Representing Fine Texture of Pencil Hardness by High-Frequency Vibrotactile Equivalence Conversion Using Ultra-Thin PZT-MEMS Vibrators.

This study aims to represent fine texture differences in pencil hardness using intensity segment modulation (ISM), a sensory equivalent conversion method of vibration from high to low frequencies. This method enables the presentation of delicate tactile sensations even with small transducers. We integrated this approach in the world's thinnest ultra-thin PZT-MEMS vibrator with a stylus-type device. The vibration waveforms of four types of pencil hardness were captured under the same conditions, and the differences in the frequency components were confirmed. We compared the fine texture feelings under raw signal, ISM, and ISM below 1 kHz conditions by conducting discrimination tests and subjective similarity evaluations. The results showed that ISM could reproduce similar feelings of the pencil hardness.

Stereohaptic Vibration: Out-of-Body Localization of Virtual Vibration Source Through Multiple Vibrotactile Stimuli on the Forearms.

This paper proposes a novel concept of "stereohaptic vibration," which employs distributed vibration to localize vibration sources outside the body. Inspired by amplitude panning, a stereophonic sound display technique, we developed a method to localize a virtual vibration source (VVS) by polarizing the perceived intensity of multiple vibration stimuli to a specific orientation. Considering the perceptual characteristics of high-frequency vibration, the perceived intensity of the VVS was allocated to multiple vibrators according to the distance and direction of the target. The velocity discrimination performance was confirmed by utilizing four stimuli around the arm and one vibration stimulus to the palm to localize the movement of a VVS throughout the arm. Discrimination experiments of the trajectory of outgoing objects with a single arm and dual arms revealed that our approach could localize in three dimensions, even outside the body. The proposed technology for localizing external virtual vibration sources is expected to enhance the virtual reality experience.

Wearable High-Resolution Haptic Display Using Suction Stimuli to Represent Cutaneous Contact Information on Finger Pad.

A high-resolution haptic display that reproduces tactile distribution information on the contact surface between a finger and an object realizes the presentation of the softness of the object and the magnitude and direction of the applied force. In this article, we developed a 32-channel suction haptic display that can reproduce tactile distribution on fingertips with high resolution. The device is wearable, compact, and lightweight, thanks to the absence of actuators on the finger. A FE analysis of the skin deformation confirmed that the suction stimulus interfered less with adjacent stimuli in the skin than when pressing with positive pressure, thus allowing more precise control of local tactile stimuli. The optimal layout with the least error was selected from three configurations dividing 62 suction holes into 32 ports.The suction pressures were determined by calculating the pressure distribution by a real-time finite element simulation of the contact between the elastic object and the rigid finger. A discrimination experiment of softness with different Young's modulus and its JND investigation suggested that the higher resolution of the suction display improved the performance of the softness presentation compared to a 16-channel suction display previously developed by the authors.

Development of a remotely controllable 4 m long aerial-hose-type firefighting robot.

In a fire outbreak, firefighters are expected to rapidly extinguish fires to stop the spread of damage and prevent secondary disasters. We proposed the concept of a dragon firefighter (DFF), which is a flying-hose-type firefighting robot. We developed a 3.6 m long DFF equipped with two nozzle units and achieved stable flight. However, the system was not yet completed because the root of the robot, which should have been operated remotely, was operated manually. In addition, the system's reliability was insufficient to successfully repeat the demonstration several times. The development of a robot demonstration system is crucial for the practical application of such a firefighting robot. In this study, we developed a demonstration system for a remotely controllable 4 m flying firehose robot for demonstration at the World Robot Summit 2020 (WRS 2020) opening ceremony in Fukushima as a milestone. This paper focuses on the following issues: 1): installation of the remotely controllable mobile base, 2): redesign of the water channels (the sizes of nozzle outlets) to get enough thrusts to fly with a fire engine, 3): development of nozzle units with a larger movable range (1.5 times larger than the conventional nozzle) in addition to waterproofing technique to improve system reliability, and 4): redesign of a passive damping mechanism to ensure better stability. Thus, a firefighting demonstration was successfully conducted at the opening ceremony of the World Robot Summit 2020 in Fukushima, Japan, and we discuss the lessons learned through the demonstration. We found that the developed DFF system incorporating a mobile base could achieve remote fire extinguishing.

Haptic Exploration During Fast Video Playback: Vibrotactile Support for Event Search in Robot Operation Videos.

