Role of phosphorylation and vanilloid ligand structure in ligand-dependent differential activations of transient receptor potential vanilloid 1.

Vanilloid analogs, which can activate transient receptor potential vanilloid 1 (TRPV1), have been classified into two types based on susceptibility to forskolin (FSK). Treatment of cells expressing TRPV1 with FSK enhances TRPV1 responses to capsaicin-type ligands while diminishing the responses to eugenol-type ligands. In this study, we determined the effect of FSK on the activation of TRPV1 stimulated with vanilloid ligands, through the influx of Ca2+ in HEK293T cells expressing TRPV1.

Divergent effects of olfactory receptors on transient receptor potential vanilloid 1 activation by capsaicin and eugenol.

We analyzed the effects of olfactory receptors (ORs) on transient receptor potential vanilloid 1 (TRPV1) activation using HEK293T cells co-expressing TRPV1 and OR51E1. We demonstrate here that the effect of OR51E1 on TRPV1 activation varies depending on the two TRPV1 ligands: capsaicin and eugenol. Notably, both of these ligands are vanilloid analogs.

Human Olfactory Receptor Sensor for Odor Reconstitution.

Among the five human senses, light, sound, and force perceived by the eye, ear, and skin, respectively are physical phenomena, and therefore can be easily measured and expressed as objective, univocal, and simple digital data with physical quantity. However, as taste and odor molecules perceived by the tongue and nose are chemical phenomena, it has been difficult to express them as objective and univocal digital data, since no reference chemicals can be defined. Therefore, while the recording, saving, transmitting to remote locations, and replaying of human visual, auditory, and tactile information as digital data in digital devices have been realized (this series of data flow is defined as DX (digital transformation) in this review), the DX of human taste and odor information is not yet in the realization stage.

Immunostick colorimetric assay for highly sensitive detection of food allergens by bio-nanocapsule-scaffolding technology.

The detection sensitivity of immunostick colorimetric assay has been increased by using a bio-nanocapsule as a scaffold for oriented immobilization of immunoglobulin Gs. This immunostick produced ∼82-folds stronger coloration in the detection of food allergens and reduced detection time by a factor of 5.

Mass-Fabrication Scheme of Highly Sensitive Wireless Electrodeless MEMS QCM Biosensor with Antennas on Inner Walls of Microchannel.

Quartz-crystal-microbalance (QCM) biosensor is a typical label-free biosensor, and its sensitivity can be greatly improved by removing electrodes and wires that would be otherwise attached to the surfaces of the quartz resonator. The wireless-electrodeless QCM biosensor was then developed using a microelectro-mechanical systems (MEMS) process, although challenges remain in the sensitivity, the coupling efficiency, and the miniaturization (or mass production). In this study, we establish a MEMS process to obtain a large number of identical ultrasensitive and highly efficient sensor chips with dimensions of 6 mm square.

Specific Binding and Endocytosis of Liposomes to HEK293T Cells via Myrisoylated Pre-S1 Peptide Bound to Sodium Taurocholate Cotransporting Polypeptide.

(1) Background: Sodium taurocholate cotransporting polypeptide (NTCP) functions as a key receptor for the hepatitis B virus (HBV) infection. Analyzing HBV and NTCP interaction is an important issue not only for basic research but also for the development of anti-HBV therapeutics. We developed here a novel model system to analyze the interaction of NTCP with liposomes instead of HBV.

Nano-visualization of the in vitro antiviral activity of black tea based on production area using a liposome-based virus membrane model.

Black tea extracts (BTEs) from four different production areas showed a higher aggregation strength for phosphatidylcholine-based liposomes containing cholesterol used as a viral membrane model. Furthermore, the anti-influenza A virus (IAV) activity of each BTE in vitro demonstrated that although Sri Lanka, Kenya, and Assam had higher anti-IAV activities, Darjeeling had a lower anti-IAV activity, showing a correlation between each BTE and the liposome aggregation strength. Moreover, the antiviral activity strength of BTEs was consistent with the antioxidant activity strength of BTEs, suggesting that the component(s) in black tea that exhibits antioxidant activity would also be the component(s) that accounts for its antiviral activity.

Effects of 3-octen-2-one on human olfactory receptor responses to vanilla flavor.

Most of the odors that humans perceive daily are complex odors. It is believed that the modulation, enhancement, and suppression of overall complex odors are caused by interactions between odor molecules. In this study, to understand the interaction between odor molecules at the level of human olfactory receptor responses, the effects of 3-octen-2-one, which has been shown to modulate vanilla flavors, were analyzed using a human olfactory receptor sensor that uses all human olfactory receptors (388 types) as sensing molecules.

