Perceptive Recommendation Robot: Enhancing Receptivity of Product Suggestions Based on Customers' Nonverbal Cues.

Service robots that coexist with humans in everyday life have become more common, and they have provided customer service in physical shops around the world in recent years. However, their potential in effective sales strategies has not been fully realized due to their low social presence. This study aims to clarify what kind of robot behavior enhances the social presence of service robots and how it affects human-robot interaction and purchasing behavior. We conducted two experiments with a sales robot, Pepper, at a retail shop in Kyoto. In Experiment 1, we showed that the robot's social presence increased and that customers looked at the robot longer when the robot understood human gaze information and was capable of shared attention. In Experiment 2, we showed that the probability of customers picking up products increased when the robot suggested products based on the humans' degree of attention from gaze and posture information. These results indicate that the robot's ability to understand and make utterances about a customer's orientation and attention effectively enhances human-robot communication and purchasing motivation.

Development of a new caged intein for multi-input conditional translation of synthetic mRNA.

mRNA medicines can be used to express therapeutic proteins, but the production of such proteins in non-target cells has a risk of adverse effects. To accurately distinguish between therapeutic target and nontarget cells, it is desirable to utilize multiple proteins expressed in each cell as indicators. To achieve such multi-input translational regulation of mRNA medicines, in this study, we engineered Rhodothermus marinus (Rma) DnaB intein to develop "caged Rma DnaB intein" that enables conditional reconstitution of full-length translational regulator protein from split fragments. By combining the caged Rma DnaB intein, the split translational regulator protein, and target protein-binding domains, we succeeded in target protein-dependent translational repression of mRNA in human cells. In addition, the caged Rma intein showed orthogonality to the previously reported Nostoc punctiforme (Npu) DnaE-based caged intein. Finally, by combining these two orthogonal caged inteins, we developed an mRNA-based logic gate that regulates translation based on the expression of multiple intracellular proteins. This study provides important information to develop safer mRNA medicines.

Tangible document sharing: handing over paper documents across a videoconferencing display.

Conventional techniques for sharing paper documents in teleconferencing tend to introduce two inconsistencies: 1) media inconsistency: a paper document is converted into a digital image on the remote site; 2) space inconsistency: a workspace deliberately inverts the partner's handwriting to make a document easy to read. In this paper, we present a novel system that eliminates these inconsistencies. The media and space inconsistencies are resolved by reproducing a real paper document on a remote site and allowing a user to handover the paper document to a remote partner across a videoconferencing display. From a series of experiments, we found that reproducing a real paper document contributes to a higher sense of information sharing. We also found that handing over a document enhances a sense of space sharing, regardless of whether the document is digital or paper-based. These findings provide insights into designing systems for sharing paper documents across distances.

Embodied, visible, and courteous: exploring robotic social touch with virtual idols.

In recent years, virtual idols have garnered considerable attention because they can perform activities similar to real idols. However, as they are fictitious idols with nonphysical presence, they cannot perform physical interactions such as handshake. Combining a robotic hand with a display showing virtual idols is the one of the methods to solve this problem. Nonetheless a physical handshake is possible, the form of handshake that can effectively induce the desirable behavior is unclear. In this study, we adopted a robotic hand as an interface and aimed to imitate the behavior of real idols. To test the effects of this behavior, we conducted step-wise experiments. The series of experiments revealed that the handshake by the robotic hand increased the feeling of intimacy toward the virtual idol, and it became more enjoyable to respond to a request from the virtual idol. In addition, viewing the virtual idols during the handshake increased the feeling of intimacy with the virtual idol. Moreover, the method of the hand-shake peculiar to idols, which tried to keep holding the user's hand after the conversation, increased the feeling of intimacy to the virtual idol.

Oppositely Charged Nanoparticles Precipitate Not Only at the Point of Overall Electroneutrality.

Precipitation of oppositely charged entities is a common phenomenon in nature and laboratories. Precipitation and crystallization of oppositely charged ions are well-studied and understood processes in chemistry. However, much less is known about the precipitation properties of oppositely charged nanoparticles. Recently, it was demonstrated that oppositely charged gold nanoparticles (AuNPs), also called nanoions, decorated with positively or negatively charged thiol groups precipitate only at the point of electroneutrality of the sample (i.e., the charges on the particles are balanced). Here we demonstrate that the precipitation of oppositely AuNPs can occur not only at the point of electroneutrality. The width of the precipitation window depends on the size and concentration of the nanoparticles. This behavior can be explained by the aggregation of partially stabilized clusters reaching the critical size for their sedimentation in the gravitational field.