Fast playback allows quick video exploration, but it also decreases the saliency of short events. We propose a haptic exploration for detection of short events during fast video playback, considering that event-related information in vibrotactile feedback can be preserved during fast playback using Time Scale Modification (TSM) methods developed for audio. We evaluate our proposal in two collision detection experiments using first-person view (FPV) videos. In the first experiment, viewers watched at a fixed playback speed, i.e., 1× or 2×, videos recorded with a camera mounted on a platform cart. In this experiment, event-related vibrations were measured at the back of the camera. In the second experiment, viewers used a media controller to adjust the playback speed in videos simulating an exploration with a mobile robot. In this experiment, event-related vibrations were generated from the measurements used in the first experiment. We show that a haptic exploration improves collision awareness under either constant or adjustable playback speed. In both experiments, the number of collisions reported without vibrotactile feedback deviated the greatest from the actual number of collisions in a video. Moreover, collision detection performance with vibrations time-scaled without Time Scale Modification (TSM) methods was not significantly different from performance without vibrations.

Introducing Whole Finger Effects in Surface Haptics: An Extended Stick-Slip Model Incorporating Finger Stiffness.

The kinematic serial chain configuration of a finger modulates the frictional properties during tactile exploration tasks. This paper analyzes and subsequently models the effects of the entire finger during sliding operations on a surface. Qualitative and quantitative study of finger movement patterns with postures, sliding directions, and contact angles first indicate the effect of finger stiffness on contact mechanics. A "stiffness ellipse" is subsequently modeled to incorporate finger pose effects, and then coupled with the lumped mass-spring-damper model of the finger pad to estimate resultant contact forces. The performance of the proposed model is verified by comparing with experimental results obtained from ten subjects. The proposed model could estimate the general tendencies of contact forces with change in postures (Extended and Flexed), sliding directions (proximal and distal), and contact angles (20°, 40° and 60°). The experimental results indicate that finger stiffness significantly modulates the contact forces, stick-slip frequency, preloading duration and initial spike during sliding. Introduction of finger posture effects could explain the change in finger normal force during tactile exploration tasks. The proposed haptic rendering model can be used to give a more natural user feedback in virtual fingertip-surface interactions.

Cationic liposomes suppress intracellular calcium ion concentration increase via inhibition of PI3 kinase pathway in mast cells.

Cationic liposomes are commonly used as vectors to effectively introduce foreign genes (antisense DNA, plasmid DNA, siRNA, etc.) into target cells. Cationic liposomes are also known to affect cellular immunocompetences such as the mast cell function in allergic reactions. In particular, we previously showed that the cationic liposomes bound to the mast cell surface suppress the degranulation induced by cross-linking of high affinity IgE receptors in a time- and dose-dependent manner. This suppression is mediated by impairment of the sustained level of intracellular Ca2+ concentration ([Ca2+]i) via inhibition of store-operated Ca2+ entry (SOCE). Here we study the mechanism underlying an impaired [Ca2+]i increase by cationic liposomes in mast cells. We show that cationic liposomes inhibit the phosphorylation of Akt and PI3 kinases but not Syk and LAT. As a consequence, SOCE is suppressed but Ca2+ release from endoplasmic reticulum (ER) is not. Cationic liposomes inhibit the formation of STIM1 puncta, which is essential to SOCE by interacting with Orai1 following the Ca2+ concentration decrease in the ER. These data suggest that cationic liposomes suppress SOCE by inhibiting the phosphorylation of PI3 and Akt kinases in mast cells.

Phosphorylation of syntaxin-3 at Thr 14 negatively regulates exocytosis in RBL-2H3 mast cells.

Recent studies have revealed that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins interact with each other, forming a SNARE complex that induces exocytosis in mast cells. Previously, we reported that syntaxin-3, a SNARE protein, regulates mast cell exocytosis and is constantly phosphorylated. In this study, we tried to identify the amino acid residue that is phosphorylated in mast cells, and to elucidate the regulatory mechanism of exocytosis by phosphorylation in syntaxin-3. We found that Thr 14 of syntaxin-3 was a phosphorylation site in mast cells. In addition, the overexpression of a constitutively dephosphorylated syntaxin-3 (T14A) mutant enhanced mast cell exocytosis. We also showed that the phosphomimetic mutation of syntaxin-3 at Thr 14 (T14E) induced structural changes in syntaxin-3, and this mutation inhibited binding of syntaxin-3 to Munc18-2. These results suggest that phosphorylated syntaxin-3 at Thr 14 negatively regulates mast cell exocytosis by impairing the interaction between syntaxin-3 and Munc18-2.

Effect of Complexin II on Membrane Fusion between Liposomes Containing Mast Cell SNARE Proteins.