Mechanical detection of interactions between proteins related to intermediate filament and transcriptional regulation in living cells.

Intermediate filaments (IF) bind to various proteins and regulate cell function in the cytoplasm. Recently, IFs were found to regulate gene expression by acting as capture scaffolds for transcription-related proteins and preventing their translocation into the nucleus. To reveal such transcriptional regulatory mechanisms controlled by IFs, a method to analyze the interaction between IFs and transcription-related proteins is necessary.

Engineering of Extracellular Vesicles for Small Molecule-Regulated Cargo Loading and Cytoplasmic Delivery of Bioactive Proteins.

Cytoplasmic delivery of functional proteins into target cells remains challenging for many biological agents to exert their therapeutic effects. Extracellular vesicles (EVs) are expected to be a promising platform for protein delivery; however, efficient loading of proteins of interest (POIs) into EVs remains elusive. In this study, we utilized small compound-induced heterodimerization between FK506 binding protein (FKBP) and FKBP12-rapamycin-binding (FRB) domain to sort bioactive proteins into EVs using the FRB-FKBP system.

Binding of Hepatitis B Virus Pre-S1 Domain-Derived Synthetic Myristoylated Peptide to Scavenger Receptor Class B Type 1 with Differential Properties from Sodium Taurocholate Cotransporting Polypeptide.

(1) Background: The myristoylated pre-S1 peptide (Myr47) synthesized to mimic pre-S1 domain (2-48) in large (L) surface protein of hepatitis B virus (HBV) prevents HBV infection to hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP). We previously demonstrated that yeast-derived nanoparticles containing L protein (bio-nanocapsules: BNCs) bind scavenger receptor class B type 1 (SR-B1). In this study, we examined the binding of Mry47 to SR-B1.

Reporter gene assay for membrane fusion of extracellular vesicles.

Extracellular vesicles (EVs) secreted by living cells are expected to deliver biological cargo molecules, including RNA and proteins, to the cytoplasm of recipient cells. There is an increasing need to understand the mechanism of intercellular cargo delivery by EVs. However, the lack of a feasible bioassay has hampered our understanding of the biological processes of EV uptake, membrane fusion, and cargo delivery to recipient cells.

Binding of Nanoparticles Harboring Recombinant Large Surface Protein of Hepatitis B Virus to Scavenger Receptor Class B Type 1.

(1) Background: As nanoparticles containing the hepatitis B virus (HBV) large (L) surface protein produced in yeast are expected to be useful as a carrier for targeting hepatocytes, they are also referred to as bio-nanocapsules (BNCs). However, a definitive cell membrane receptor for BNC binding has not yet been identified. (2) Methods: By utilizing fluorescence-labeled BNCs, we examined BNC binding to the scavenger receptor class B type 1 (SR-B1) expressed in HEK293T cells.

HBV Pre-S1-Derived Myristoylated Peptide (Myr47): Identification of the Inhibitory Activity on the Cellular Uptake of Lipid Nanoparticles.

The Myr47 lipopeptide, consisting of hepatitis B virus (HBV) pre-S1 domain (myristoylated 2-48 peptide), is an effective commercialized anti-HBV drug that prevents the interaction of HBV with sodium taurocholate cotransporting polypeptide (NTCP) on human hepatocytes, an activity which requires both N-myristoylation residue and specific amino acid sequences. We recently reported that Myr47 reduces the cellular uptake of HBV surface antigen (HBsAg, subviral particle of HBV) in the absence of NTCP expression. In this study, we analyzed how Myr47 reduces the cellular uptake of lipid nanoparticles (including liposomes (LPs) and HBsAg) without NTCP expression.

Polymerized Albumin Receptor of Hepatitis B Virus for Evading the Reticuloendothelial System.

Various strategies, such as optimization of surface chemistry, size, shape, and charge, have been undertaken to develop nanoparticles (NPs) as DDS (drug delivery system) nanocarriers for evading the reticuloendothelial system (RES) in vivo. We previously developed a hollow NP composed of hepatitis B virus (HBV) surface antigen L proteins and lipid bilayers, hereinafter referred to as bio-nanocapsule (BNC), as a nonviral DDS nanocarrier. Such a BNC harbors the HBV-derived human hepatic cell-specific infection mechanism, and intravenously injected BNCs by themselves were shown to avoid clearance by RES-rich organs and accumulate in target tissues.