Comprehensive Evaluation of Lipid Nanoparticles and Polyplex Nanomicelles for Muscle-Targeted mRNA Delivery.

The growing significance of messenger RNA (mRNA) therapeutics in diverse medical applications, such as cancer, infectious diseases, and genetic disorders, highlighted the need for efficient and safe delivery systems. Lipid nanoparticles (LNPs) have shown great promise for mRNA delivery, but challenges such as toxicity and immunogenicity still remain to be addressed. In this study, we aimed to compare the performance of polyplex nanomicelles, our original cationic polymer-based carrier, and LNPs in various aspects, including delivery efficiency, organ toxicity, muscle damage, immune reaction, and pain. Our results showed that nanomicelles (PEG-PAsp(DET)) and LNPs (SM-102) exhibited distinct characteristics, with the former demonstrating relatively sustained protein production and reduced inflammation, making them suitable for therapeutic purposes. On the other hand, LNPs displayed desirable properties for vaccines, such as rapid mRNA expression and potent immune response. Taken together, these results suggest the different potentials of nanomicelles and LNPs, supporting further optimization of mRNA delivery systems tailored for specific purposes.

Enhancement of bone regeneration by coadministration of angiogenic and osteogenic factors using messenger RNA.

BACKGROUND: Bone defects remain a challenge today. In addition to osteogenic activation, the crucial role of angiogenesis has also gained attention. In particular, vascular endothelial growth factor (VEGF) is likely to play a significant role in bone regeneration, not only to restore blood supply but also to be directly involved in the osteogenic differentiation of mesenchymal stem cells. In this study, to produce additive angiogenic-osteogenic effects in the process of bone regeneration, VEGF and Runt-related transcription factor 2 (Runx2), an essential transcription factor for osteogenic differentiation, were coadministered with messenger RNAs (mRNAs) to bone defects in the rat mandible. METHODS: The mRNAs encoding VEGF or Runx2 were prepared via in vitro transcription (IVT). Osteogenic differentiation after mRNA transfection was evaluated using primary osteoblast-like cells, followed by an evaluation of the gene expression levels of osteogenic markers. The mRNAs were then administered to a bone defect prepared in the rat mandible using our original cationic polymer-based carrier, the polyplex nanomicelle. The bone regeneration was evaluated by micro-computerized tomography (µCT) imaging, and histologic analyses. RESULTS: Osteogenic markers such as osteocalcin (Ocn) and osteopontin (Opn) were significantly upregulated after mRNA transfection. VEGF mRNA was revealed to have a distinct osteoblastic function similar to that of Runx2 mRNA, and the combined use of the two mRNAs resulted in further upregulation of the markers. After in vivo administration into the bone defect, the two mRNAs induced significant enhancement of bone regeneration with increased bone mineralization. Histological analyses using antibodies against the Cluster of Differentiation 31 protein (CD31), alkaline phosphatase (ALP), or OCN revealed that the mRNAs induced the upregulation of osteogenic markers in the defect, together with increased vessel formation, leading to rapid bone formation. CONCLUSIONS: These results demonstrate the feasibility of using mRNA medicines to introduce various therapeutic factors, including transcription factors, into target sites. This study provides valuable information for the development of mRNA therapeutics for tissue engineering.

Patterning Perovskite Quantum Dots Using Photopolymerization.

All-inorganic cesium lead halide perovskite quantum dots (QDs) have several potential applications, owing to their unique optical and electronic properties. However, patterning perovskite QDs using conventional methods is difficult because of the ionic nature of QDs. Here, we demonstrate a unique approach, in which perovskite QDs are patterned in polymer films through the photocuring of monomers under patterned light illumination. The pattern illumination creates the transient polymer concentration difference, which drives the QDs to form patterns; hence controlling polymerization kinetics is essential for the generation of the QD pattern. For the patterning mechanism, a light projection system equipped with a digital micromirror device (DMD) is developed; thus, light intensity, an important factor to determine polymerization kinetics, is precisely controlled per position on the photocurable solution, resulting in the understanding of the mechanism and the formation of distinct QD patterns. The demonstrated approach assisted by the DMD-equipped projection system can form desired perovskite QD patterns solely by patterned light illumination, paving the way for the development of patterning methods for perovskite QDs and other nanocrystals.