Mast cells are involved in allergic responses and undergo exocytotic release of inflammatory mediators in response to antigen stimulation. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in this membrane fusion process; some SNARE-binding proteins regulate SNARE-dependent liposome membrane fusion. SNARE-binding protein complexin II is expressed in mast cells, where it positively regulates exocytotic release after antigen stimulation. We found that complexin II suppressed SNARE-dependent membrane fusion between mast cell SNARE-containing liposomes. This inhibitory effect of complexin II was abolished when we used a structurally divergent mutant (R59H) complexin II, where Arg59 is substituted with histidine. These results suggest that complexin II negatively regulates SNARE-dependent exocytotic membrane fusion in mast cells, and this inhibitory effect is dependent upon Arg59.

Effects of PIP2 on membrane fusion between mast cell SNARE liposomes mediated by synaptotagmin 2.

Recent studies have revealed that SNARE proteins are involved in exocytotic release in mast cells. Previously, we reported that mast cell SNARE proteins induce membrane fusion between liposomes. Moreover, we found that synaptotagmin 2, a candidate Ca2+ sensor for mast cell exocytosis, enhanced SNARE-mediated membrane fusion via Ca2+ and phosphatidylserine. Phosphatidylinositol 4,5-bisphosphate (PIP2) is an acidic phospholipid like phosphatidylserine. In the present study, we investigated whether PIP2 is involved in the enhancement effect of synaptotagmin 2 on SNARE-mediated membrane fusion. PIP2 did not show any significant effect on SNARE-mediated membrane fusion by itself. In the presence of Ca2+, synaptotagmin 2 enhanced SNARE-mediated membrane fusion between liposomes containing PIP2. However, even in the presence of Ca2+, when we used 100% PC liposomes, synaptotagmin 2 did not show any significant effect on SNARE-mediated membrane fusion. These results indicated that PIP2 is involved in the enhancement effect of synaptotagmin 2 on membrane fusion between liposomes containing mast cell SNARE proteins.

Orai-2 is localized on secretory granules and regulates antigen-evoked Ca²âº mobilization and exocytosis in mast cells.

The increase in intracellular Ca(2+) through the Ca(2+) channel is an indispensable step for the secretion of inflammatory mediators by mast cells. It was recently reported that Orai-1 is responsible for the Ca(2+) influx that is activated by depletion of stored Ca(2+). There are three isoforms of Orai: Orai-1, Orai-2, and Orai-3; however, isoforms other than Orai-1 are poorly understood. We found that Orai-2 is expressed and localized on secretory granules in RBL-2H3. Ca(2+) release from Ca(2+) store, induced by antigen stimulation, was significantly attenuated by knockdown of Orai-2, while that induced by thapsigargin was not affected. Furthermore, exocytotic release induced by antigen stimulation was inhibited in knockdown cells. This observation suggests a new role of Orai isoforms in secretory cells.

Inhibitory effects of a cationic liposome on allergic reaction mediated by mast cell activation.

Several studies have shown that cationic liposomes exert immunomodulatory effects with low immunogenicity and toxicity, and offer advantages such as easy preparation and targeting. Cationic liposomes not only transport DNA to immune cells but also enhance the function of antigen presenting cells such as dendritic cells and macrophages. Here, we investigated the effect of a particular cationic liposome on mast cell function during allergic reaction. We found that the cationic liposomes bound to the mast cell surface suppressed degranulation induced by cross-linking of high affinity immunoglobulin E receptors in a time- and dose-dependent manner. The suppression of degranulation was mediated by impairment of the sustained level of intracellular Ca(2+) concentration ([Ca(2+)]i) derived from the inhibition of store-operated Ca(2+) entry. The decrease in sustained elevation of [Ca(2+)]i led to the suppression of phosphorylation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins such as SNAP-23, syntaxin-4, which are necessary for membrane fusion between secretory granules and the plasma membrane during degranulation. Furthermore, the cationic liposomes suppressed vascular permeability elevation induced by mast cell activation in mice. These results showed that cationic liposomes possess the novel property of inhibiting mast cell activation, suggesting the possibility of developing cationic liposomes as anti-allergic effectors.

Detection of asymmetric distribution of phospholipids by fluorescence resonance energy transfer.

It is well established that the plasma membrane exhibits an asymmetric distribution of lipids between the inner and outer leaflets of the lipid bilayer. Recent studies suggest that the asymmetric distribution changes locally and temporarily, accompanied by cellular events. However, available methods to detect lipid asymmetry lack spatio-temporal resolution. As a technique of potential use for real-time imaging of lipid asymmetry, we a novel method that utilizes fluorescence resonance energy transfer (FRET) between NBD-labeled phospholipids (donor) and extracellular rhodamine (acceptor). When cell apoptosis was induced by staurosporine, the fluorescence intensity of NBD-labeled phosphatidylserine decreased owing to FRET from NBD to rhodamine. This method provides a simple way to detect lipid asymmetry and may be useful for observing dynamic changes in asymmetric distribution of lipids.

