account of the most relevant and recent aspects of the synthesis and application of mannosylated bioactive formulations for MR-mediated delivery in treatments of cancer and other infectious diseases. It further highlights recent findings related to the necessary structural features of the mannose-containing ligands for successful binding to the MR.

Quantifying Protein Dynamics by Kymograph Analysis.

Time-lapse imaging of the subcellular localization and dynamic behavior of proteins is critical to understand their biological functions in cells. With the advent of various methodologies and computational tools, the precise tracking and quantification of protein spatiotemporal dynamics have become feasible. Kymograph analysis, in particular, has been extensively adopted for the quantitative assessment of proteins, vesicles, and organelle movements. However, conventional kymograph analysis, which is based on a single linear trajectory, may not comprehensively capture the complexity of proteins that alter their course during intracellular transport and activity. In this chapter, we introduced an advanced protocol for whole-cell kymograph analysis that allows for three-dimensional (3D) tracking of protein dynamics. This method was validated through the analysis of tip-focused endocytosis and exocytosis processes in growing tobacco pollen tubes by employing both the advanced whole-cell and classical kymograph methods. In addition, we enhanced this method by integrating pseudo-colored kymographs that enables the direct visualization of changes in protein fluorescence intensity with fluorescence recovery after photobleaching to advance our understanding of protein localization and dynamics. This comprehensive method offers a novel insight into the intricate dynamics of protein activity within the cellular context.

Direct observation of cortactin protecting Arp2/3-actin filament branch junctions from GMF-mediated destabilization

How cells tightly control the formation and turnover of branched actin filament arrays to drive cell motility, endocytosis, and other cellular processes is still not well understood. Here, we investigated the mechanistic relationship between two binding partners of the Arp2/3 complex, glia maturation factor (GMF) and cortactin. Individually, GMF and cortactin have opposite effects on the stability of actin filament branches, but it is unknown how they work in concert with each other to govern branch turnover. Using TIRF microscopy, we observe that GMF’s branch destabilizing activities are potently blocked by cortactin (IC50 = 1.3 nM) and that this inhibition requires direct interactions of cortactin with Arp2/3 complex. The simplest model that would explain these results is competition for binding Arp2/3 complex. However, we find that cortactin and GMF do not compete for free Arp2/3 complex in solution. Further, we use single molecule analysis to show that cortactin’s on-rate (3 ×107 s−1 M−1) and off-rate (0.03 s−1) at branch junctions are minimally affected by excess GMF. Together, these results show that cortactin binds with high affinity to branch junctions, where it blocks the destabilizing effects of GMF, possibly by a mechanism that is allosteric in nature. In addition, the affinities we measure for cortactin at actin filament branch junctions (Kd = 0.9 nM) and filament sides (Kd = 206 nM) are approximately 20-fold stronger than previously reported. These observations contribute to an emerging view of molecular complexity in how Arp2/3 complex is regulated through the integration of multiple inputs.

Integrated transcriptomic and proteomic analyses of two sugarcane (Saccharum officinarum Linn.) varieties differing in their lodging tolerance

BackgroundLodging seriously affects sugarcane stem growth and sugar accumulation, reduces sugarcane yield and sucrose content, and impedes mechanization. However, the molecular mechanisms underlying sugarcane lodging tolerance remain unclear. In this study, comprehensive transcriptomic and proteomic analyses were performed to explore the differential genetic regulatory mechanisms between upright (GT42) and lodged (GF98-296) sugarcane varieties.ResultsThe stain test showed that GT42 had more lignin and vascular bundles in the stem than GF98-296. The gene expression analysis revealed that the genes that were differentially expressed between the two varieties were mainly involved in the phenylpropanoid pathway at the growth stage. The protein expression analysis indicated that the proteins that were differentially expressed between the two varieties were related to the synthesis of secondary metabolites, the process of endocytosis, and the formation of aminoacyl-tRNA. Time-series analysis revealed variations in differential gene expression patterns between the two varieties, whereas significant protein expression trends in the two varieties were largely consistent, except for one profile. The expression of CYP84A, 4CL, and CAD from the key phenylpropanoid biosynthetic pathway was enhanced in GT42 at stage 2 but suppressed in GF98-296 at the growth stage. Furthermore, the expression of SDT1 in the nicotinate and nicotinamide metabolism was enhanced in GT42 cells but suppressed in GF98-296 cells at the growth stage.ConclusionOur findings provide reference data for mining lodging tolerance-related genes that are expected to facilitate the selective breeding of sugarcane varieties with excellent lodging tolerance.