Development of Synthetic mRNAs Encoding Split Cytotoxic Proteins for Selective Cell Elimination Based on Specific Protein Detection.

For the selective elimination of deleterious cells (e.g., cancer cells and virus-infected cells), the use of a cytotoxic gene is a promising approach. DNA-based systems have achieved selective cell elimination but risk insertional mutagenesis. Here, we developed a synthetic mRNA-based system to selectively eliminate cells expressing a specific target protein. The synthetic mRNAs used in the system are designed to express an engineered protein pair that are based on a cytotoxic protein, Barnase. Each engineered protein is composed of an N- or C-terminal fragment of Barnase, a target protein binding domain, and an intein that aids in reconstituting full-length Barnase from the two fragments. When the mRNAs are transfected to cells expressing the target protein, both N- and C-terminal Barnase fragments bind to the target protein, causing the intein to excise itself and reconstitute cytotoxic full-length Barnase. In contrast, when the target protein is not present, the reconstitution of full-length Barnase is not induced. Four candidate constructs containing split Barnase were evaluated for the ability to selectively eliminate target protein-expressing cells. One of the candidate sets demonstrated highly selective cell death. This system will be a useful therapeutic tool to selectively eliminate deleterious cells.

Glycocalyx Components Detune the Cellular Uptake of Gold Nanoparticles in a Size- and Charge-Dependent Manner.

Functionalized nanoparticles (NPs) are widely used in targeted drug delivery and biomedical imaging due to their penetration into living cells. The outer coating of most cells is a sugar-rich layer of the cellular glycocalyx, presumably playing an important part in any uptake processes. However, the exact role of the cellular glycocalyx in NP uptake is still uncovered. Here, we in situ monitored the cellular uptake of gold NPs─functionalized with positively charged alkaline thiol (TMA)─into adhered cancer cells with or without preliminary glycocalyx digestion. Proteoglycan (PG) components of the glycocalyx were treated by the chondroitinase ABC enzyme. It acts on chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate and slowly on hyaluronate. The uptake measurements of HeLa cells were performed by applying a high-throughput label-free optical biosensor based on resonant waveguide gratings. The positively charged gold NPs were used with different sizes [d = 2.6, 4.2, and 7.0 nm, small (S), medium (M), and large(L), respectively]. Negatively charged citrate-capped tannic acid (CTA, d = 5.5 nm) NPs were also used in control experiments. Real-time biosensor data confirmed the cellular uptake of the functionalized NPs, which was visually proved by transmission electron microscopy. It was found that the enzymatic digestion facilitated the entry of the positively charged S- and M-sized NPs, being more pronounced for the M-sized. Other enzymes digesting different components of the glycocalyx were also employed, and the results were compared. Glycosaminoglycan digesting heparinase III treatment also increased, while glycoprotein and glycolipid modifying neuraminidase decreased the NP uptake by HeLa cells. This suggests that the sialic acid residues increase, while heparan sulfate decreases the uptake of positively charged NPs. Our results raise the hypothesis that cellular uptake of 2-4 nm positively charged NPs is facilitated by glycoprotein and glycolipid components of the glycocalyx but inhibited by PGs.

Anti-Inflammatory Therapy for Temporomandibular Joint Osteoarthritis Using mRNA Medicine Encoding Interleukin-1 Receptor Antagonist.

