De Novo Glycan Display on Cell Surfaces Using HaloTag: Visualizing the Effect of the Galectin Lattice on the Lateral Diffusion and Extracellular Vesicle Loading of Glycosylated Membrane Proteins.

Glycans cover the cell surface to form the glycocalyx, which governs a myriad of biological phenomena. However, understanding and regulating glycan functions is extremely challenging due to the large number of heterogeneous glycans that engage in intricate interaction networks with diverse biomolecules. Glycocalyx-editing techniques offer potent tools to probe their functions. In this study, we devised a HaloTag-based technique for glycan manipulation, which enables the introduction of chemically synthesized glycans onto a specific protein (protein of interest, POI) and concurrently incorporates fluorescent units to attach homogeneous, well-defined glycans to the fluorescence-labeled POIs. Leveraging this HaloTag-based glycan-display system, we investigated the influence of the interactions between Gal-3 and various N-glycans on protein dynamics. Our analyses revealed that glycosylation modulates the lateral diffusion of the membrane proteins in a structure-dependent manner through interaction with Gal-3, particularly in the context of the Gal-3-induced formation of the glycan network (galectin lattice). Furthermore, N-glycan attachment was also revealed to have a significant impact on the extracellular vesicle-loading of membrane proteins. Notably, our POI-specific glycan introduction does not disrupt intact glycan structures, thereby enabling a functional analysis of glycans in the presence of native glycan networks. This approach complements conventional glycan-editing methods and provides a means for uncovering the molecular underpinnings of glycan functions on the cell surface.

Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma-PK). Tma-PK was expressed in Escherichia coli and purified from the cells. Tma-PK exhibited higher thermostability than human PK. The purified Tma-PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma-PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma-PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus, the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents.

Extracellular Microvesicles Modified with Arginine-Rich Peptides for Active Macropinocytosis Induction and Delivery of Therapeutic Molecules.

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), transfer bioactive molecules from donor to recipient cells in various pathophysiological settings, thereby mediating intercellular communication. Despite their significant roles in extracellular signaling, the cellular uptake mechanisms of different EV subpopulations remain unknown. In particular, plasma membrane-derived MVs are larger vesicles (100 nm to 1 µm in diameter) and may serve as efficient molecular delivery systems due to their large capacity; however, because of size limitations, receptor-mediated endocytosis is considered an inefficient means for cellular MV uptake. This study demonstrated that macropinocytosis (lamellipodia formation and plasma membrane ruffling, causing the engulfment of large fluid volumes outside cells) can enhance cellular MV uptake. We developed experimental techniques to induce macropinocytosis-mediated MV uptake by modifying MV membranes with arginine-rich cell-penetrating peptides for the intracellular delivery of therapeutic molecules.

Detection of SARS-CoV-2 spike protein D614G mutation using µTGGE.

BACKGROUND: The accurate and expeditious detection of SARS-CoV-2 mutations is critical for monitoring viral evolution, assessing its impact on transmission, virulence, and vaccine efficacy, and formulating public health interventions. In this study, a detection system utilizing micro temperature gradient gel electrophoresis (µTGGE) was developed for the identification of the D614 and G614 variants of the SARS-CoV-2 spike protein. METHODS: The in vitro synthesized D614 and G614 gene fragments of the SARS-CoV-2 spike protein were amplified via polymerase chain reaction and subjected to µTGGE analysis. RESULTS: The migration patterns exhibited by the D614 and G614 variants on the polyacrylamide gel were distinctly dissimilar and readily discernible by µTGGE. In particular, the mid-melting pattern of D614 was shorter than that of G614. CONCLUSIONS: Our results demonstrate the capability of µTGGE for the rapid, precise, and cost-effective detection of SARS-CoV-2 spike protein D614 and G614 variants without the need for sequencing. Therefore, this approach holds considerable potential for use in point-of-care mutation assays for SARS-CoV-2 and other pathogens.

Increase in the solubility of uvsY using a site saturation mutagenesis library for application in a lyophilized reagent for recombinase polymerase amplification.

BACKGROUND: Recombinase uvsY from bacteriophage T4, along with uvsX, is a key enzyme for recombinase polymerase amplification (RPA), which is used to amplify a target DNA sequence at a constant temperature. uvsY, though essential, poses solubility challenges, complicating the lyophilization of RPA reagents. This study aimed to enhance uvsY solubility. METHODS: Our hypothesis centered on the C-terminal region of uvsY influencing solubility. To test this, we generated a site-saturation mutagenesis library for amino acid residues Lys91-Glu134 of the N-terminal (His)6-tagged uvsY. RESULTS: Screening 480 clones identified A116H as the variant with superior solubility. Lyophilized RPA reagents featuring the uvsY variant A116H demonstrated enhanced performance compared to those with wild-type uvsY. CONCLUSIONS: The uvsY variant A116H emerges as an appealing choice for RPA applications, offering improved solubility and heightened lyophilization feasibility.