Enhancing the production of recombinant human TGF-β1 through an understanding of TGF-β1 synthesis, signaling, and endocytosis in CHO cells.

To enhance the production of recombinant human transforming growth factor-beta1 (rhTGF-β1) in Chinese hamster ovary (CHO) cells, rhTGF-β1 was first characterized for endocytosis, signaling pathway, and overall maturation process. The mature rhTGF-β1 used for clinical application was internalized into CHO cells and inhibited the growth of CHO cells in a dose-dependent manner. However, mature rhTGF-β1 was mostly produced in the form of latent rhTGF-β1 in cultures of recombinant CHO (rCHO) cells producing rhTGF-β1 (CHO-rhTGF-β1). The concentration of active mature rhTGF-β1 in the culture supernatant of CHO-rhTGF-β1 cells was not high enough to compromise yield. In addition, a significant amount of unprocessed precursors was produced by CHO-rhTGF-β1 cells. Overexpression of PACEsol, a soluble form of furin, in CHO-rhTGF-β1 cells was effective for the proteolytic cleavage of unprocessed precursors. The highest mature rhTGF-β1 concentration (6.4μgmL-1 ) was obtained with the PACEsol-expressing clone, which was approximately 45% higher than that of the parental clone (P<0.01). Thus, a comprehensive understanding of the intrinsic properties of rhTGF-β1 with respect to the overall maturation process, signaling pathway, and endocytosis is essential for effectively enhancing the production of mature rhTGF-β1 in CHO cells.

Inhibiting Multidrug Resistance with Transferrin-Targeted Polymersomes through Optimization of Ligand Density.

Transferrin-conjugated polymersomes, transferrin-biotin/avidin/biotin-Pluronic F127-poly(lactic acid) (Tf-F127-PLA), were successfully prepared through a biotin-avidin bridging technique to study their ability to inhibit multidrug resistance of cancer cells. Hydrophilic doxorubicin (DOX) was selected as the model drug to be loaded into Tf-F127-PLA polymersomes. DOX loaded in Tf-F127-PLA polymersomes was released fast initially, followed by a slow release. The effect of the transferrin ligand density of Tf-F127-PLA/DOX polymersomes on their targeting properties was studied by both cytotoxicity and cellular uptake assays against A549 lung cancer cells. It was shown that Tf-F127-PLA/DOX polymersomes had better targeting ability than nontargeted drug-loaded polymersomes. Furthermore, Tf-F127-PLA/DOX polymersomes with 2% Tf molar content have more effective antitumor activity and a higher cellular uptake than those with 4 and 5% Tf molar content. 2% Tf-F127-PLA/DOX polymersomes also exhibited better anticancer ability in multidrug resistant cancer cells A549/ADR than nontargeted PLA-F127-PLA/DOX polymersomes. It was further proved that the endocytosis of polymersomes by A549/ADR cells was an energy-dependent endocytosis process, which was related to clathrin, macrocytosis, and caveolin. Also, the endocytosis of Tf-F127-PLA/DOX polymersomes was proven to be mediated by the transferrin receptor.