Messenger RNA (mRNA) is an emerging drug modality for protein replacement therapy. As mRNA efficiently provides protein expression in post-mitotic cells without the risk of insertional mutagenesis, direct delivery of mRNA can be applied, not only as an alternative to gene therapy, but also for various common diseases such as osteoarthritis (OA). In this study, using an mRNA-encoding interleukin-1 receptor antagonist (IL-1Ra), we attempted anti-inflammatory therapy in a rat model of the temporomandibular joint (TMJ) OA, which causes long-lasting joint pain with chronic inflammation. For the intra-articular injection of mRNA, a polyplex nanomicelle, our original polymer-based carrier, was used to offer the advantage of excellent tissue penetration with few immunogenic responses. While the protein expression was transient, a single administration of IL-1Ra mRNA provided sustained pain relief and an inhibitory effect on OA progression for 4 weeks. The mRNA-loaded nanomicelles provided the encoded protein diffusely in the disc and articular cartilage without upregulation of the expression levels of the pro-inflammatory cytokines IL-6 and tumor necrosis factor-α (TNF-α). This proof-of-concept study demonstrates how anti-inflammatory proteins delivered by mRNA delivery using a polyplex nanomicelle could act to alleviate OA, stimulating the development of mRNA therapeutics.

A protocol to construct RNA-protein devices for photochemical translational regulation of synthetic mRNAs in mammalian cells.

Here, we describe a protocol for the translational regulation of transfected messenger RNAs (mRNAs) using light in mammalian cells. We detail the steps for photocaged ligand synthesis, template DNA preparation, and mRNA synthesis. We describe steps for mRNA transfection, treatment of cells with a photocaged ligand followed by light irradiation, and analysis of the transgene expression. The protocol enables spatiotemporally regulated transgene expression without the risk of insertional mutagenesis. For complete details on the use and execution of this protocol, please refer to Nakanishi et al. (2021).

Self-assembly of graphene oxide flakes for smart and multifunctional coating with reversible formation of wrinkling patterns.

One of the main purposes of smart and multifunctional coatings is to have the versatility to be applied in a wide range of applications. However, the functions of smart materials are often highly limited. In particular, the stimuli-responsive lateral expansion of coatings based on 2D materials has not been reported before. This manuscript describes small two-dimensional graphene oxide (GO) flakes (e.g., thin sheets with a thickness of a few nanometers and much larger lateral dimensions) that act as elementary agents for the formation of smart and multifunctional coatings. The coating can be self-assembled from the GO flakes and disassembled flexibly when required. The coating is stimuli-responsive: upon localized contact with water, it expands and forms wrinkling patterns throughout its whole surface. Evaporating the water allows the wrinkles to disappear; hence, the process is reversible. This stimuli-responsiveness can be controlled to be reduced or completely switched off by temperature or pressure. These features are fundamentally due to the reversible intermolecular interactions among the flakes and favorable packing structure of the coating. The smart coating is shown to be useful for patterned fluidic systems of the desired shapes and the development of channels between fluidic reservoirs via the shortest path. Importantly, these results showed that a simple collection of uniquely 2D elementary agents with small nanoscale thickness can self-assemble into macroscopic materials that perform interactive and multifunctional operations.

Synthetic mRNA for ex vivo therapeutic applications.

Synthetic mRNA is attracting much attention as a new drug modality. Now, there is great interest in the next applications of mRNA medicines, especially for therapeutic purposes of various diseases. Not only in vivo applicable mRNA medicines, there are many researches using ex vivo or in vitro mRNA transfection, some of which are already used in the preclinical or clinical settings. In this short article, the ex vivo mRNA applications are reviewed, in the hope of providing insight to develop future mRNA medicines.

Versatile Design of Intracellular Protein-Responsive Translational Regulation System for Synthetic mRNA.

Synthetic mRNA (mRNA) enables transgene expression without the necessity of nuclear import and the risk of insertional mutagenesis, which makes it an attractive tool for medical applications such as vaccination and protein replacement therapy. For further improvement of mRNA therapeutics, cell-selective translation is desirable, because transgene expression in nontarget cells sometimes causes adverse effects. In this study, we developed an intracellular protein-responsive translational regulation system based on Caliciviral VPg-based translational activator (CaVT) combined with inteins and target protein-binding nanobodies. This system enabled both translational activation and repression in a target protein-dependent manner. Importantly, the target protein can be altered by simply exchanging the nanobodies. The versatile design for target protein-responsive translational regulation holds promise for producing mRNA therapeutics with high safety.

Patterning Silver Nanowires by Inducing Transient Concentration Gradients in Reaction Mixtures.