Endocytosis-like uptake of surface-modified drug nanocarriers into giant unilamellar vesicles.

We had previously developed surface-modified poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for use as a cellular drug delivery system. The cellular uptake of PLGA-NPs was mediated predominantly by endocytosis, and this uptake was increased by surface modifications with polymers, such as chitosan (CS) and polysorbate 80 (P80). In the present study, we prepared a cell-sized giant unilamellar vesicle (GUV) that mimics a cell membrane to investigate the interaction between cell membranes and NPs. Endocytosis-like uptake of NPs into a GUV was observed when the NPs were modified with nonionic surfactant P80 probably due to change in viscoelasticity and enhanced fusion activity of the membrane induced by P80. In contrast, unmodified NPs and those modified with CS were not internalized into a GUV. These results suggest that surface properties of PLGA-NPs are an important formulation parameter for their interaction with lipid membranes.

The suppression of IgE-mediated histamine release from mast cells following exocytic exclusion of biodegradable polymeric nanoparticles.

The objective of this study is to evaluate the effect of polymeric nanoparticles (NPs) on the allergic response of mast cells that release inflammatory mediators such as histamine through exocytosis. Submicron-sized biodegradable poly(DL-lactide-co-glycolide) (PLGA) NPs were prepared by the emulsion solvent diffusion method. Here, we examined the interactions of the mast cells with two types of PLGA NPs, unmodified NPs and NPs modified with chitosan (CS), a biodegradable cationic polymer. The cellular uptake of NPs increased by CS modification due to electrostatic interactions with the plasma membrane. NPs were taken up by mast cells through an endocytic pathway (endocytic phase) and then the cellular uptake was saturated and maintained plateau level by the exclusion of NPs through exocytosis (exocytic phase). Antigen-induced histamine release from mast cells was inhibited during the exocytic phase. The extent of histamine release inhibition was related to the amount of excluded NPs. Exocytic exclusion of NPs competitively antagonize the antigen-induced exocytotic release of histamine by highjacking exocytosis machinery such as SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, since histamine release was recovered in mast cells that overexpress SNAP-23. The inhibitory effect of the allergic response by PLGA NPs was also evaluated in vivo using the mouse model for systemic anaphylaxis. The administration of NPs suppressed the antigen-induced systemic allergic response in vivo. In conclusion, PLGA NP itself has actions to inhibit the allergic responses mediated by mast cells.

Biosurfactant mannosyl-erythritol lipid inhibits secretion of inflammatory mediators from RBL-2H3 cells.

BACKGROUND: Biosurfactant mannosyl-erythritol lipids (MELs) are glycolipids produced by microbes that have various biological activities. It has been reported that MELs exhibit excellent surface-activity and also various bioactivities, such as induction of cell differentiation and apoptosis. However, little is known about their action related to drug discovery or drug seeds. METHODS: We investigated the effects of MELs on the secretion of inflammatory mediators from mast cells that play a central role in allergic responses. Mast cells secrete three kinds of inflammatory mediators and we quantified these secreted mediators by photometer or ELISA. The action mechanisms of MELs were studied by Ca(2+)-sensitive fluorescence dye and Western blotting of phosphorylated proteins. RESULTS: MELs inhibited exocytotic release by antigen stimulation in a dose-dependent manner. We also found that MELs inhibited antigen-induced secretion of leukotriene C(4) and cytokine TNF-α (tumor necrosis factor-α). The inhibitory action of MELs on mediator secretion was mediated by inhibition of Ca(2+) increase, phosphorylation of MAP kinases and SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) that serve as a molecular machinery for exocytotic membrane fusion. CONCLUSIONS: MELs have anti-inflammatory action inhibiting the secretion of inflammatory mediators from mast cells. GENERAL SIGNIFICANCE: MELs affects two of major intracellular signaling pathways including Ca(2+) increase and MAP kinases. MELs also inhibited the phosphorylation of SNARE proteins that is crucial for not only exocytosis but also intracellular vesicular trafficking.

Effects of synaptotagmin 2 on membrane fusion between liposomes that contain SNAREs involved in exocytosis in mast cells.

Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca(2+) concentration, which triggers the exocytotic release of inflammatory mediators such as histamine. Recent research, including our own, has revealed that SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in exocytosis. Although exocytosis in mast cells is Ca(2+) dependent, the target molecule that interacts with Ca(2+) is not clear. Synaptotagmin is a Ca(2+) sensor and regulates exocytosis in neuronal cells. However, the role of synaptotagmin 2, a member of the synaptotagmin family, in exocytosis in mast cells remains controversial. In this study, we investigated the role of synaptotagmin 2 by a liposome-based fusion assay. SNARE proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressed in Escherichia coli and purified as GST-tagged or His-tagged fusion proteins. These SNARE proteins were incorporated by a detergent dialysis method. Membrane fusion between liposomes was monitored by fluorescence resonance energy transfer between fluorescent-labeled phospholipids. In the presence of Ca(2+), low synaptotagmin 2 concentration inhibited membrane fusion between SNARE-containing liposomes, while high synaptotagmin 2 concentration enhanced membrane fusion. This enhancement required phosphatidylserine as a membrane component. These results suggest that synaptotagmin 2 regulates membrane fusion of SNARE-containing liposomes involved in exocytosis in mast cells, and that this regulation is dependent on synaptotagmin 2 concentration, Ca(2+), and phosphatidylserine.

Liprin-α is involved in exocytosis and cell spreading in mast cells.

The active zone is a specialized region of the presynaptic plasma membrane where the neurotransmitter release occurs by exocytosis. Mast cells also release inflammatory mediators by exocytosis resulting in induction of allergic responses. In our previous reports, we found that active zone proteins, Munc13-1 and ELKS regulates exocytosis of mast cell positively. In this study, we investigated the involvement of liprin-α, another active zone protein, in exocytosis in mast cells. We found that three isoforms of liprin-α, liprin-α1, -α2 and -α3 were expressed. Immunocytochemical experiments revealed that liprin-α1 resided both in the cytoplasm and on the plasma membrane. Upon stimulation with antigen, the area of a cell increased remarkably due to cell spreading and the distribution of liprin-α1 became punctuated. Interestingly, knockdown of liprin-α1 caused decrease in exocytotic release and cell spreading. These results suggest that liprin-α1 facilitates exocytosis and cell spreading, and these events might have correlated each other in mast cells.

Latrotoxin-induced exocytosis in mast cells transfected with latrophilin.

α-Latrotoxin (α-LTX) is known to cause massive exocytosis from presynaptic nerve terminals. We investigated the effects of α-LTX on exocytotic release from mast cells, typical non-neuronal secretory cells. When we transfected mast cells with latrophilin, a specific receptor for α-LTX, α-LTX caused intracellular Ca(2+) to increase and led to exocytosis in the presence of extracellular Ca(2+). On the other hand, neither Ca(2+) increase nor exocytosis was observed in the absence of extracellular Ca(2+). These results indicate that α-LTX, together with latrophilin, works as a Ca(2+) ionophore. However, α-LTX had additional effects on signal transduction in mast cells. We found that inhibitors of protein kinase C (PKC) partially suppressed exocytosis. Furthermore, several soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including SNAP-23, were phosphorylated by α-LTX. These results suggest that exocytosis induced by α-LTX can be explained by (1) elevation of intracellular Ca(2+), (2) phosphorylation of SNARE proteins including SNAP-23, syntaxin-4 and VAMP-8 through PKC-dependent and -independent pathways. Our study may provide a new system to investigate the action of α-LTX and the mechanism of exocytosis in mast cells.

Artificial exocytotic system that secretes intravesicular contents upon Ca2+ influx.

Exocytosis is a crucial process of secreting various signaling molecules such as neurotransmitters, hormones, and other chemical mediators into the extracellular space. Exocytotic release is caused by membrane fusion of intracellular vesicles with the plasma membrane triggered by an increase in intracellular Ca(2+). In the present study, we developed an artificial system of exocytosis that secretes intravesicular contents upon Ca(2+) influx. We prepared artificial secretory cells using cell-sized giant unilamellar liposomal vesicles (GUVs) that contain small liposomes (SUVs) that correspond to secretory vesicles. To observe exocytosis-like secretion in an artificial system, we labeled both an intra-SUV solution and an SUV membrane with a soluble fluorescent dye and a rhodamine-labeled phospholipid, respectively. To induce membrane fusion between SUVs and a GUV as observed in exocytosis, the Ca(2+) concentration of intra-GUV was elevated by incorporating ionomycin (a Ca(2+) ionophore) into the GUV membrane. We succeeded in inducing exocytosis-like secretion by Ca(2+) elevation in a GUV together with the osmolarity difference between the intra-GUV and extra-GUV solutions.