Folic acid-decorated vitamin E Poly(ethylene glycol) monoplatinum ester with disulfide bond as theranostic nanoparticle for drug resistant tumor diagnosis and treatment

Vitamin E Poly(ethylene glycol) monoplatinum ester (TPGS) nanoparticles have attracted much attention in recent years for overcome multidrug resistance. Herein, a well-defined folic acid (FA)-conjugated and disulfide bond-linked polymer (FA-SS-TPGS) was synthesized. These polymer nanoparticles were utilized as theranostic agents for tumor-targeted magnetic resonance imaging (MRI) and chemotherapy. By loading doxorubicin (DOX) and superparamagnetic iron oxide (SPIO) particles into TPGS nanoparticles, FA-SS-TPGS@DOX/SPIO nanoparticles are obtained. In vitro drug release studies revealed that under a reducing environment in the presence of glutathione (GSH), approximately 100% of the doxorubicin (DOX) was released from the disulfide bond-linked theranostic nanoparticles within 24 h. DOX and SPIO were efficiently delivered into HepG2-ADM cells due to the folate receptor-mediated endocytosis process of the nanoparticles. Additionally, the presence of glutathione (GSH) triggered the cleaving of the disulfide bonds, further facilitating the delivery of DOX and SPIO into the cells. Furthermore, the FA-SS-TPGS @DOX-SPIO nanoparticles exhibited strong MRI contrast enhancement properties. In conclusion, FA-SS-TPGS@DOX/SPIO are potential nanoparticles for tumor-targeted MRI and chemotherapy, which can also overcome multidrug resistance.

Small Plasma Membrane-Targeted Fluorescent Dye for Long-Time Imaging and Protein Degradation Analyses.

Plasma membrane (PM)-targeted fluorescent dyes have become an important tool to visualize morphological and dynamic changes in the cell membrane. However, most of these PM dyes are either too large and thus might potentially perturb the membrane and affect its functions or exhibit a short retention time on the cell membrane. The rapid internalization problem is particularly severe for PM dyes based on cationic and neutral hydrophobic fluorescent dyes, which can be easily transported into the cells by transmembrane potential and passive diffusion mechanisms. In this paper, we report a small but highly specific PM fluorescent dye, PM-1, which exhibits a very long retention time on the plasma membrane with a half-life of approximately 15 h. For biological applications, we demonstrated that PM-1 can be used in combination with protein labeling probes to study ectodomain shedding and endocytosis processes of cell surface proteins and successfully demonstrated that native transmembrane human carbonic anhydrase IX (hCAIX) is degraded via the ectodomain shedding mechanism. In contrast, hCAIX undergoes endocytic degradation in the presence of sheddase inhibitors. We believe that PM-1 can be a versatile tool to provide detailed insights into the dynamic processes of the cell surface proteins.

Emerging roles of proximal tubular endocytosis in renal fibrosis.

Endocytosis is a crucial component of many pathological conditions. The proximal tubules are responsible for reabsorbing the majority of filtered water and glucose, as well as all the proteins filtered through the glomerular barrier via endocytosis, indicating an essential role in kidney diseases. Genetic mutations or acquired insults could affect the proximal tubule endocytosis processes, by disturbing or overstressing the endolysosomal system and subsequently activating different pathways, orchestrating renal fibrosis. This paper will review recent studies on proximal tubular endocytosis affected by other diseases and factors. Endocytosis plays a vital role in the development of renal fibrosis, and renal fibrosis could also, in turn, affect tubular endocytosis.

Comprehensive pan-cancer analysis of expression profiles and prognostic significance for NUMB and NUMBL in human tumors.

NUMB has been initially identified as a critical cell fate determinant that modulates cell differentiation via asymmetrical partitioning during mitosis, including tumor cells. However, it remains absent that a systematic assessment of the mechanisms underlying NUMB and its homologous protein NUMBLIKE (NUMBL) involvement in cancer. This study aimed to investigate the prognostic significance for NUMB and NUMBL in pan-cancer. In this study, using the online databases TIMER2.0, gene expression profiling interactive analysis, cBioPortal, the University of ALabama at Birmingham CANcer data analysis Portal, SearchTool for the Retrieval of Interacting Genes/Proteins, and R software, we focused on the relevance between NUMB/NUMBL and oncogenesis, progression, mutation, phosphorylation, function and prognosis. This study demonstrated that abnormal expression of NUMB and NUMBL were found to be significantly associated with clinicopathologic stages and the prognosis of survival. Besides, genetic alternations of NUMB and NUMBL focused on uterine corpus endometrial carcinoma, and higher genetic mutations of NUMBL were correlated with more prolonged overall survival and disease-free survival in different cancers. Moreover, S438 locus of NUMB peptide fragment was frequently phosphorylated in 4 cancer types and relevant to its phosphorylation sites. Furthermore, endocytosis processing and neurogenesis regulation were involved in the functional mechanisms of NUMB and NUMBL separately. Additionally, the pathway enrichment suggested that NUMB was implicated in Hippo, Neurotrophin, Thyroid hormone, and FoxO pathways, while MAPK, Hippo, Rap1, mTOR, and Notch pathways were related to the functions of NUMBL. This study highlights the predictive roles of NUMB and NUMBL in pan-cancer, suggesting NUMB and NUMBL might be served as potential biomarkers for diagnosis and prognosis in various malignant tumors.