Inkjet-Based Intracellular Delivery System that Effectively Utilizes Cell-Penetrating Peptides for Cytosolic Introduction of Biomacromolecules through the Cell Membrane.

In the drug delivery system, the cytosolic delivery of biofunctional molecules such as enzymes and genes must achieve sophisticated activities in cells, and microinjection and electroporation systems are typically used as experimental techniques. These methods are highly reliable, and they have high intracellular transduction efficacy. However, a high degree of proficiency is necessary, and induced cytotoxicity is considered as a technical problem. In this research, a new intracellular introduction technology was developed through the cell membrane using an inkjet device and cell-penetrating peptides (CPPs). Using the inkjet system, the droplet volume, droplet velocity, and dropping position can be accurately controlled, and minute samples (up to 30 pL/shot) can be carried out by direct administration. In addition, CPPs, which have excellent cell membrane penetration functions, can deliver high-molecular-weight drugs and nanoparticles that are difficult to penetrate through the cell membrane. By using the inkjet system, the CPPs with biofunctional cargo, including peptides, proteins such as antibodies, and exosomes, could be accurately delivered to cells, and efficient cytosolic transduction was confirmed.

Application of recombinant human pyruvate kinase in recombinase polymerase amplification.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. In this study, we attempted to use pyruvate kinase instead of creatine kinase (CK) that has been consistently used as an ATP-regenerating enzyme in RPA. Human pyruvate kinase M1 (PKM) was expressed in Escherichia coli and purified from the cells. RPA with PKM was performed at 41°C with the in vitro synthesized urease subunit ß (ureB) DNA from Ureaplasma parvum serovar 3 as a standard DNA. The optimal concentrations of PKM and phosphoenolpyruvate were 20 ng/µL and 10 mM, respectively. The RPA reaction with PKM was more sensitive than that with CK. PKM exhibited higher thermostability than CK, suggesting that the RPA reagents with PKM are preferable to those with CK for onsite use.

Ultrafast sensitivity-controlled and specific detection of extracellular vesicles using optical force with antibody-modified microparticles in a microflow system.

Extracellular vesicles (EVs), including nanoscale exosomes and ectosomes, hold promise as biomarkers that provide information about the cell of origin through their cargo of nucleic acids and proteins, both on their surface and within. Here, we develop a detection method of EVs based on light-induced acceleration of specific binding between their surface and antibody-modified microparticles, using a controlled microflow with three-dimensional analysis by confocal microscopy. Our method successfully detected 103-104 nanoscale EVs in liquid samples as small as a 500 nanoliters within 5 minutes, with the ability to distinguish multiple membrane proteins. Remarkably, we achieved the specific detection of EVs secreted from living cancer cell lines with high linearity, without the need for a time-consuming ultracentrifugation process that can take several hours. Furthermore, the detection range can be controlled by adjusting the action range of optical force using a defocused laser, consistent with the theoretical calculations. These findings demonstrate an ultrafast, sensitive, and quantitative approach for measuring biological nanoparticles, enabling innovative analyses of cell-to-cell communication and early diagnosis of various diseases, including cancer.

Recombinase polymerase amplification using novel thermostable strand-displacing DNA polymerases from Aeribacillus pallidus and Geobacillus zalihae.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), and strand-displacing DNA polymerase (Pol). Component instability and the need to store commercial kits in a deep freezer until use are some limitations of RPA. In a previous study, Bacillus stearothermophilus Pol (Bst-Pol) was used as a thermostable strand-displacing DNA polymerase in RPA. Here, we attempted to optimize the lyophilization conditions for RPA with newly isolated thermostable DNA polymerases for storage at room temperature. We isolated novel two thermostable strand-displacing DNA polymerases, one from a thermophilic bacterium Aeribacillus pallidus (H1) and the other from Geobacillus zalihae (C1), and evaluated their performances in RPA reaction. Urease subunit ß (UreB) DNA from Ureaplasma parvum serovar 3 was used as a model target for evaluation. The RPA reaction with H1-Pol or C1-Pol was performed at 41 °C with the in vitro synthesized standard UreB DNA. The minimal initial copy numbers of standard DNA from which the amplified products were observed were 600, 600, and 6000 copies for RPA with H1-Pol, C1-Pol, and Bst-Pol, respectively. Optimization was carried out using RPA components, showing that the lyophilized RPA reagents containing H1-Pol exhibited the same performance as the corresponding liquid RPA reagents. In addition, lyophilized RPA reagents with H1-Pol showed almost the same activity after two weeks of storage at room temperature as the freshly prepared liquid RPA reagents. These results suggest that lyophilized RPA reagents with H1-Pol are preferable to liquid RPA reagents for onsite use.