Binding of liposomes composed of phosphatidylcholine to scavenger receptor class B type 1 and its modulation by phosphatidic acid in HEK293T cells.

In this study, we developed a method to analyze liposomal binding to a cell membrane receptor using fluorescence-labeled liposomes and demonstrated that scavenger class B type 1 (SR-B1) plays a crucial role in binding of liposomes containing phosphatidylcholine (PC) to HEK293T cell membrane and phosphatidic acid (PA) can modulate it. Site-directed mutagenesis of SR-B1 revealed that S112F and T175A mutations in its ectodomain abrogated binding and endocytosis of PC liposomes in HEK293T cells. K151A and K156A mutations attenuated their binding and endocytosis too.

Id2 Represses Aldosterone-Stimulated Cardiac T-Type Calcium Channels Expression.

Aldosterone excess is a cardiovascular risk factor. Aldosterone can directly stimulate an electrical remodeling of cardiomyocytes leading to cardiac arrhythmia and hypertrophy. L-type and T-type voltage-gated calcium (Ca) channels expression are increased by aldosterone in cardiomyocytes.

Real-Time Luminescence Assay for Cytoplasmic Cargo Delivery of Extracellular Vesicles.

Extracellular vesicles (EVs) have been considered to deliver biological cargos between cells and mediate intercellular communication and potential drug delivery carriers. However, the mechanisms that underlie the biological process of EV uptake and cytoplasmic cargo release in recipient cells are largely unknown. Quantitative and real-time assays for the assessment of cargo delivery efficiency inside recipient cells have not been feasible.

Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor.

Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins.

Enhancing antibody-dependent cellular phagocytosis by Re-education of tumor-associated macrophages with resiquimod-encapsulated liposomes.

Tumor-associated macrophages (TAMs) exist in nearly all tumors, and form a major part of the tumor microenvironment. TAMs are divided into two groups: tumor-suppressing M1 type and tumor-promoting M2 type. Most TAMs are educated by the tumor cells to become M2 type, which support tumor growth and make immunotherapy ineffective.

Influence of Nivolumab for Intercellular Adhesion Force between a T Cell and a Cancer Cell Evaluated by AFM Force Spectroscopy.

The influence of nivolumab on intercellular adhesion forces between T cells and cancer cells was evaluated quantitatively using atomic force microscopy (AFM). Two model T cells, one expressing high levels of programmed cell death protein 1 (PD-1) (PD-1 Jurkat) and the other with low PD-1 expression levels (PD-1 Jurkat), were analyzed. In addition, two model cancer cells, one expressing programmed death-ligand 1 (PD-L1) on the cell surface (PC-9, PD-L1) and the other without PD-L1 (MCF-7, PD-L1), were also used.

A regulatory role of scavenger receptor class B type 1 in endocytosis and lipid droplet formation induced by liposomes containing phosphatidylethanolamine in HEK293T cells.

We have recently reported that phosphatidylethanolamine (PE)-containing liposomes are endocytosed and then induce lipid droplets (LDs) in HEK293T cells. In this study, we elucidated a mechanism responsible for endocytosis of PE-containing liposomes and induction of LDs. By using fluorescence-labeled liposomes and flow cytometry, we found that PE-containing liposomes were very efficiently internalized in HEK293T cells.

Digital Pathology Platform for Respiratory Tract Infection Diagnosis via Multiplex Single-Particle Detections.

The variability of bioparticles remains a key barrier to realizing the competent potential of nanoscale detection into a digital diagnosis of an extraneous object that causes an infectious disease. Here, we report label-free virus identification based on machine-learning classification. Single virus particles were detected using nanopores, and resistive-pulse waveforms were analyzed multilaterally using artificial intelligence.

Development of a universal method for the measurement of binding affinities of antibody drugs towards a living cell based on AFM force spectroscopy.

A universal method to measure the binding affinities of antibody drugs towards their targets on the surface of living cells was developed based on atomic force microscopy (AFM) analysis. Nivolumab, an antibody drug targeting programmed cell death 1 (PD-1), was mainly used as a model for this evaluation. The surface of a tip-less AFM cantilever was coated with nano-capsules, on which immunoglobulin G-binding ZZ domains of protein A were exposed, and nivolumab molecules were immobilized on the cantilever through binding between the antibody Fc domains and the ZZ domains, which controlled the molecular orientation of the antibodies.