Integrated analysis of mRNAs and lncRNAs reveals candidate marker genes and potential hub lncRNAs associated with growth regulation of the Pacific Oyster, Crassostrea gigas

BackgroundThe Pacific oyster, Crassostrea gigas, is an economically important shellfish around the world. Great efforts have been made to improve its growth rate through genetic breeding. However, the candidate marker genes, pathways, and potential lncRNAs involved in oyster growth regulation remain largely unknown. To identify genes, lncRNAs, and pathways involved in growth regulation, C. gigas spat was cultured at a low temperature (15 ℃) to yield a growth-inhibited model, which was used to conduct comparative transcriptome analysis with spat cultured at normal temperature (25 ℃).ResultsIn total, 8627 differentially expressed genes (DEGs) and 1072 differentially expressed lncRNAs (DELs) were identified between the normal-growth oysters (cultured at 25 ℃, hereinafter referred to as NG) and slow-growth oysters (cultured at 15 ℃, hereinafter referred to as SG). Functional enrichment analysis showed that these DEGs were mostly enriched in the AMPK signaling pathway, MAPK signaling pathway, insulin signaling pathway, autophagy, apoptosis, calcium signaling pathway, and endocytosis process. LncRNAs analysis identified 265 cis-acting pairs and 618 trans-acting pairs that might participate in oyster growth regulation. The expression levels of LNC_001270, LNC_003322, LNC_011563, LNC_006260, and LNC_012905 were inducible to the culture temperature and food abundance. These lncRNAs were located at the antisense, upstream, or downstream of the SREBP1/p62, CDC42, CaM, FAS, and PIK3CA genes, respectively. Furthermore, the expression of the trans-acting lncRNAs, including XR_9000022.2, LNC_008019, LNC_015817, LNC_000838, LNC_00839, LNC_011859, LNC_007294, LNC_006429, XR_002198885.1, and XR_902224.2 was also significantly associated with the expression of genes enriched in AMPK signaling pathway, insulin signaling pathway, autophagy, apoptosis, calcium signaling pathway, and endocytosis process.ConclusionsIn this study, we identified the critical growth-related genes and lncRNAs that could be utilized as candidate markers to illustrate the molecular mechanisms underlying the growth regulation of Pacific oysters.

Nuclear Transport of the Molecular Drug via Nanocarrier-Based Nonendocytic Cellular Uptake.

Although subcellular targeting can enhance the therapeutic performance of most drugs, such targeting requires appropriate carrier-based delivery that can bypass endosomal/lysosomal trafficking. Recent works show that nanocarriers can be designed for direct cell membrane translocation and nonendocytic uptake, bypassing the usual endocytosis processes. Here we show that this approach can be adapted for the rapid cell nucleus delivery of molecular drugs. In particular, a guanidinium-terminated nanocarrier is used to create a weak interaction-based carrier-drug nanoassembly for direct membrane translocation into the cytosol. The rapid and extensive entry of a drug-loaded nanocarrier into the cell without any vesicular coating and affinity of the drug to the nucleus allows their nucleus labeling. Compared to endocytotic uptake that requires more than hours for cell uptake followed by predominant lysosomal entrapment, this nonendocytic uptake labels the nucleus within a few minutes without any lysosomal trafficking. This approach may be utilized for nanocarrier-based subcellular targeting of drugs for more effective therapy.