Dodecaborate-Encapsulated Extracellular Vesicles with Modification of Cell-Penetrating Peptides for Enhancing Macropinocytotic Cellular Uptake and Biological Activity in Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a radiation therapy for cancer. In BNCT, the internalization of boron-10 atoms by cancer cells induces cell death through the generation of α particles and recoiling lithium-7 nuclei when irradiated with low-energy thermal neutrons. In this study, we aimed to construct exosomes [extracellular vesicles (EVs)]-based drug delivery technology in BNCT. Because of their pharmaceutical advantages, such as controlled immune responses and effective usage of cell-to-cell communication, EVs are potential next-generation drug delivery carriers. In this study, we successfully developed polyhedral borane anion-encapsulated EVs with modification of hexadeca oligoarginine, which is a cell-penetrating peptide, on the EV membrane to induce the actin-dependent endocytosis pathway, macropinocytosis, which leads to efficient cellular uptake and remarkable cancer cell-killing BNCT activity. The simple and innovative technology of the EV-based delivery system with "cassette" modification of functional peptides will be applicable not only for BNCT but also for a wide variety of therapeutic methodologies.

Macropinocytosis-Inducible Extracellular Vesicles Modified with Antimicrobial Protein CAP18-Derived Cell-Penetrating Peptides for Efficient Intracellular Delivery.

The antimicrobial protein CAP18 (approximate molecular weight: 18 000), which was first isolated from rabbit granulocytes, comprises a C-terminal fragment that has negatively charged lipopolysaccharide binding activity. In this study, we found that CAP18 (106-121)-derived (sC18)2 peptides have macropinocytosis-inducible biological functions. In addition, we found that these peptides are highly applicable for use as extracellular vesicle (exosomes, EV)-based intracellular delivery, which is expected to be a next-generation drug delivery carrier. Here, we demonstrate that dimerized (sC18)2 peptides can be easily introduced on EV membranes when modified with a hydrophobic moiety, and that they show high potential for enhanced cellular uptake of EVs. By glycosaminoglycan-dependent induction of macropinocytosis, cellular EV uptake in targeted cells was strongly increased by the peptide modification made to EVs, and intriguingly, our herein presented technique is efficiently applicable for the cytosolic delivery of the biologically cell-killing functional toxin protein, saporin, which was artificially encapsulated in the EVs by electroporation, suggesting a useful technique for EV-based intracellular delivery of biofunctional molecules.

Preparation and Antiviral Activity of Some New C3- and CS-Symmetrical Tri-Substituted Triazine Derivatives Having Benzylamine Substituents.

We report the preparation of new C3- and CS-symmetrical molecules constructed on a triazine (TAZ) template. Anti-herpes simplex virus type 1 (anti-HSV-1) and cytotoxic activities against Vero cells of synthesized TAZ derivatives were evaluated. The results suggested that the presence of an electron-donating group(s) on the benzene ring in benzylamine groups on the TAZ template is an important structural factor for expressing a high level of anti-HSV-1 activity and low cytotoxicity for these C3 types of TAZ derivatives. Among the tested TAZ derivatives, compounds 4f and 7h showed the highest anti HSV-1 activities (EC50=0.98 and 1.23 µM, respectively) and low cytotoxic activities to Vero cells (50% cytotoxic concentration (CC50)=292.2 and >200 µM, respectively).

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia.

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue organization. Drosophila melanogaster has emerged as a popular experimental model system in cancer biology to study the genetic and cellular mechanisms of tumorigenesis. In particular, genetic tools for Drosophila imaginal discs (developing epithelia in larvae) enable the creation of transformed pro-tumor cells within a normal epithelial tissue, a situation similar to the initial stages of human cancer. A recent study of tumorigenesis in Drosophila wing imaginal discs, however, showed that tumor initiation depends on the tissue-intrinsic cytoarchitecture and the local microenvironment, suggesting that it is important to consider the region-specific susceptibility to tumorigenic stimuli in evaluating tumor phenotypes in imaginal discs. To facilitate phenotypic analysis of tumor progression in imaginal discs, here we describe a protocol for genetic experiments using the GAL4-UAS system to induce neoplastic tumors in wing imaginal discs. We further introduce a diagnosis method to classify the phenotypes of clonal lesions induced in imaginal epithelia, as a clear classification method to discriminate various stages of tumor progression (such as hyperplasia, dysplasia, or neoplasia) had not been described before. These methods might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.

Sirtuin inhibitor Ex-527 causes neural tube defects, ventral edema formations, and gastrointestinal malformations in Xenopus laevis embryos.

Chemical reagent Ex-527 is widely used as a major inhibitor of Sirtuin enzymes, which are a family of highly conserved protein deacetylases and have been linked with caloric restriction and aging by modulating energy metabolism, genomic stability, and stress resistance. However, the extent to which Ex-527 controls early developmental events of vertebrate embryos remains to be understood. Here, we report an examination of Ex-527 effects during Xenopus early development, followed by a confirmation of expressions of xSirt1 and xSirt2 in embryonic stages and enhancement of acetylation by Ex-527. First, we found that reductions in size of neural plate at neurula stages were induced by Ex-527 treatment. Second, tadpoles with short body length and large edematous swellings in the ventral side were frequently observed. Moreover, Ex-527-treated embryos showed severe gastrointestinal malformations in late tadpole stages. Taken together with these results, we conclude that the Sirtuin family start functioning at early embryonic stages and is required for various developmental events.