Patterning nanocrystals in polymer films is essential for the widespread use of nanocrystals in various fields from optics to electronics; therefore, the development of patterning methods for nanocrystals is an important task. Here, we report a unique approach for patterning silver nanowires (AgNWs) using a thermodynamic driving force induced by transient concentration gradients in reaction mixtures. The procedure starts with the preparation of a photocurable monomer solution containing homogeneously dispersed AgNWs. Ultraviolet illumination through a straight-line mask reduces the polymerization rate of monomers in the masked area, decreasing the polymer concentration in comparison with that in the unmasked area. Such transient polymer concentration gradients yield imbalances in the chemical potentials of AgNWs, inducing the migration of AgNWs to form a straight-line pattern of AgNWs. The pattern of AgNWs was visualized via photoluminescence imaging under a laser scanning confocal microscope and compared with the light patterns applied to the mixture. These observations revealed that the magnitude of the AgNW migration is enhanced as the transient concentration gradient increases by thickening the mask to decrease the intensity of light passing through the mask. The structural features of the AgNW pattern were reproduced using numerical simulations based on a set of reaction-diffusion equations, which suggested the key role of the polymerization kinetics characterized by the Trommsdorff-Norrish effect. Moreover, as the AgNW pattern becomes clearer, the electrical resistance along the patterns decreases and more complex patterns can be produced, indicating the potential of the method. Overall, the present patterning method constitutes a simple approach that only requires illumination through a mask to generate the AgNW pattern, which renders it a promising alternative for patterning nanocrystals in polymer films.

Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells.

Synthetic mRNAs, which are produced by in vitro transcription, have been recently attracting attention because they can express any transgenes without the risk of insertional mutagenesis. Although current synthetic mRNA medicine is not designed for spatiotemporal or cell-selective regulation, many preclinical studies have developed the systems for the translational regulation of synthetic mRNAs. Such translational regulation systems will cope with high efficacy and low adverse effects by producing the appropriate amount of therapeutic proteins, depending on the context. Protein-based regulation is one of the most promising approaches for the translational regulation of synthetic mRNAs. As synthetic mRNAs can encode not only output proteins but also regulator proteins, all components of protein-based regulation systems can be delivered as synthetic mRNAs. In addition, in the protein-based regulation systems, the output protein can be utilized as the input for the subsequent regulation to construct multi-layered gene circuits, which enable complex and sophisticated regulation. In this review, I introduce what types of proteins have been used for translational regulation, how to combine them, and how to design effective gene circuits.

Purification of Specific Cell Populations Differentiated from Stem Cells Using MicroRNA-Responsive Synthetic Messenger RNAs.

Pluripotent stem cells have the potential to differentiate into various cell types that can be used for basic biological studies, drug discovery, and regenerative medicine. To obtain reliable results using the differentiated cells, the contamination of nontarget cells should be avoided. microRNAs (miRNAs) can serve as indicators to distinguish target and nontarget cells, because the activities of miRNAs are different among cell types.In this chapter, we introduce a method to purify target cells using synthetic messenger RNAs (mRNAs) that respond to cell-specific miRNAs. The method is composed of five steps: mRNA sequence design, template DNA preparation by PCR, in vitro mRNA transcription, mRNA transfection into cells, and fluorescence-activated cell sorting. This synthetic mRNA-based cell purification method will advance various applications of pluripotent stem cells.

Interfacial structures of particle-stabilized emulsions examined by ultrasonic scattering analysis with a core-shell model.

Pickering emulsions comprising liquid droplets stabilized by solid microparticles have gained much attention in the field of cosmetics, inks, and drug delivery systems. To ensure that microparticles in Pickering emulsions are localized at the surface of liquid droplets, ultrasonic spectroscopy analysis combined with scattering function theory was conducted in this study. Two specific cases were investigated: (1) silica particles and liquid droplets independently dispersed in liquid and (2) silica particles effectively localized at the surface of the droplets. It was found that the core-shell model was effective for analyzing nanoparticles anchored at the surface of oil droplets. Conversely, it was found that an effective shell comprised of solid particles was no longer observed as the particle size or the distance between solid particles increased. When a large solid particle was applied, the ultrasonic spectra resembled those of conventional surfactant-stabilized emulsions without solid stabilizers.